ceruletide and Pancreatitis

Acute pancreatitis (AP) is one of the most common diseases in gastroenterology, affecting 2% of all hospitalized patients. Nevertheless, neither the etiology nor the pathophysiology of the disease is fully characterized, and no specific or effective treatment has been developed. Heparanase (Hpa) is an endoglycosidase that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPGs) into shorter oligosaccharides, activity that is highly implicated in cell invasion associated with cancer metastasis and inflammation. Given that AP is a typical inflammatory disease, we investigated whether Hpa plays a role in AP. Our results provide keen evidence that Hpa expression and activity are significantly increased following cerulein-induced AP in wild type mice. In parallel to the classic manifestations of AP, namely elevation of amylase and lipase levels, pancreas edema and inflammation as well as induction of cytokines and signaling molecules, have been detected in this experimental model of the disease. Noteworthy, these features were far more profound in transgenic mice overexpressing heparanase (Hpa-Tg), suggesting that these mice can be utilized as a model system to reveal the molecular mechanism by which Hpa functions in AP. Further support for the involvement of Hpa in the pathogenesis of AP emerged from our observation that treatment of experimental AP with PG545 or SST0001(= Ronepastat), two potent Hpa inhibitors, markedly attenuated the biochemical, histological and immunological manifestations of the disease. Hpa, therefore, emerges as a potential new target in AP, and Hpa inhibitors are hoped to prove beneficial in AP along with their promising efficacy as anti-cancer compounds.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Glucuronidase; Humans; Pancreatitis

Acute pancreatitis refers to the sudden inflammation of the pancreas. It is associated with premature activation and release of digestive enzymes into the pancreatic interstitium and systemic circulation, resulting in pancreatic tissue autodigestion and multiple organ dysfunction, as well as with increased cytokine production, ultimately leading to deleterious local and systemic effects. Although mechanisms involved in pathogenesis of acute pancreatitis have not been completely elucidated, oxidative stress is regarded as a major risk factor. In human acute pancreatitis, lipid peroxide levels in pancreatic tissues increase. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (C22:6n-3), exerts anti-inflammatory and antioxidant effects on various cells. Previous studies have shown that DHA activates peroxisome proliferator-activated receptor-γ and induces catalase, which inhibits oxidative stress-mediated inflammatory signaling required for cytokine expression in experimental acute pancreatitis using cerulein. Cerulein, a cholecystokinin analog, induces intra-acinar activation of trypsinogen in the pancreas, which results in human acute pancreatitis-like symptoms. Therefore, DHA supplementation may be beneficial for preventing or inhibiting acute pancreatitis development. Since DHA reduces serum triglyceride levels, addition of DHA to lipid-lowering drugs like statins has been investigated to reduce hypertriglyceridemic acute pancreatitis. However, high DHA concentrations increase cytosolic Ca

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Docosahexaenoic Acids; Humans; Hypertriglyceridemia; Pancreatitis

Hydrogen sulfide (H2S) and substance P play a key role in inflammation. Using animal models of inflammation of different etiologies such as acute pancreatitis, sepsis, burns, and joint inflammation, studies have recently shown an important role of the proinflammatory action of H2S and substance P. Also, H2S contributes to inflammation in different conditions via substance P. This chapter reviews methods and key data that have led to our current understanding of the role of H2S and substance P in inflammation.

Topics: Acute Disease; Animals; Burns; Carrageenan; Ceruletide; Disease Models, Animal; Drug Synergism; Edema; Endotoxemia; Hydrogen Sulfide; Lipopolysaccharides; Lung Injury; Mice; Pancreatitis; Substance P

Oxidative stress is considered to be an important regulator of the pathogenesis of acute pancreatitis. Reactive oxygen species (ROS) regulate the activation of inflammatory cascades, the recruitment of inflammatory cells and tissue damage in acute pancreatitis. A hallmark of the inflammatory response in pancreatitis is the induction of cytokine expression, which is regulated by a number of signaling molecules including oxidant-sensitive transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases (MAPKs). Cross-talk between ROS and pro-inflammatory cytokines is mediated by NF-κB, AP-1, STAT3, and MAPKs; this crosstalk amplifies the inflammatory cascade in acute pancreatitis. Therapeutic studies have shown that antioxidants and natural compounds can have beneficial effects for patients with pancreatitis and can also influence the expression of proinflammatory cytokines in cerulein-induced pancreatitis. Since oxidative stress may activate inflammatory signaling pathways and contribute to the development of pancreatitis, antioxidant therapy may alleviate the symptoms or prevent the development of pancreatitis. Since chronic administration of high doses of antioxidants may have deleterious effects, dosage levels and duration of antioxidant treatment should be carefully determined.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Disease Models, Animal; Inflammation Mediators; Oxidative Stress; Pancreas; Pancreatitis; Reactive Oxygen Species; Signal Transduction

Melatonin, the main product of the pineal gland, is also released from the gastrointestinal endocrine-neurocrine (EE) cells. The concentrations of melatonin produced in the gut exceeds that originating from central nervous system. In spite of the presence of melatonin receptors in the pancreatic tissue little is known about the role of this indole in the pancreas. Our experimental studies have shown that exogenous melatonin, as well as this produced endogenously from its precursor; L-tryptophan, strongly stimulates pancreatic amylase secretion when given intraperitoneally, or into the gut lumen. This was accompanied by significant increases of CCK plasma level. Above pancreatostimulatory effects of luminal administration of melatonin, were completely reversed by bilateral vagotomy, capsaicin deactivation of sensory nerves or pretreatment of the rats with CCK1 receptor antagonist; tarazepide as well as serotonin antagonist; ketanserin. Melatonin, as well as its precursor; L-tryptophan, effectively protects the pancreas against the damage induced by caerulein overstimulation or ischemia/reperfusion. The beneficial effects of melatonin or L-tryptophan on acute pancreatitis could be related to the ability of melatonin to scavenge the free radicals, to activate antioxidative enzymes and to modulate the cytokine production.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Cholecystokinin; Free Radical Scavengers; Gastrointestinal Tract; Melatonin; Pancreas; Pancreatitis; Receptor, Cholecystokinin A; Reperfusion Injury; Tryptophan

In acute pancreatitis, multiple organ failure in the early phase and infectious complications in the late phase are contributors to mortality. To analyze the mechanism of aggravation of acute pancreatitis is to investigate the mechanism of organ dysfunction and infection. As strategy to elucidate the mechanism, various animal experimental models are utilized. Caerulein-induced pancreatitis and bile salt-induced pancreatitis (duct injection model) are frequently employed for mild edematous pancreatitis and severe necrotizing pancreatitis, respectively. It is important to select an appropriate experimental model that corresponds to the purpose of study.

Topics: Acute Disease; Animals; Apoptosis; Arginine; Bacterial Translocation; Bile Acids and Salts; Ceruletide; Disease Models, Animal; Disease Progression; Duodenum; Ethionine; Humans; Ligation; Lipopolysaccharides; Macrophage Activation; Pancreatitis; Severity of Illness Index

Topics: Acute Disease; Animals; CD11b Antigen; CD11c Antigen; Ceruletide; Chemokines; Endothelium; Humans; Intercellular Adhesion Molecule-1; Liver; Neuropeptides; Pancreatitis; Reactive Oxygen Species; Respiratory Distress Syndrome; Selectins; Signal Transduction; Transcription, Genetic

Autodigestion by proteolytic enzymes is thought to represent the critical mechanism by which acute pancreatitis is initiated. Where and why pancreatic proteases, which are physiologically stored and secreted as inactive precursor zymogens, are activated within the pancreas has remained controversial. Here we present data which indicate that: the lysosomal protease cathepsin B can activate trypsinogen in vitro in a manner that is similar to trypsinogen activation by enterokinase; that cathepsin B colocalizes with trypsinogen in the secretory compartment of the rat pancreas and of the human pancreas; that trypsinogen activation begins in a secretory compartment that is distinct from mature zymogen granules; and that the inhibition of cathepsin B can either increase or decrease premature trypsinogen activation depending on the concentration of the inhibitor, its specificity and its site of action in the pancreatic acinar cell. These observations elucidate some of the complex relations between cysteine and serine proteases in the pancreas with respect to their mechanisms of activation, their subcellular sites of action, and their possible role in the onset of pancreatitis.

Topics: Acute Disease; Animals; Cathepsin B; Ceruletide; Cytoplasmic Granules; Dogs; Enteropeptidase; Enzyme Activation; Humans; Lysosomes; Mice; Pancreas; Pancreatitis; Rats; Trypsin; Trypsinogen

In the last decade, the role of oxidative stress has been extensively evaluated in different experimental models of acute pancreatitis. This review shows that there is strong evidence that this stress occurs as an early phenomenon in pancreatic tissue in the course of caerulein-induced acute pancreatitis. Oxidative stress was documented in pancreatic tissue by means of methods showing generation of reactive oxygen species (e.g., chemiluminescence) and accumulation of products of reactive oxygen species-mediated lipid peroxidation. with concomitant depletion of enzymatic and low molecular weight antioxidants. Features of acinar cell injury and inflammation, especially pancreatic edema, show a marked improvement following treatment with a broad spectrum of antioxidants, platelet activating factor antagonists, or donors of nitric oxide (NO). Unfortunately, in most cases these beneficial effects are temporary and generally restricted to an early phase of the disease. However, results of well-designed clinical trials should finally evaluate the importance of oxidative stress-oriented treatment in acute pancreatitis in humans.

Topics: Acute Disease; Animals; Ceruletide; Gastrointestinal Agents; Humans; Oxidative Stress; Pancreatitis

Topics: Acute Disease; Animals; Cathepsin B; Ceruletide; Disease Models, Animal; Enzyme Activation; Humans; In Vitro Techniques; Lysosomes; Pancreas; Pancreatitis; Trypsinogen

Circumstantial evidence suggests that gallstone-induced pancreatitis is triggered by obstruction of the pancreatic duct. In this report I will review the results of studies conducted during the last decade that have employed the diet-induced, secretagogue-induced, and duct obstruction-induced models of experimental pancreatitis to investigate the early events that lead to the development of acute pancreatitis. In each of these models, digestive enzyme zymogens and the lysosomal hydrolase cathepsin B were found to become colocalized. These observations have led to the hypothesis that intra-acinar cell activation of digestive enzyme zymogens by lysosomal hydrolases may be an important critical event in the development of acute pancreatitis. Recent morphologic studies evaluating the initial 24 hours after ligation of the opossum pancreatic duct indicate that the earliest lesions in this model of hemorrhagic pancreatitis occur in acinar cells.

Topics: Acute Disease; Amino Acids; Amylases; Animals; Ceruletide; Cholelithiasis; Choline Deficiency; Disease Models, Animal; Edema; Enzyme Precursors; Ethionine; Hydrolases; Mice; Pancreatitis; Protein Biosynthesis; Proteins; Rabbits

Intravenous infusion of the synthetic cholecystokinin analogue cerulein at a dose of 0.25 micrograms/kg/h causes maximal stimulation of pancreatic exocrine secretion. The infusion of supramaximal doses of cerulein (5 and 10 micrograms/kg/h) induces a significant increase in pancreatic enzymes in blood, and interstitial edema and inflammatory cell infiltration. This model of hormone-induced pancreatitis works in rats, mice, dogs and hamsters. Besides intravenous infusion, repeated intraperitoneal injections can also be used for induction of pancreatitis. In the early phase of cerulein-induced pancreatitis, large autophagic vacuoles result from fusion of zymogen granules within the acinar cell. This is accompanied by an increase in lysosomal enzyme activity and activation of trypsinogen which finally leads to cellular necrosis. All animals survive the induction of pancreatitis. The pancreas completely regenerates within 6 days after induction of pancreatitis. This model of experimental pancreatitis favors the analysis of intracellular events in the early phase of pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Humans; Infusions, Intravenous; Pancreatitis; Proteins; Regeneration

A membrane-bound system through which secretory and lysosomal proteins travel in a vectorial fashion is essential for the preserved integrity of pancreatic acinar cells. This system is composed of an ordered array of compartments, such as the rough endoplasmic reticulum, the Golgi complex, lysosomes, and secretory granules. As a principle, in acute pancreatitis the final steps of this transport seem to be disturbed. Caerulein-induced pancreatitis is a valuable experimental model for studying altered intracellular transport, and compartmentation of lysosomal and digestive enzymes. The formation of enlarged secretory vacuoles containing lysosomal and digestive enzymes is paralleled by the activation of lysosomes and degradation of cellular organelles in autophagosomes. On the level of secretory and autophagic vacuoles, activation of serine proteases occurs, which in addition to increasing lysosomal enzyme activities can represent the initial stage for acinar cell destruction and the development of pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Humans; Lysosomes; Pancreatitis

The authors examined the effect of long acting somatostatin analogue (Sandostatin, Sandoz) on acute experimental pancreatitis and on the subsequent regeneration. Acute injury to the pancreas was produced by an intraductal intervention (ligature of the bile duct and intraductal injection of taurocholic acid) and by a metabolic route (supramaximal dose of caerulein by repeated subcutaneous injections). The effect of the drug on the acute injury was examined at 6 and 24 hours following the intervention and the effect on regeneration was examined on day 3 and 5 in all cases by determination of plasma enzyme levels and examination of the pancreatic tissue. Long acting somatostatin analogue did not prove to be effective in the serious acute pancreatitis produced by the intraductal intervention. However, in the acute phase of the caerulein induced pancreatitis, it had a beneficial effect as seen by it's ability to moderate the serum enzyme levels. During the examination of pancreatic regeneration was found that in caerulein induced pancreatitis the weight of the pancreas decreases due to atrophy and that this was not affected by long acting somatostatin analogue. As a matter of fact, the somatostatin counteracted the caerulein induced DNA increase, and therefore acted against the reactive hyperplasia. Therefore, the favorable effect of long acting somatostatin analogue is witnessed only in the caerulein induced acute injury but it does not accelerate the rate of pancreatic regeneration following injury. Due to this fact, protracted administration of this agent can not be rationalized.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Drug Evaluation, Preclinical; Octreotide; Pancreas; Pancreatitis; Rats; Rats, Inbred Strains

Topics: Animals; Ceruletide; Choline Deficiency; Diet; Disease Models, Animal; Humans; Pancreas; Pancreatitis

Acute experimental pancreatitis (induced by cerulein) recently has been reported to cause marked diaphragmatic dysfunction, which may contribute to respiratory distress in this setting. In cerulein-induced acute pancreatitis, expression of inducible nitric oxide synthase is induced to produce a large amount of nitric oxide. Nitric oxide excessively produced has been implicated in diaphragmatic dysfunction induced by a variety of etiologies. The aims of the current study were, first, to examine whether nitric oxide overproduced through inducible nitric oxide synthase is involved in cerulein-induced impairment of diaphragmatic function, and second, if demonstrated, to assess effects of ONO-1714, an inducible nitric oxide synthase inhibitor, on diaphragmatic dysfunction associated with cerulein-induced acute pancreatitis.. Prospective, randomized animal study.. University research laboratory.. Ninety-one male Sprague-Dawley rats, weighing 200-250 g.. Rats were randomly divided into seven groups (n = 8 each): CONT-SAL, CAER-SAL, CONT-ONO, CAER-DEX, CAER-AMI, CAER-ONOhigh, and CAER-ONOlow. Groups labeled CAER received two consecutive intraperitoneal doses (50 microg/kg) of cerulein, whereas groups labeled CONT received two consecutive intraperitoneal injections of saline. Groups labeled SAL received intraperitoneal saline before cerulein or saline. The group labeled DEX received 2 mg/kg intraperitoneal dexamethasone, and the group labeled AMI received 100 mg/kg intraperitoneal aminoguanidine. The groups labeled ONO, ONOhigh, and ONOlow received ONO-1714 at 0.1 mg/kg, 0.1 mg/kg, and 0.03 mg/kg, respectively, before cerulein or saline.. Diaphragmatic contractility and fatigability were assessed in vitro by using muscle strips excised from the costal diaphragms 6 hrs after the first dose of cerulein or saline. Expression of inducible nitric oxide synthase protein in the diaphragm was assessed by immunohistochemistry by using anti-inducible nitric oxide synthase antibody. Plasma concentrations of nitrite plus nitrate and diaphragmatic concentrations of malondialdehyde were measured. With another set of rats (n = 5 each group), diaphragmatic inducible nitric oxide synthase activity was determined. Twitch and tetanic tensions and tensions generated during fatigue trial were lower in group CAER-SAL than in group CONT-SAL. Cerulein increased diaphragmatic malondialdehyde and plasma nitrite plus nitrate concentrations. Positive immunostaining for inducible nitric oxide synthase protein was found in group CAER-SAL. Dexamethasone and aminoguanidine attenuated the diaphragmatic mechanical damages. A high dose of ONO-1714 attenuated cerulein-induced impairment of diaphragmatic contractility and endurance capacity, although a low dose of the drug failed to do so.. Cerulein-induced diaphragmatic dysfunction was attributable, in part, to nitric oxide overproduced via inducible nitric oxide synthase. Pretreatment with ONO-1714 at a dose of 0.1 mg/kg attenuated diaphragmatic dysfunction associated with cerulein-induced pancreatitis in rats assessed by contractile profiles and endurance capacity. This beneficial effect of ONO-1714 may be attributable, in part, to inhibition of diaphragmatic lipid peroxidation induced by nitric oxide-derived free radicals.

Topics: Amidines; Analysis of Variance; Animals; Ceruletide; Diaphragm; Disease Models, Animal; Enzyme Inhibitors; Heterocyclic Compounds, 2-Ring; Immunohistochemistry; Lipid Peroxidation; Male; Malondialdehyde; Muscle Contraction; Nitric Oxide; Pancreatitis; Prospective Studies; Rats; Rats, Sprague-Dawley; Respiration Disorders; Statistics, Nonparametric

Faecal elastase 1 (FE1) and the pancreolauryl test (PLT) are widely used for the non-invasive diagnosis of exocrine pancreatic insufficiency (EPI). Whether one of these two tests is superior for the detection of mild-to-moderate EPI is the subject of controversy. The aim of this study was to compare the diagnostic performance of the PLT and FE1 for the detection of EPI in patients with chronic pancreatitis.. Forty consecutive patients (27 males, 13 females, 23-72 years) with chronic pancreatitis based on imaging procedures (computed tomography, endoscopic retrograde pancreatography and endoscopic ultrasound) were admitted to the study. A secretin-caerulein test (SCT) was performed after an overnight fast by giving secretin (1 U/kg/h) and caerulein (100 ng/kg/h) intravenously over 90 min. Duodenal contents were aspirated at 15 min intervals and analysed for pH, bicarbonate, amylase, lipase and elastase. EPI was graded on the basis of the results of the SCT as absent, mild, moderate or severe. A serum PLT was performed in accordance with a modified protocol previously described. A commercial ELISA was used for determination of FE1. The cut-off values were > or = 4.5 mg/l for PLT and > or = 200 microg/g for FE1. and 13 severe) on the basis of the results of the SCT. The sensitivity of the PLT for diagnosing EPI of all degrees of severity was 82% (27/33), compared with 50% for FE1 (16/ 33). In patients with severe EPI, the PLT was abnormal in 100% (13/13) and FE1 was abnormal in 85% (11/13) of the cases. The sensitivity decreases for both tests in the group of mild/moderate EPI (PLT 70% (14/20), FE1 35% (7/20)). In all seven patients with normal exocrine pancreatic function, both PLT and FE1 were also normal.. The PLT is more sensitive than FE1 for the diagnosis of mild-to-moderate EPI, and is therefore more appropriate for completing the staging of chronic pancreatitis.

Topics: Adult; Aged; Ceruletide; Chronic Disease; Feces; Female; Fluoresceins; Humans; Male; Middle Aged; Pancreas; Pancreatic Elastase; Pancreatic Function Tests; Pancreatitis; Secretin; Sensitivity and Specificity; Severity of Illness Index

In order to determine whether the doses of cerulein generally used in the secretin-cerulein test are supramaximal, the pancreatic secretion of enzymes and bicarbonate in response to intravenous infusion of cerulein plus secretin was studied in 6 subjects on two separate days at the respective doses of 50 ng/kg/h and 0.5 CU/kg/h on one day, and 100 ng/kg/h and 1 CU/kg/h on the other. In all subjects studied, the infusion of cerulein at the dose of 100 ng/kg/h caused a pancreatic enzyme response significantly higher than that produced by 50 ng/kg/h, demonstrating that the doses of cerulein generally used in clinical practice to explore the exocrine pancreatic function are not supramaximal.

Topics: Adult; Bicarbonates; Ceruletide; Chronic Disease; Chymotrypsin; Clinical Trials as Topic; Exocrine Pancreatic Insufficiency; Humans; Infusions, Intravenous; Lipase; Male; Middle Aged; Pancreatic Function Tests; Pancreatitis; Random Allocation

Secretin (1 CU/Kg) plus Caerulein (100 ng/Kg) or Cholecystokinin (1 or 2 IvyU/Kg) were given by rapid intravenous injection (Schedule 1) or by continuous infusion (Schedule 2) to 63 control subjects (C) and 69 patients affected by chronic pancreatitis (CP). Duodenal juice was collected for two and four 30-minute periods in schedule 1 and schedule 2, respectively. Volume, bicarbonate, and enzyme content were measured. Secretin-Daerulein, by rapid intravenous injection, showed a strong overlapping between C and CP values and led to some side-effects. Secretin-Caerulein by continuous intravenous infusion gave almost identical results as the Secretin-Cholecystokinin.

Topics: Adolescent; Adult; Aged; Bicarbonates; Ceruletide; Cholecystokinin; Chronic Disease; Clinical Trials as Topic; Duodenum; Female; Humans; Injections, Intravenous; Male; Middle Aged; Pancreas; Pancreatic Juice; Pancreatitis; Secretin

In the present study, we aimed to investigate the effects of Fenbufen treatment on the SAP model induced by caerulein and lipopolysaccharide.. Severe acute pancreatitis (SAP) is an extremely dangerous disease with high mortality, which is associated with inflammatory response and acinar cell death. The caspase family plays an important role in cell death, such as caspase-1 and caspase-11 in pyroptosis. In recent years, caspases have been shown to be a novel pharmacological target of Fenbufen.. Effects of Fenbufen on pancreatic tissue damage and serum levels of lipase and amylase in SAP in mice; Effect of Fenbufen on caspase-1 pathway in SAP in mice; Effect of Fenbufen on caspase-1/caspase-11-mediated pyroptosis of PACs in SAP in mice; Effect of Fenbufen on isolated PACs and caspase-1/caspase-11-mediated pyroptosis in vitro.. In vivo, eighteen female C57BL/6 mice were randomly divided into 3 groups: the NC group, the SAP group, and the Fenbufen +SAP group with 6 mice in each group. The SAP model was induced by intraperitoneal injection of caerulein and lipopolysaccharide. The pathological changes in pancreatic and the serum levels of lipase and amylase and the relative gene and protein expressions in each group were compared. In vitro, pancreatic acinar cells were assigned to 5 groups: medium group, SAP group, Fenbufen 100μM group, Fenbufen 200μM group, and Fenbufen 400μM group. The cell damage and the relative gene and protein expressions in each group were evaluated.. Our results showed that Fenbufen ameliorated the severity of SAP and decreased the serum levels of lipase and amylase. Meanwhile, the in vivo and in vitro data demonstrated that Fenbufen inhibited the activation of caspase-1 and caspase-11, decreasing the levels of IL-1β, IL-18, and GSDMD. In in vitro experiments, we found that by inhibiting the activation of caspase-1 and caspase-11, Fenbufen significantly reduced lactate dehydrogenase (LDH) excretion by acinar cells.. In general, our data showed that Fenbufen could protect the pancreatic acinar cell from injury by inhibiting pyroptosis.

Topics: Acute Disease; Amylases; Animals; Caspase 1; Caspases; Ceruletide; Female; Lipase; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Pancreatitis; Pyroptosis

Acute pancreatitis (AP) is an acute inflammatory condition of pancreas with high morbidity and mortality, which has no effective medical treatment. Chaiqin chengqi decoction (CQCQD) has been clinically used for AP for many years in China. However, the underlying mechanisms are still unknown.. To investigate the mechanism of CQCQD on gasdermin D (GSDMD) -mediated pyroptosis in AP.. In this study, network pharmacology was used to screen the potential mechanism of CQCQD protecting against AP and then we focused to investigate the mechanism of CQCQD on GSDMD mediated pyroptosis. Mouse models of AP were conducted by caerulein and L-arginine. In order to clarify the mechanism of CQCQD, two kinds of GSDMD gene knockout mice (Gsdmd. In the caerulein-induced AP model, CQCQD ameliorated pancreatic pathological injury, attenuated systemic inflammation and serum enzymatic levers. Moreover, network pharmacology analysis showed GSDMD mediated pyroptosis was one of the core targets of CQCQD protecting against AP. Additionally, CQCQD appreciably decreased the levels of pyroptosis-related proteins N-terminal GSDMD, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3, and cleaved Caspase-1. Furthermore, the protective effect of CQCQD was neutralized in Gsdmd. CQCQD could reduce pancreatic necrosis and inflammatory response via inhibiting GSDMD-mediated pyroptosis in acinar cells of AP. Rhein may be the key active ingredient of CQCQD in suppressing pyroptosis.

Topics: Acute Disease; Animals; Ceruletide; Gasdermins; Humans; Mice; Molecular Docking Simulation; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis; Pyroptosis

Gut microbiota dysbiosis induced by acute pancreatitis (AP) exacerbates pancreatic injury and systemic inflammatory responses. The alleviation of gut microbiota dysbiosis through faecal microbiota transplantation (FMT) is considered a potential strategy to reduce tissue damage and inflammation in many clinical disorders. Here, we aim to investigate the effect of gut microbiota and microbiota-derived metabolites on AP and further clarify the mechanisms associated with pancreatic damage and inflammation.. AP rat and mouse models were established by administration of caerulein or sodium taurocholate in vivo. Pancreatic acinar cells were exposed to caerulein and lipopolysaccharide in vitro to simulate AP.. Normobiotic FMT alleviated AP-induced gut microbiota dysbiosis and ameliorated the severity of AP, including mitochondrial dysfunction, oxidative damage and inflammation. Normobiotic FMT induced higher levels of NAD. Gut microbiota-derived NMN alleviates the severity of AP by activating the SIRT3-PRDX5 pathway. Normobiotic FMT could be served as a potential strategy for AP treatment.

Topics: Acute Disease; Animals; Ceruletide; Dysbiosis; Gastrointestinal Microbiome; Inflammation; Mice; NAD; Nicotinamide Mononucleotide; Pancreatitis; Rats; Sirtuin 3

Acute pancreatitis (AP) is an acute inflammatory abdominal disease frequently associated with intestinal barrier dysfunction. Biochanin A (BCA), a dietary isoflavone, has gained increasing interest with its pronounced biological activities. However, its potential beneficial effects on AP have not been demonstrated. Herein, we explored the protective effect of BCA on caerulein-induced AP in BALB/c mice and underlying mechanisms. BCA alleviated AP as evidenced by reduced serum amylase and lipase levels, pancreatic edema, pancreatic myeloperoxidase activity, and improved pancreatic morphology. Amelioration of pancreatic damage by BCA was associated with reduced levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 in both pancreas and colon. Moreover, BCA attenuated AP-associated barrier damage by upregulating the expression of tight junction proteins zonulin occluding (ZO)-1, ZO-2, occludin, and claudin-1. Concomitantly, the translocation of pathogenic bacteria Escherichia coli (E. coli) to pancreas was reduced by BCA. More importantly, reduction of E. coli dissemination by BCA inhibited the TLR4-MAPK/NF-κB signaling and NLRP3 inflammasome activation, thereby protecting against AP and related intestinal injury. Consistently, TLR4 inhibition by TAK-242 pre-treatment counteracted the anti-inflammatory effects of BCA in acinar cells. Taken together, our study extends beneficial effects of BCA to AP prevention, and dietary BCA supplement may be a potential strategy to safeguard AP.

Topics: Acute Disease; Animals; Ceruletide; Escherichia coli; Mice; NF-kappa B; Pancreatitis; Toll-Like Receptor 4

Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear.. To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca. We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO. AZA impaired the ductal HCO

Topics: Acute Disease; Animals; Bicarbonates; Cell Membrane; Ceruletide; Cystic Fibrosis Transmembrane Conductance Regulator; Mice; Pancreatitis

Topics: Animals; Cathepsin B; Ceruletide; Mice; Pancreas; Pancreatitis; Secretagogues; Trypsin; Trypsinogen

Acute pancreatitis (AP) is a frequent abdominal inflammatory disease. Despite the high morbidity and mortality, the management of AP remains unsatisfactory. Disulfiram (DSF) is an FDA-proved drug with potential therapeutic effects on inflammatory diseases. In this study, we aim to investigate the effect of DSF on pancreatic acinar cell necrosis, and to explore the underlying mechanisms. Cell necrosis was induced by sodium taurocholate or caerulein, AP mice model was induced by nine hourly injections of caerulein. Network pharmacology, molecular docking, and molecular dynamics simulation were used to explore the potential targets of DSF in protecting against cell necrosis. The results indicated that DSF significantly inhibited acinar cell necrosis as evidenced by a decreased ratio of necrotic cells in the pancreas. Network pharmacology, molecular docking, and molecular dynamics simulation identified RIPK1 as a potent target of DSF in protecting against acinar cell necrosis. qRT-PCR analysis revealed that DSF decreased the mRNA levels of RIPK1 in freshly isolated pancreatic acinar cells and the pancreas of AP mice. Western blot showed that DSF treatment decreased the expressions of RIPK1 and MLKL proteins. Moreover, DSF inhibited NF-κB activation in acini. It also decreased the protein expression of TLR4 and the formation of neutrophils extracellular traps (NETs) induced by damage-associated molecular patterns released by necrotic acinar cells. Collectively, DSF could ameliorate the severity of mouse acute pancreatitis by inhibiting RIPK-dependent acinar cell necrosis and the following formation of NETs.

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Disulfiram; Mice; Molecular Docking Simulation; Necrosis; Pancreatitis; Receptor-Interacting Protein Serine-Threonine Kinases

Acute pancreatitis can eventually lead to morbidity and mortality. The present study aimed to identify the differentially expressed microRNAs (miRNAs) that are related to acute pancreatitis and explore the in vitro functional role of miR-92b in acute pancreatitis.. Bioinformatics analysis was used to identify differentially expressed miRNAs in caerulein- induced acute pancreatitis samples when compared to normal controls. The role of miR-92b in acute pancreatitis was examined by in vitro functional assays.. MiRNA-network analysis revealed 12 miRNAs that function as "core regulatory miRNAs". Further validation studies revealed that six miRNAs (miR-216a, miR-216b, miR-217, miR- 92b, miR-375 and miR-148a) were differentially expressed in the serum samples from patients with acute pancreatitis. These six miRNAs have fair diagnostic potential for severe acute pancreatitis. Caerulein induced cell injury and inflammatory response and repressed miR-92b expression in AR42J cells. MiR-92b overexpression attenuated caerulein-induced cell injury and inflammatory responses in AR42J cells. Luciferase reporter assay showed that mitogen-activated protein kinase 4 (MAP2K4) was a direct target of miR-92b. MiR-92b overexpression repressed MAP2K4 expression, while caerulein up-regulated MAP2K4 expression in AR42J cells. The rescue experiments showed that enforced expression of MAP2K4 partially reversed the miR-92b-mediated protective effects on caerulein-induced AR42J cell injury.. In conclusion, we identified miR-216a, miR-216b, miR217, miR-92b, miR-375 and miR-148a as new candidate biomarkers for acute pancreatitis. Further in vitro functional studies revealed that miR-92b attenuated caerulein-induced cell injury and inflammatory responses in AJ42R cells partially via targeting MAP2K4.

Topics: Acute Disease; Biomarkers; Ceruletide; Humans; MicroRNAs; Pancreatitis

To explore the possible mechanism of Dachaihu Decoction (DCHD) in the treatment of AP, and use in vivo experiments to verify.. The targets and active ingredients of DCHD in the treatment of AP were obtained through network pharmacology, and the preliminary verification was carried out by molecular docking. Caerulein was used to develop the AP rat model. H&E staining was performed to observe variations in pancreatic tissue. Western blot and RT-qPCR were conducted to evaluate the associated proteins and mRNA.. The network pharmacology and molecular docking results showed that the key targets (EGFR, TNF, SRC, VEGFA and CTNNB1) and key active components (beta-sitosterol, stigmasterol, baicalein, quercetin, and kaempferol) of DCHD in the treatment of AP had good binding. H&E staining revealed that rat pancreatic tissues considerably damaged post caerulein intervention, and it has also been suggested that DCHD ameliorates damage to pancreatic tissue. Simultaneously, EGFR, TNF, SRC, VEGFA protein, and mRNA expression levels were increased in the model group compared to the blank group (. DCHD protects pancreatic tissues and improves symptoms in AP rats by upregulating CTNNB1 protein and mRNA while inhibiting EGFR, TNF, SRC, and VEGFA protein and mRNA expression.

Topics: Acute Disease; Animals; Artificial Intelligence; Ceruletide; Drugs, Chinese Herbal; ErbB Receptors; Molecular Docking Simulation; Pancreatitis; Rats; RNA, Messenger

Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.

Topics: Animals; Ceruletide; Chymotrypsin; Humans; Mice; Mice, Inbred C57BL; Mutation; Pancreatitis; Trypsin; Trypsinogen

Acute pancreatitis (AP) is a disease characterized by local and systemic inflammation with an increasing incidence worldwide. Receptor-interacting serine/threonine protein kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), and innate immune cell macrophages have been reported to be involved in the pathogenesis of AP. However, the mechanisms by which RIPK3 and MLKL regulate pancreatic injury, as well as the interactions between injured pancreatic acinar cells and infiltrating macrophages in AP, remain poorly defined. In the present study, experimental pancreatitis was induced in C57BL/6J, Ripk3

Topics: Acute Disease; Animals; Ceruletide; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Transcription Factors

Acute pancreatitis (AP) is one of the life-threatening diseases of the digestive system. MicroRNA has been asserted to be a regulator of AP. This paper explored the miR-374a-5p expression in AP patients and investigated the efficacy of AR42J cells. In this study, 60 healthy people, 58 MAP patients and 58 SAP patients were included, and the serum miR-374a-5p levels of the subjects were detected by RT-qPCR technology. The pancreatitis cell model was structured by stimulating AR42J cells with cerulein. Next, cell viability and apoptosis were detected by CCK-8 assay and flow cytometry. ELISA was used to measure the concentration of cytokines, such as TNF-α, IL-6, and IL-1β. The data showed that miR-374a-5p was downregulated in samples from AP patients, while showing discriminative power for AP populations. Attenuated miR-374a-5p were negatively bound up with patients' Ranson score and APACHE II score. Besides, miR-374a-5p was declined in cerulein-treated AR42J cells and forced elevation of miR-374a-5p was beneficial to increase cell viability, and inhibit cell apoptosis and inflammation. The present study found that miR-374a-5p was reduced in AP serum samples, and up-regulated expression level of miR-374a-5p in cell models had a protective effect on cerulein-induced inhibition of cell function and inflammatory response.

Topics: Acinar Cells; Acute Disease; Apoptosis; Ceruletide; Humans; MicroRNAs; Pancreatitis

Necroptosis is a necrotic form of regulated cell death, which is primarily mediated by the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) pathway in a caspase-independent manner. Necroptosis has been found to occur in virtually all tissues and diseases evaluated, including pancreatitis. Celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium wilfordii (thunder god vine), possesses potent anti-inflammatory and anti-oxidative activities. Yet, it is unclear whether celastrol has any effects on necroptosis and necroptotic-related diseases. Here we showed that celastrol significantly suppressed necroptosis induced by lipopolysaccharide (LPS) plus pan-caspase inhibitor (IDN-6556) or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). In these in vitro cellular models, celastrol inhibited the phosphorylation of RIPK1, RIPK3, and MLKL and the formation of necrosome during necroptotic induction, suggesting its possible action on upstream signaling of the necroptotic pathway. Consistent with the known role of mitochondrial dysfunction in necroptosis, we found that celastrol significantly rescued TSI-induced loss of mitochondrial membrane potential. TSI-induced intracellular and mitochondrial reactive oxygen species (mtROS), which are involved in the autophosphorylation of RIPK1 and recruitment of RIPK3, were significantly attenuated by celastrol. Moreover, in a mouse model of acute pancreatitis that is associated with necroptosis, celastrol administration significantly reduced the severity of caerulein-induced acute pancreatitis accompanied by decreased phosphorylation of MLKL in pancreatic tissues. Collectively, celastrol can attenuate the activation of RIPK1/RIPK3/MLKL signaling likely by attenuating mtROS production, thereby inhibiting necroptosis and conferring protection against caerulein-induced pancreatitis in mice.

Topics: Acute Disease; Animals; Apoptosis; Caspases; Ceruletide; Mice; Necroptosis; Pancreatitis; Pentacyclic Triterpenes; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases

We aimed to show whether the serum level of Human Epididymitis Protein 4 increases in rats with an experimental acute pancreatitis model created by cerulein.. This study included 24 male Sprague-Dawley rats which were randomly divided into 4 groups each containing 6 rats.. the group treated with saline, Group 1: pancreatitis group created with cerulein at a total dose of 80 µg/kg, Group 2: pancreatitis group created with cerulein at a total dose of 120 µg/kg, Group 3: pancreatitis group created with cerulein at a total dose of 160 µg/kg.. There were statistically significant differences between edema, acinar necrosis, fat necrosis, and perivascular inflammation scores among the study groups. While the degree of all histopathological findings is lowest in the control group, pancreatic parenchyma damage increases as the amount of injected cerulein increases. There was no statistically significant difference between alanine aminotransferase, aspartate aminotransferase, and Human Epididymis Protein 4 values between study groups. On the other hand, there was a statistically significant difference between amylase and lipase values. The lipase value of the control group was significantly lower than the lipase value of the second and third groups. The amylase value of the control group was significantly lower than all other groups. The highest Human Epididymis Protein 4 value was measured as 104 pmol/L in the first pancreatitis group, where the severity of pancreatitis was mild.. In the present study, it was concluded that the Human Epididymis Protein 4 value increased in the case of mild pancreatitis, but there is no correlation between the severity of pancreatitis and the Human Epididymis Protein 4 value.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Epididymitis; Humans; Lipase; Male; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Rats, Wistar

Acute pancreatitis (AP) is an inflammatory disease with high mortality. Previous study has suggested that circular RNAs are dysregulated and involved in the regulation of inflammatory responses in AP. This study aimed to investigate the function and regulatory mechanism underlying mmu_circ_0000037 in caerulein-induced AP cellular model.. Caerulein-treated MPC-83 cells were used as an in vitro cellular model for AP. The expression levels of mmu_circ_0000037, microRNA (miR)-92a-3p, and protein inhibitor of activated STAT1 (Pias1) were detected by quantitative real-time polymerase chain reaction. Cell viability, amylase activity, apoptosis, and inflammatory response were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Amylase Assay Kit, flow cytometry, and enzyme-linked immunosorbent assays. The protein level was quantified by western blot analysis. The target interaction between miR-92a-3p and mmu_circ_0000037 or Pias1 were predicted by StarbaseV3.0 and validated by dual-luciferase reporter assay and RNA immunoprecipitation assay.. Mmu_circ_0000037 and Pias1 levels were decreased, whereas miR-92a-3p expression was elevated in caerulein-induced MPC-83 cells. Overexpression of mmu_circ_0000037 protected MPC-83 cells from caerulein-induced the decrease of cell viability, as well as the promotion of amylase activity, apoptosis and inflammation. MiR-92a-3p was targeted by mmu_circ_0000037, and miR-92a-3p overexpression rescued the effect of mmu_circ_0000037 on caerulein-induced MPC-83 cell injury. Pias1 was confirmed as a target of miR-92a-3p and mmu_circ_0000037 regulated the expression of Pias1 by sponging miR-92a-3p.. Mmu_circ_0000037 relieves caerulein-induced inflammatory injury in MPC-83 cells by targeting miR-92a-3p/Pias1 axis, providing a theoretical basis for the treatment of AP.

Topics: Acute Disease; Amylases; Ceruletide; Humans; MicroRNAs; Pancreatitis; Protein Inhibitors of Activated STAT; RNA, Circular; Small Ubiquitin-Related Modifier Proteins

Hypertriglyceridemia (HTG) is one of the common causes of acute pancreatitis (AP). Hyperlipidemic acute pancreatitis (HTG-AP) is associated with higher mortality owing to its tendency for greater severity and rapid progression. The purpose of this study was to explore the mechanism of involvement of tumor necrosis factor receptor-related factor 6 (TRAF6) in pyroptosis during HTG-AP.. The HTG environment was simulated with palmitic acid treatment in vitro and a high-fat diet in vivo. Cerulein was used to establish the HTG-AP model, followed by genetic and pharmacological inhibition of TRAF6. Pyroptosis activation, inflammatory reaction, and the interaction between TRAF6 and pyroptosis in HTG-AP were assessed.. HTG was found to aggravate the development of pancreatitis, accompanied by increased pyroptosis and enhanced inflammatory response in HTG-AP models. Mechanistically, TRAF6 downregulation decreased the activation of pyroptosis in cerulein-induced HTG-AP.. Collectively, inhibition of TRAF6 improved HTG-AP and the associated inflammation by alleviating pyroptosis.

Topics: Acute Disease; Animals; Ceruletide; Hypertriglyceridemia; Inflammation; Pancreatitis; Pyroptosis; Rats; TNF Receptor-Associated Factor 6

Acute pancreatitis (AP) is an inflammatory process unexpectedly occurring in the pancreas, imposing a substantial burden on healthcare systems. Herein, we aimed to clarify the mechanism of action of phospholipase D2 (PLD2) in cerulein-treated AR42J cells, affording valuable insights into the treatment of AP.. The levels of PLD2, miR-5132-5p, inflammatory factors (interleukin [IL]-10, IL-6, and tumor necrosis factor-α), caspase-3 activity, and apoptosis-related proteins (Bax and Bcl-2) in cerulein-treated AR42J cells were detected using reverse transcription-quantitative polymerase chain, caspase-3 activity, and Western blot analysis. Protein levels of nuclear Factor erythroid 2-Related Factor 2 (Nrf2) and nuclear factor-k-gene binding (NF-κB) were detected by Western blot analysis. TargetScan predicted upstream microRNAs (miRNAs) of PLD2, and the interaction between miR-5132-5p and PLD2 was verified using a luciferase assay.. In cerulein-treated AR42J cells, PLD2 levels were downregulated, while miR-5132-5p expression was upregulated. Overexpression of PLD2 attenuated the cerulein-mediated facilitatory effect on inflammation and apoptosis in AR42J cells by regulating the Nrf2/NFκB pathway. Luciferase reporter analysis revealed that miR-5132-5p targeted PLD2, and miR-5132-5p negatively regulated PLD2. Upregulation of miR-5132-5p expression exacerbated inflammation and apoptosis and reversed the protective effect of PLD2 overexpression on AP.. PLD2 targeted by miR-5132-5p can attenuate cerulein-induced AP in AR42J cells via the Nrf2/NFκB pathway, providing therapeutic targets for patients with AP.

Topics: Acute Disease; Animals; Caspase 3; Ceruletide; Inflammation; MicroRNAs; NF-E2-Related Factor 2; NF-kappa B; Pancreatitis; Rats

Acute pancreatitis (AP) is an inflammatory condition of the pancreas characterized by oxidative stress and inflammation in its pathophysiology. Acetyl-11-keto-β-boswellic acid (AKBA) is an active triterpenoid with antioxidant activity. This article seeks to assess the impact of AKBA on AP and investigate its underlying mechanisms.. Our results confirm that AKBA exerts protective effects against AP in mice by inhibiting oxidative stress in macrophages through the Nrf2/HO-1 Pathway.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Lipase; Macrophages; Mice; Molecular Docking Simulation; NF-E2-Related Factor 2; Oxidative Stress; Pancreatitis; Reactive Oxygen Species

Acute pancreatitis (AP) is a severe disease with high morbidity and mortality in which inflammation and coagulation play crucial roles. The development of inflammation leads to vascular injury, endothelium and leukocytes stimulation, and an increased level of tissue factor, which results in the activation of the coagulation process. For this reason, anticoagulants may be considered as a therapeutic option in AP. Previous studies have shown that pretreatment with heparin, low-molecular-weight heparin (LMWH), or acenocoumarol inhibits the development of AP. The aim of the present study was to check if pretreatment with warfarin affects the development of edematous pancreatitis evoked by cerulein. Warfarin (90, 180, or 270 µg/kg/dose) or saline were administered intragastrically once a day for 7 days consecutively before the induction of AP. AP was evoked by the intraperitoneal administration of cerulein. The pre-administration of warfarin at doses of 90 or 180 µg/kg/dose reduced the histological signs of pancreatic damage in animals with the induction of AP. Additionally, other parameters of AP, such as an increase in the serum activity of lipase and amylase, the plasma concentration of D-dimer, and interleukin-1β, were decreased. In addition, pretreatment with warfarin administered at doses of 90 or 180 µg/kg/dose reversed the limitation of pancreatic blood flow evoked by AP development. Warfarin administered at a dose of 270 µg/kg/dose did not exhibit a preventive effect in cerulein-induced AP. Conclusion: Pretreatment with low doses of warfarin inhibits the development of AP evoked by the intraperitoneal administration of cerulein.

Topics: Acute Disease; Animals; Ceruletide; Heparin, Low-Molecular-Weight; Inflammation; Pancreatitis; Rats; Rats, Wistar; Warfarin

The pathogenesis of acute pancreatitis mainly involves NLRP3 inflammasome-mediated pancreatic cell injury, although regulators of this inflammasome machinery are still not fully identified. Membrane-associated RING-CH 9 (MARCH9) is a member of MARCH-type finger proteins, which regulates innate immunity through catalyzing polyubiquitination of critical immune factors. The aim of present research is to examine the function of MARCH9 in acute pancreatitis.. Cerulein-induced acute pancreatitis was established on pancreatic cell line AR42J and rat model. Reactive oxygen species (ROS) accumulation and NLRP3 inflammasome-dependent cell pyroptosis in pancreas were examined by flow cytometry.. MARCH9 was downregulated by cerulein, but overexpressing MARCH9 could inhibit NLRP3 inflammasome activation and ROS accumulation, thus suppressing pancreatic cell pyroptosis and mitigating pancreatic injury. We further uncovered that the mechanism underlying such an effect of MARCH9 is through mediating the ubiquitination of NADPH oxidase-2, whose deficiency reduces cellular ROS accumulation and inflammasome formation.. Our results suggested that MARCH9 suppresses NLRP3 inflammasome-mediated pancreatic cell injury through mediating the ubiquitination and degradation of NADPH oxidase-2, which compromises ROS generation and NLRP3 inflammasomal activation.

Topics: Acute Disease; Animals; Ceruletide; Inflammasomes; NADPH Oxidases; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatic Hormones; Pancreatitis; Pyroptosis; Rats; Reactive Oxygen Species

Acute pancreatitis (AP), which is characterized by self-digestion of the pancreas by its own prematurely activated digestive proteases, is a major reason for hospitalization. The autodigestive process causes necrotic cell death of pancreatic acinar cells and the release of damage associated molecular pattern which activate macrophages and drive the secretion of pro-inflammatory cytokines. The MYD88/IRAK signaling pathway plays an important role for the induction of inflammatory responses. Interleukin-1 receptor associated kinase-3 (IRAK3) is a counter-regulator of this pathway. In this study, we investigated the role of MYD88/IRAK using Irak3-/- mice in two experimental animal models of mild and severe AP. IRAK3 is expressed in macrophages as well as pancreatic acinar cells where it restrains NFκB activation. Deletion of IRAK3 enhanced the migration of CCR2

Topics: Acute Disease; Adaptor Proteins, Signal Transducing; Animals; Ceruletide; Disease Models, Animal; Inflammation; Mice; Mice, Inbred C57BL; Myeloid Differentiation Factor 88; Necrosis; Pancreas; Pancreatitis; Patient Acuity; Signal Transduction; Systemic Inflammatory Response Syndrome

With the number of patients with acute pancreatitis (AP) increasing year by year, it is pressing to explore new key genes and markers for the treatment of AP. miR-455-3p/solute carrier family 2 member 1 (Slc2a1) obtained through bioinformatics analysis may participate in the progression of AP.. The C57BL/6 mouse model of AP was constructed for subsequent studies. Through bioinformatics analysis, the differentially expressed genes related to AP were screened and hub genes were identified. A caerulein-induced AP animal model was constructed to detect the pathological changes of mouse pancreas by HE staining. The concentrations of amylase and lipase were measured. Primary mouse pancreatic acinar cells were isolated and subjected to microscopy to observe their morphology. The enzymatic activities of trypsin and amylase were detected. The secretion of inflammatory cytokines in mouse were measured with the ELISA kits of TNF-. A total of five (Fyn, Gadd45a, Sdc1, Slc2a1, and Src) were identified by bioinformatics analysis, and miR-455-3p/Slc2a1 were further studied. HE staining results showed that the AP models were successfully established by caerulein induction. In mice with AP, the expression of miR-455-3p was reduced, while that of Slc2a1 was increased. In the caerulein-induced cell model, the expression of Slc2a1 was significantly reduced after intervention of miR-455-3p mimics, whereas increased after miR-455-3p inhibitor treatment. miR-455-3p decreased the secretion of inflammatory cytokines in the cell supernatant, reduced the activity of trypsin and amylase, and alleviated the cell damage induced by caerulein. In addition, Slc2a1 3'UTR region was bound by miR-455-3p, and its protein expression was also regulated.. miR-455-3p alleviated caerulein-induced mouse pancreatic acinar cell damage by regulating the expression of Slc2a1.

Topics: Acinar Cells; Acute Disease; Amylases; Animals; Ceruletide; Cytokines; Mice; Mice, Inbred C57BL; MicroRNAs; Pancreatitis; Trypsin

This research discussed the specific mechanism by which PIAS1 affects acute pancreatitis (AP).. PIAS1, Foxa2, and FTO expression was assessed in Cerulein-induced AR42J cells and mice. Loss- and gain-of-function assays and Cerulein induction were conducted in AR42J cells and mice for analysis. The relationship among PIAS1, Foxa2, and FTO was tested. Cell experiments run in triplicate, and eight mice for each animal group.. Cerulein-induced AP cells and mice had low PIAS1 and Foxa2 and high FTO. Cerulein induced pancreatic injury in mice and inflammation and oxidative stress in pancreatic tissues, which could be reversed by PIAS1 or Foxa2 upregulation or FTO downregulation. PIAS1 elevated SUMO modification of Foxa2 to repress FTO transcription. FTO upregulation neutralized the ameliorative effects of PIAS1 or Foxa2 upregulation on Cerulein-induced AR42J cell injury, inflammation, and oxidative stress.. PIAS1 upregulation diminished FTO transcription by increasing Foxa2 SUMO modification, thereby ameliorating Cerulein-induced AP.

Topics: Acute Disease; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Animals; Ceruletide; Down-Regulation; Hepatocyte Nuclear Factor 3-beta; Inflammation; Mice; Pancreatitis; Sumoylation; Up-Regulation

Acute pancreatitis (AP) is an inflammatory disease of the exocrine pancreas. Disruptions in organelle homeostasis, including macroautophagy/autophagy dysfunction and endoplasmic reticulum (ER) stress, have been implicated in human and rodent pancreatitis. Syntaxin 17 (STX17) belongs to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) subfamily. The Qa-SNARE STX17 is an autophagosomal SNARE protein that interacts with SNAP29 (Qbc-SNARE) and the lysosomal SNARE VAMP8 (R-SNARE) to drive autophagosome-lysosome fusion. In this study, we investigated the role of STX17 in the pathogenesis of AP in male mice or rats induced by repeated intraperitoneal injections of cerulein. We showed that cerulein hyperstimulation induced AP in mouse and rat models, which was characterized by increased serum amylase and lipase activities, pancreatic edema, necrotic cell death and the infiltration of inflammatory cells, as well as markedly decreased pancreatic STX17 expression. A similar reduction in STX17 levels was observed in primary and AR42J pancreatic acinar cells treated with CCK (100 nM) in vitro. By analyzing autophagic flux, we found that the decrease in STX17 blocked autophagosome-lysosome fusion and autophagic degradation, as well as the activation of ER stress. Pancreas-specific STX17 knockdown using adenovirus-shSTX17 further exacerbated pancreatic edema, inflammatory cell infiltration and necrotic cell death after cerulein injection. These data demonstrate a critical role of STX17 in maintaining pancreatic homeostasis and provide new evidence that autophagy serves as a protective mechanism against AP.

Topics: Acute Disease; Animals; Autophagy; Ceruletide; Disease Models, Animal; Edema; Humans; Male; Mice; Pancreatitis; Rats; SNARE Proteins

Ursolic acid (UA) is found in many plants, and has been reported to have anti-protease, antioxidant, anti-inflammatory, antimicrobial, nephroprotective, hepatoprotective, and cardioprotective effects.. The purpose of this study was to investigate the effects of ursolic acid in cerulein-induced acute pancreatitis (AP).. Thirty-two Wistar albino rats were randomly assigned to 4 equal groups: Sham, acute pancreatitis, treatment, and ursolic acid group.. Serum amylase levels in the AP and treatment groups were significantly higher than in the others (p < 0.05). In addition, serum IL-1β, IL-6, and TNF-α levels were significantly higher in the AP group in comparison with the treatment group. Although pancreatic tissue total oxidant activity in the AP and treatment groups was similar, pancreatic tissue total antioxidant capacity was significantly higher in the treatment group than in the AP group.. Damage to the pancreas and remote organs in AP was observed to be reduced by UA. In addition, oxidative stress was observed to be decreased by the effect of UA.. El ácido ursólico se encuentra en numerosas plantas y se ha informado que tiene efectos antiproteasas, antioxidantes, antiinflamatorios, antimicrobianos, nefroprotectores, hepatoprotectores y cardioprotectores.. Este estudio tuvo como objetivo investigar los efectos del ácido ursólico en la pancreatitis aguda inducida por ceruleína.. Treinta y dos ratas albinas Wistar fueron asignadas aleatoriamente a cuatro grupos iguales: grupo simulado, grupo de pancreatitis aguda, grupo de tratamiento y grupo de ácido ursólico.. Los niveles de amilasa sérica en los grupos de pancreatitis aguda y de tratamiento fueron significativamente más altos que en los otros grupos (p < 0.05). Además, los niveles séricos de IL-1β, IL-6 y TNF-α fueron significativamente más altos en el grupo de pancreatitis aguda en comparación con el grupo de tratamiento. Aunque la actividad oxidante total del tejido pancreático en ambos grupos fue similar, la capacidad antioxidante total del tejido pancreático en el grupo de tratamiento fue significativamente mayor.. Se observó que el ácido ursólico reduce el daño al páncreas y órganos remotos en la pancreatitis aguda, al igual que el estrés oxidativo.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Pancreatitis; Rats; Rats, Wistar; Triterpenes; Ursolic Acid

Nafamostat mesilate (NM), a synthetic broad-spectrum serine protease inhibitor, has been commonly used for treating acute pancreatitis (AP) and other inflammatory-associated diseases in some East Asia countries. Although the potent inhibitory activity against inflammation-related proteases (such as thrombin, trypsin, kallikrein, plasmin, coagulation factors, and complement factors) is generally believed to be responsible for the anti-inflammatory effects of NM, the precise target and molecular mechanism underlying its anti-inflammatory activity in AP treatment remain largely unknown.. The protection of NM against pancreatic injury and inhibitory effect on the NOD-like receptor protein 3 (NLRP3) inflammasome activation were investigated in an experimental mouse model of AP. To decipher the molecular mechanism of NM, the effects of NM on nuclear factor kappa B (NF-κB) activity and NF-κB mediated NLRP3 inflammasome priming were examined in lipopolysaccharide (LPS)-primed THP-1 cells. Additionally, the potential of NM to block the activity of histone deacetylase 6 (HDAC6) and disrupt the association between HDAC6 and NLRP3 was also evaluated.. NM significantly suppressed NLRP3 inflammasome activation in the pancreas, leading to a reduction in pancreatic inflammation and prevention of pancreatic injury during AP. NM was found to interact with HDAC6 and effectively inhibit its function. This property allowed NM to influence HDAC6-dependent NF-κB transcriptional activity, thereby blocking NF-κB-driven transcriptional priming of the NLRP3 inflammasome. Furthermore, NM exhibited the potential to interfere the association between HDAC6 and NLRP3, impeding HDAC6-mediated intracellular transport of NLRP3 and ultimately preventing NLRP3 inflammasome activation.. Our current work has provided valuable insight into the molecular mechanism underlying the immunomodulatory effect of NM in the treatment of AP, highlighting its promising application in the prevention of NLRP3 inflammasome-associated inflammatory pathological damage.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Ceruletide; Histone Deacetylase 6; Inflammasomes; Inflammation; Mice; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; NLR Proteins; Pancreatitis

There is an unmet clinical need for effective, targeted interventions to prevent post-ERCP pancreatitis (PEP). We previously demonstrated that the serine-threonine phosphatase, calcineurin (Cn) is a critical mediator of PEP and that the FDA-approved calcineurin inhibitors, tacrolimus (Tac) or cyclosporine A, prevented PEP. Our recent observations in preclinical PEP models demonstrating that Cn deletion in both pancreatic and hematopoietic compartments is required for maximal pancreas protection, highlighted the need to target both systemic and pancreas-specific Cn signaling. We hypothesized that rectal administration of Tac would effectively mitigate PEP by ensuring systemic and pancreatic bioavailability of Tac. We have tested the efficacy of rectal Tac in a preclinical PEP model and in cerulein-induced experimental pancreatitis.. C57BL/6 mice underwent ductal cannulation with saline infusion to simulate pressure-induced PEP or were given seven, hourly, cerulein injections to induce pancreatitis. To test the efficacy of rectal Tac in pancreatitis prevention, a rectal Tac suppository (1 mg/kg) was administered 10 min prior to cannulation or first cerulein injection. Histological and biochemical indicators of pancreatitis were evaluated post-treatment. Pharmacokinetic parameters of Tac in the blood after rectal delivery compared to intravenous and intragastric administration was evaluated.. Rectal Tac was effective in reducing pancreatic injury and inflammation in both PEP and cerulein models. Pharmacokinetic studies revealed that the rectal administration of Tac helped achieve optimal blood levels of Tac over an extended time compared to intravenous or intragastric delivery.. Our results underscore the effectiveness and clinical utility of rectal Tac for PEP prophylaxis.

Topics: Administration, Rectal; Animals; Anti-Inflammatory Agents, Non-Steroidal; Ceruletide; Cholangiopancreatography, Endoscopic Retrograde; Mice; Mice, Inbred C57BL; Pancreatitis; Tacrolimus

Various organosulfur compounds, such as dimethyl trisulfide (DMTS), display anti-inflammatory properties. We aimed to examine the effects of DMTS on acute pancreatitis (AP) and its mechanism of action in both in vivo and in vitro studies. AP was induced in FVB/n mice or Wistar rats by caerulein, ethanol-palmitoleic acid, or L-ornithine-HCl. DMTS treatments were administered subcutaneously. AP severity was assessed by pancreatic histological scoring, pancreatic water content, and myeloperoxidase activity measurements. The behaviour of animals was followed. Pancreatic heat shock protein 72 (HSP72) expression, sulfide, and protein persulfidation were measured. In vitro acinar viability, intracellular Ca

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Mice; Pancreas; Pancreatitis; Rats; Rats, Wistar; Sulfides

Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Iron is an essential element for life and is involved in many metabolic processes. Ferroptosis is a type of regulated cell death that is triggered by iron and oxidative stress. A well-established mouse AP model was adopted to study the role of iron and ferroptosis in the pathogenesis of pancreatitis. Mice were injected with cerulein to induce AP, and pancreatic tissue samples were analyzed to determine the pathology, cell death, iron deposition, expression of iron transporters, and lipid peroxidation. The role of iron was studied by giving mice extra iron or iron chelator. In vitro studies with acinar cells with ferroptosis activator and inhibitor were also performed to assess the inflammatory response. Iron was found accumulated in the pancreatic tissue of mice who suffered cerulein-induced pancreatitis. Cell death and lipid peroxidation increased in these tissues and could be further modulated by iron dextran or iron chelator. Mice given Hemin through gavage had reduced levels of GSH in pancreatic tissue and increased inflammatory response. Studies with acinar cells showed increased levels of lipid peroxidation and ferroptosis-specific mitochondrial damage when treated with ferroptosis inducer and inflammatory cytokines.

Topics: Acute Disease; Animals; Ceruletide; Ferroptosis; Iron; Iron Chelating Agents; Mice; Pancreatitis

Ubiquitin-binding associated protein 2 (UBAP2) is reported to promote macropinocytosis and pancreatic adenocarcinoma (PDAC) growth, however, its role in normal pancreatic function remains unknown. We addressed this knowledge gap by generating UBAP2 knockout (U2KO) mice under a pancreas-specific Cre recombinase (Pdx1-Cre). Pancreatic architecture remained intact in U2KO animals, but they demonstrated slight glucose intolerance compared to controls. Upon cerulein challenge to induce pancreatitis, U2KO animals had reduced levels of several pancreatitis-relevant cytokines, amylase and lipase in the serum, reduced tissue damage, and lessened neutrophil infiltration into the pancreatic tissue. Mechanistically, cerulein-challenged U2KO animals revealed reduced NF-κB activation compared to controls. In vitro promoter binding studies confirmed the reduction of NF-κB binding to its target molecules supporting UBAP2 as a new regulator of inflammation in pancreatitis and may be exploited as a therapeutic target in future to inhibit pancreatitis.

Topics: Acute Disease; Adenocarcinoma; Animals; Ceruletide; Glucose; Inflammation; Mice; NF-kappa B; Pancreas; Pancreatic Neoplasms; Pancreatitis

Acute pancreatitis (AP) is the most common gastrointestinal disease leading to hospitalizations and unexpected deaths. The development of AP leads to damage of the pancreatic microcirculation with a cascade of subsequent events resulting, among others, in coagulopathy. Previous research showed that anticoagulants can be important therapeutic agents. Heparin and acenocoumarol can alleviate the course of AP, as well as accelerate healing and post-inflammatory regeneration of the pancreas. The aim of this study was to determine whether warfarin, a drug with more stable effects than acenocoumarol, affects the healing and regeneration of the pancreas in the cerulein-induced AP. AP was evoked in Wistar male rats by intraperitoneal administration of cerulein. The first dose of warfarin (45, 90 or 180 μg/kg) was administered 24 hours after the first dose of cerulein and the doses of warfarin were repeated once a day in subsequent 10 days. The severity of AP was assessed immediately after the last dose of cerulein, as well as at days 1, 2, 3, 5, and 10 after AP induction. Treatment with warfarin dose-dependently increased international normalized ratio (INR) and attenuated the severity of pancreatitis in histological examination and accelerated pancreatic recovery. These effects were accompanied with a faster reduction in the AP-evoked increase in serum activity of amylase and lipase, the serum concentration of pro-inflammatory interleukin-1β, and the plasma level of D-Dimer. In addition, treatment with warfarin decreased pancreatic weight (an index of pancreatic edema) and improved pancreatic blood flow in rats with AP. The therapeutic effect was particularly pronounced after the administration of warfarin at a dose of 90 μg/kg. We conclude that treatment with warfarin accelerated regeneration of the pancreas and recovery in the course of cerulein-induced mild-edematous acute pancreatitis.

Topics: Acenocoumarol; Acute Disease; Animals; Ceruletide; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Warfarin

Acute pancreatitis is a common and serious inflammatory condition currently lacking disease modifying therapy. The cholinergic anti-inflammatory pathway (CAP) is a potent protective anti-inflammatory response activated by vagus nerve-dependent α7 nicotinic acetylcholine receptor (α7nAChR) signaling using splenic CD4. The effect of galantamine (1-6 mg/kg-body weight) on caerulein-induced acute pancreatitis was evaluated in mice. Two hours following 6 hourly doses of caerulein (50 µg/kg-body weight), organ and serum analyses were performed with accompanying pancreatic histology. Experiments utilizing vagotomy, gene knock out (KO) technology and the use of nAChR antagonists were also performed.. Galantamine attenuated pancreatic histologic injury which was mirrored by a reduction in serum amylase and pancreatic inflammatory cytokines and an increase the anti-inflammatory cytokine IL-10 in the serum. These beneficial effects were not altered by bilateral subdiaphragmatic vagotomy, KO of either choline acetyltransferase. Galantamine improves acute pancreatitis via a mechanism which does not involve previously established physiological and molecular components of the CAP. As galantamine is an approved drug in widespread clinical use with an excellent safety record, our findings are of interest for further evaluating the potential benefits of this drug in patients with acute pancreatitis.

Topics: Acetylcholinesterase; Acute Disease; alpha7 Nicotinic Acetylcholine Receptor; Animals; Anti-Inflammatory Agents; Body Weight; Ceruletide; Cytokines; Galantamine; Humans; Mice; Pancreatitis

Pancreatitis is characterized by acinar cell death and persistent inflammation. Ferroptosis is a type of lipid peroxidation-dependent necrosis, which is negatively regulated by glutathione peroxidase 4. We studied how trypsin, a serine protease secreted by pancreatic acinar cells, affects the contribution of ferroptosis to triggering pancreatitis.. In vitro, the mouse pancreatic acinar cell line 266-6 and mouse primary pancreatic acinar cells were used to investigate the effect of exogenous trypsin on ferroptosis sensitivity. Short hairpin RNAs were designed to silence gene expression, whereas a library of 1080 approved drugs was used to identify new ferroptosis inhibitors in 266-6 cells. In vivo, a Cre/LoxP system was used to generate mice with a pancreas-specific knockout of Gpx4 (Pdx1-Cre;Gpx4. Supraphysiological doses of trypsin (500 or 1000 ng/mL) alone did not trigger significant cell death in 266-6 cells and mouse primary pancreatic acinar cells, but did increase the sensitivity of these cells to ferroptosis upon treatment with cerulein, L-arginine, alcohol, erastin, or RSL3. Proteasome 26S subunit, non-adenosine triphosphatase 4-dependent lipid peroxidation caused ferroptosis in pancreatic acinar cells by promoting the proteasomal degradation of glutathione peroxidase 4. The drug screening campaign identified the antipsychotic drug olanzapine as an antioxidant inhibiting ferroptosis in pancreatic acinar cells. Mice lacking pancreatic Gpx4 developed more severe pancreatitis after cerulein infection or ethanol feeding than control mice. Conversely, olanzapine administration protected against pancreatic ferroptotic damage and experimental pancreatitis in Gpx4-deficient mice.. Trypsin-mediated sensitization to ferroptotic damage increases the severity of pancreatitis in mice, and this process can be reversed by olanzapine.

Topics: Animals; Ceruletide; Disease Models, Animal; Ferroptosis; Mice; Pancreatitis; Trypsin

Circular RNAs (circRNAs) have been demonstrated to play important roles in acute pancreatitis (AP). Herein, this study aimed to investigate the role and mechanism of circRNAs utrophin (circ_UTRN) in AP.. In vitro cultured rat pancreatic acinar cell line AR42J was exposed to caerulein (10 nmol/L) to mimic an AP cell model. The levels of circ_UTRN and microRNA (miR)-320-3p and protein tyrosine kinase 2 (PTK2) were examined using quantitative real-time polymerase chain reaction and Western blot assays. Cell apoptosis was analysed by flow cytometry and Western blot assays. ELISA was employed to detect the levels of tumour necrosis factor-α (TNF-α), IL-1β and IL-6. The binding interaction between miR-320-3p and circ_UTRN or PTK2 was verified using dual-luciferase reporter assay.. The expression of circ_UTRN was decreased by caerulein in pancreatic acinar cells, ectopic overexpression of circ_UTRN reduced inflammation and promoted apoptosis in caerulein-mediated pancreatic acinar cells. In a mechanical study, circ_UTRN served as a sponge of miR-320-3p, and miR-320-3p directly targeted PTK2. Rescue assay suggested that the promotion of apoptosis and inhibition of inflammation induced by circ_UTRN re-expression in caerulein-mediated pancreatic acinar cells were partially abolished by miR-320-3p overexpression or PTK2 knockdown. Besides that, miR-320-3p inhibition impaired caerulein-induced cell apoptosis arrest and inflammation via targeting PTK2.. Up-regulation of circ_UTRN in pancreatic acinar cells attenuates caerulein-evoked cell apoptosis arrest and inflammation enhancement via miR-320-3p/PTK2, suggesting that circ_UTRN/miR-320-3p/PTK2 axis might be engaged in caerulein-induced AP.

Topics: Animals; Apoptosis; Cell Proliferation; Ceruletide; Focal Adhesion Kinase 1; Inflammation; MicroRNAs; Pancreatitis; Rats; RNA, Circular; Signal Transduction

Circular RNA hsa_circ_0073748 (circ_0073748) is upregulated in patients with acute pancreatitis (AP), a clinically common sudden inflammatory response. MicroRNA (miR)-132-3p is a stress-induced factor with high conservation between species. Herein, expression and role of circ_0073748 and miR-132-3p in caerulein-induced pancreatitis were studied. Expression levels of circ_0073748, miR-132-3p, TNF receptor associated factor 3 (TRAF3), Bcl-2 and Bcl-2-associated X protein (Bax) were examined by reverse transcription-quantitative PCR and Western blotting. Cell proliferation was measured by MTS and EdU assays. Flow cytometry and assay kits detected apoptosis, inflammatory, and oxidative responses. Western blotting detected nuclear factor (NF)-κB signaling pathway. Circ_0073748 was upregulated and miR-132-3p was downregulated in AP patients' plasma and human pancreatic ductal HPDE6-C7 cells with caerulein induction. Interfering circ_0073748 and reinforcing miR-132-3p improved cell viability, EdU incorporation, and superoxide dismutase (SOD) activity of caerulein-treated HPDE6-C7 cells but suppressed malonaldehyde (MDA), IL-6 and TNF-α levels and apoptosis rate. Moreover, TRAF3 downregulation was allied with circ_0073748 silencing and miR-132-3p overexpression in caerulein-induced HPDE6-C7 cells. Mechanically, circ_0073748 was identified as a sponge for miR-132-3p to modulate TRAF3 expression, thus establishing a competitive endogenous RNA (ceRNA) regulation model. Notably, circ_0073748 blockage could suppress expressions of phosphorylated P65 (p-P65) and p-IκB in caerulein-induced HPDE6-C7 cells by promoting miR-132-3p and inhibiting TRAF3. Silencing circ_0073748 and upregulating miR-132-3p could alleviate caerulein-induced HPDE6-C7 injury and inactivate canonical NF-κB signal by inhibiting TRAF3. Circ_0073748/miR-132-3p/TRAF3 ceRNA pathway might be one underlying mechanism and therapeutic target of caerulein-induced AP.

Topics: Acute Disease; Apoptosis; Ceruletide; Humans; MicroRNAs; NF-kappa B; Pancreatitis; TNF Receptor-Associated Factor 3

T7K24R mice carry mutation p.K24R in mouse cationic trypsinogen (isoform T7), which is analogous to the human hereditary pancreatitis-associated mutation p.K23R. The mutation renders trypsinogen more prone to autoactivation. We recently reported that T7K24R mice exhibit increased severity of acute pancreatitis induced by repeated cerulein injections. The objective of the present study was to test whether trypsinogen mutant mice are prone to develop chronic pancreatitis, as observed in patients. We characterized the natural course of cerulein-induced pancreatitis in T7K24R mice and the C57BL/6N parent strain from the acute episode to 3 months post-attack. As expected, an acute episode of pancreatitis in C57BL/6N mice was followed by rapid recovery and histological restitution. In stark contrast, T7K24R mice developed progressive chronic pancreatitis with acinar cell atrophy, persistent macrophage infiltration, and diffuse fibrosis. The nadir of pancreas damage occurred on days 5-6 after the acute episode and was accompanied by digestive dysfunction. Remarkably, histological recovery was markedly delayed and permanent, chronic changes were still detectable 1-3 months after the acute pancreatitis episode. We conclude that during cerulein-induced acute pancreatitis in T7K24R mice, trypsin triggers an autonomous inflammatory program resulting in chronic disease progression, even after the cessation of cerulein-mediated injury. We propose that this uniquely trypsin-dependent mechanism explains the development of hereditary chronic pancreatitis in humans. Trypsin inhibition during acute attacks should prevent or delay progression to chronic disease.

Topics: Acute Disease; Animals; Ceruletide; Humans; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Trypsinogen

The anti-inflammatory and anti-apoptotic properties of crocetin have been widely demonstrated in numerous diseases. However, the exact role and mechanism of crocetin in acute pancreatitis have not been elucidated. Thus, this paper aims at exploring whether crocetin could be used to alleviate acute pancreatitis and further demonstrating the underlying mechanisms. Cell viability of caerulein-induced pancreatic exocrine cell line AR42J treated with crocetin was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). Apoptosis and inflammation of these treated cells were detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), western blot and enzyme linked immunosorbent assay (ELISA). The expression of sirtuin-1 (SIRT1) was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. After knockdown of SIRT1, cell viability, apoptosis and inflammation were measured again by corresponding kits. Finally, the NF-κB nuclear translocation and proteins in the NF-κB signaling were examined. Crocetin remarkably suppressed the apoptosis and inflammation of caerulein-induced AR42J cells. The decreased expression of SIRT1 was increased in caerulein-induced AR42J cells after exposure to crocetin. After knockdown of SIRT1, the alleviative effects of crocetin were found to be canceled in these cells. Furthermore, SIRT1 knockdown promoted the NF-κB signal transduction. On the whole, we presented the first evidence for the importance of SIRT1-NF-κB axis in acute pancreatitis and proposed that crocetin alleviates the caerulein-induced apoptosis and inflammation in AR42J cells by activating SIRT1 via NF-κB.

Topics: Acute Disease; Apoptosis; Carotenoids; Ceruletide; Humans; Inflammation; NF-kappa B; Pancreatitis; Sirtuin 1; Vitamin A

Acute pancreatitis (AP) is an acute inflammatory disease that can lead to death. Mir-325-3p is strongly and abnormally expressed in many diseases, necessitating exploration of its function and mechanism in AP.. Blood samples from AP patients and mice were analyzed. The expression levels of miR-325-3p in AP patients and mouse were detected. Whether miR-325-3p targets RIPK3 gene was predicted by TargetScan online database and dual luciferase reporter assay. In vitro experiments verified the effect of miR-325-3p overexpression on caerulein-induced MPC83 pancreatic acinar cancer cell line. In vivo experiments verified the effect of overexpression of miR-325-3p on the disease degree of pancreatic tissues in AP mice.. Analysis of blood samples from AP patients and experiments in mice demonstrated that expression of miR-325-3p was significantly reduced during the process of AP in humans and mice. Predicted using the TargetScan online database and through dual luciferase reporter assay detection, miR-325-3p directly targets the RIPK3 gene. In vitro experiments revealed that overexpression of miR-325-3p reversed caerulein-induced apoptosis and necroptosis in MPC83 pancreatic acinar cancer cell line. We used Z-VAD-FMK to assess necroptosis and demonstrated that miR-325-3p targets necroptosis to reduce cell damage. In subsequent experiments in mice, we verified that overexpression of miR-325-3p reduces inflammation, edema, hemorrhage, and necrosis in acute pancreatitis. Characteristic western blot, immunohistochemistry, and transmission electron microscopy results revealed that overexpression of miR-325-3p reduces the severity of acute pancreatitis by inhibiting pancreatic necroptosis in AP mice.. The current research results indicate that miR-325-3p directly targets RIPK3 and exerts a protective role in mouse AP. Necroptosis is still the primary mechanism of RIPK3 regulation. MiR-325-3p inhibits acute pancreatitis by targeting RIPK3-dependent necroptosis, which may represent a novel treatment method for acute pancreatitis.

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Humans; Mice; MicroRNAs; Pancreatitis; Receptor-Interacting Protein Serine-Threonine Kinases

Triptolide (TP), the main active ingredient of

Topics: Acute Disease; Animals; Antioxidants; Ceruletide; China; Disease Models, Animal; Diterpenes; Epoxy Compounds; Gene Expression Regulation; Hep G2 Cells; Humans; Inflammation; Male; Mice; Mice, Inbred ICR; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Phenanthrenes; Reactive Oxygen Species

The excessive inflammatory response mediated by macrophage is one of the key factors for the progress of acute pancreatitis (AP). Paeonol (Pae) was demonstrated to exert multiple anti-inflammatory effects. However, the role of Pae on AP is not clear. In the present study, we aimed to investigate the protective effect and mechanism of Pae on AP in vivo and vitro. In the caerulein-induced mild acute pancreatitis (MAP) model, we found that Pae administration reduced serum levels of amylase, lipase, IL-1β and IL-6 and alleviated the histopathological manifestations of pancreatic tissue in a dose-dependent manner. And Pae decrease the ROS generated, restore mitochondrial membrane potential (ΔΨm), inhibit M1 macrophage polarization and NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) in vitro. In addition, specific NLRP3 inhibitor MCC950 eliminated the protective effect of Pae on AP induced by caerulein in mice. Correspondingly, the inhibitory effect of Pae on ROS generated and M1 polarization was not observed in BMDMs with MCC950 in vitro. Taken together, our datas for the first time confirmed the protective effects of Pae on AP via the NLRP3 inflammasomes Pathway.

Topics: Acetophenones; Acute Disease; Animals; Ceruletide; Inflammasomes; Macrophages; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis; Reactive Oxygen Species

Proper mitochondrial function and adequate cellular ATP are necessary for normal pancreatic protein synthesis and sorting, maintenance of intracellular organelles and enzyme secretion. Inorganic phosphate is required for generating ATP and its limited availability may lead to reduced ATP production causing impaired Ca

Topics: Acute Disease; Adenosine Triphosphate; Animals; Ceruletide; Cholecystokinin; Hypophosphatemia; Ion Channels; Mice; Pancreas; Pancreatitis; Phosphates

Mounting evidence suggests that long non-coding RNAs (lncRNAs) and microRNAs exert a critical regulatory role in acute pancreatitis. The present study aimed to explore the role of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in acute pancreatitis (AP) that was induced by caerulein in rat pancreatic acinar cells (AR42J). The potential target sites of lncRNA NEAT1 and miR-365a-3p were predicted using starBase and were confirmed using dual-luciferase reporter assay. Reverse transcription-quantitative polymerase chain reaction was performed to assess lncRNA NEAT1 and miR-365a-3p expression levels in AP induced by caerulein. Cell Counting Kit-8 and flow cytometry assays were performed to assess AR42J cell viability. Western blotting was performed to evaluate the expression of apoptosis-related proteins. Interleukin (IL)-1β, IL-6, and tumor necrosis factor-α levels were detected by ELISA. The results of the dual-luciferase reporter assay confirmed that miR-365a-3p could bind to NEAT1. LncRNA NEAT1 was upregulated in AR42J cells treated with 10 nmol/l caerulein, and miR-365a-3p was expressed at low levels in an AP model. Overexpression of miR-365a-3p suppressed the apoptosis and inflammatory response of AR42J cells induced by caerulein. Importantly, inhibition of lncRNA NEAT1 decreased apoptosis and inflammation in caerulein-treated AR42J cells, while these effects were reverted upon co-transfection with a miR-365a-3p inhibitor. In conclusion, lncRNA NEAT1 was involved in AP progression by sponging miR-365a-3p and may thus be a novel target for treating patients with AP.

Topics: Acute Disease; Animals; Apoptosis; Ceruletide; Down-Regulation; MicroRNAs; Pancreatitis; Rats; RNA, Long Noncoding

Innate immunity and metabolites link to the pathogenesis and severity of acute pancreatitis (AP). However, liver metabolism and its role in immune response and AP progression remain elusive. We investigated the function of liver metabolism in the pathogenesis of AP.. Circulating ketone body β-hydroxybutyrate (βOHB) levels were determined in AP clinical cohorts and caerulein-induced AP (CER-AP) mouse models receiving seven (Cer*7) or twelve (Cer*12) injection regimens at hourly intervals. Liver transcriptomics and metabolomics were compared between CER-AP (Cer*7) and CER-AP (Cer*12). Inhibition of fatty acid β-oxidation (FAO)-ketogenesis, or supplementation of βOHB was performed in mouse models of AP. The effect and mechanism of βOHB were examined in vitro.. Elevated circulating βOHB was observed in patients with non-severe AP (SAP) but not SAP. These findings were replicated in CER-AP (Cer*7) and CER-AP (Cer*12), which manifested as limited and hyperactive immune responses, respectively. FAO-ketogenesis was activated in CER-AP (Cer*7), while impaired long-chain FAO and mitochondrial function were observed in the liver of CER-AP (Cer*12). Blockage of FAO-ketogenesis (Cpt1a antagonism or Hmgcs2 knockdown) worsened, while supplementation of βOHB or its precursor 1,3-butanediol alleviated the severity of CER-AP. Mechanistically, βOHB had a discernible effect on pancreatic acinar cell damage, instead, it greatly attenuated the activation of pancreatic and systemic proinflammatory macrophages via class I histone deacetylases.. Our findings reveal that hepatic ketogenesis is activated as an endogenous protective programme to restrain AP progression, indicating its potential therapeutic value.. This work was supported by the National Natural Science Foundation of China, Shanghai Youth Talent Support Programme, and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant.

Topics: 3-Hydroxybutyric Acid; Acute Disease; Adolescent; Animals; Ceruletide; China; Disease Models, Animal; Humans; Ketone Bodies; Macrophage Activation; Mice; Pancreatitis

Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.

Topics: Acinar Cells; Animals; Ceruletide; Mice; Pancreas; Pancreatic Neoplasms; Pancreatitis

The expression of amino acid transporters is known to vary during acute pancreatitis (AP) except for LAT1 (. To evaluate the role of LAT1 in the development of and recovery from AP.. AP was induced with caerulein (cae) injections in female and male mice expressing LAT1 or after its knockout (LAT1 Cre/LoxP). The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight, assessing microscopical tissue architecture, mRNA and protein expression, protein synthesis, and enzyme activity levels, as well as by testing the recruitment of immune cells by FACS and ELISA.. The initial injury, evaluated by measurements of plasma amylase, lipase, and trypsin activity, as well as the gene expression of dedifferentiation markers, did not differ between the groups. However, early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice. Especially in females, we observed less mTOR reactivation and dysfunctional autophagy. The later regeneration phase was clearly delayed in female LAT1 knockout mice, which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein (rbpjl) and basic helix-loop-helix family member A15 (mist1). Amylase mRNA and protein levels remained lower, and, strikingly, female LAT1 knockout mice presented signs of fibrosis lasting until day seven. In contrast, pancreas morphology had returned to normal in wild-type littermates.. LAT1 supports the regeneration of acinar cells after AP. Female mice lacking LAT1 exhibited more pronounced alterations than male mice, indicating a sexual dimorphism of amino acid metabolism.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Female; Large Neutral Amino Acid-Transporter 1; Male; Mammals; Mice; Mice, Knockout; Pancreas; Pancreatitis; RNA, Messenger; TOR Serine-Threonine Kinases

Severe acute pancreatitis (SAP), as a typical acute inflammatory injury disease, is one of the acute gastrointestinal diseases with a remarkable mortality rate. Macrophages, typical inflammatory cells involved in SAP, play an important role in the pathogenesis of SAP, which are separated into proinflammation M1 and antiinflammation M2. Growth and differentiation factor 11 (GDF11), as a member of the TGF-β family also called BMP-11, has been discovered to suppress inflammation. However, the mechanism by which GDF11 inhibits inflammation and whether it can ameliorate SAP are still elusive. The present research aimed to investigate the roles of GDF11 in SAP and the potential immunomodulatory effect of macrophage polarization. The mouse and rat SAP model were constructed by caerulein and retrograde injection of sodium taurocholate respectively. The effects of GDF11 on SAP were observed by serology, histopathology and tissue inflammation, and the effects of GDF11 on the polarization of macrophages in vivo were observed. Raw264.7 and THP1 crells were used to study the effect of GDF11 on macrophage polarization in vitro. To further investigate the causal link underneath, our team first completed RNA and proteome sequencing, and utilized specific suppressor to determine the implicated signal paths. Herein, we discovered that GDF11 alleviated the damage of pancreatic tissues in cerulein induced SAP mice and SAP rats induced by retrograde injection of sodium taurocholate, and further found that GDF11 facilitated M2 macrophage polarization and diminished M1 macrophage polarization in vivo and in vitro. Subsequently, we further found that the regulation of GDF11 on macrophage polarization through TGFβR1/smad2 pathway. Our results revealed that GDF11 ameliorated SAP and diminished M1 macrophage polarization and facilitated M2 macrophage polarization. The Role of GDF11 in modulating macrophage polarization might be one of the mechanisms by which GDF11 played a protective role in pancreatic tissues during SAP.

Topics: Acute Disease; Animals; Ceruletide; Growth Differentiation Factors; Humans; Inflammation; Macrophage Activation; Macrophages; Mice; Pancreatitis; Rats; RAW 264.7 Cells; Receptor, Transforming Growth Factor-beta Type I; Smad2 Protein; Taurocholic Acid; THP-1 Cells

This study was to investigate the changes of autophagy in pancreatic tissue cells from hyperlipidemic acute pancreatitis (HLAP) rats and the molecular mechanism of autophagy to induce inflammatory injury in pancreatic tissue cells. Male Sprague Dawley (SD) rats were intraperitoneally injected with caerulein to establish acute pancreatitis (AP) model and then given a high fat diet to further prepare HLAP model. The HLAP rats were treated with autophagy inducer rapamycin or inhibitor 3-methyladenine. Pancreatic acinar (AR42J) cells were treated with caerulein to establish HLAP cell model. The HLAP cell model were treated with rapamycin or transfected with vascular endothelial growth factor (VEGF) siRNA. The inflammatory factors in serum and cell culture supernatant were detected by ELISA method. The histopathological changes of pancreatic tissue were observed by HE staining. The changes of ultrastructure and autophagy in pancreatic tissue were observed by electron microscopy. The expression levels of Beclin-1, microtubule- associated protein light chain 3-II (LC3-II), mammalian target of rapamycin complex 1 (mTORC1), and VEGF were measured by immunohistochemistry and Western blot. The results showed that, compared with control group, the autophagy levels and inflammatory injury of pancreatic tissue cells from HLAP model rats were obviously increased, and these changes were aggravated by rapamycin treatment, but alleviated by 3-methyladenine treatment. In HLAP cell model, rapamycin aggravated the autophagy levels and inflammatory injury, whereas VEGF siRNA transfection increased mTORC1 protein expression, thus alleviating the autophagy and inflammatory injury of HLAP cell model. These results suggest that VEGF-induced autophagy plays a key role in HLAP pancreatic tissue cell injury, and interference with VEGF-mTORC1 pathway can reduce the autophagy levels and alleviate the inflammatory injury. The present study provides a new target for prevention and treatment of HLAP.

Topics: Acute Disease; Animals; Autophagy; Ceruletide; Male; Mammals; Mechanistic Target of Rapamycin Complex 1; Microtubule-Associated Proteins; Pancreatitis; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Sirolimus; Vascular Endothelial Growth Factor A

Circular RNAs (circRNAs) have been found to be involved in the progression of acute pancreatitis (AP). The objective of our study was to investigate the effects of circ_0000284 on caerulein-induced AR42J cell injury. To mimic AP in vitro, rat pancreatic acinar AR42J cells were treated with caerulein. The expression of circ_0000284 and miR-10a-5p was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Enzyme-linked immunosorbent assay (ELISA) was employed to determine the content of inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor α (TNF-α). Western blotting was applied to analyze the levels of Wnt/β-catenin pathway-related and apoptosis-related proteins. Cell viability and apoptosis were monitored by Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The target connection between circ_0000284 and miR-10a-5p was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. AP induced inflammation in patients, and caerulein treatment increased apoptosis and inflammation in AR42J cells. Circ_0000284 was upregulated in serum of AP patients and caerulein-induced AR42J cells, while Wnt/β-catenin pathway was inactivated. Knockdown of circ_0000284 could decrease apoptosis and inflammation in caerulein-induced AR42J cells, which was attenuated by miR-10a-5p inhibition or Wnt signaling pathway antagonist Dickkopf-related protein 1 (DKK1). MiR-10a-5p was sponged by circ_000028 and was downregulated in caerulein-induced AR42J cells. Circ_0000284 depletion could protect caerulein-induced AR42J cells from apoptosis and inflammation by upregulating miR-10a-5p expression and activating Wnt/β-catenin pathway, underscoring a potential target for AP therapy.

Topics: Acute Disease; Animals; beta Catenin; Ceruletide; Humans; Inflammation; MicroRNAs; Pancreatitis; Rats; Wnt Signaling Pathway

Necroptosis is a form of regulated necrosis mainly controlled by receptor-interacting protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Necroptosis has important roles in defensing against pathogenic infections, but it is also implicated in various inflammatory diseases including pancreatitis. Baicalin, a flavonoid from Scutellaria baicalensis Georgi, has been shown to possess anti-inflammatory and anti-pyroptosis properties, yet it is unclear whether baicalin can inhibit necroptosis and confer protection against necroptosis-related diseases. Here we reported that baicalin significantly inhibited necroptosis in macrophages induced by lipopolysaccharide plus pan-caspase inhibitor (IDN-6556), or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). Mechanistically, baicalin did not inhibit the phosphorylation of RIPK1, RIPK3 and MLKL, nor membrane translocation of p-MLKL, during necroptotic induction, but instead inhibited p-MLKL oligomerization that is required for executing necroptosis. As intracellular reactive oxygen species (ROS) has been reported to be involved in p-MLKL oligomerization, we assessed the effects of N-acetyl-L-cysteine (NAC), an ROS scavenger, on necroptosis and found that NAC significantly attenuated TSI-induced necroptosis and intracellular ROS production concomitantly with reduced levels of oligomerized p-MLKL, mirroring the effect of baicalin. Indeed, inhibitory effect of baicalin was associated with reduced TSI-induced superoxide (indicating mitochondrial ROS) production and increased mitochondrial membrane potential within cells during necroptosis. Besides, oral administration of baicalin significantly reduced the severity of caerulein-induced acute pancreatitis in mice, an animal model of necroptosis-related disease. Collectively, baicalin can inhibit necroptosis through attenuating p-MLKL oligomerization and confers protection against caerulein-induced pancreatitis in mice.

Topics: Acute Disease; Animals; Apoptosis; Ceruletide; Flavonoids; Mice; Necroptosis; Necrosis; Pancreatitis; Protein Kinases; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases

Hinokitiol is a natural bio-active tropolone derivative with promising antioxidant and anti-inflammatory properties. This study was conducted to evaluate the ameliorative effects of hinokitiol against acute pancreatitis induced by cerulein. Mice were pre-treated with hinokitiol intraperitoneally for 7 days (50 and 100 mg/kg), and on the final day of study, cerulein (6 × 50 μg/kg) was injected every hour for six times. Six hours after the last dose of cerulein, blood was collected from the mice through retro-orbital plexus for biochemical analysis. After blood collection, mice were euthanized and the pancreas was harvested for studying effects on oxidative stress, pro-inflammatory cytokines, immunohistochemistry and histopathology of tissue sections. Hinokitiol treatment significantly reduced edema of the pancreas and reduced the plasma levels of lipase and amylase in mice with cerulein-induced acute pancreatitis. It also attenuated the oxidative and nitrosative stress related damage as evident from the reduced malondialdehyde (MDA) and nitrite levels, which were significantly increased in the mice with acute pancreatitis. Furthermore, hinokitiol administration significantly reduced the pancreatitis-evoked decrease in the activity of catalase, glutathione (GSH) and superoxide dismutase (SOD) in the pancreatic tissue. Pre-treatment with hinokitiol significantly reduced the elevated levels of pro-inflammatory cytokines like interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) as well as increased the levels of anti-inflammatory cytokine interleukin-10 (IL-10) in the pancreatic tissue of mice with acute pancreatitis. The immunohistochemical expression of nuclear factor kappa light chain enhancer of activated B cells (NF-κB), cyclooxygenase (COX-2) and TNF-α were significantly decreased by hinokitiol in mice with cerulein-induced acute pancreatitis. In conclusion, the results of the present study demonstrate that hinokitiol has significant potential to prevent cerulein-induced acute pancreatitis.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Ceruletide; Cytokines; Disease Models, Animal; Mice; Monoterpenes; NF-kappa B; Pancreas; Pancreatitis; Tropolone; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) is a frequent cause for hospitalization. However, molecular determinants that modulate severity of experimental pancreatitis are only partially understood.. To investigate the role of secreted protein acidic and rich in cysteine (SPARC) during cerulein-induced AP in mice.. AP was induced by repeated cerulein injections in SPARC knock-out mice (SPARC. Upon cerulein induction, SPARC expression was robustly induced in pancreatic stellate cells (PSCs) but not in acinar cells. Genetic SPARC ablation resulted in attenuated severity of AP with significantly reduced levels of pancreatic necrosis, apoptosis, immune cell infiltration, and reduced fibrosis upon chronic stimulation. However, the release of amylase upon cerulein stimulation in primary acinar cell culture from SPARC. SPARC mediates the severity of AP. The potential link between SPARC and the CCL2 axis could open new avenues for tailored therapeutic interventions in AP patients and warrants further investigations.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Cysteine; Mice; Osteonectin; Pancreatitis

Cerulein‑induced pancreatitis resembles human acute pancreatitis in terms of pathological events, such as enzymatic activation and inflammatory cell infiltration in the pancreas. Cerulein is a cholecystokinin analog that increases levels of reactive oxygen species (ROS) and interleukin‑6 (IL‑6) expression level in pancreatic acinar cells. Serum levels of resistin, which is secreted from adipocytes, are reportedly higher in patients with acute pancreatitis than in healthy individuals. Previously, it was shown that the adipokine resistin can aggravate the cerulein‑induced increase in ROS levels and IL‑6 expression level in pancreatic acinar cells. Peroxisome proliferator‑activated receptor‑gamma (PPAR‑γ) is a key regulator of the transcription and expression of antioxidant enzymes, including heme oxygenase 1 (HO‑1) and catalase. α‑lipoic acid, a naturally occurring dithiol antioxidant, can prevent cerulein‑induced pancreatic damage in rats. In the present study, it was aimed to investigate whether α‑lipoic acid can attenuate the cerulein/resistin‑induced increase in IL‑6 expression and ROS levels via PPAR‑γ activation in pancreatic acinar AR42J cells. The anti‑inflammatory mechanism of α‑lipoic acid was determined using reverse transcription‑quantitative PCR, western blot analysis, enzyme‑linked immunosorbent assay, immunofluorescence staining and fluorometry. Treatment with cerulein and resistin increased ROS levels and IL‑6 expression level, which were inhibited by α‑lipoic acid in pancreatic acinar cells. α‑lipoic acid increased the nuclear translocation and expression level of PPAR‑γ and the expression levels of its target genes: HO‑1 and catalase. The PPAR‑γ antagonist GW9662 and HO‑1 inhibitor zinc protoporphyrin reversed the inhibitory effect of α‑lipoic acid on cerulein/resistin‑induced increase in ROS and IL‑6 levels. In conclusion, α‑lipoic acid inhibits the cerulein/resistin‑induced increase in ROS production and IL‑6 expression levels by activating PPAR‑γ and inducing the expression of HO‑1 and catalase in pancreatic acinar cells.

Topics: Acinar Cells; Acute Disease; Animals; Antioxidants; Catalase; Ceruletide; Humans; Interleukin-6; Pancreas; Pancreatitis; PPAR gamma; Rats; Reactive Oxygen Species; Resistin; Thioctic Acid

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Humans; Mice; Pancreas; Pancreatitis

Severe acute pancreatitis (SAP) is the most serious subtype of acute pancreatitis, manifested as multiple-organ failure resulting in high morbidity and mortality. Based on the role of tripartite motif-containing protein 29 (TRIM29) in immune responses, we aimed to explore its effect on SAP.. Peripheral blood monocyte cells from the SAP or non-SAP patients, as well as bone marrow-derived macrophages from wild-type, TRIM29 -/- , or stimulator of interferon genes (STING) -/- mice after injecting 50 mg/kg of cerulein to induce SAP, were isolated to analyze the role of TRIM29 and STING in the SAP.. Tripartite motif-containing protein 29 was significantly reduced in SAP patients. Compared with wild-type mice, TRIM29 deficiency mice displayed more severe symptom of acute pancreatitis after cerulein injection, which were lost in TRIM29 -/- STING -/- mice. Moreover, interferon and its related genes, as well as STING degradation, were decreased in TRIM29 -/- mice.. Our study demonstrated that TRIM29 negatively regulated the severity of SAP by degrading STING at its downstream, suggesting that TRIM29 and STING might serve as therapeutic targets for SAP.

Topics: Acute Disease; Animals; Ceruletide; Interferons; Macrophages; Mice; Pancreatitis; Transcription Factors

hyperlipidemia acute pancreatitis (HTG-AP) is a major hidden danger affecting human health, however, whether there is a protective effect of resveratrol on HTG-AP is unclear. Therefore our study was aimed to investigate the preventive effect and the underlying mechanism of resveratrol in the HTG-AP mice model.. This research was divided into two parts. In the first part, mice were adaptively fed with normal chow or HFD for 6 weeks. From the second week, resveratrol-treated mice were in intragastric administration with resveratrol (45 mg/kg/d) for 4 weeks. In the second part, the procedures were the same as the first part. After the last intragastric administration with resveratrol, all mice were intraperitoneal injections of cerulean.. We found resveratrol effectively inhibited pancreatic pathological injury in the HFD, AP, and HTG-AP mice. Resveratrol reduced the LPS, IL-6, TNF-α, and MCP-1 expressions in the HFD mice. Resveratrol also reduced TNF-α, MDA, and MCP-1 expressions and increased SOD and T-AOC expressions in the AP and HTG-AP mice. Furthermore, resveratrol suppressed the NF-κB pro-inflammatory signaling pathway in pancreatic tissues in the AP and HTG-AP mice. Moreover, resveratrol improved the gut microbiota in the HFD mice.. The resveratrol pre-treatment could attenuate pancreas injury, inflammation, and oxidative stress in the HTG-AP mice, via restraining the NF-κB signaling pathway and regulating gut microbiota. Therefore, Our study proved that the resveratrol pre-treatment had a preventive effect on HTG-AP.

Topics: Acute Disease; Animals; Ceruletide; Diet, High-Fat; Gastrointestinal Microbiome; Humans; Mice; NF-kappa B; Pancreatitis; Resveratrol; Signal Transduction; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) is an inflammatory process of the pancreas resulting from biliary obstruction or alcohol consumption. Approximately, 10-20% of AP can evolve into severe AP (SAP). In this study, we sought to explore the physiological roles of the transcription factor serum response factor (SRF), annexin A2 (ANXA2), and nuclear factor-kappaB (NF-κB) in SAP.. C57BL/6 mice and rat pancreatic acinar cells (AR42J) were used to establish an AP model in vivo and in vitro by cerulein with or without lipopolysaccharide (LPS). Production of pro-inflammatory cytokines (IL-1β and TNF-α) were examined by ELISA and immunoblotting analysis. Hematoxylin and eosin (HE) staining and TUNEL staining were performed to evaluate pathological changes in the course of AP. Apoptosis was examined by flow cytometric and immunoblotting analysis. Molecular interactions were tested by dual luciferase reporter, ChIP, and Co-IP assays.. ANXA2 was overexpressed in AP and correlated to the severity of AP. ANXA2 knockdown rescued pancreatic acinar cells against inflammation and apoptosis induced by cerulein with or without LPS. Mechanistic investigations revealed that SRF bound with the ANXA2 promoter region and repressed its expression. ANXA2 could activate the NF-κB signaling pathway by inducing the nuclear translocation of p50. SRF-mediated transcriptional repression of ANXA2-protected pancreatic acinar cells against AP-like injury through repressing the NF-κB signaling pathway.. Our study highlighted a regulatory network consisting of SRF, ANXA2, and NF-κB that was involved in AP progression, possibly providing some novel targets for treating SAP.

Topics: Acute Disease; Animals; Annexin A2; Ceruletide; Lipopolysaccharides; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreas; Pancreatitis; Rats; Serum Response Factor; Signal Transduction

Acute pancreatitis (AP) is a common cause of a clinically acute abdomen. Crosstalk between acinar cells and leukocytes (especially macrophages) plays an important role in the development of AP. However, the mechanism mediating the interaction between acinar cells and macrophages is still unclear. This study was performed to explore the role of acinar cell extracellular vesicles (EVs) in the crosstalk between acinar cells and macrophages involved in the pathogenesis of AP. EVs derived from caerulein-treated acinar cells induced macrophage infiltration and aggravated pancreatitis in an AP rat model. Further research showed that acinar cell-derived EV miR-183-5p led to M1 macrophage polarization by downregulating forkhead box protein O1 (FoxO1), and a dual-luciferase reporter assay confirmed that FoxO1 was directly inhibited by miR-183-5p. In addition, acinar cell-derived EV miR-183-5p reduced macrophage phagocytosis. Acinar cell-derived EV miR-183-5p promoted the pancreatic infiltration of M1 macrophages and increased local and systemic damage

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Down-Regulation; Extracellular Vesicles; Macrophages; MicroRNAs; Nerve Tissue Proteins; Pancreatitis; Rats

Severe acute pancreatitis can easily lead to systemic inflammatory response syndrome and death. Macrophages are known to be involved in the pathophysiology of acute pancreatitis (AP), and macrophage activation correlates with disease severity. In this study, we examined the role of ubiquitin-specific protease 25, a deubiquitinating enzyme and known regulator of macrophages, in the pathogenesis of AP.. We used L-arginine, cerulein, and choline-deficient ethionine-supplemented diet-induced models of AP in Usp25. Our results show that Usp25 deficiency exacerbates pancreatic and lung injury, neutrophil and macrophage infiltration, and systemic inflammatory responses in L-arginine, cerulein, and choline-deficient ethionine-supplemented diet-induced models of AP. Bone marrow Usp25. Usp25 deficiency in macrophages enhances TBK1-NF-κB signaling, and the induction of inflammatory chemokines and type I interferon-related genes exacerbates pancreatic and lung injury in AP.

Topics: Acute Disease; Animals; Arginine; Ceruletide; Choline; Cytokines; Deubiquitinating Enzymes; Disease Models, Animal; Ethionine; Interferon Type I; Lung Injury; Macrophages; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreatitis; Signal Transduction; Ubiquitin Thiolesterase; Ubiquitin-Specific Proteases

Acute pancreatitis is a severe inflammatory disease of the pancreas. In experimental acute pancreatitis, cerulein induces the expression of interleukin‑6 (IL‑6) by activating Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 in pancreatic acinar cells. Ligands of peroxisome proliferator activated receptor‑γ (PPAR‑γ) and suppressor of cytokine signaling (SOCS) 3 inhibit IL‑6 expression by suppressing JAK2/STAT3 in cerulein‑stimulated pancreatic acinar AR42J cells. Lutein, an oxygenated carotenoid, upregulates and activates PPAR‑γ to regulate inflammation in a renal injury model. The present study aimed to determine whether lutein activated PPAR‑γ and induced SOCS3 expression in unstimulated AR42J cells, and whether lutein inhibited activation of JAK2/STAT3 and IL‑6 expression via activation of PPAR‑γ and SOCS3 expression in cerulein‑stimulated AR42J cells. The anti‑inflammatory mechanism of lutein was determined using reverse transcription‑quantitative PCR, western blot analysis and enzyme‑linked immunosorbent assay in AR42J cells stimulated with or without cerulein. In another experiment, cells were treated with lutein and the PPAR‑γ antagonist GW9662 or the PPAR‑γ agonist troglitazone prior to cerulein stimulation to determine the involvement of PPAR‑γ activation. The results indicated that lutein increased PPAR‑γ and SOCS3 levels in unstimulated cells. Cerulein increased phospho‑specific forms of JAK2 and STAT3, and mRNA and protein expression of IL‑6, but decreased SOCS3 levels in AR42J cells. Cerulein‑induced alterations were suppressed by lutein or troglitazone. GW9662 alleviated the inhibitory effect of lutein on JAK2/STAT3 activation and IL‑6 expression in cerulein‑stimulated cells. In conclusion, lutein inhibited the activation of JAK2/STAT3 and reduced IL‑6 levels via PPAR‑γ‑mediated SOCS3 expression in pancreatic acinar cells stimulated with cerulein.

Topics: Acinar Cells; Acute Disease; Ceruletide; Humans; Interleukin-6; Lutein; Pancreatitis; PPAR gamma; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Troglitazone

Polo-like kinase 1 (Plk1) plays an important role in cell-cycle regulation. Recent work has suggested that Plk1 could be a biomarker of gemcitabine response in pancreatic ductal adenocarcinoma (PDAC). Although targeting Plk1 to treat PDAC has been attempted in clinical trials, the results were not promising, and the mechanisms of resistance to Plk1 inhibition is poorly understood. In addition, the role of Plk1 in PDAC progression requires further elucidation. Here, we showed that Plk1 was associated with poor outcomes in patients with PDAC. In an inducible transgenic mouse line with specific expression of Plk1 in the pancreas, Plk1 overexpression significantly inhibited caerulein-induced acute pancreatitis and delayed development of acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Bioinformatics analyses identified the regulatory networks in which Plk1 is involved in PDAC disease progression, including multiple inflammation-related pathways. Unexpectedly, inhibition or depletion of Plk1 resulted in upregulation of PD-L1 via activation of the NF-κB pathway. Mechanistically, Plk1-mediated phosphorylation of RB at S758 inhibited the translocation of NF-κB to nucleus, inactivating the pathway. Inhibition of Plk1 sensitized PDAC to immune checkpoint blockade therapy through activation of an antitumor immune response. Together, Plk1 suppresses PDAC progression and inhibits NF-κB activity, and targeting Plk1 can potentiate the efficacy of immunotherapy in PDAC.. Inhibition of Plk1 induces upregulation of PD-L1 expression in pancreatic ductal adenocarcinoma, stimulating antitumor immunity and sensitizing tumors to immunotherapy.

Topics: Acute Disease; Animals; B7-H1 Antigen; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; Ceruletide; Humans; Immune Checkpoint Inhibitors; Mice; NF-kappa B; Pancreatic Neoplasms; Pancreatitis; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins

Premature intracellular trypsinogen activation has long been considered a key initiator of acute pancreatitis (AP). Cathepsin B (CTSB) activates trypsinogen, while cathepsin L (CTSL) inactivates trypsin(ogen), and both proteins play a role in the onset of AP.. AP was induced by 7 hourly intraperitoneal injections of cerulein (50 μg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (Ctsb. Double deletion of Ctsb and Cstl did not affect pancreatic development or mouse growth. After 7 times cerulein injections, double Ctsb and Ctsl deficiency in mouse pancreases increased trypsin activity to the same extent as that in Ctsl-deficient mice, while Ctsb deficiency decreased trypsin activity but did not affect the severity of AP. Ctsb. Double deletion of Ctsb and Ctsl in the mouse pancreas altered intrapancreatic trypsin activity but did not affect disease severity and inflammatory response after cerulein-induced AP.

Topics: Acute Disease; Amylases; Animals; Cathepsin B; Ceruletide; Mice; Mice, Knockout; Pancreas; Pancreatitis; Trypsin; Trypsinogen

Pachymic acid (PA), a natural triterpenoid, possesses the capacity to repress inflammatory and profibrotic responses. However, the role of PA in pancreatic fibrosis remains unclear. Here the effect of PA on anti-fibrogenic response was investigated using in vivo and in vitro pancreatitis models. We demonstrated that PA treatment repressed TGF-β-induced pancreatic stellate cells (PSCs) activation in vitro, as evidenced by decreased expression of Collagen I, α-smooth muscle actin, and fibronectin. PA decreased Cerulein-induced acinar injury and pancreatic fibrosis in an experimental pancreatitis model. Mechanistically, PA repressed Cerulein or (TGF-β)-induced activation of nuclear factor (NF)-κB signaling and thus decreased NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome activation in PSCs. Pharmacological inhibition of NLRP3 repressed TGF-β-induced activation of PSCs. More important, NLRP3 activator partially attenuated the effect of PA on inhibiting PSCs activation. Collectively, these data demonstrate that PA represses PSCs activation and pancreatic fibrosis through repressing NF-κB/NLRP3 signaling.

Topics: Actins; Ceruletide; Collagen Type I; Fibronectins; Fibrosis; Humans; Inflammasomes; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis; Transforming Growth Factor beta; Triterpenes

In this research, we screened out two genes upregulated in mice with acute pancreatitis (AP) by gene sequencing: microRNA (miR)-320-3p and matrix metalloprotease 8 (MMP8). This study was designed to determine whether miR-320-3p and MMP8 participate in AP development and explore the mechanisms, with a new idea for clinical diagnosis and treatment of AP. Expression of miR-320-3p, DNA methyltransferase 3a (DNMT3a), and MMP8 in mouse pancreatic tissues and AR42J cells was tested by RT-qPCR and western blot assays. Pancreatic pathological changes, serum amylase and lipase, and inflammatory factors in mouse serum and cell supernatant were measured by hematoxylin-eosin staining, automation analyzer, and enzyme-linked immunosorbent assay, respectively. Cell proliferation and apoptosis were determined by CCK-8 assay and flow cytometry. The interaction between miR-320-3p, DNMT3a, and MMP8 was verified by luciferase activity assay, ChIP-qPCR, and MSP assay. High expression of miR-320-3p and MMP8, and low expression of DNMT3a were observed in pancreatic tissues of AP mice and caerulein-induced AP cellular model. Downregulation of miR-320-3p alleviated injury of mouse pancreas, reduced the levels of serum amylase and lipase, and blocked inflammatory factor levels in AP mice. In caerulein-induced AP cellular models, inhibiting miR-320-3p facilitated proliferation and inhibited apoptosis. Upregulation of MMP8 resulted in the opposite results, which could be reversed by simultaneous inhibition of miR-320-3p. miR-320-3p targeted DNMT3a, and downregulating miR-320-3p promoted DNMT3a expression. Moreover, DNMT3a promoted DNA methylation in MMP8 promoter region, thereby inhibiting MMP8 expression in AP mouse and cellular models. This research suggests that miR-320-3p inhibits DNMT3a to reduce MMP8 methylation and increase MMP8 expression, thereby promoting AP progression.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Cell Proliferation; Ceruletide; DNA Methylation; DNA Methyltransferase 3A; Eosine Yellowish-(YS); Hematoxylin; Lipase; Luciferases; Matrix Metalloproteinase 8; Mice; MicroRNAs; Pancreatitis

Topics: Acute Disease; ADAM17 Protein; Animals; Carcinogens; Ceruletide; Cytokines; Disintegrins; Endopeptidases; Fibrosis; Interleukin-6; Ketones; Mice; Nicotine; Nitrosamines; Pancreatitis; Peptide Hydrolases; Tumor Necrosis Factor-alpha

The aim of the study was to evaluate whether a new and successful treatment opportunity can be provided in acute pancreatitis and may prevent symptomatic treatments and show its effect through etiopathogenesis. Therefore, we want to investigate the efficacy of golimumab in an experimental rat model of cerulein-induced acute pancreatitis.. A total of 35 rats, including 7 rats in each group, were distributed into 5 groups (sham, acute pancreatitis, placebo, acute pancreatitis+golimumab 5 mg/kg, and acute pancreatitis+golimumab 10 mg/kg). An experimental cerulein-induced acute pancreatitis model was accomplished by intraperitoneal cerulein injections. After sacrification, rat blood samples were collected for amylase, IL-6, and IL-1beta measurements. Histopathological analysis of the pancreas was performed with Tunel and hematoxylin and eosin staining.. Amylase, IL-6, and IL-1beta levels were found to be increased in the acute pancreatitis group. IL-1beta, amylase, IL-6 levels, and pancreatic inflammation were all significantly decreased in golimumab groups (P < .01). Moreover, in both golimumab groups, golimumab treatment significantly reduced apoptosis in pancreatic tissues (P < .05). Golimumab treatment was found to significantly reduce edema formation, inflammation, vacuolization, and fat necrosis of pancreatic tissues (P < .05).. Firstly in the literature, we investigated the efficacy of golimumab in the experimental acute pancreatitis model. In the light of our findings, it could be suggested that golimumab may be an effective and safe therapeutic option in the treatment of patients with acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Disease Models, Animal; Inflammation; Interleukin-6; Pancreas; Pancreatitis; Rats

Acute pancreatitis (AP) is mainly caused by acinar cells releasing various inflammatory factors, causing inflammatory storms and leading to severe pancreatitis. Detection methods and treatment targets for pancreatitis are lacking, raising the urgency of identifying diagnostic markers and therapeutic targets for AP. MicroRNAs (miRNAs) have recently been identified as molecular markers for various biological processes such as tumors, immunity, and metabolism, and the involvement of miRNAs in inflammatory responses has been increasingly studied. To explore the role of miRNAs in AP is the primary objective of this study. By using qPCR on our cerulein-induced pancreatitis cell model, it is worth noting that the change of miR-146a-5p expression in inflammation-related miRNAs in AP was predominant. Next, ELISA, CCK8, and flow cytometry were used to inspect the impact of miR-146a-5p on pancreatitis. BiBiServ bioinformatics anticipated binding ability of miR-146a-5p and 3'-untranslated region (3'UTR) of TNF receptor-associated factor 6 (TRAF6), and the dual-luciferase assay verified the combination of the two. TRAF6 knockdown verified the effect of TRAF6 on the progression of pancreatitis. Finally, rescue experiments verified the capability of miR-146a-5p and TRAF6 interaction on the Toll-like receptor 9 (TLR9)/NOD-like receptor protein 3 (NLRP3) signaling pathway and cell function. The expression of miR-146a-5p decreased in cerulein-induced AR42J pancreatic acinar cells. Functional experiments verified that miR-146a-5p facilitated the proliferation of AR42J pancreatic acinar cells and inhibited their apoptosis. Bioinformatic predictions and dual-luciferase experiments verified the actual binding efficiency between miR-146a-5p and 3'UTR of TRAF6. Our study confirmed that knockdown of TRAF6 restrained the progression of pancreatitis, and knockdown of TRAF6 rescued pancreatitis caused by miR-146a-5p downregulation by the TLR9/NLRP3 signaling pathway. Therefore, downregulation of miR-146a-5p in the induced pancreatitis cell model promotes the progression of pancreatitis via the TLR9/TRAF6/NLRP3 signaling pathway. There is potential for miR-146a-5p to serve as a diagnostic marker and therapeutic nucleic acid drug for AP.

Topics: 3' Untranslated Regions; Acute Disease; Animals; Ceruletide; Down-Regulation; MicroRNAs; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis; Rats; Signal Transduction; TNF Receptor-Associated Factor 6; Toll-Like Receptor 9

Acute pancreatitis (AP) is a major, globally increasing gastrointestinal disease and a biliary origin is the most common cause. However, the effects of bile acids (BAs), given systemically, on the pancreas and on disease severity remains elusive. In this study, we have investigated the roles of different circulating BAs in animal models for AP to elucidate their impact on disease severity and the underlying pathomechanisms. BAs were incubated on isolated acini and AP was induced through repetitive injections of caerulein or L-arginine; pancreatic duct ligation (PDL); or combined biliopancreatic duct ligation (BPDL). Disease severity was assessed using biochemical and histological parameters. Serum cholecystokinin (CCK) concentrations were determined via enzyme immunoassay. The binding of the CCK1 receptor was measured using fluorescence-labeled CCK. In isolated acini, hydrophobic BAs mitigated the damaging effects of CCK. The same BAs further enhanced pancreatitis in L-arginine- and PDL-based pancreatitis, whereas they ameliorated pancreatic damage in the caerulein and BPDL models. Mechanistically, the binding affinity of the CCK1 receptor was significantly reduced by hydrophobic BAs. The hydrophobicity of BAs and the involvement of CCK seem to be relevant in the course of AP. Systemic BAs may affect the severity of AP by interfering with the CCK1 receptor.

Topics: Acute Disease; Animals; Arginine; Bile Acids and Salts; Ceruletide; Cholecystokinin; Disease Models, Animal; Hydrophobic and Hydrophilic Interactions; Mice; Pancreas; Pancreatitis

Hair follicle-derived mesenchymal stem cell (HF-MSC)-based therapies protect against acute pancreatitis (AP). However, accumulating evidence suggests that MSC-based therapy mainly involves the secretion of MSC-derived small extracellular vesicles (MSC-sEVs) through paracrine effects. Thus, the present research investigated the therapeutic effect of HF-MSC-sEVs in AP and the underlying mechanisms.. SEVs were purified from cultured HF-MSC supernatant. The effects of sEVs in vitro were analyzed on caerulein-simulated pancreatic acinar cells (PACs). The therapeutic potential of sEVs in vivo was examined in a caerulein-induced AP model. The organ distribution of sEVs in mice was determined by near-infrared fluorescence (NIRF) imaging. Serum specimens and pancreatic tissues were collected to analyze the inhibition of inflammation and pyroptosis in vivo, as well as the appropriate infusion route: intraperitoneal (i.p.) or intravenous (i.v.) injection.. HF-MSC-sEVs were taken up by PACs and improved cell viability in vitro. In vivo, sEVs were abundant in the pancreas, and the indicators of pancreatitis, including amylase, lipase, the inflammatory response, myeloperoxidase (MPO) expression and histopathology scores, in sEV-treated mice were markedly improved compared with those in the AP group, especially via tail vein injection. Furthermore, we revealed that sEVs observably downregulated the levels of crucial pyroptosis proteins in both PACs and AP tissue.. We innovatively demonstrated that HF-MSC-sEVs could alleviate inflammation and pyroptosis in PACs in AP, suggesting a refreshing cell-free remedy for AP.

Topics: Acute Disease; Animals; Ceruletide; Extracellular Vesicles; Hair Follicle; Inflammation; Mice; Pancreatitis

Acute pancreatitis (AP) is an inflammatory disease with very poor outcomes. However, the order of induction and coordinated interactions of systemic inflammatory response syndrome (SIRS) and compensatory anti-inflammatory response syndrome (CARS) and the potential mechanisms in AP are still unclear.. An integrative analysis was performed based on transcripts of blood from patients with different severity levels of AP (GSE194331), as well as impaired lung (GSE151572), liver (GSE151927) and pancreas (GSE65146) samples from an AP experimental model to identify inflammatory signals and immune response-associated susceptibility genes. An AP animal model was established in wild-type (WT) mice and Tlr2-deficient mice by repeated intraperitoneal injection of cerulein. Serum lipase and amylase, pancreas impairment and neutrophil infiltration were evaluated to assess the effects of. In summary, we discovered SIRS and CARS were stimulated in parallel, not activated consecutively. In addition, among the novel susceptibility genes,

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Ceruletide; Disease Models, Animal; Disease Progression; Lipase; Mice; Mice, Inbred C57BL; Pancreatitis; Systemic Inflammatory Response Syndrome; Toll-Like Receptor 2

Acute pancreatitis (AP) is an inflammatory disease of the pancreas. A growing number of studies have shown that long noncoding RNAs (lncRNAs) play an important role in AP progression. Here, we aimed to elucidate the role of Small Nucleolar RNA Host Gene 11(SNHG11) and its underlying molecular mechanisms behind AP progression. The in vivo and in vitro AP cell models were established by retrograde injection of sodium taurocholate and caerulein stimulation into AR42J cells and HPDE6-C7 cells, respectively. A bioinformatics website predicted the relationship between SNHG11, miR-7-5p, and Phospholipase C Beta 1(PLCB1) and validated it with a dual-luciferase reporter assay and an RNA immunoprecipitation (RIP) assay. AR42J cells and HPDE6-C7 cells were transfected with an overexpression of plasmids or shRNA to investigate the effects of the SNHG11/miR-7-5p/PLCB1 axis on cell proliferation and apoptosis, inflammatory cytokine secretion, and acute pancreatitis. Low expression of SNHG11 and PLCB1 and high expression of miR-7-5p were observed in AP pancreatic tissue and AP cell models. SNHG11 overexpression inhibited apoptosis and inflammatory responses induced by caerulein. Simultaneously, we discovered that SNHG11 regulates PLCB1 expression by sponging miR-7-5p. PLCB1 overexpression abrogated inflammatory damage exacerbated by miR-7-5p enrichment. In addition, the SNHG11/miR-7-5p/PLCB1 axis could be involved in caerulein-induced inflammatory injury by participating in the p38MAPK signaling pathway. The overexpressed SNHG11/miR-7-5p/PLCB1 axis can inhibit AP progression by participating in the p38MAPK signaling pathway, thereby providing a potential therapeutic target and therapeutic direction for AP therapy.

Topics: Acute Disease; Ceruletide; Humans; MicroRNAs; p38 Mitogen-Activated Protein Kinases; Pancreatitis; Phospholipase C beta; RNA, Long Noncoding

Acute pancreatitis is a clinical picture with a wide range of symptoms from mild inflammation to multiorgan failure and death. The aim of this study is to investigate the effects of Adalimumab (ADA) on inflammation and apoptosis in a cerulein-induced acute pancreatitis model in rats.. Experimental cerulein-induced acute pancreatitis model was created by applying 4 intraperitoneal cerulein injections at 1-h intervals. A total of 40 rats, 8 in each group, were randomly distributed into five groups. In the groups that ADA treatment was given, two different doses of ADA were administered 5 mg/kg and 20 mg/kg as low and high doses, respectively. The rats were sacrificed 12 h after the last intraperitoneal administration of ADA. Blood samples were obtained from each rat for amylase, IL-6, and IL-1β measurements. Hematoxylin and Eosin (H&E) stains were used to undertake the histopathological analysis of the pancreas. The terminal deoxynucleotidyl transferase-mediated nick-end-labeling (TUNEL) method was used to evaluate apoptosis.. : Plasma amylase, IL-6, and IL-1β levels were significantly elevated in acute pancreatitis groups (p < 0.05). It was determined that both low (5 mg/kg) and high doses (20 mg/kg) of ADA ameliorated the parameters (plasma amylase, IL-6, and IL-1β) (p < 0.05). Although significant improvements were detected in the Schoenberg scoring system and the apoptotic index from the TUNEL method after highdose ADA treatment, no significant amelioration was observed in the histopathological examinations in the low-dose ADA group.. : It has been determined that the administration of high-dose ADA effectively alleviated the symptoms of acute pancreatitis and reduced the level of apoptosis. In line with the findings of our study, we have predicted that high-dose (20 mg/kg) ADA can be used as an effective and safe drug in the treatment of patients with acute pancreatitis.

Topics: Acute Disease; Adalimumab; Amylases; Animals; Ceruletide; Disease Models, Animal; Inflammation; Interleukin-6; Pancreatitis; Rats; Rats, Wistar

Alcohol consumption is always the main cause of acute pancreatitis (AP). It has been reported that alcohol exerts direct damage to the pancreas. However, the specific role of alcohol during AP needs to be investigated. This study aims to examine the effects of alcohol in cerulein-induced AP and the role of the AMPK pathway.. Human subjects from operations, cerulein-induced AP rat, and cerulein-stimulated AR42J cell line were enrolled in this study. Electron microscopy was employed for observation of cell morphology, immunohistochemistry for identification of cells, ELISA for detection of inflammation factors, Annexin V/PI double staining for evaluation of cell apoptosis, immunofluorescence for assessment of autophagic flux, oil red O staining for examination of lipid droplet accumulation, and Western blot for measurement of expressions of proteins related to autophagy, apoptosis, and AMPK signal pathway. PI3K inhibitor 3-MA and AMPK inhibitor BML-275 were utilized for investigation of the relationship between impaired autophagic flux and the AMPK pathway by inhibiting or stimulating the formation of autophagosome.. Alcohol consumption caused lipid droplet accumulation in the pancreas, and it also activated AMPK signaling pathway, thus aggravating the autophagic flux during AP. Alcohol up-regulated the expressions of anti-apoptotic proteins during the induction of AP to inhibit cell apoptosis and enhance cell necrosis. Inhibition of autophagosome formation by AMPK inhibitor BML-275 ameliorated the decreased cell viability caused by alcohol and cerulein in vitro.. Alcohol aggravates AP progression by impairing autophagic flux and enhancing cell autophagy through the AMPK signaling pathway.

Topics: Adenylate Kinase; Animals; Autophagy; Cell Line; Central Nervous System Depressants; Ceruletide; Ethanol; Humans; Pancreas; Pancreatitis; Pancreatitis, Alcoholic; Phosphoinositide-3 Kinase Inhibitors; Pyrazoles; Pyrimidines; Rats; Signal Transduction

Obesity is an important risk factor for severe acute pancreatitis. The necrosis of epididymal adipose tissue occurs in severe acute pancreatitis. Adipose tissue macrophages play an important role in metabolic related inflammation. Therefore, we explored the potential mechanisms between adipose tissue macrophages and obesity-related severe acute pancreatitis.. Severe acute pancreatitis mice model was induced by caerulein with lipopolysaccharide. The severity of severe acute pancreatitis was evaluated according to the morphological, general, and biochemical change. We assessed the injury of epididymal white adipose tissue, pancreas, and adipose tissue macrophages in obese mice and lean mice with severe acute pancreatitis. Outcomes of caerulein-induced severe acute pancreatitis were studied in lean and obese mice with or without lipase inhibitor orlistat.. Fat necrosis and pancreatic injury increased in the SAP groups. High levels of serum free fatty acid and triglyceride were increased significantly in the SAP group. The NLRP3-caspase1 inflammasome signal pathway in adipose tissue macrophages markedly enhanced in the SAP groups compared with control group. Free fatty acid can trigger macrophages inflammation through NLRP3-caspase1. Lipase inhibited by orlistat remarkably decreased in adipose tissue necrosis, and the levels of serum lipase, amylase, and pancreatic tissue damage decreased in the orlistat group compared with the SAP group. The NLRP3-caspase1 inflammasome pathway in adipose tissue macrophages markedly decreased in the orlistat groups compared with SAP group. The levels of serum free fatty acid and triglyceride were decreased significantly in the orlistat group.. Inflammation increases in adipose tissue macrophages of obese mice with severe acute pancreatitis. Free fatty acid generated via adipocyte lipolysis worsens inflammation in adipose tissue macrophages and the outcome of severe acute pancreatitis in obese mice through the NLRP3-caspase1 inflammasome pathway.

Topics: Acute Disease; Adipose Tissue; Animals; Caspase 1; Ceruletide; Fatty Acids, Nonesterified; Inflammasomes; Inflammation; Lipase; Macrophages; Mice; Necrosis; NLR Family, Pyrin Domain-Containing 3 Protein; Obesity; Orlistat; Pancreatitis; Triglycerides

Acute pancreatitis (AP) is an inflammatory gastrointestinal disorder affecting the pancreas. Previous study reported that tetraspanin 1 (TSPAN1) expression was significantly upregulated in the pancreas of AP patients. However, the underlying molecular mechanism of TSPAN1 in the pathogenesis of AP remains unclear. Thus, the aim of the present study was to investigate the potential role of TSPAN1 in development of AP. RT-qPCR was carried out to quantify the relative mRNA levels of TSPAN1 and anterior gradient-2 (AGR2). The CCK-8 assay was used to detect the cell viability. The TUNEL assay was performed to visualize the apoptotic cells. Western blot was performed to determine the expressions of proteins related to endoplasmic reticulum (ER) stress and apoptosis. ELISA kits were adopted to detect the concentration of inflammatory cytokines including TNF-α and IL-6. Finally, immunoprecipitation (IP) was used to verify the interaction between TSPAN1 and AGR2. TSPAN1 was upregulated in serum of AP patients and AP cell models. TSPAN1 silencing promoted the cell proliferation and inhibited inflammatory response in cerulein-induced AR42J cells. Moreover, TSPAN1 induced endoplasmic reticulum stress by binding AGR2. Interestingly, the overexpression of AGR2 abolished the effects of TSPAN1 silencing on cell proliferation and inflammatory response in cerulein-induced AR42J cells. In summary, TSPAN1 silencing protects against cerulein-induced pancreatic acinar cell injury through inhibiting ER stress-mediated by AGR2. Hence, TSPAN1 may serve as a promising therapeutic target for AP treatment.

Topics: Acinar Cells; Acute Disease; Ceruletide; Humans; Mucoproteins; Oncogene Proteins; Pancreas; Pancreatitis; Tetraspanins

Acute pancreatitis (AP), an inflammatory disorder of the pancreas, is a complicated disease without specific drug therapy. (R)-4,6-dimethoxy-3-(4-methoxy phenyl)-2,3-dihydro-1H-indanone [(R)-TML104] is a synthesized analog of the natural product resveratrol sesquiterpenes (±) -isopaucifloral F. This study aimed to investigate the effect and underlying mechanism of (R)-TML104 on AP. The experimental AP model was induced by caerulein hyperstimulation in BALB/c mice. (R)-TML104 markedly attenuated caerulein-induced AP, as evidenced by decreased pancreatic edema, serum amylase levels, serum lipase levels, and pancreatic myeloperoxidase activity. In addition, (R)-TML104 significantly inhibited the expression of pancreatic chemokines C-C motif chemokine ligand 2 and macrophage inflammatory protein-2 and the infiltration of neutrophils and macrophages. Mechanistically, (R)-TML104 activated AMP-activated protein kinase and induced sirtuin 1 (SIRT1) expression. (R)-TML104 treatment markedly induced the SIRT1-signal transducer and activator of transcription 3 (STAT3) interaction and reduced acetylation of STAT3, thus inhibiting the inflammatory response mediated by the interleukin 6-STAT3 pathway. The effect of (R)-TML104 on SIRT1-STAT3 interaction was reversed by treatment with a SIRT1 inhibitor selisistat (EX527). Together, our findings indicate that (R)-TML104 alleviates experimental pancreatitis by reducing the infiltration of inflammatory cells through modulating SIRT1.

Topics: Acute Disease; Animals; Ceruletide; Mice; Pancreas; Pancreatitis; Resveratrol; Sirtuin 1

Topics: Acute Disease; Animals; Caspase 3; Caspases; Ceruletide; Humans; Interleukin-18; Interleukin-1beta; Intracellular Signaling Peptides and Proteins; Male; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatitis; Pyroptosis; Rats; TNF Receptor-Associated Factor 6

Acute pancreatitis (AP) is an acute inflammatory condition of the pancreas. Previous studies have shown that rutaecarpine (RUT), an important alkaloid component of Evodia rutaecarpa, exhibits certain protective effects against AP in rats by upregulating calcitonin gene-related peptide (CGRP). However, the molecular mechanism of RUT in AP remains unknown. This study aimed to investigate the effects of RUT on cerulein-induced AP in vivo and in vitro, and to explore the underlying molecular mechanisms. In cerulein/LPS-treated wild-type mice, but not CGRP gene knock-out mice, RUT significantly ameliorated pancreatic inflammation by alleviating histopathological changes, reducing IL-6 and TNF-α levels, and increasing in IL-10 levels. Moreover, RUT improved AP by suppressing the MAPK and NF-κB signaling pathways. These effects were mostly mediated through CGRP. Cell-based studies revealed that RUT significantly improved cell viability while suppressing the apoptosis of AR42J cells with cerulein-induced AP, downregulating IL-6 and TNF-α, stimulating IL-10 release, and inhibiting MAPK, NF-κB, and STAT3 signaling activation, all in a CGRP-dependent manner. RUT ameliorated cerulein/LPS-induced AP inflammatory responses in mice and AR42J cells in a CGRP-dependent manner and thus may represent a potential therapeutic option for AP patients. Our study provides valuable insights for AP drug development.

Topics: Acute Disease; Animals; Calcitonin; Calcitonin Gene-Related Peptide; Ceruletide; Humans; Indole Alkaloids; Mice; NF-kappa B; Pancreatitis; Quinazolines; Rats

Severe acute pancreatitis (SAP) is a necrotic pancreatic inflammation associated with high mortality rate (up to 70%). Bone marrow (BM) mesenchymal stem cells (MSCs) have been investigated in pancreatic cellular regeneration, but still their effects are controversial. Therefore, the present study is aimed at examining the enrichment of the stem cells with ascorbic acid (AA) and N-acetylcysteine (NAC) and explore their combined action on the expression of the inflammatory cytokines: interleukin 1

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Blood Glucose; Body Weight; Caspase 3; Cell Proliferation; Ceruletide; Fluorescent Dyes; Insulin; Interleukin-1beta; Islets of Langerhans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; NF-kappa B; Organic Chemicals; Pancreatitis; Proliferating Cell Nuclear Antigen; Rats; Regeneration; Tumor Necrosis Factor-alpha

As a calcium-regulated protein, CaMK II is closely related to cell death, and it participates in the development of pathological processes such as reperfusion injury, myocardial infarction, and oligodendrocyte death. The function of CaMK II activation in acute pancreatitis (AP) remains unclear. In our study, we confirmed that the expression of p-CaMK II was increased significantly and consistently in injured pancreatic tissues after caerulein-induced AP. Then, we found that KN93, an inhibitor of CaMK II, could mitigate the histopathological manifestations in pancreatic tissues, reduce serum levels of enzymology, and decrease oxidative stress products. Accordingly, we elucidated the effect of KN93 in vitro and found that KN93 had a protective effect on the pancreatic acinar cell necroptosis pathway by inhibiting the production of ROS and decreasing the expression of RIP3 and p-MLKL. In addition, we identified the protective effect of KN93 on AP through another mouse model induced by pancreatic duct ligation (PDL). Together, these data demonstrated that CaMK II participates in the development of AP and that inhibiting CaMK II activation could protect against AP by reducing acinar cell necroptosis, which may provide a new idea target for the prevention and treatment of AP in the clinic.

Topics: Acinar Cells; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Ceruletide; Male; Mice; Mice, Inbred C57BL; Necroptosis; Pancreatitis; Protective Agents; Reactive Oxygen Species; Sulfonamides

Previous studies have provided conflicting results regarding whether the serum ghrelin concentration can reflect the severity of acute pancreatitis (AP). The present study examined the correlation between the serum ghrelin concentration and AP severity in animal models and investigated whether altered ghrelin expression in pancreatic acinar cells influences IKKβ/NF-κB signaling and pro-inflammatory cytokine production.. Mild or severe AP was induced in rats by intraperitoneal injection of cerulein or retrograde cholangiopancreatic duct injection of sodium taurocholate, respectively. After successful model induction, serum ghrelin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were determined by enzyme-linked immunosorbent assay, and IKKβ/NF-κB activation was assessed by immunohistochemistry. Subsequently, stable overexpression or knockdown of ghrelin in AR42J cells was achieved by lentiviral transfection. After transfected cells and control cells were treated with cerulein for 24 h, the TNF-α and IL-1β levels in the supernatants were determined by enzyme-linked immunosorbent assay, and the expression levels of p-p65, IKKβ, and p-IKKβ were detected by Western blotting.. In rat AP models, AP severity was correlated with increased IKKβ/NF-κB activation, pro-inflammatory cytokine production, and ghrelin secretion. The levels of pro-inflammatory cytokines TNF-α and IL-1β as well as IKKβ/NF-κB signaling activity were increased upon knockdown of ghrelin in the AP acinar cell model and decreased with ghrelin overexpression.. Serum ghrelin is related to the severity of AP. Ghrelin may play a protective role in the pathogenesis of AP by inhibiting the pro-inflammatory cytokines and the activation of the IKKβ/NF-κB signaling pathway.

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Cytokines; Ghrelin; I-kappa B Kinase; NF-kappa B; Pancreas; Pancreatitis; Rats; Signal Transduction; Tumor Necrosis Factor-alpha

Serum amyloid A (SAA) is an early and sensitive biomarker of inflammatory diseases, but its role in acute pancreatitis (AP) is still unclear. Here, we used a caerulein-induced mouse model to investigate the role of SAA in AP and other related inflammatory responses. In our study, we found that the expression of a specific SAA isoform, SAA3, was significantly elevated in a caerulein-induced AP animal model. In addition, SAA3-knockout (Saa3

Topics: Acute Disease; Animals; Ceruletide; Mice; Necroptosis; Pancreatitis; Serum Amyloid A Protein

Acute pancreatitis (AP) is a dysfunctional pancreas disease marked by severe inflammation. Long non-coding RNAs (lncRNAs) involving in the regulation of inflammatory responses have been frequently mentioned. The purpose of this study was to ensure the function and action mode of lncRNA maternally expressed gene 3 (MEG3) in caerulein-induced AP cell model. HPDE cells were treated with caerulein to establish an AP model in vitro. The expression of MEG3, miR-195-5p, and fibroblast growth factor receptor 2 (FGFR2) was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and apoptosis were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry assay, respectively. The expression of CyclinD1, B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), FGFR2, P65, phosphorylated P65 (p-P65), alpha inhibitor of nuclear factor kappa beta (NF-κB) (IκB-α), and phosphorylated IκB-α (p-IκB-α) at the protein level was quantified by western blot. The concentrations of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were monitored by enzyme-linked immunosorbent assay (ELISA). The targeted relationship between miR-195-5p and MEG3 or FGFR2 was forecasted by the online software starBase v2.0 and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. As a result, the expression of MEG3 and FGFR2 was decreased in caerulein-induced HPDE cells, while the expression of miR-195-5p was increased. MEG3 overexpression inhibited cell apoptosis and inflammatory responses that were induced by caerulein. Mechanically, miR-195-5p was targeted by MEG3 and abolished the effects of MEG3 overexpression. FGFR2 was a target of miR-195-5p, and MEG3 regulated the expression of FGFR2 by sponging miR-195-5p. FGFR2 overexpression abolished miR-195-5p enrichment-aggravated inflammatory injuries. Moreover, the NF-κB signaling pathway was involved in the MEG3/miR-195-5p/FGFR2 axis. Collectively, MEG3 participates in caerulein-induced inflammatory injuries by targeting the miR-195-5p/FGFR2 regulatory axis via mediating the NF-κB pathway in HPDE cells.

Topics: Cells, Cultured; Ceruletide; Dose-Response Relationship, Drug; Humans; Inflammation; MicroRNAs; NF-kappa B; Pancreas; Pancreatitis; Receptor, Fibroblast Growth Factor, Type 2; RNA, Long Noncoding

Acute pancreatitis (AP) is a common acute abdominal disease with high mortality and mortality rates. Increasing evidences clarified that Traditional Chinese Medicine (TCM) adjuvant therapy for AP can be used and it gives a positive effect. Quercetin (3,3',4',5,7-pentahydroxyflavone, QE) is a type of flavone compound with positive effect on cancer and inflammation prevention. The current study aims to identify the effect of QE on AP and potential molecular effect. In this case, caerulein (CAE) induced AP cell and mice model were used. QE alleviated inflammatory mediators TNF-α, IL-6, and IL-10 in experiments. In addition, miR-216b was increased based on QE treatment. In further study, MAP2K6 of p38/MAPK signaling pathway was identified as a direct target of miR-216b, and QE inhibited p38/MAPK signaling pathway through up-regulating miR-216b. Our study also first confirmed that long non-coding RNA NEAT1 is a direct target of miR-216b and can be suppressed by QE. Because of the target, NEAT1, miR-216b, and MAP2K6 formed a competitive endogenous RNA (ceRNA) network. Besides direct target mediated by QE, it also decreased TNF-α which down-regulated TRAF2 and MAP3K5 located on upstream of p38/MAPK signaling and formed a feedback loop. In conclusion, QE has a protective effect on AP through inhibiting p38/MAPK signaling pathway by up-regulating miR-216b and suppressing TNF-α.

Topics: Animals; Cell Line; Ceruletide; Disease Models, Animal; Down-Regulation; MAP Kinase Kinase 6; Mice; Mice, Inbred C57BL; MicroRNAs; p38 Mitogen-Activated Protein Kinases; Pancreas; Pancreatitis; Quercetin; Rats; RNA, Long Noncoding; Signal Transduction; Up-Regulation

Borneol is a commonly used flavouring substance in traditional Chinese medicine, which possesses several pharmacological activities including analgesic, antiinflammatory, and antioxidant properties. The aim of this study was to investigate the effects of borneol on cerulein-induced acute pancreatitis (AP) model. Swiss albino mice were pretreated with borneol (100 and 300 mg/kg) daily for 7 days, before six consecutive injections of cerulein (50 μg/kg/hr, intraperitoneally). The protective effect of borneol was studied by biochemical, enzyme linked immunosorbent assay, histological, immunoblotting, and immunohistochemical analysis. Oral administration of borneol significantly attenuated pancreatic damage by reducing amylase, lipase levels and histological changes. Borneol attenuated cerulein-induced oxidative-nitrosative stress by decreasing malondialdehyde, nitrite levels, and elevating reduced glutathione levels. Pancreatic inflammation was ameliorated by inhibiting myeloperoxidase activity and pro-inflammatory cytokine (Interleukins and TNF-α) levels. Furthermore, borneol administration significantly increased nuclear factor E2-related factor 2 (Nrf2), superoxide dismutase (SOD1) expression and reduced phospho-NF-κB p65 expression. Treatment with borneol significantly inhibited TNF-α, IL-1β, IL-6, and inducible nitric oxide synthase expression in cerulein-induced AP mouse model. Together, these results indicate that borneol which is currently used as US-FDA approved food adjuvant has the potential to attenuate cerulein-induced AP possibly by reducing the oxidative damage and pancreatic inflammation by modulating Nrf2/NF-κB pathway.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Camphanes; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Inflammation; Male; Mice; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Signal Transduction

Agents that modulate the activity of high-voltage gated calcium channels (HVCCs) exhibit experimentally and clinically significant effect by relieving visceral pain. Among these agents, the toxins Phα1β and ω-conotoxin MVIIA effectively reduce chronic pain in rodent models. The molecular mechanisms underlying the chronic pain associated with acute pancreatitis (AP) are poorly understood. Hypercalcemia is a risk factor; the role of cytosolic calcium is considered to be a modulator of pancreatitis. Blockade of Ca

Topics: Abdominal Pain; Analgesics; Animals; Anti-Inflammatory Agents; Behavior, Animal; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Ceruletide; Disease Models, Animal; Exploratory Behavior; Hyperalgesia; Inflammation Mediators; Male; Neuropeptides; omega-Conotoxins; Pain Threshold; Pancreas; Pancreatitis; Rats, Wistar; Spider Venoms; Spinal Cord

Acute pancreatitis (AP) is a potential gastrointestinal problem most commonly associated with pancreatic inflammation and acinar cells injury. Nimbolide (NB), isolated from the tree Azadirachta indica, possesses antioxidant and anti-inflammatory effects. Here, we aimed to investigate the pancreatic protective effects of NB in ameliorating cerulein-induced pancreatic inflammation and apoptosis in AP model and evaluate the potential mechanism of action. AP was induced in Swiss albino mice by six-hourly intraperitoneal exposures of cerulein (50 µg/kg/hr) and pre-treatment of NB (0.3 and 1 mg/kg) 7 days prior to the cerulein exposure. Various parameters associated with AP in plasma and pancreatic tissues were evaluated. Severity of AP was effectively ameliorated by NB as shown by reducing pancreatic edema, plasma amylase and lipase levels, MPO levels and in cerulein-induced histological damage. Further, the antioxidant effect of NB was associated with a significant inhibition of oxidative-nitrosative stress in Raw 264.7 cells and cerulein-induced AP mice. Moreover, NB suppressed proinflammatory cytokines, iNOS and nitrotyrosine expression. In addition, NB inhibited NF-κB activation and increased SIRT1 expression in cerulein challenged mice. Furthermore, NB also inhibited pancreatic apoptosis by downregulating cleaved caspase 3 and Bax while upregulating Bcl2 expression in cerulein-treated mice. Inhibition of pancreatic inflammation and apoptosis resulted in attenuation of cerulein-induced AP. These results suggest that NB exerts strong anti-pancreatitis effects against cerulein-induced AP by combating inflammatory and apoptosis signaling via SIRT1 activation.

Topics: alpha-Amylases; Animals; Apoptosis; Ceruletide; Edema; Injections, Intraperitoneal; Limonins; Male; Mice; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; RAW 264.7 Cells; Signal Transduction; Sirtuin 1

Topics: Animals; cdc42 GTP-Binding Protein; Cells, Cultured; Ceruletide; Disease Models, Animal; Flavonoids; Gene Expression Regulation; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mice, Inbred C57BL; MicroRNAs; Pancreatitis; Phytotherapy; Rats; Scutellaria baicalensis; Severity of Illness Index

Acute pancreatitis is a common inflammatory disease. MicroRNAs have been implicated in the pathogenesis of acute pancreatitis.. The purpose of this study was to investigate the precise roles of miR-193a-5p and miR-320-5p in AP.. The levels of miR-193a-5p, miR-320-5p and tumor necrosis factor receptor-associated factor 3 were detected by quantitative real-time polymerase chain reaction. Cell apoptosis was determined using flow cytometry. Enzyme-linked immunosorbent assay was performed to measure TNF-α, IL-6, IL-1β and IL-8 production, amylase activity, and malondialdehyde content. Targeted relationship between miR-193a-5p or miR-320-5p and TRAF3 was confirmed by the dual-luciferase reporter and RNA immunoprecipitation assays.. Our data showed that miR-193a-5p and miR-320-5p were down-regulated in acute pancreatitis serum and caerulein-treated AR42J cells. The increased expression of miR-193a-5p or miR-320-5p alleviated caerulein-induced cell injury in AR42J cells. Tumor necrosis factor receptor-associated factor 3 was a direct target of miR-193a-5p and miR-320-5p in AR42J cells. Moreover, miR-193a-5p and miR-320-5p regulated caerulein-induced AR42J cells injury through targeting tumor necrosis factor receptor-associated factor 3.. The present findings demonstrated that miR-193a-5p and miR-320-5p protected AR42J cells against caerulein-induced cell injury by targeting tumor necrosis factor receptor-associated factor 3, highlighting their roles as potential therapeutic targets for acute pancreatitis treatment.

Topics: Adult; Animals; Case-Control Studies; Cell Line; Ceruletide; Female; Humans; Male; MicroRNAs; Middle Aged; Pancreatitis; Rats; TNF Receptor-Associated Factor 3

Acute pancreatitis (AP) is an inflammatory reaction of pancreatic tissue self-digestion, edema, hemorrhage, and even necrosis after the activation of pancreatic enzymes in the pancreas caused by a variety of etiologies. This study was aimed to explore the functions and mechanism of Wip1 in AP. Twenty male SD rats were randomly assigned into 2 groups (control group: saline treatment; AP group: cerulein treatment). And cerulein-treated AR42J cells were conducted as AP model in vitro. The levels of amylase were detected by using the Beckman biochemical analyzer. The levels of IFNβ and TNFα were analyzed by ELISA. The autophagosomes were observed by transmission electron microscopy. The Wip1-specific shRNAs were transfected to AR42J cells to silence the expression of Wip1. The levels of Wip1 were measured by qRT-PCR and Western blot. The levels of STING/TBK1/IRF3 and LC3 were measured by Western blot. The AP model was successfully constructed by cerulein administration. Wip1 was notably upregulated in AP models. Autophagy and STING pathway activation were involved in the development of AP. Wip1 inhibition counteracts the promotion effect on inflammatory response induced by cerulein in AR42J Cells. Wip1 inhibition inhibited the activity of the STING/TBK1/IRF3 and reduced LC3 levels in AP. This study preliminarily explored that Wip1 could regulate autophagy and participate in the development of AP through the STING/TBK1/IRF3 signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy; Cell Line; Ceruletide; Cytokines; Disease Models, Animal; Inflammation Mediators; Interferon Regulatory Factor-3; Male; Membrane Proteins; Pancreas; Pancreatitis; Protein Phosphatase 2C; Protein Serine-Threonine Kinases; Rats, Sprague-Dawley; Signal Transduction; Up-Regulation

Acute pancreatitis (AP) is an inflammatory, complicated pancreatic disease, carrying significant morbidity and mortality. However, the molecular and cellular mechanisms involved in AP pathogenesis remain to be elucidated. Here, we explore the role of FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) in AP progression. Caerulein with or without LPS- induced or taurolithocholic acid 3-sulfate (TLC-S)-induced AP mouse models and cell models were performed for the validation of FENDRR expression in vivo and in vitro, respectively. Histopathological examinations of pancreatic tissues were performed to evaluate the severity of AP. Transmission electron microscopy was utilized to visualize the autophagic vacuoles. siRNA specifically targeting FENDRR was further applied. Flow cytometry was employed to assess cell apoptosis. ELISA, immunoflureoscence, and western blotting analysis were also performed to determine the levels of inflammatory cytokines and autophagy activity. RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays were carried out to reveal the epigenetic regulation of FENDRR on ATG7. Additionally, silencing FENDRR was also verified in AP mouse models. Higher FENDRR and impaired autophagy were displayed in both AP mouse models and cell models. FENDRR knockdown dramatically attenuated caerulein- or TLC-S-induced AR42J cells apoptosis and autophagy suppression. Further mechanistic experiments implied that the action of FENDRR is moderately attributable to its repression of ATG7 via direct interaction with the epigenetic repressor PRC2. Moreover, the silencing of FENDRR significantly induced the promotion of ATG7, thus alleviating the development of AP in vivo. Our study highlights FENDRR as a novel target that may contribute to AP progression, suggesting a therapeutic target for AP treatment.

Topics: Animals; Autophagy; Autophagy-Related Protein 7; Cell Line; Ceruletide; Cytokines; Disease Models, Animal; Epigenesis, Genetic; Inflammation Mediators; Lipopolysaccharides; Male; Mice, Inbred C57BL; Pancreas; Pancreatitis; Polycomb Repressive Complex 2; RNA, Long Noncoding; Signal Transduction

Protecting the intestinal mucosa from being destroyed helps reduce the inflammation caused by acute pancreatitis (AP). In this study, whether okra pectin (OP) could attenuate the inflammation of AP through protecting the intestinal barrier was investigated.. OP was obtained from crude okra pectin (COP) through the purification by DEAE cellulose 52 column. Supplementation with OP or COP in advance reduced the severity of AP, as revealed by lower serum amylase and lipase levels, abated pancreatic edema, attenuated myeloperoxidase activity and pancreas histology. OP or COP inhibited the production of pancreatic proinflammatory cytokines, including tumor necrosis factor-α and interleukin-6. In addition, the upregulation of AP-related proteins including ZO-1, occludin, the antibacterial peptide-defensin-1 (DEFB1) and cathelicidin-related antimicrobial peptide (CRAMP), as well as the histological examination of colon injuries, demonstrated that OP or COP provision could effectively maintain intestinal barrier function. Ultimately, dietary OP or COP supplementation could inhibit AP-induced intestinal inflammation. For the above, the effect of OP was better than COP.. Dietary OP supplementation could be considered as a preventive method that effectively interferes with intestinal damage and attenuates inflammatory responses trigged by AP. © 2020 Society of Chemical Industry.

Topics: Abelmoschus; Animals; Ceruletide; Cytokines; Fruit; Humans; Intestinal Mucosa; Male; Mice; Occludin; Pancreatitis; Pectins; Plant Extracts; Tumor Necrosis Factor-alpha; Zonula Occludens-1 Protein

We previously showed that Lactobacillus brevis-derived polyphosphate (poly P) exerts a curative effect on intestinal inflammation. However, whether or not poly P improves the inflammation and injury of distant organs remains unclear.. We aimed to investigate the change in the intestinal microbiome and to evaluate the protective effect of poly P on injuries in a cerulein-induced acute pancreatitis (AP) mouse.. Poly P was orally administered to BALB/C mice every day for 24 days, and then mice were intraperitoneally injected with cerulein. Before cerulein injection, stool samples were collected and analyzed by 16S rRNA gene sequencing. Mice were sacrificed at 24 h after the last cerulein injection; subsequently, the serum, pancreas, and colon were collected.. The microbial profile differed markedly between poly P and control group. Notably, the levels of beneficial bacteria, including Alistipes and Candidatus_Saccharimonas, were significantly increased, while those of the virulent bacteria Desulfovibrio were decreased in the poly P group. The elevations of the serum amylase and lipase levels by cerulein treatment were suppressed by the pre-administration of poly P for 24 days, but not for 7 days. The numbers of cells MPO-positive by immunohistology were decreased and the levels of MCP-1 significantly reduced in the AP + Poly P group. An immunofluorescence analysis showed that the ZO-1 and occludin in the colon was strongly augmented in the epithelial cell membrane layer in the AP + Poly P group.. Poly P attenuates AP through both modification of the intestinal microbiome and enhancement of the intestinal barrier integrity.

Topics: Animals; Ceruletide; Cytokines; Gastrointestinal Microbiome; Gene Expression Regulation; Inflammation; Levilactobacillus brevis; Male; Mice; Mice, Inbred BALB C; Pancreatitis; Polyphosphates; RNA, Bacterial; RNA, Ribosomal, 16S

AR42J are immortalized pancreatic adenocarcinoma cells that share similarities with pancreatic acinar cells. AR42J are often used as a cell-culture model of cerulein (CN)-induced acute pancreatitis (AP). Nevertheless, it is controversial how to treat AR42J for reliable induction of AP-like processes. Gene knockout and/or overexpression often remain challenging, as well. In this study, we demonstrate conditions for a reliable induction of proinflammatory markers upon CN treatment in AR42J and high transfection efficacy using Glyoxalase-I (Glo-I) as a target of interest.. Effects of dexamethasone (dexa) and CN on cell morphology and amylase secretion were analyzed via ELISA of supernatant. IL-6, TNF-α and NF-κB-p65 were measured via qRT-PCR, ELISA and Western Blot (WB). Transfection efficacy was determined by WB, qRT-PCR and immune fluorescence of pEGFP-N1-Glo-I-Vector and Glo-I-siRNA.. Treatment of AR42J with 100 nm dexa is mandatory for differentiation to an acinar-cell-like phenotype and amylase production. CN resulted in secretion of amylase but did not influence amylase production. High levels of CN-induced amylase secretion were detected between 3 and 24 hours of incubation. Treatment with LPS alone or in combination with CN did not influence amylase release compared to control or CN. CN treatment resulted in increased TNF-α production but not secretion and did not influence IL-6 mRNA. CN-induced stimulation of NF-κB was found to be highest on protein levels after 6h of incubation. Transient transfection was able to induce overexpression on protein and mRNA levels, with highest effect after 12 to 24 hours. Gene-knockdown was achieved by using 30 pmol of siRNA leading to effective reduction of protein levels after 72 hours. CN did not induce amylase secretion in AR42J cell passages beyond 35.. AR42J cells demonstrate a reliable in-vitro model of CN-induced AP but specific conditions are mandatory to obtain reproducible data.

Topics: Animals; Cell Line, Tumor; Cell Shape; Ceruletide; Dexamethasone; Gene Knockdown Techniques; Interleukin-6; Models, Biological; NF-kappa B; Pancreatitis; Rats; RNA, Small Interfering; Tumor Necrosis Factor-alpha

Pancreatic ductal adenocarcinoma (PDAC) is highly metastatic and represents one of the deadliest forms of human cancers. Previous studies showed that activation of Yes-associated protein 1 (YAP1) plays a key role in malignant transformation in the pancreas. In this study, we found that YAP1 regulates the expression of epithelial cell transforming 2 (ECT2), a guanine nucleotide exchange factor for Rho-like GTPases. By immunohistochemistry analysis of human tissues, we show that ECT2 is highly expressed in primary PDAC and liver metastasis but not in normal pancreas. These correlations were also observed in a mouse model of PDAC, where pancreatic transformation is driven by mutants of

Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Cell Line; Cell Movement; Cell Proliferation; Cell Survival; Ceruletide; Humans; Neoplasms, Experimental; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins; Transcription Factors; Up-Regulation; YAP-Signaling Proteins

Acute pancreatitis (AP), an inflammatory disorder of the pancreas with a high hospitalization rate, frequently leads to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). However, therapeutic targets for effective treatment and early intervention of AP are still urgently required to be identified. Here, we have observed that the expression of pancreatic lincRNA-EPS, a long intergenic non-coding RNA, is dynamically changed during both caerulein-induced AP (Cer-AP) and sodium taurocholate-induced severe AP (NaTc-SAP). The expression pattern of lincRNA-EPS is negatively correlated with the typical inflammatory genes such as IL-6, IL-1β, CXCL1, and CXCL2. Further studies indicate that knockout of lincRNA-EPS aggravates the pathological symptoms of AP including more induction of serum amylase and lipase, severe edema, inflammatory cells infiltration and acinar necrosis in both experimental AP mouse models. Besides these intrapancreatic effects, lincRNA-EPS also protects against tissue damages in the extra-pancreatic organs such as lung, liver, and gut in the NaTc-SAP mouse model. In addition, we have observed more serum pro-inflammatory cytokines TNF-α and IL-6 in the lincRNA-EPS

Topics: Animals; Ceruletide; Disease Models, Animal; HEK293 Cells; HMGB1 Protein; Humans; Inflammation; Inflammation Mediators; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Targeted Therapy; Necrosis; NF-kappa B; Pancreas; Pancreatitis; RNA, Long Noncoding; Severity of Illness Index; Taurocholic Acid

Mesotrypsin is a low-abundance human trypsin isoform with a unique evolutionary mutation that conferred resistance to trypsin inhibitors and restricted substrate specificity. Mesotrypsin degrades the serine protease inhibitor Kazal type 1 (SPINK1) and thereby might increase risk for pancreatitis. Here, we report a mouse model designed to test the role of mesotrypsin in pancreatitis. We introduced the human mesotrypsin evolutionary signature mutation into mouse cationic trypsinogen (isoform T7), resulting in a Gly to Arg change at the corresponding position 199. In biochemical experiments using purified proteins, the p.G199R T7 mutant recapitulated all salient features of human mesotrypsin. T7G199R mice developed normally with no spontaneous pancreatitis or other obvious phenotypic changes. Cerulein-induced acute pancreatitis in C57BL/6N and T7G199R mice showed similar severity with respect to inflammatory parameters and acinar cell necrosis while plasma amylase activity was higher in T7G199R mice. Neither SPINK1 degradation nor elevated intrapancreatic trypsin activation was apparent in T7G199R mice. The results indicate that in T7G199R mice the newly created mesotrypsin-like activity has no significant impact on cerulein-induced pancreatitis. The observations suggest that human mesotrypsin is unimportant for pancreatitis; a notion that is consistent with published human genetic studies.

Topics: Animals; Ceruletide; Chymotrypsin; Disease Models, Animal; Gene Expression Regulation; Glycoproteins; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mutation; Pancreatitis; Prostatic Secretory Proteins; Trypsin; Trypsin Inhibitor, Kazal Pancreatic; Trypsinogen

Cathepsin B (CTSB), nod-like receptor family pyrin domain-containing 3 (NLRP3), and caspase-1 play an important role in the development of Acute Pancreatitis (AP). Besides, the relationship between the proteins remains poorly understood. In addition, whereas previous studies have found caspase-1 activation in AP, pyroptosis, a caspase-1 induced cell death mode, has never been proposed and proved in AP.. We induced AP in mice by intraperitoneal injection of cerulein. Mice in the inhibitor group of CTSB were pretreated with injection of CA-074me, while mice in the inhibitor group of caspase-1 were of Ac-YVAD-CHO, 1 h earlier. We evaluated the inflammation of the pancreas and the detected expression of activated CTSB, NLRP3, ASC, caspase-1p20, IL-1β and IL-18. TUNEL staining was used to detect acinar cell death.. The inflammation of the pancreas in the two inhibitor groups was significantly reduced compared with that in the AP group. We observed that CA-074me not only inhibits CTSB, but also suppresses the expression and activity of NLRP3, ASC and caspase-1. We found that CA-074me further inhibits the downstream event of caspase-1, including pro-inflammatory cytokine secretion and pyroptosis. Whereas Ac-YVAD-CHO inhibited caspase-1 and decreased pro-inflammatory cytokine secretion and pyroptosis, it did not down-regulate the expression and activity ofCTSB, NLRP3 and ASC.. The results indicate that CTSB may aggravate AP by activating the NLRP3 inflammasome and promoting Caspase-1-induced pyroptosis. These provide clues about the pathophysiological mechanisms of AP, shedding light on new ideas and potential targets for the prevention and treatment of AP.

Topics: Animals; Caspase 1; Cathepsin B; Ceruletide; Inflammasomes; Interleukin-1beta; Male; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatitis; Pyroptosis

Acute pancreatitis (AP) and recurring AP are serious health care problems causing excruciating pain and potentially lethal outcomes due to sepsis. The validated caerulein- (CAE) induced mouse model of acute/recurring AP produces secondary persistent hypersensitivity and anxiety-like behavioral changes for study.. To determine efficacy of acetyl-L-carnitine (ALC) to reduce pain-related behaviors and brain microglial activation along the pain circuitry in CAE-pancreatitis.. Pancreatitis was induced with 6 hly intraperitoneal (i.p.) injections of CAE (50 µg/kg), 3 d a week for 6 wk in male C57BL/6J mice. Starting in week 4, mice received either vehicle or ALC until experiment's end. Mechanical hyper-sensitivity was assessed with von Frey filaments. Heat hypersensitivity was determined with the hotplate test. Anxiety-like behavior was tested in week 6 using elevated plus maze and open field tests. Microglial activation in brain was quantified histologically by immunostaining for ionized calcium-binding adaptor molecule 1 (Iba1).. Mice with CAE-induced pancreatitis had significantly reduced mechanical withdrawal thresholds and heat response latencies, indicating ongoing pain. Treatment with ALC attenuated inflammation-induced hypersensitivity, but hypersensitivity due to abdominal wall injury caused by repeated intraperitoneal injections persisted. Animals with pancreatitis displayed spontaneous anxiety-like behavior in the elevated plus maze compared to controls. Treatment with ALC resulted in increased numbers of rearing activity events, but time spent in "safety" was not changed. After all the abdominal injections, pancreata were translucent if excised at experiment's end and opaque if excised on the subsequent day, indicative of spontaneous healing. Post mortem histopathological analysis performed on pancreas sections stained with Sirius Red and Fast Green identified wide-spread fibrosis and acinar cell atrophy in sections from mice with CAE-induced pancreatitis that was not rescued by treatment with ALC. Microglial Iba1 immunostaining was significantly increased in hippocampus, thalamus (intralaminar nuclei), hypothalamus, and amygdala of mice with CAE-induced pancreatitis compared to naïve controls but unchanged in the primary somatosensory cortex compared to naïves.. CAE-induced pancreatitis caused increased pain-related behaviors, pancreatic fibrosis, and brain microglial changes. ALC alleviated CAE-induced mechanical and heat hypersensitivity but not abdominal wall injury-induced hypersensitivity caused by the repeated injections.

Topics: Acetylcarnitine; Acute Disease; Animals; Brain; Ceruletide; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Microglia; Pain; Pancreas; Pancreatitis

Chaiqin chengqi decoction (CQCQD) and its derivatives have been widely used in China for the early management of patients with acute pancreatitis (AP). Numerous studies demonstrate the anti-inflammatory and anti-oxidative effects of CQCQD and derivatives, but whether these effects can be attributed to suppressing neurogenic inflammation, has never been studied.. To investigate the effects of CQCQD on substance P (SP)-neurokinin 1 receptor (NK1R) based neurogenic inflammation in an experimental AP model.. For AP patients on admission, pain score was accessed by visual analog scale (VAS); the levels of serum SP and expressions of pancreatic SP and NK1R were also determined. For in vivo study, mice received 7 intraperitoneal injections of cerulein (50 μg/kg) at hourly intervals to induce AP, whilst controls received normal saline injections. In the treatment groups, CQCQD (10 g/kg, 200 μl) was intragastrically given at the third, fifth, and seventh of the cerulein injection or the NK1R antagonist CP96345 (5 mg/kg) was intraperitoneally injected 30 min before the first cerulein administration. The von Frey test was performed to evaluate pain behavior. Animals were sacrificed at 12 h from the first cerulein/saline injection for severity assessment. Pharmacology network analysis was used to identify active ingredients of CQCQD for AP and pain. In vitro, freshly isolated pancreatic acinar cells were pre-treated with CQCQD (5 mg/ml), CP96345 (1 μM), or selected active compounds of CQCQD (12.5, 25, and 50 μM) for 30 min, followed by SP incubation for another 30 min.. The VAS score as well as the levels of serum SP and expressions of pancreatic SP-NK1R were up-regulated in moderately severe and severe patients compared with those with mild disease. CQCQD, but not CP96345, consistently and significantly ameliorated pain, pancreatic necrosis, and systemic inflammation in cerulein-induced AP as well as inhibited NK1R internalization of pancreatic acinar cells. These effects of CQCQD were associated with reduction of pancreatic SP-NK1R and neuron activity in pancreas, dorsal root ganglia, and spinal cord. Baicalin, emodin, and magnolol, the top 3 active components of CQCQD identified via pharmacology network analysis, suppressed NK1R internalization and NF-κB signal pathway activation in isolated pancreatic acinar cells.. CQCQD ameliorated cerulein-induced AP and its associated pain via inhibiting neuron activation-mediated pancreatic acinar cell SP-NK1R signaling pathways and its active compounds baicalin, emodin, and magnolol contributed to this effect.

Topics: Acinar Cells; Analgesics; Animals; Anti-Inflammatory Agents; Biphenyl Compounds; Ceruletide; Drugs, Chinese Herbal; Emodin; Flavonoids; Ganglia, Spinal; Humans; Lignans; Male; Mice, Inbred C57BL; Neurons; Pain; Pancreas; Pancreatitis; Receptors, Neurokinin-1; Signal Transduction; Spinal Cord; Substance P

Acute pancreatitis (AP) is an inflammatory disorder of the pancreas that is associated with substantial morbidity and mortality. Chaiqin chengqi decoction (CQCQD) has been proven clinically to be an effective treatment for AP for decades in West China Hospital. Quality control for CQCQD containing many hundreds of characteristic phytochemicals poses a challenge for developing robust quality assessment metrics.. To evaluate quality consistency of CQCQD with a multi-strategy based analytical method, identify potential quality-markers (Q-markers) based on drug properties and effect characteristics, and endeavor to establish CQCQD as a globally-accepted medicine.. A typical analysis of constitutive medicinal plant materials was performed following the Chinese Pharmacopoeia. The extraction process was optimized through an orthogonal array (L. The chemical markers and other physical and chemical indices in the original materials met Chinese Pharmacopoeia standards. A total of 22 co-existing fingerprint peaks were selected and the similarity varied between 0.946 and 0.990. Batch D10 possessed the highest similarity index. HCA classified the 10 batches into 2 main groups: 7 batches represented by D10 and 3 batches represented by D1. During the initial Q-marker screen stage, 22 compounds were detected in both plant materials and decoctions, while 13 compounds were identified in plasma. Network pharmacology predicted the potential targets and pathway of AP related to the 22 compounds. All 10 batches showed reduced necrosis below 60% with the best effect achieved by D10 (~40%). The spectrum-efficacy relationship analyzed by Pearson correlation analysis indicated that emodin, rhein, aloe emodin, geniposide, hesperridin, chrysin, syringin, synephrine, geniposidic acid, magnolol, physcion, sinensetin, and baicalein showed positive correlation with pancreatic acinar cell death protection. Similar to the in vitro evaluation, batch D10 significantly reduced total histopathological scores and biochemical severity indices at a clinically-equivalent dose but batch D1 did not. The content of naringin, narirutin and baicalin in batches D1, D5 and D9 consistently exceeds the upper limit of the predicted value. Eight markers whose lower limit is predicted to be close to 0 contributed less to the material basis for AP protection.. Despite qualified materials used for CQCQD preparation, the clinical effect depends on appropriate content range of Q-markers. Emodin, rhein, aloe emodin, magnolol, hesperidin, synephrine, baicalein, and geniposide are considered as vital Q-markers in the primary screen. This study proposed a feasible platform for producing highly consistent batches of CQCQD in future study.

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Mice; Necrosis; Pancreas; Pancreatitis; Quality Control

Acute pancreatitis (AP) is a common inflammatory disorder with high incidence and mortality. AMPK-SIRT1 pathway is involved in a variety of diseases, but its role in AP remains elusive. This study was aimed to explore the role of AMPK-SIRT1 pathway in AP.. AP models in vivo and vitro were constructed by intraperitoneal administration of L-arginine and caerulein-stimulated respectively. Rat serum amylase, IL-6 and TNF-α were determined by ELISA. The expression levels of AMPK, SIRT1, Beclin-1, LC3 and p62 were determined by qRT-PCR and western blot. The number of autophagosome was checked by transmission electron microscope.. Compared with NC rats, serum amylase, IL-6 and TNF-α were increased in AP rats. The expressions of AMPK and SIRT1 were decreased, while Beclin-1, LC3II/Iratio and p62 were markedly increased in AP rats. After activation of AMPK by metformin, expressions of p-AMPKα, SIRT1 were significantly raised, while expressions of Beclin-1, LC3 II/I, p62, TNF-α, IL-6 were reduced, and the number of autophagosome was decreased significantly in caerulein-stimulated AR42J cells. The inhibition of AMPK by compound C obtained opposite results.. During AP occurrence, p-AMPK and SIRT1 were down-regulated, leading to the accumulation of p62, increase of autophagic vacuoles, damage of autophagy, and the occurrence of inflammation. It hinted that activation of AMPK restored impaired autophagy and inhibited inflammation reaction by up-regulating SIRT1. Our findings might provide important theoretical basis for explaining the pathogenesis of AP and investigating therapeutic target to treat and prevent AP.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Ceruletide; Gene Expression Regulation; Inflammation; Pancreatitis; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirtuin 1

Milk fat globule-EGF factor 8 (MFG-E8) is a secreted glycoprotein that regulates tissue homeostasis, possesses potent anti-inflammatory properties, and protects against tissue injury. The human pancreas expresses MFG-E8; however, the role of MFG-E8 in the pancreas remains unclear. We examined the expression of MFG-E8 in the pancreas at baseline and during cerulein-induced acute pancreatitis in mice and determined whether MFG-E8 attenuates the progression of pancreatitis, a serious inflammatory condition that can be life-threatening. We administered cerulein to wild-type (WT) and

Topics: Acinar Cells; Animals; Antigens, Surface; Ceruletide; Mice, Inbred C57BL; Mice, Knockout; Milk Proteins; Pancreas, Exocrine; Pancreatitis; Recombinant Proteins; RNA, Messenger; Severity of Illness Index

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease's progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson's trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFβ, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Disease Models, Animal; Fibrosis; Inflammation; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Pancreatitis, Chronic; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Acute pancreatitis is a common inflammatory disorder of the exocrine pancreas with no specific therapy. Intracellular nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) salvage pathway, is involved in many inflammatory disorders. In this study, we investigated the role of NAMPT in experimental acute pancreatitis.. Acute pancreatitis was induced in mice using three disparate models: (1) caerulein hyperstimulation, (2) ethanol plus palmitoleic acid, and (3) retrograde biliopancreatic ductal infusion of sodium taurocholate. The NAMPT inhibitor FK866 and NAMPT downstream product nicotinamide mononucleotide (NMN) was administered. Serum and pancreas were collected and analyzed biochemically and histologically. Bone marrow derived macrophages were isolated, cultured with cytokines or pancreatic acini, then analyzed by quantitative PCR and non-targeted metabolomics.. The levels of pancreatic NAMPT and NAD were down-regulated upon acute pancreatitis. NAMPT inhibitor FK866 suppressed M1 macrophage polarization while NMN boosted it. In co-culture of macrophages with acinar cells, inhibition of NAMPT prevented M1-like macrophage differentiation induced by injured pancreatic acini. The injured pancreatic acinar milieu induced a unique metabolic signature linked to macrophage polarization, and inhibition of NAMPT reversed these metabolites changes. Furthermore, NMN supplementation aggravated caerulein hyperstimulation pancreatitis and alcoholic pancreatitis, and inhibition of NAMPT protected against caerulein hyperstimulation, alcoholic and biliary acute pancreatitis and reducing pancreatic macrophage infiltration in vivo.. NAMPT inhibition protects against acute pancreatitis via preventing M1 macrophage polarization and restoring the metabolites related to macrophage polarization and that NAMPT could be a promising therapeutic target for acute pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Cytokines; Macrophages; Mice; NAD; Nicotinamide Mononucleotide; Nicotinamide Phosphoribosyltransferase; Pancreatitis; Sirtuin 1

Acute pancreatitis (AP) lacks targeted prevention and treatment measures. Some key points in the pathogenesis of AP remain unclear, such as early activation of pancreatic enzymes. Several recent reports have shown the protective effect of hydrogen on several AP animal models, and the mechanism is related to antioxidant activity. Heat shock protein 60 (Hsp60) is known to accompany pancreatic enzymes synthesis and secretion pathway of in pancreatic acinar cells, while role of hsp60 in AP remains a topic. Aim of this study was to investigate effect of hydrogen pretreatment on AP and the mechanisms, focusing on pancreatic oxidative stress and Hsp60 expression.. 80 mice were randomly assigned into four groups: HAP group, AP group, HNS group, and NS group and each group were set 3 observation time point as 1 h, 3 h and 5 h (n = 6-8). Mouse AP model was induced by intraperitoneal injection of 50 μg/kg caerulein per hour for 6 injections both in AP and HAP groups, and mice in NS group and HNS group given normal saline (NS) injections at the same way as control respectively. Mice in HAP group and HNS group were treated with hydrogen-rich gases inhalation for 3 days before the first injection of caerulein or saline, while mice in AP group and NS group in normal air condition. Histopathology of pancreatic tissue, plasma amylase and lipase, plasma IL-1 and IL-6, pancreatic glutathione (GSH) and malondialdehyde (MDA), and Hsp60 mRNA and protein expression were investigated. Comparisons were made by one-way analysis of variance.. The pancreatic pathological changes, plasma amylase and lipase activity, and the increase of plasma IL-1 and IL-6 levels in AP mice were significantly improved by the hydrogen-rich gases pretreatment, Meanwhile, the pancreatic GSH content increased and the pancreatic MDA content decreased. And, the hydrogen-rich gases pretreatment improved the Hsp60 protein expression in pancreatic tissues of AP mice at 1 h and 5 h.. Pre-inhalation of hydrogen-rich gases have a good protective effect on AP mice, and the possible mechanisms of reduced oxidative stress and the early increased pancreatic Hsp60 protein deserve attention.

Topics: Administration, Inhalation; Animals; Ceruletide; Chaperonin 60; Disease Models, Animal; Female; Gases; Gastrointestinal Agents; Hydrogen; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Pancreas; Pancreatitis; Random Allocation

Acute pancreatitis (AP) is a severe and potentially fatal disease caused predominantly by alcohol excess and gallstones, which lacks a specific therapy. The role of Receptor-Interacting Protein Kinase 1 (RIPK1), a key component of programmed necrosis (Necroptosis), is unclear in AP. We assessed the effects of RIPK1 inhibitor Necrostatin-1 (Nec-1) and RIPK1 modification (RIPK1

Topics: Acinar Cells; Alcohols; Animals; Bile Acids and Salts; Calcium; Ceruletide; Imidazoles; Indoleamine-Pyrrole 2,3,-Dioxygenase; Indoles; Male; Mice, Inbred C57BL; Pancreas; Pancreatitis; Protective Agents; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases

/Objectives: The pathogenesis of hyperglycemia during acute pancreatitis (AP) remains unknown due to inaccessibility of human tissues and lack of animal models. We aimed to develop an animal model to study the mechanisms of hyperglycemia and impaired glucose tolerance in AP.. We injected ferrets with intraperitoneal cerulein (50 μg/kg, 9 hourly injections) or saline. Blood samples were collected for glucose (0, 4, 8, 12, 24h); TNF-α, IL-6 (6h); amylase, lipase, insulin, glucagon, pancreatic polypeptide (PP), glucagon-like peptide-1 (GLP-1), and gastric inhibitory polypeptide (GIP) (24h). Animals underwent oral glucose tolerance test (OGTT), mixed meal tolerance test (MMTT) at 24h or 3 months, followed by harvesting pancreas for histopathology and immunostaining.. Cerulein-injected ferrets exhibited mild pancreatic edema, neutrophil infiltration, and elevations in serum amylase, lipase, TNF-α, IL-6, consistent with AP. Plasma glucose was significantly higher in ferrets with AP at all time points. Plasma glucagon, GLP-1 and PP were significantly higher in cerulein-injected animals, while plasma insulin was significantly lower compared to controls. OGTT and MMTT showed abnormal glycemic responses with higher area under the curve. The hypoglycemic response to insulin injection was completely lost, suggestive of insulin resistance. OGTT showed low plasma insulin; MMTT confirmed low insulin and GIP; abnormal OGTT and MMTT responses returned to normal 3 months after cerulein injection.. Acute cerulein injection causes mild acute pancreatitis in ferrets and hyperglycemia related to transient islet cell dysfunction and insulin resistance. The ferret cerulein model may contribute to the understanding of hyperglycemia in acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Blood Glucose; Ceruletide; Ferrets; Gastric Inhibitory Polypeptide; Glucagon; Glucagon-Like Peptide 1; Humans; Hyperglycemia; Insulin; Insulin Resistance; Interleukin-6; Lipase; Pancreatitis; Tumor Necrosis Factor-alpha

Symptoms associated with acute pancreatitis can be debilitating, and treatment remains a challenge. This study aimed to investigate the efficacy of selectively inhibiting the soluble form of TNF (solTNF) using the biologic XPro1595 in a mouse model of acute pancreatitis.. Acute pancreatitis was induced in adult male C57Bl/6J mice by administering cerulein (8 injections of 50 µg/kg I.P., spaced an hour apart), with XPro1595 (10 mg/kg, S.C.) or vehicle being administered approximately 18 h after the last injection. Serum was collected 6 or 18 h after the last cerulein injection, pancreatic tissue was collected 2 and 7 days post-induction, and brain hippocampal tissue was collected at 7 days post-induction. The animal's pain level was assessed 3, 5 and 7 days post-induction.. The induction of acute pancreatitis promoted a strong increase in serum amylase levels, which had receded back to baseline levels by the next morning. XPro1595 treatment began after amylase levels had subsided at 18 h, and prevented pancreatic immune cell infiltration, that subsequently prevented tissue disruption and acinar cell death. These improvements in pathology were associated with a significant reduction in mechanical hypersensitivity (neuropathic pain). XPro1595 treatment also prevented an increase in hippocampal astrocyte reactivity, that may be associated with the prevention of neuropathic pain in this mouse model.. Overall, we observed that selectively inhibiting solTNF using XPro1595 improved the pathophysiological and neurological sequelae of cerulein-induced pancreatitis in mice, which provides support of its use in patients with pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Humans; Male; Mice; Pancreas; Pancreatitis; Tumor Necrosis Factor-alpha

Topics: Acinar Cells; Animals; Cell Lineage; Ceruletide; Disease Models, Animal; Homeostasis; Humans; Methyltransferases; Mice; Pancreas; Pancreatitis; Regeneration; Stem Cells

Topics: Animals; Ceruletide; Disease Models, Animal; Enzyme Activation; Inflammation Mediators; Macrophages; Mice, Inbred C57BL; Pancreas; Pancreatitis; Patient Acuity; Peptide Hydrolases; Proof of Concept Study; Recurrence; Time Factors

Obesity is one of the most important risk factors for acute pancreatitis. Based on the effect of sleeve gastrectomy (SG) on improving body weight and blood lipids, we investigated whether SG is beneficial in improving pancreatitis in obese rats.. Two studies were used to evaluate the effect of SG on the first onset of pancreatitis and acute episodes of recurrent pancreatitis in obese rats. A high-fat diet (HFD) for 8 weeks resulted in obesity in rats. Study 1: Obese rats were treated with SG and sham surgery. Pancreatitis was induced by intraperitoneal injection of cerulein at 6 weeks after surgery. The severity of pancreatitis was assessed by histological examination, cytokines, and infiltration of inflammatory cells. Study 2 performed the same procedure as in study 1, except that rats were intraperitoneally injected with a small dose of cerulein three times a week for 6 weeks before surgery to induce recurrent pancreatitis.. The body weight, food intake, and blood lipids of SG rats in study 1 and study 2 were significantly lower than those of sham rats during the 6 weeks after surgery. Compared with sham rats, SG rats in both studies had fewer inflammatory cytokines, inflammatory cell infiltration, and pathological injury in the pancreas after cerulein-induced acute pancreatitis.. SG reduces the severity of the first onset of pancreatitis and the seriousness of acute episodes of recurrent pancreatitis. The improvement of lipid metabolism and body weight by SG may play an important role in this effect.

Topics: Acute Disease; Animals; Ceruletide; Gastrectomy; Obesity; Obesity, Morbid; Pancreatitis; Rats

Nod-like receptor pyrin domain containing 3 (NLRP3) is one of the well characterized inflammasome that controls the maturation of pro-inflammatory cytokines and thereby the inflammation in pancreas which could be a promising target for anti-inflammatory drugs. The present study is aimed to explore whether luteolin can target the NLRP3 inflammasome and modulate its activity through the signaling protein, HSP70 in the ethanol-cerulein model of experimental pancreatitis.. Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats received normal diet. Groups 3 and 4 rats received isocalorically adjusted diet containing ethanol for 5 weeks and cerulein (20 μg/kg body weight i.p., thrice weekly for the last 3 weeks of the experimental period). Additionally, group 2 and 4 rats received 2 mg/kg body weight of luteolin orally from third week.. Luteolin co-administration decreased the serum levels of HSP70, oxidative stress markers, myeloperoxidase, GSH/GSSG and GST with concomitant downregulation in the mRNA expression of HSP70, caspase-1, ASC-NLRP3 and NF-κB. Spearman's rank correlation test showed that serum HSP70 has positive correlation with the expression of ASC-NLRP3, caspase-1, NF-κB and 4-hydroxynonenal and negative correlation with GSH:GSSG ratio.. The modulating effect of luteolin on the expression of HSP70, NF-κB and thereby on ASC-NLRP3 complex may be claimed for its pancreato-protective activity.

Topics: Animals; Apoptosis; Body Weight; Caspase 1; Caspase Activation and Recruitment Domain; Ceruletide; Ethanol; Glutathione Disulfide; HSP70 Heat-Shock Proteins; Inflammasomes; Luteolin; Male; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis; Rats; Rats, Wistar

The management of acute pancreatitis (AP) remains a challenge to clinicians worldwide for limited effective interventions. Retinoid orphan receptor gamma t (RORγt) is a therapeutic target for several diseases; however, it is unclear whether inhibiting RORγt can ameliorate AP. The relative expression of RORγt, IL-17 and IL-23 in the peripheral blood mononuclear cells of AP patients was measured by RT-PCR. An AP mouse model was induced by ceruletide, and SR1001 was injected before ceruletide administration. RORγt+ cells, T helper 17 cells (Th17), regulatory T cells (Tregs) and γδ T cells were assessed in the pancreas and spleen by flow cytometry. Higher RORγt expression in patients indicated the potential role of RORγt in AP progression. Analyses of the IL-17/IL-23 axis confirmed its role. SR1001 significantly alleviated AP histologically in the mouse model. Serum levels of amylase, IL-6, TNFalpha, IL-17 and IL-23 decreased upon SR1001 treatment. SR1001 selectively decreased the number of RORγt+, Th17, Tregs and γδ T cells in the pancreas but not the spleen. Collectively, these results showed that SR1001 exerted therapeutic effects on AP by suppressing IL-17-secreting Th17 and γδ T cells in the pancreas. Thus, SR1001 may be a promising drug for the treatment of AP in the clinic.

Topics: Acute Disease; Adult; Aged; Animals; Case-Control Studies; Ceruletide; Disease Models, Animal; Disease Progression; Female; Humans; Interleukin-17; Intraepithelial Lymphocytes; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred C57BL; Middle Aged; Nuclear Receptor Subfamily 1, Group F, Member 3; Pancreatitis; Sulfonamides; Th17 Cells; Thiazoles

Molecular signalling mediated by the phosphatidylinositol-3-kinase (PI3K)-Akt axis is a key regulator of cellular functions. Importantly, alteration of the PI3K-Akt signalling underlies the development of different human diseases, thus prompting the investigation of the pathway as a molecular target for pharmacologic intervention. In this regard, recent studies showed that small molecule inhibitors of PI3K, the upstream regulator of the pathway, reduced the development of inflammation during acute pancreatitis, a highly debilitating and potentially lethal disease. Here we investigated whether a specific reduction of Akt activity, by using either pharmacologic Akt inhibition, or genetic inactivation of the Akt1 isoform selectively in pancreatic acinar cells, is effective in ameliorating the onset and progression of the disease. We discovered that systemic reduction of Akt activity did not protect the pancreas from initial damage and only transiently delayed leukocyte recruitment. However, reduction of Akt activity decreased acinar proliferation and exacerbated acinar-to-ductal metaplasia (ADM) formation, two critical events in the progression of pancreatitis. These phenotypes were recapitulated upon conditional inactivation of Akt1 in acinar cells, which resulted in reduced expression of 4E-BP1, a multifunctional protein of key importance in cell proliferation and metaplasia formation. Collectively, our results highlight the critical role played by Akt1 during the development of acute pancreatitis in the control of acinar cell proliferation and ADM formation. In addition, these results harbour important translational implications as they raise the concern that inhibitors of PI3K-Akt signalling pathways may negatively affect the regeneration of the pancreas. Finally, this work provides the basis for further investigating the potential of Akt1 activators to boost pancreatic regeneration following inflammatory insults. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Acinar Cells; Adaptor Proteins, Signal Transducing; Animals; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Ceruletide; Disease Models, Animal; Male; Metaplasia; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatic Ducts; Pancreatitis; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

Acute pancreatitis is an inflammatory process of the pancreatic gland that may lead to dysregulation of the trans-sulfuration pathway. The aims of this work were firstly to study the methionine cycle as well as the trans-sulfuration pathway using metabolomic and proteomic approaches identifying the causes of this dysregulation in an experimental model of acute pancreatitis; and secondly to reveal the effects of S-adenosylmethionine administration on these pathways. Acute pancreatitis was induced by cerulein in mice, and a group of animals received S-adenosylmethionine treatment. Cerulein-induced acute pancreatitis rapidly caused marked depletion of methionine, S-adenosylmethionine, 5'-methylthioadenosine, cystathionine, cysteine, and glutathione levels in pancreas, but S-adenosylhomocysteine and homocysteine remained unchanged. Protein steady-state levels of S-adenosylhomocysteine-hydrolase and cystathionine gamma-lyase diminished but methylthioadenosine phosphorylase levels increased in pancreas with acute pancreatitis. Although cystathionine β-synthase protein levels did not change with acute pancreatitis, Nos2 mRNA and protein levels were markedly up-regulated and caused tyrosine nitration of cystathionine β-synthase in pancreas. S-adenosylmethionine administration enhanced Nos2 mRNA expression and cystathionine β-synthase nitration and triggered homocysteine accumulation in acute pancreatitis. Furthermore, S-adenosylmethionine administration promoted enrichment of the euchromatin marker H3K4me3 in the promoters of Tnf-α, Il-6, and Nos2 and enhanced the mRNA up-regulation of these genes. Accordingly, S-adenosylmethionine administration increased inflammatory infiltrate and edema in pancreas with acute pancreatitis. In conclusion, tyrosine-nitration of cystathionine β-synthase blockades the trans-sulfuration pathway in acute pancreatitis promoting homocysteine accumulation upon S-adenosylmethionine treatment.

Topics: Animals; Ceruletide; Cystathionine; Cystathionine beta-Synthase; Cysteine; Disease Models, Animal; Glutathione; Homocysteine; Male; Mice; Nitric Oxide Synthase Type II; Pancreatitis; S-Adenosylmethionine; Up-Regulation

Pancreatitis starts with primarily sterile local inflammation that induces systemic inflammatory response syndrome, followed by compensatory anti-inflammatory response syndrome (CARS). We investigated the mechanisms of these processes in mice and human serum.. We induced severe acute pancreatitis by partial duct ligation with caerulein stimulation or intraperitoneal injection of l-arginine in mice with deletion of interleukin (IL)12B, NLRP3, or IL18 and in mice given MCC950, a small molecule inhibitor of the NLRP3-inflammasome. Pancreata were collected from mice and analyzed by histology, and cytokine levels were measured in serum samples. We measured activation of adaptive immune responses in mice with pancreatitis by flow cytometry analysis of T cells (CD25 and CD69) isolated from the spleen. Differentiation of T-helper (Th1) cells, Th2 cells, and T-regulatory cells was determined by nuclear staining for TBET, GATA3, and FOXP3. We performed transcriptome analysis of mouse lymph nodes and bone marrow-derived macrophages after incubation with acini. We measured levels of cytokines in serum samples from patients with mild and severe acute pancreatitis.. Activation of the adaptive immune response in mice was initiated by macrophage-derived, caspase 1-processed cytokines and required activation of NLRP3 (confirmed in serum samples from patients with pancreatitis). Spleen cells from mice with pancreatitis had increases in Th2 cells but not in Th1 cells. Bone marrow-derived macrophages secreted IL1B and IL18, but not IL12, after co-incubation with pancreatic acini. T-cell activation and severity of acute pancreatitis did not differ significantly between IL12B-deficient and control mice. In contrast, NLRP3- or IL18-deficient mice had reduced activation of T cells and no increase in Th2 cell-mediated responses compared with control mice. The systemic type 2 immune response was mediated by macrophage-derived cytokines of the IL1 family. Specifically, IL18 induced a Th2 cell-mediated response in the absence of IL12. MCC950 significantly reduced neutrophil infiltration, T-cell activation, and disease severity in mice.. In mice with severe pancreatitis, we found systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome developed in parallel. Infiltrating macrophages promote inflammation and simultaneously induce a Th2 cell-mediated response via IL18. Inhibition of NLRP3 reduces systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome and might be used to treat patients with severe pancreatitis.

Topics: Acinar Cells; Adaptive Immunity; Animals; Arginine; Cells, Cultured; Ceruletide; Cytokines; Disease Models, Animal; Furans; Heterocyclic Compounds, 4 or More Rings; Humans; Indenes; Inflammasomes; Injections, Intraperitoneal; Interleukin-18; Macrophages; Mice; Mice, Knockout; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatitis; Primary Cell Culture; Sulfonamides; Sulfones; Systemic Inflammatory Response Syndrome; Th2 Cells

Enteric viruses that inhabit the intestine have profound effects on innate and adaptive immunity of the gut and thus distant organs. Acute pancreatitis (AP) is a common abdominal inflammatory disease, in which gut bacteria play an indispensable part, particularly in the severe form with local and systemic complications. So far, little is known about the role of enteric viruses in the pathophysiology of AP. In this study, we evaluated the effect of enteric virus depletion by oral anti-viral cocktail (AVC) on caerulein (Cae)-hyperstimulation induced experimental AP and underlying mechanisms. We found that AVC treatment alleviated experimental AP, accompanied by suppressed innate immune cell infiltration and TLR9 expression and signaling in pancreas and intestine. Furthermore, AVC administration reduced AP-induced interleukin-6 (IL-6) production, IL-6-activated signal transducers and activators of transcription 3 (STAT3) signaling. Concordantly, expression of AP-induced STAT3-responsive chemokines, especially monocyte chemotactic protein-1 (MCP-1) and chemokine (C-X-C motif) ligand 1 (CXCL1) was reduced, thereby contributing to modulated pancreatic immune milieu. Treatment of mice with a toll-like receptor 9 (TLR9) agonist abolished the protective effect of AVC by activation of IL6/STAT3 signaling and downstream chemokine production. Conversely, treatment of mice with TLR9 antagonists, mimicking AVC, exerted protective effects against AP. Collectively, these results suggest that depletion of enteric viruses protects mice from experimental AP through inhibiting TLR9 signaling. Our study therefore implies a previously unrecognized role of enteric viruses in AP.

Topics: Animals; Antiviral Agents; Ceruletide; Chemokines; Female; Interleukin-6; Intestinal Mucosa; Mice, Inbred BALB C; Pancreatitis; Signal Transduction; STAT3 Transcription Factor; Toll-Like Receptor 9; Virulence; Viruses

No specific therapy exists for acute pancreatitis (AP), and current treatment remains entirely supportive. Adipose stem cells (ASCs) have significant immunomodulatory and regenerative activities. We hypothesized that systemic administration of ASCs would mitigate inflammation in AP.. AP was induced in mice by 6 hourly intraperitoneal injections of cerulein. Twenty-four hours after AP induction, mice were randomized into four systemic treatment groups: sham group (no acute pancreatitis), vehicle, human ASCs, and human ASC-conditioned media. Mice were sacrificed at 48 h, and blood and organs were collected and analyzed. Pancreatic injury was quantified histologically using a published score (edema, inflammation, and necrosis). Pancreatic inflammation was also studied by immunohistochemistry and PCR.. When using IV infusion of Hoechst-labeled ASCs, ASCs were found to localize to inflamed tissues: lungs and pancreas. Mice treated with ASCs had less severe AP, as shown by a significantly decreased histopathology score (edema, inflammation, and necrosis) (p = 0.001). ASCs infusion polarized pancreatic macrophages toward an anti-inflammatory M2 phenotype. ASC-conditioned media reduced pancreatic inflammation similarly to ASCs only, highlighting the importance of ASCs secreted factors in modulating inflammation.. Intravenous delivery of human ASCs markedly reduces pancreatic inflammation in a murine model of AP ASCs which represent an effective therapy for AP.

Topics: Adipose Tissue; Animals; Ceruletide; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Stem Cell Transplantation; Stromal Cells

Acute pancreatitis (AP) is a disorder of the pancreas marked by profound inflammation and oxidative stress. Phytoconstituents presents an important toolbox of preventive strategies to combat inflammatory disorders. To this end, we selected the active constituent of Crocus sativus, crocin for evaluation against cerulein-induced AP, owing to its promising antiinflammatory activity in acute as well as chronic inflammatory conditions. The animals were randomly divided into five groups comprising of normal control, cerulein control, crocin low dose (30 mg/kg), crocin high dose (100 mg/kg), and crocin control (100 mg/kg). Various biochemical parameters and the levels of inflammatory cytokines and p65-NFκB were measured. The mechanism was investigated by histology and immunohistochemistry. We found that crocin significantly reduced the pancreatic edema, amylase, and lipase levels. It abrogated the oxidative stress incurred by cerulein challenge. We found that crocin modulated the pancreatic inflammatory cytokine levels. Crocin perturbed the nuclear translocation of p65-NFκB. Crocin reverted the pancreatic histology associated with AP. Furthermore, it upregulated the expression of Nrf-2 and downregulated the expression of IL-6, TNF-α, nitrotyrosine, and NFκB. Cumulatively, these results indicate that crocin has promising potential to prevent cerulein induced AP and regular intake of saffron can prove beneficial for the pancreatic health.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Carotenoids; Ceruletide; Crocus; Cytoprotection; Male; Mice; Oxidative Stress; Pancreas; Pancreatitis; Phytotherapy

Acute pancreatitis (AP) is one of the leading causes of hospital admission for gastrointestinal disorders. Although lipid peroxides are produced in AP, it is unknown if targeting lipid peroxides prevents AP. This study aimed to investigate the role of mitochondrial aldehyde dehydrogenase 2 (ALDH2), a critical enzyme for lipid peroxide degradation, in AP and the possible underlying mechanisms. Cerulein was used to induce AP in C57BL/6 J male mice and pancreatic acinar cells were used to elucidate underlying mechanisms in vitro. Pancreatic enzymes in the serum, lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and Bcl-2, Bax and cleaved caspase-3 were measured. ALDH2 activation with a small-molecule activator, Alda-1, reduced the levels of the pancreatic enzymes in the serum and the lipid peroxidation products MDA and 4-HNE. In addition, Alda-1 decreased Bax and cleaved caspase-3 expression and increased Bcl-2 expression in vivo and in vitro. In conclusion, ALDH2 activation by Alda-1 has a protective effect in cerulein-induced AP by mitigating apoptosis in pancreatic acinar cells by alleviating lipid peroxidation.

Topics: Aldehyde Dehydrogenase, Mitochondrial; Aldehydes; Animals; Apoptosis; Benzamides; Benzodioxoles; Cell Line; Ceruletide; Lipid Peroxidation; Male; Malondialdehyde; Mice, Inbred C57BL; Pancreas; Pancreatitis; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Severity of Illness Index; Small Molecule Libraries

In this study, the aim was to examine the potential protective effects of Myrtus communis subsp. communis leaf ethanol extract (MC) treatment against acute pancreatitis (AP) in rats. Thirty-two rats were grouped as the saline-pretreated control (C), MC-pretreated control (MC), saline-pretreated AP (AP), and MC-pretreated AP (MC + AP) groups. To induce AP, cerulein was administered (50 µg/kg) two times. The rats were given MC for 14 days before cerulein injection. Six hours after the final cerulein injection, the rats were sacrificed. Pancreatic damage was associated with an increase in the serum activity of lipase and amylase, the pancreatic activity of myeloperoxidase, and the pancreatic level of malondialdehyde, interleukin-1β, and interleukin-6. AP also led to a decrease in the pancreatic level of anti-inflammatory interleukin-10 and glutathione. Pretreatment with MC before the induction of AP significantly reduced the pancreatic damage observed during the histological examination as well as reversed the biochemical changes evoked by AP. PRACTICAL APPLICATIONS: Acute pancreatitis is characterized by high mortality (average about 5%; severe cases may reach about 30%). The current treatment for acute pancreatitis is mainly symptomatic. The introduction of herbal drugs may lead to the development of a new strategy in the treatment of this disease. This study revealed that MC reduced pancreatic injury by decreasing pro-inflammatory cytokines, increasing antioxidant capacity and anti-inflammatory cytokine, IL-10. To the authors' knowledge, this research is the first report showing that MC inhibits the development of AP. This observation suggests that MC may be useful in the prevention and the treatment of AP in clinical settings.

Topics: Acute Disease; Animals; Ceruletide; Myrtus; Pancreatitis; Plant Extracts; Rats

In this study, we investigated the anti-inflammatory effects of silymarin on cerulein-induced acute pancreatitis (AP) in mice.. Cerulein (50 μg/kg) was injected intraperitoneally once hourly for 6 hours to induce AP. To investigate the prophylactic effects of silymarin, dimethyl sulfoxide or silymarin (25, 50, and 100 mg/kg) was injected intraperitoneally 1 hour before cerulein injection. To investigate the therapeutic effects of silymarin, dimethyl sulfoxide or silymarin (100 mg/kg) was injected intraperitoneally 1, 3, or 5 hours after the first cerulein injection. Blood, pancreas, and lungs were harvested 6 hours after the last cerulein injection.. Pre- and posttreatment with silymarin decreased the pancreas weight/body weight ratio and serum amylase activity. Furthermore, silymarin treatment inhibited pancreas and lung injury and neutrophil infiltration during cerulein-induced AP. In addition, silymarin inhibited increased secretion of proinflammatory cytokines such as interleukin 1β, interleukin 6, and tumor necrosis factor α. Finally, mitogen-activated protein kinases (MAPKs) and nuclear factor-κB were activated by cerulein, and only p38 in MAPK was inhibited by silymarin.. These findings suggest that silymarin attenuates the severity of AP through inhibition of p38 MAPKs and that silymarin could be a potential prophylactic and therapeutic agent for the treatment of AP.

Topics: Acute Disease; Amylases; Animals; Antioxidants; Ceruletide; Cytokines; Inflammation Mediators; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; NF-kappa B; Organ Size; Pancreas; Pancreatitis; Protective Agents; Severity of Illness Index; Silymarin

The mechanisms underlying pathogenesis of acute pancreatitis (AP) are still not completely understood. An early, critical feature of AP is aberrant calcium (Ca) signaling, termed Ca overload, within pancreatic acinar cells. This study aimed to develop a model system in rats for AP induction to study the contribution of the Na-Ca exchanger 1 (NCX1) ion channel in AP pathogenesis.. To establish a rat model of AP induction, cerulein or L-arginine were intraperitoneally injected and tissue was histologically analyzed by hematoxylin and eosin staining. A cell culture-based model for AP induction was similarly created through cerulein treatment of AR42J cells. Induction of AP was also examined following exposure to the NXC1-targeted inhibitor KB-R7943. The expression of each gene was detected by Western blotting, immunofluorescence, immunohistochemistry, or quantitative reverse transcription polymerase chain reaction. Transcriptional regulation by nuclear factor (NF)-κB was detected using an NCX1 promoter-fusion dual luciferase reporter system. Cytosolic Ca was measured using a fluorescent calcium indicator.. We found that cerulein induced NCX1 expression via activation of nuclear factor NF-κB, which potentially binds to the NCX1 promoter to induce its transcription.. Our findings reveal a regulatory pathway through NF-κB/NCX1 governing Ca overload in AP development, thus providing potential targets for AP treatment.

Topics: Acinar Cells; Acute Disease; Animals; Calcium; Calcium Signaling; Cell Line, Tumor; Ceruletide; Gene Expression Regulation; Intracellular Space; Male; NF-kappa B; Pancreas; Pancreatitis; Rats, Sprague-Dawley; Sodium-Calcium Exchanger; Up-Regulation

Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein.

Topics: Animals; Ceruletide; Chymotrypsin; Enzyme Activation; Female; Male; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peptide Hydrolases; Serum Albumin, Bovine; Sodium Chloride; Trypsin

Topics: Amylases; Animals; Ceruletide; Disease Models, Animal; Endothelin-1; Endothelin-Converting Enzymes; Gene Expression Regulation; Humans; Inflammation; Mice; Oncogenes; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); Receptor, Endothelin A; Receptor, Endothelin B

Acute pancreatitis (AP) is a severe pancreatic disorder that remains associated with high mortality due to a lack of effective drugs and management strategies. This study aimed to investigate the molecular pathogenic mechanisms of AP involving p53 and endoplasmic reticulum (ER) stress pathways.. Expression of PRSS1 and p53 in human AP tissues was detected by immunohistochemistry and Western blotting. AP was induced with caerulein in humanized PRSS1 transgenic mice, and its severity was verified by histological imaging, evaluation of edema, serum amylase, and trypsin activity assays. A transferase-mediated d-UTP nick end-labeling assay was performed to evaluate acinar cell apoptosis associated with AP. The expression of ER stress genes was assessed by quantitative RT-PCR (qRT-PCR) and Western blotting.. PRSS1 and p53 were highly expressed in human AP tissues. Expression of human PRSS1 in caerulein-treated mice induced significant acinar cell apoptosis and AP progression. P53 knockout significantly suppressed AP progression in humanized PRSS1 transgenic mice. The ER stress pathway was activated by PRSS1 and mediated the progression of AP in mouse pancreatic tissues. Application of a p53 inhibitor effectively ameliorated caerulein-induced AP in PRSS1 transgenic mice, while a p53 activator promoted the progression of AP.. P53, which was activated by the ER stress pathway, promoted the progression of AP in mice expressing PRSS1 by inducing acinar cell apoptosis.

Topics: Acinar Cells; Animals; Apoptosis; Ceruletide; Endoplasmic Reticulum Stress; Humans; Mice; Mice, Transgenic; Pancreatitis; Trypsin; Tumor Suppressor Protein p53

The aim of this study was to investigate the effects of eupatilin on protein kinase D1 (PKD1) and nuclear factor kappa B (NF-κB) signaling pathways in cerulein-induced in vitro pancreatitis.. We used collagenase digestion to isolate pancreatic acinar cells from male C57BL/6 mice. In vitro acute pancreatitis was induced by treatment with a supramaximal dose of cerulein. Eupatilin was pretreated before stimulation with cerulein.. Eupatilin significantly reduced cerulein-induced amylase release in pancreatic acini. Eupatilin treatment downregulated cerulein-induced expression of interleukin (IL)-1β, IL-6, and CC chemokine ligands 2 and 5, but it upregulated expression of IL-4 and IL-10. We demonstrated that eupatilin pretreatment attenuated cerulein-induced necrosis in isolated pancreatic acinar cells. This effect of eupatilin was confirmed by lactic dehydrogenase assay, fluorescence-activated cell sorting analysis, and cytopathologic analysis. Eupatilin inhibited cerulein-induced activation of PKD1/NF-κB and the nuclear translocation of NF-κB.. Our data demonstrated that eupatilin is a potential therapeutic candidate for the treatment of pancreatitis through its ability to reduce cellular necrosis and inflammatory responses by inhibition of the PKD1/NF-κB signaling pathway.

Topics: Acinar Cells; Active Transport, Cell Nucleus; Acute Disease; Amylases; Animals; Cell Nucleus; Ceruletide; Cytokines; Flavonoids; Male; Mice, Inbred C57BL; NF-kappa B; Pancreatitis; Protein Kinase C; Signal Transduction

Acute pancreatitis (AP) is a sudden inflammation of the pancreas that may be life-threatening disease with high mortality rates, particularly in the presence of systemic inflammatory response and multiple organ failure. Oxidative stress has been shown to be involved in the pathophysiology of acute pancreatitis.. This study is designed to investigate the possible effect of mesna on an experimental model of cerulein-induced acute pancreatitis.. Animals were divided into five groups: Group 1 served as a control group given the saline; group II (mesna group) received mesna at a dose of (100 mg/kg per dose, i.p.) four times; group III (acute pancreatitis group) received cerulein at a dose of (20 µg/kg/dose, s.c.) four times with 1-h intervals; group VI, cerulein + mesna, was treated with mesna at a dose of (100 mg/kg, i.p.) 15 min before each cerulein injection.. Animals with acute pancreatitis showed elevated serum amylase and lipase levels. Biochemical parameters showed increased pancreatic tumor necrosis factors-α (TNF-α) and interleukin-1β (IL-1β) levels. A disturbance in oxidative stress markers was evident by elevated pancreatic lipid peroxides (TBARS) and decline in pancreatic antioxidants' concentrations including reduced glutathione (GSH); superoxide dismutase (SOD); and glutathione peroxidase (GSH-Px). Histological examination confirmed pancreatic injury. Pre-treatment with mesna was able to abolish the changes in pancreatic enzymes, oxidative stress markers (TBARS, SOD, GSH and GSH-Px), pancreatic inflammatory markers (TNF-α, IL-1β) as well as histological changes.. Mesna mitigates AP by alleviating pancreatic oxidative stress damage and inhibiting inflammation.

Topics: Animals; Antioxidants; Ceruletide; Cholagogues and Choleretics; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Interleukin-1beta; Mesna; Oxidative Stress; Pancreas; Pancreatitis; Protective Agents; Rats; Treatment Outcome; Tumor Necrosis Factor-alpha

In this study, we focused on the function of nuclear factor E2-related factor 2 (Nrf2) in acute pancreatitis (AP), which has been shown to have protective effects in gliomas, hepatocytes, and astrocytes.. Acute pancreatitis cell line and animal model were induced by administration of lipopolysaccharide and cerulein into the cell supernatant or intraperitoneal injection. Oxidative stress status was evaluated by measuring the level of amylase, C-reactive protein, malondialdehyde, superoxide dismutase, and myeloperoxidase. Morphological alterations in the pancreas were evaluated by hematoxylin-eosin staining, the wet-to-dry weight ratio, and the pathology injury scores. Western blot, reverse transcription-polymerase chain reaction, and immunofluorescence staining were performed to analyze the expression of Nrf2, Heme oxygenase 1, and NAD(P)H: quinone oxidoreductase 1.. Overexpression of Nrf2 inhibits oxidative stress and inflammatory responses by inducting the expression of superoxide dismutase as well as reducing the level of amylase, malondialdehyde, and myeloperoxidase in the AR42J rat pancreatic acinar cells in AP. Importantly, overexpression of Nrf2 displayed the same protective effect in vivo. Data from an AP rat model showed that Nrf2 could relieve pancreatic damage.. These results indicated that Nrf2 has a protective role in lipopolysaccharide and cerulein-induced cytotoxicity, providing potential therapeutic strategies for the treatment of AP.

Topics: Amylases; Animals; Cell Line; Ceruletide; Disease Models, Animal; Heme Oxygenase (Decyclizing); Inflammation Mediators; Lipopolysaccharides; Male; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidative Stress; Pancreas, Exocrine; Pancreatitis; Rats, Sprague-Dawley; Signal Transduction; Up-Regulation

At the initial stage of carcinogenesis, when RasV12-transformed cells are surrounded by normal epithelial cells, RasV12 cells are apically extruded from epithelia through cell competition with the surrounding normal cells. In this study, we demonstrate that expression of cyclooxygenase (COX)-2 is upregulated in normal cells surrounding RasV12-transformed cells. Addition of COX inhibitor or COX-2-knockout promotes apical extrusion of RasV12 cells. Furthermore, production of Prostaglandin (PG) E

Topics: Animals; Anticarcinogenic Agents; Cell Line, Transformed; Cell Transformation, Neoplastic; Ceruletide; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Disease Models, Animal; Dogs; Epithelial Cells; Female; Genes, ras; Ibuprofen; Madin Darby Canine Kidney Cells; Male; Mice, Inbred C57BL; Mice, Transgenic; Pancreatitis; Signal Transduction

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein-induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21-treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein-induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein-induced pancreatic injury and improves cerulein-induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up-regulate Sirtuin-1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein-induced pancreatic injury and weakens the regulative effect in FGF21-activated autophagy in mice. These results showed that FGF21 protects against cerulein-induced AP by activation of Sirtuin-1-autophagy axis.

Topics: Animals; Autophagosomes; Autophagy; Cells, Cultured; Ceruletide; Fibroblast Growth Factors; Male; Mice, Inbred C57BL; Pancreas; Pancreatitis; Signal Transduction; Sirtuin 1

We reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis.. We induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca. The expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca. In cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca

Topics: Acinar Cells; Amylases; Animals; Annexins; Autophagy; Calcium; Cell Survival; Ceruletide; Cholecystokinin; Exocytosis; Interferon Regulatory Factor-2; Mice, Knockout; Pancreas; Pancreatitis; Peptide Fragments; S100 Calcium Binding Protein G; Signal Transduction; Up-Regulation

Early death in severe acute pancreatitis (SAP) is caused by pancreatic necrosis and multiple-organ failure due to microcirculation disorder. The aim of this study was to prove that recombinant human-soluble thrombomodulin (rTM) has therapeutic effects on SAP by preventing pancreatic necrosis and organ failure.. Male Wister rats were used. Cerulein was administered intraperitoneally 4 times every 1 hour, and lipopolysaccharide was administered intraperitoneally 3 hours after. One hour after administration of lipopolysaccharide, rTM was injected intravenously. Rats were observed for 24 hours after starting the experiment, and the survival rate was evaluated. All surviving rats were killed, and the blood sample, liver, and pancreas were excised. Serum amylase, aspartate aminotransferase, alanine aminotransferase, and high mobility group box 1 were measured, and the liver and pancreas were examined histologically. For the evaluation of microcirculation, von Willebrand factor staining was performed.. Serum amylase, aspartate aminotransferase, and alanine aminotransferase were significantly decreased. The survival rate was significantly improved to 100%. Moreover, serum high mobility group box 1 was decreased. Liver injury and pancreatic necrosis became less severe, and microcirculation was preserved histologically.. Early administration of rTM prevents organ failure by maintenance of microcirculation and improves prognoses of SAP.

Topics: Alanine Transaminase; Amylases; Animals; Aspartate Aminotransferases; Biomarkers; Ceruletide; Disease Models, Animal; Drug Evaluation, Preclinical; Endothelial Cells; HMGB1 Protein; Humans; Lipopolysaccharides; Liver; Male; Microvessels; Pancreas; Pancreatitis; Pancreatitis, Acute Necrotizing; Rats; Rats, Wistar; Recombinant Proteins; Solubility; Thrombomodulin

MicroRNAs have been considered to be closely related with the development of severe acute pancreatitis (SAP), and microRNA-375 (miR-375) was believed to be a marker of SAP. We aim to investigate the role of miR-375 in regulating SP.. Cerulein and lipopolysaccharide were used to establish the models of SAP. AR42J cell line was chosen for study in vitro. Flow cytometry was applied for assessing apoptosis. The contents of inflammatory factors were detected with related enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction assays. Hematoxylin and eosin staining was applied to observe the pathological changes of pancreatic tissues. Immunohistochemistry analysis was conducted for investigating the expression of light chain 3.. The level of miR-375 in pancreatitis tissues and cell lines was upregulated. Overexpression of miR-375 promoted inflammation and the apoptosis of acinar cells through inhibiting autophagy. The binding site between miR-375 and ATG7 was identified, and miR-375 could directly regulate the ATG7. microRNA-375 suppressed autophagy and promoted inflammation and the apoptosis of acinar cells via targeting ATG7.. We proved that miR-375 could inhibit autophagy and promote inflammation and the apoptosis of acinar cells through regulating ATG7. This study first proves that miR-375 modulates the development of SAP through targeting ATG7.

Topics: Acinar Cells; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 7; Binding Sites; Cell Line; Ceruletide; Disease Models, Animal; Humans; Lipopolysaccharides; MicroRNAs; Pancreatitis; Protein Binding; Rats; RNA Stability; RNA, Messenger; Up-Regulation

Trypsinogen activation is the hallmark of acute pancreatitis (AP) independent of intra-acinar NF-κB activation and inflammation. We previously found that dopamine (DA) receptor 2 (DRD2) activation controls inflammation during AP via PP2A-dependent NF-κB activation. In this study, we sought to examine whether DRD2 signaling mediates trypsinogen activation and the underlying mechanisms. Pancreatic acinar cells were stimulated with cholecystokinin-8 in vitro. AP was induced by intraperitoneal injections of caerulein and LPS or l-arginine. Pancreatitis severity was assessed biochemically and histologically. We found that activation of DRD2 by quinpirole, a potent DRD2 agonist, resulted in the reduction of trypsinogen activation and the upregulation of HSP70 in vitro and in vivo. Mechanistically, we found that quinpirole induced dephosphorylation of heat shock factor 1 (HSF1), a master transcription factor of HSP70, leading to increased nuclear translocation of HSF1 in a PP2A-dependent pathway. Furthermore, DRD2 activation restored lysosomal pH and, therefore, maintained lysosomal cathepsin B activity in a HSP70-dependent manner. VER155008, a potent HSP70 antagonist, abolished the protective effects observed with DRD2 activation in vitro and in two experimental models of AP. Our data showed that besides controlling NF-κB activation, DRD2 activation prevented trypsinogen activation during acute pancreatitis via PP2A-dependent upregulation of HSP70 and further support that DRD2 agonist could be a promising therapeutic strategy for treating AP.

Topics: Animals; Ceruletide; Dopamine Agonists; Gene Expression Regulation; Heat Shock Transcription Factors; HSP72 Heat-Shock Proteins; Lysosomes; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Phosphorylation; Protein Phosphatase 2; Quinpirole; Receptors, Dopamine D2; Trypsinogen; Up-Regulation

IL-17A combined with TNF-α plays a vital role in inflammatory response and interference of the synergistic effect is an effective strategy for treating inflammatory diseases. Ellipticine, a natural alkaloid, has biological activities on anti-tumor and anti-HIV. However, it is still unknown whether ellipticine can inhibit IL-17A and TNF-α-mediated signaling and has treatment effect on PALI. Here, we reported that ellipticine significantly inhibited the production of pro-inflammatory cytokines and chemokines in pulmonary epithelial cell BEAS-2B treated with IL-17A and TNF-α, but not IL-17A or TNF-α alone. Meanwhile, ellipticine attenuated NF-κB and MAPKs activation in response to IL-17A and TNF-α treatment, inhibited Act1 and TRAF6-mediated NF-κB activation, and blocked the interaction of Act1 with TRAF6. Furthermore, we found that ellipticine significantly alleviated CAE and LPS-induced SAP/PALI. Ellipticine treatment dramatically reduced inflammatory cells infiltration, MPO activity, serum amylase and lipase activity and the protein concentration of BALF. Collectively, our findings indicate that ellipticine inhibits the synergistic effect of IL-17A and TNF-α by targeting on Act1 and TRAF6 interaction and is a potential therapeutic agent for the treatment of SAP/PALI.

Topics: Acute Lung Injury; Adaptor Proteins, Signal Transducing; Amylases; Animals; Anti-Inflammatory Agents; Cell Line, Transformed; Ceruletide; Disease Models, Animal; Ellipticines; Epithelial Cells; Gene Expression Regulation; HEK293 Cells; Humans; Interleukin-17; Lipase; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; NF-kappa B; Pancreatitis; Peroxidase; Signal Transduction; TNF Receptor-Associated Factor 6; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) is a healthcare challenge with considerable mortality. Treatment is limited to supportive care, highlighting the need to investigate disease drivers and prognostic markers. Activin A is an established mediator of inflammatory responses, and its serum levels correlate with AP severity. We hypothesized that activin A is independent of body mass index (BMI) and is a targetable promoter of the AP inflammatory response.. We assessed whether BMI and serum activin A levels are independent markers to determine disease severity in a cohort of patients with AP. To evaluate activin A inhibition as a therapeutic, we used a cerulein-induced murine model of AP and treated mice with activin A-specific neutralizing antibody or immunoglobulin G control, both before and during the development of AP. We measured the production and release of activin A by pancreas and macrophage cell lines and observed the activation of macrophages after activin A treatment.. BMI and activin A independently predicted severe AP in patients. Inhibiting activin A in AP mice reduced disease severity and local immune cell infiltration. Inflammatory stimulation led to activin A production and release by pancreas cells but not by macrophages. Macrophages were activated by activin A, suggesting activin A might promote inflammation in the pancreas in response to injury.. Activin A provides a promising therapeutic target to interrupt the cycle of inflammation and tissue damage in AP progression. Moreover, assessing activin A and BMI in patients on hospital admission could provide important predictive measures for screening patients likely to develop severe disease.

Topics: Activins; Animals; Anti-Inflammatory Agents; Biomarkers; Body Mass Index; Cell Line; Ceruletide; Cohort Studies; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Humans; Macrophage Activation; Macrophages; Mice; Pancreas; Pancreatitis; Patient Admission; Predictive Value of Tests; Severity of Illness Index

Compared with the general population, individuals with diabetes have a higher risk of developing severe acute pancreatitis, a highly debilitating and potentially lethal inflammation of the exocrine pancreas. In this study, we investigated whether 1-deoxysphingolipids, atypical lipids that increase in the circulation following the development of diabetes, exacerbate the severity of pancreatitis in a diabetic setting.. We analysed whether administration of an L-serine-enriched diet to mouse models of diabetes, an established method for decreasing the synthesis of 1-deoxysphingolipids in vivo, reduced the severity of acute pancreatitis. Furthermore, we elucidated the molecular mechanisms underlying the lipotoxicity exerted by 1-deoxysphingolipids towards rodent pancreatic acinar cells in vitro.. We demonstrated that L-serine supplementation reduced the damage of acinar tissue resulting from the induction of pancreatitis in diabetic mice (average histological damage score: 1.5 in L-serine-treated mice vs 2.7 in the control group). At the cellular level, we showed that L-serine decreased the production of reactive oxygen species, endoplasmic reticulum stress and cellular apoptosis in acinar tissue. Importantly, these parameters, together with DNA damage, were triggered in acinar cells upon treatment with 1-deoxysphingolipids in vitro, suggesting that these lipids are cytotoxic towards pancreatic acinar cells in a cell-autonomous manner. In search of the initiating events of the observed cytotoxicity, we discovered that 1-deoxysphingolipids induced early mitochondrial dysfunction in acinar cells, characterised by ultrastructural alterations, impaired oxygen consumption rate and reduced ATP synthesis.. Our results suggest that 1-deoxysphingolipids directly damage the functionality of pancreatic acinar cells and highlight that an L-serine-enriched diet may be used as a promising prophylactic intervention to reduce the severity of pancreatitis in the context of diabetes.

Topics: Acinar Cells; Animals; Apoptosis; Ceruletide; Diabetes Mellitus, Experimental; Disease Models, Animal; DNA Damage; Endoplasmic Reticulum Stress; In Vitro Techniques; Mice; Mitochondria; Oxygen Consumption; Pancreas; Pancreatitis; Reactive Oxygen Species; Serine; Severity of Illness Index; Sphingolipids

Acute pancreatitis (AP), an inflammatory condition of pancreas, destructs the exocrine cells by releasing various pro-inflammatory cytokines that activates the stellate cells. However, the underlying molecular mechanism remains unclear. The present study investigated the role of retinoblastoma (Rb), hydrogen sulphide and nuclear factor-κB (NF-κB) in the regulation of exocrine cell proliferation under inflammatory condition.. The randomly grouped male swiss mice were administered with 6 consecutive hourly i.p injections of caerulein to induce AP. Palbociclib (PD) (25 mg/kg body weight), a CDK4/6 inhibitor, was administered 1 h after the first cerulein injection intraperitoneally to block the RB pathway by inhibiting the activity of the CDK4/6 complexes and DL propargylglycine (PAG) which blocks the endogenous H

Topics: Animals; Antineoplastic Agents; Ceruletide; DNA; Gene Expression Regulation; Hydrogen Sulfide; Lung Injury; Male; Mice; NF-kappa B; Pancreas; Pancreatitis; Phosphorylation; Piperazines; Protein Binding; Pyridines; Random Allocation; Retinoblastoma Protein; Tumor Necrosis Factor-alpha

Topics: Animals; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cells, Cultured; Ceruletide; Homeodomain Proteins; Metaplasia; Mice; Mice, Transgenic; NF-kappa B; Pancreatitis; Proto-Oncogene Proteins p21(ras); Resveratrol; Signal Transduction; Trans-Activators

Calcineurin is a ubiquitously expressed central Ca. We performed studies with mice with hematopoietic-specific or pancreas-specific deletion of protein phosphatase 3, regulatory subunit B, alpha isoform (PPP3R1, also called CNB1), in mice with deletion of CNB1 (Cnb1. Mice with hematopoietic-specific deletion of CNB1 developed the same level of local pancreatic inflammation as control mice after administration of caerulein or infusion of radiocontrast into biliopancreatic ducts. Cnb1. Hematopoietic and neutrophil expression of calcineurin promotes pancreatitis-associated lung inflammation, whereas pancreatic calcineurin promotes local pancreatic inflammation. The findings indicate that the protective effects of blocking or deleting calcineurin on pancreatitis are mediated by the source of its expression. This information should be used in the development of strategies to inhibit calcineurin for the prevention of pancreatitis and pancreatitis-associated lung inflammation.

Topics: Acinar Cells; Acute Lung Injury; Animals; Bone Marrow Cells; Calcineurin; Calcineurin Inhibitors; Calcium-Binding Proteins; Cells, Cultured; Ceruletide; Cytokines; Disease Models, Animal; Female; Humans; Male; Mice; Mice, Transgenic; Muscle Proteins; Neutrophils; NFATC Transcription Factors; Pancreas; Pancreatitis; Primary Cell Culture

Disturbances in pancreatic microcirculation, beginning with vasoconstriction, are crucial in early pancreatitis and progression to necrotizing pancreatitis. Thus, vascular-targeted treatment aiming to restore a sufficient level of microcirculation through vasodilation would possibly reduce the severity of pancreatitis. Lidocaine is an anti-arrhythmic and local anesthetic drug, which also acts as a vasodilator at higher concentrations.. To evaluate the efficacy of intra-arterial infusion of lidocaine into the celiac trunk in treatment of cerulein-induced acute pancreatitis.. Wistar rats (n = 20) were randomly divided into 2 equal groups: the control group (NaCl group, n = 10) and the study group (lidocaine group, n = 10). All subjects underwent surgical intervention with intra-arterial infusion of 0.9% NaCl (control group) or 1% lidocaine hydrochloride (study group) into the celiac trunk. Blood samples were collected 5 times at regular intervals from each rat for amylase and lipase measurements. Histopathological analysis of the pancreas was performed.. A total number of 16 rats (control group n = 7, study group n = 9) were included. In the postoperative course, the study group (lidocaine group) revealed lower values of serum amylase and lipase levels compared to the control group (NaCl group), except the values at the 1st treatment point, which appeared 1 h after intraoperative drug injection. Significantly lower treatment endpoint levels of pancreatic enzymes were seen in the lidocaine group. Moreover, no differences were observed between the 1st and the last treatment point in the control group; however, these differences were significant for both enzymes in the study group. Histopathology revealed reduced pancreatitis severity in the study group compared to the controls.. Intra-arterial lidocaine infusion into the celiac trunk decreases pancreatitis severity. What is more, this study demonstrates the relevance of early vasodilation in the therapy of acute pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Infusions, Intra-Arterial; Lidocaine; Pancreas; Pancreatitis; Random Allocation; Rats; Rats, Wistar; Treatment Outcome

This study is to investigate the protective effect of Acetyl-α-boswellic acid and Acetyl-β-boswellic mixture(α/β-ABA), which is the active ingredients isolated from Frankincense, on actue pancreatitis and its mechanism. Our experimental results showed that 2 μM α/β-ABA reduced production of NO, TNF-α, IL-6, IL-10 and IL-1β in RAW264.7 cells that were stimulated with lipopolysaccharide (LPS) which indicates its anti-inflammatory role. In pancreatitis model induced by caerulein, intra-gastrical administration of 100 mg/kg α/β-ABA relieved inflammatory cells infiltration significantly and attenuated the serum elevation of amylase TNF-α and IL-6 remarkably in mice. Furthermore, α/β-ABA down-regulated mitogen-activated protein kinase (MAPK) family phosphorylated proteins in pancreas, including phosphorylated p38, ERK1/2 and JNK, to reduce the serum inflammatory factors. Finally, α/β-ABA alleviated the pancreatic edema and inflammatory cell infiltration in pancreatitis mice model. This study suggests that α/β-ABA may be targeted for drug development against pancreatitis via modulating MAPKs pathway.

Topics: Animals; Cell Survival; Ceruletide; Cytokines; Disease Models, Animal; Down-Regulation; Edema; Inflammation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Pancreatitis; RAW 264.7 Cells; Triterpenes

Acute pancreatitis (AP), a sudden inflammatory process of pancreas, is painful and may contribute to death. The aberrant expression of miR-27a-5p has been reported in many types of cancers and diseases including AP. Thus, it is urgent to manifest the functions and mechanism of miR-27a-5p in AP. The levels of miR-27a-5p, tumor necrosis factor (TNF) receptor-associated factor 3 (Traf3) in serum of AP patient, or cerulein-treated AR42J cells were detected by qRT-PCR. Functionally, the apoptotic rate, the protein levels of Bcl-2 and Bax, the caspase-3 activity, and the levels of IL-1β, IL-6, and TNF-α in cerulein-treated AR42J cells were measured by flow cytometry, Western blot, caspase-3 activity assay, and qRT-PCR and ELISA assay, respectively. In addition, the putative target of miR-27a-5p was predicted by TargetScan online database, and the dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify this interaction. Cerulein-treated mouse AP model was established to explore the role of miR-27a-5p in AP in vivo. The level of miR-27a-5p was notably downregulated in AP patients and cerulein-treated AR42J cells. The functional experiments indicated that miR-27a-5p mimics attenuated the promotion effects on cell apoptosis and the inflammatory response in AR42J cells caused by cerulein. The interaction between miR-27a-5p and Traf3 was predicted by TargetScan online database and validated by dual luciferase reporter assay and RIP assay. Following qRT-PCR results exhibited that Traf3 was apparently enhanced in cerulein-treated AR42J cells. The further functional experiments disclosed that Traf3 overexpression relieved the inhibitory effects on cell apoptosis and the inflammatory response induced by miR-27a-5p mimics in cerulein-treated AR42J cells. Moreover, miR-27a-5p alleviated cerulein-induced injury in vivo. In this study, we established the cerulein-treated AR42J cells as AP model in vitro. We validated that miR-27a-5p was significantly downregulated, and Traf3 was strikingly upregulated in AP patient and/or cerulein-treated AR42J cells. The further mechanistical and functional experiments unraveled that miR-27a-5p regulated Traf3 to relieve the cerulein-induced cell apoptosis and inflammatory injury of AR42J cells. Therefore, this novel regulatory network may provide therapeutic target for AP patients.

Topics: Animals; Apoptosis; Cell Line; Ceruletide; Humans; Inflammation Mediators; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Pancreatitis; Rats; TNF Receptor-Associated Factor 3

The aim of this study was to investigate the changes of pancreatic microvascular vasomotion and blood distribution pattern in acute pancreatitis (AP), and whether Angiotensin (Ang)-(1-7) treatment could restore pancreatic microcirculation profiles.. Mice were randomly separated into control, AP, and Ang-(1-7)-treated AP (A-AP) group. Acute pancreatitis was induced in mice by intraperitoneal injection of cerulein and lipopolysaccharide. Pancreatitis was confirmed by histopathology, serum amylase, and high-sensitive C-reactive protein. Pancreatic microvascular vasomotion and blood distribution pattern in AP progression were assessed by laser Doppler. Meanwhile, ultrastructural changes of pancreatic microcirculation, including microvascular cavity and wall and endothelial mitochondria, were evaluated by transmission electron microscopy.. Acute pancreatitis mice exhibited pathological pancreatic injuries with lower blood distribution pattern and decreased average blood perfusion, relative velocity, effective frequency, and amplitude of microvascular vasomotion. The pancreatic pathological injuries in Ang-(1-7)-treated mice were significantly alleviated. Consistently, Ang-(1-7) treatment led to a restoration in pancreatic microcirculation profiles. Furthermore, non-Ang-(1-7)-treated mice showed an irregular microvascular wall, narrow cavity, and swelling mitochondria, and these ultrastructural impairments were reversed by Ang-(1-7) administration.. Pancreatic microcirculation profiles are abnormal in the progression of AP. Angiotensin-(1-7) administration could restore functional status of pancreatic microcirculation.

Topics: Angiotensin I; Animals; Ceruletide; Endothelium, Vascular; Lipopolysaccharides; Male; Mice, Inbred C57BL; Microcirculation; Microscopy, Electron, Transmission; Pancreas; Pancreatitis; Peptide Fragments

Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis.. We performed studies with LSL-Kras. Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D. In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Energy Metabolism; Fibrosis; Humans; Interleukins; Intramolecular Oxidoreductases; Mice, Transgenic; Mutation; Octamer Transcription Factors; Pancreas; Pancreatic Neoplasms; Pancreatitis; Prostaglandin D2; Proto-Oncogene Proteins p21(ras); Time Factors; Transcription Factors

Topics: Acinar Cells; Activating Transcription Factor 6; Adult; Animals; Apoptosis; Apoptosis Regulatory Proteins; Case-Control Studies; Ceruletide; Disease Models, Animal; Endoplasmic Reticulum Stress; Female; Gene Knockdown Techniques; Humans; Male; Mice, Knockout; Middle Aged; Mitochondrial Proteins; Pancreas; Pancreatitis; Transcriptional Activation; Trypsin; Tumor Suppressor Protein p53

Impaired or hyperactive pancreas regeneration after injury would cause exocrine insufficiency or recurrent / chronic pancreatitis and potentially carcinogenesis. Macrophages are the most abundant immune cells in the regenerative pancreas, however their phenotype and role remain poorly defined.. Using caerulein-induced acute pancreatitis (AP) model, we examined the dynamic landscape of pancreatic macrophages throughout the acute inflammation to regeneration phases by flow cytometric and RNA-seq analyses. Liposome depletion of macrophages, Il4ra. We found that M1 macrophages dominated in the pro-inflammatory phase of AP, while M2-like macrophages dominated during pancreas repair/regeneration. Depletion of macrophages at early or late regenerative stage dramatically blocked the acinar-ductal metaplasia (ADM) or delayed inflammation resolution, respectively. Moreover, alternative activation of macrophages was partially dependent on IL-4RA signaling, and ECM/AKT activation in pancreatic macrophages facilitated inflammation resolution during tissue regeneration.. Our findings illustrate a dynamic phenotype and function of macrophages during AP repair/regeneration, helping us better understand the mechanism of pancreatic regeneration and providing clues for novel therapeutic strategy.

Topics: Animals; Cell Polarity; Ceruletide; Disease Models, Animal; Gene Expression Profiling; Liposomes; Liver Regeneration; Macrophages; Mice; Pancreatitis; Phenotype; Receptors, Cell Surface; Sequence Analysis, RNA; Wound Healing

Acute pancreatitis (AP) refers to inflammation in the pancreas, which may lead to death in severe cases. Coenzyme Q10 (Q10), generally known to generate energy, plays an important role as an anti-oxidant and anti-inflammatory effector. Here, we showed the effect of Q10 on inflammatory response in murine AP model. For this study, we induced AP by injection of cerulein intraperitoneally or pancreatic duct ligation (PDL) in mice. The level of cytokines and digestive enzymes were measured in pancreas, and blood. All pancreatic tissues were excised for investigation such as histological changes, infiltration of immune cells. Administration of Q10 attenuated the severity of AP and its associated pulmonary complication as shown by reduction of acinar cell death, parenchymal edema, inflammatory cell infiltration and alveolar thickening in both cerulein-induced AP and PDL-induced AP. Moreover, reduction of the cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were observed in pancreas and pancreatic acinar cells by Q10. Furthermore, Q10 reduced the infiltration of immune cells such as monocytes and neutrophils and augmentation of chemokines such as CC chemokine-2 (CCL2) and C-X-C chemokine-2 (CXCL2) in pancreas of AP mice. In addition, Q10 deactivates the phosphorylation of extracellular signal-regulated kinase (ERK) and c-jun NH

Topics: Animals; Anti-Inflammatory Agents; Ceruletide; Cytokines; Female; Lung; MAP Kinase Signaling System; Mice, Inbred C57BL; Monocytes; Neutrophil Infiltration; Neutrophils; Pancreas; Pancreatitis; Ubiquinone

Severe acute pancreatitis (SAP) is a non-bacterial inflammatory disease that clinically causes a very high rate of mortality. Dihydrokaempferol (DHK) is a natural flavonoid extracted from Bauhinia championii. Our research aimed to establish the treatment function of DHK on SAP-induced pancreas injury and delve into its potential mechanism. In this study, SAP was induced by caerulein (CER) and Lipopolysaccharide (LPS). DHK was administered orally at different doses of 20, 40, or 80 mg/kg. Results from serum amylase/lipase, pancreas hematoxylin-eosin staining technique, pancreas malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) showed the therapeutic effect of DHK in a mice SAP model. MTT revealed DHK alleviated CER + LPS induced cytotoxicity in a dose-dependent manner in the pancreatic acinar cells of mice. Next, we verified DHK suppressed the level of Keap1 and promoted transcriptional activation of nuclear Nrf2 in the presence of CER + LPS. The molecular docking study suggested that there is a potential interaction between DHK and Keap1. To further look at the role of Keap1 using in vitro and in vivo models, Keap1 overexpression adenovirus (ad-Keap1) was performed. The results revealed that ad-Keap1suppressed the nuclear translocation of Nrf2 which is enhanced by DHK, and suppressed the antioxidative functionality of DHK both in mice and cell models. Collectively, this research demonstrated that DHK bettered the SAP induced pancreas injury by regulating the Keap1/Nrf2 pathway and regulating oxidative stress injury.

Topics: Animals; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Flavonoids; Glutathione; Kelch-Like ECH-Associated Protein 1; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; NF-E2-Related Factor 2; Oxidative Stress; Pancreatitis; Reactive Oxygen Species; Severity of Illness Index

Acute pancreatitis (AP) is the inflammatory response of the exocrine pancreas to various causes. Modafinil has significant anti-inflammation and anti-oxidation effects. No experiment has assessed the effects of modafinil on AP. Thus, the study aims to study the effects of modafinil on AP and its potential mechanism in vivo and vitro. 5% sodium taurocholate was retrograde injected into pancreatic duct to establish AP rat model. The severity of AP was detected by HE staining, serum amylase and lipase levels. The inflammation, oxidative stress and apoptosis were detected separately by ELISA, MDA and SOD kits, tunnel staining and Western blotting in rats. Besides, SNIP1 expression was analyzed by qPCR and Western blotting. In vivo, AR42J cells were stimulated by cerulein and lipopolysaccharide to establish AP cell model. Flow cytometry examined cell apoptosis. After the plasmids silencing SNIP1 were transfected into AP cells, the inhibitory effects of modafinil on inflammation, oxidative stress and apoptosis were significantly reversed. The results indicated that modafinil showed significant curative and therapeutic effects by regulating SNIP1 level.

Topics: Acute Disease; Animals; Cell Line; Ceruletide; Inflammation; Modafinil; Pancreatitis; Rats; RNA-Binding Proteins

Acute pancreatitis (AP) is associated with impaired acinar cell autophagic flux, intracellular zymogen activation, cell necrosis and inflammation. Activation of the cholinergic system of vagus nerve has been shown to attenuate AP, but the effect of organ-intrinsic cholinergic system on pancreatitis remains unknown. In this study, we aim to examine the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation within the pancreas during AP. In vivo, AP was induced by caerulein plus LPS or ethanol plus palmitoleic acid in mice. In vitro, pancreatic acini were isolated and subjected to cholecystokinin (CCK) stimulation. Mice or acini were pre-treated with PNU-282987 (selective α7nAChR agonist) or methyllycaconitine citrate salt (selective α7nAChR antagonist). Pancreatitis severity, acinar cell injury, autophagic flux, and transcription factor EB (TFEB) pathway were analyzed. Both caerulein plus LPS in vivo and CCK in vitro led to an up-regulation of α7nAChR, indicating activation of pancreas-intrinsic α7nAChR signaling during AP. PNU-282987 decreased acinar cell injury, trypsinogen activation and pancreatitis severity. Conversely, methyllycaconitine citrate salt increased acinar cell injury and aggravated AP. Moreover, activation of α7nAChR by PNU-282987 promoted autophagic flux as indicated by reduced p62, increased LysoTracker staining and decreased number of autolysosomes with undegraded contents. Furthermore, PNU-282987 treatment significantly increased TFEB activity in pancreatic acinar cells. α7nAChR activation also attenuated pancreatic inflammation and NF-κB activation. Our results showed that activation of α7nAChR protected against experimental pancreatitis through enhancing TFEB-mediated acinar cell autophagy, suggesting that activation of pancreas-intrinsic α7nAChR may serve as an endogenous protective mechanism during AP.

Topics: Aconitine; alpha7 Nicotinic Acetylcholine Receptor; Animals; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Benzamides; Bridged Bicyclo Compounds; Ceruletide; Ethanol; Fatty Acids, Monounsaturated; Injections, Intraperitoneal; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Pancreatitis; Signal Transduction

Chaiqin chengqi decoction (CQCQD) is a Chinese herbal formula derived from dachengqi decoction. CQCQD has been used for the management of acute pancreatitis (AP) in the West China Hospital for more than 30 years. Although CQCQD has a well-established clinical efficacy, little is known about its bioactive ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory effects.. Toll-like receptor 4 (TLR4) and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pro-inflammatory signaling pathways, play a central role in AP in determining the extent of pancreatic injury and systemic inflammation. In this study, we screened the bioactive ingredients using a pharmacological sub-network analysis based on the TLR4/NLRP3 signaling pathways followed by experimental validation.. The main CQCQD bioactive compounds were identified by UPLC-QTOF/MS. The TLR4/NLRP3 targets in AP for CQCQD active ingredients were confirmed through a pharmacological sub-network analysis. Mice received 7 intraperitoneal injections of cerulein (50 μg/kg; hourly) to induce AP (CER-AP), while oral gavage of CQCQD (5, 10, 15 and 20 g/kg; 3 doses, 2 hourly) was commenced at the 3rd injection of cerulein. Histopathology and biochemical indices were used for assessing AP severity, while polymerase chain reaction, Western blot and immunohistochemistry analyses were used to study the mechanisms. Identified active CQCQD compounds were further validated in freshly isolated mouse pancreatic acinar cells and cultured RAW264.7 macrophages.. The main compounds from CQCQD belonged to flavonoids, iridoids, phenols, lignans, anthraquinones and corresponding glycosides. The sub-network analysis revealed that emodin, rhein, baicalin and chrysin were the compounds most relevant for directly regulating the TLR4/NLRP3-related proteins TLR4, RelA, NF-κB and TNF-α. In vivo, CQCQD attenuated the pancreatic injury and systemic inflammation of CER-AP and was associated with reduced expression of TLR4/NLRP3-related mRNAs and proteins. Emodin, rhein, baicalin and chrysin significantly diminished pancreatic acinar cell necrosis with varied effects on suppressing the expression of TLR4/NLRP3-related mRNAs. Emodin, rhein and chrysin also decreased nitric oxide production in macrophages and their combination had synergistic effects on alleviating cell death as well as expression of TLR4/NLRP3-related proteins.. CQCQD attenuated the severity of AP at least in part by inhibiting the TLR4/NLRP3 pro-inflammatory pathways. Its active ingredients, emodin, baicalin, rhein and chrysin contributed to these beneficial effects.

Topics: Acinar Cells; Animals; Anti-Inflammatory Agents, Non-Steroidal; Ceruletide; Drugs, Chinese Herbal; Emodin; Flavonoids; Inflammasomes; Male; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis; RAW 264.7 Cells; Toll-Like Receptor 3

Acute pancreatitis (AP) is a sudden inflammatory process of the pancreas that may also involve surrounding tissues and/or remote organs. Inflammation and parenchymal cell death are common pathological features of this condition and determinants of disease severity. Polyethylene glycols (PEGs) are non-immunogenic, non-toxic water-soluble polymers widely used in biological, chemical, clinical and pharmaceutical settings.. To evaluate the protective effect of a 35-kDa molecular weight PEG (PEG35) on the pancreatic damage associated to cerulein-induced acute pancreatitis. Wistar rats were assigned at random to a control group, a cerulein-induced AP group and a PEG35 treatment group. AP was induced by five hourly intraperitoneal injections of cerulein (50 μg/kg/bw), while the control animals received saline solution. PEG35 was administered intraperitoneally 10 minutes before each cerulein injection in a dose of 10 mg/kg. After AP induction, samples of pancreatic tissue and blood were collected for analysis. AR42J pancreatic acinar cells were treated with increasing concentrations of PEG35 prior to exposure with tumor necrosis factor α (TNFα), staurosporine or cerulein. The severity of AP was determined on the basis of plasma levels of lipase, lactate dehydrogenase activity, pancreatic edema and histological changes. To evaluate the extent of the inflammatory response, the gene expression of inflammation-associated markers was determined in the pancreas and in AR42J-treated cells. Inflammation-induced cell death was also measured in models of. Administration of PEG35 significantly improved pancreatic damage through reduction on lipase levels and tissue edema in cerulein-induced AP rats. The increased associated inflammatory response caused by cerulein administration was attenuated by a decrease in the gene expression of inflammation-related cytokines and inducible nitric oxide synthase enzyme in the pancreas. In contrast, pancreatic tissue mRNA expression of interleukin 10 was markedly increased. PEG35 treatment also protected against inflammation-induced cell death by attenuating lactate dehydrogenase activity and modulating the pancreatic levels of apoptosis regulator protein BCL-2 in cerulein hyperstimulated rats. Furthermore, the activation of pro-inflammatory markers and inflammation-induced cell death in pancreatic acinar cells treated with TNFα, cerulein or staurosporine was significantly reduced by PEG35 treatment, in a dose-dependent manner.. PEG35 ameliorates pancreatic damage in cerulein-induced AP and AR42J-treated cells through the attenuation of the inflammatory response and associated cell death. PEG35 may be a valuable option in the management of AP.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Pancreas; Pancreatitis; Polyethylene Glycols; Rats; Rats, Wistar

Astaxanthin (ATX) is a naturally occurring carotenoid and a potent antioxidant. Various anti-inflammatory effects of ATX have been examined. We aimed to investigate the protective effect of ATX and its mechanism in a cerulein-induced acute pancreatitis rat model.. The rats were randomized into 2 main groups as control (C) and acute pancreatitis group (AP). AP group was subsequently divided into subgroups as AP+vehicle (AP), AP+ATX, and ATX+peroxisome proliferator-activated receptor-alpha antagonist GW6471 (ATX+GW) groups. To induce AP, the rats were administered cerulein (50 µg/kg, intraperitonally [ip]) at 1 hour intervals, whereas the C group received saline. The AP group was treated with vehicle olive oil, ATX 40 mg/kg/orally, or GW6471 and ATX (GW1 mg/kg/ip; ATX; 40 mg/kg/peroral). Treatments were administered after the 1st cerulein injection. At the 7th hour after the final injection, the rats were killed and the pancreatic tissue was used for the determination of malondialdehyde (MDA), glutathione (GSH), and myeloperoxidase (MPO) activities and luminol-lucigenin chemiluminescence levels. Serum amylase, lipase, and histopathological analyses were performed.. Elevated serum lipase and amylase levels in the vehicle-treated AP group (p<0.01) decreased in the ATX and ATX+GW groups (p<0.05). In the AP groups, GSH was reduced and MDA, MPO, luminol, and lucigenin levels were increased (p<0.05-0.001). ATX reversed these changes (p<0.05-0.001). The vehicle-treated group revealed significant severe cytoplasmic degeneration and vacuolization, whereas ATX ameliorated these destructions. GW6471 did not abolish the positive effects of ATX biochemically or histologically.. ATX has a potent protective effect on AP via its radical scavenging and antioxidant properties. Therefore, we believe that ATX may have therapeutic potential.

Topics: Acute Disease; Animals; Antioxidants; Ceruletide; Disease Models, Animal; Oxidative Stress; Pancreas; Pancreatitis; Rats; Xanthophylls

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.

Topics: Acinar Cells; Adenocarcinoma; Animals; Antineoplastic Agents; Calcium; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Dedifferentiation; Cell Transformation, Neoplastic; Cells, Cultured; Ceruletide; Deoxycytidine; Disease Progression; Gemcitabine; GTP-Binding Proteins; Histone Deacetylase Inhibitors; Mice; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); RGS Proteins; Signal Transduction

To investigate the effect and mechanism of galactose on cerulean-induced pancreatic acinar cell injury. Acute pancreatitis cell injury model was established by arbusin-induced pancreatic acinar cell AR42J injury; galactose (25, 50, 100 mmol / L) was used to treat the injured cells, and the optimal concentration was 50 mmol / L; cell counting kit (CCK-8), enzyme linked immunosorbent assay (ELISA) to detect cell survival rate and necrosis rate; flow cytometry and Western blotting (Western blot) to detect cell apoptosis and autologous phage-related gene (Beclin1) and microtubule-associated protein 1 light chain 3 (LC3), apoptosis-related protein B-cell lymphoma / leukemia-2 (Bcl-2), Bcl-2-related X gene (Bax), and fibroblasts Expression of growth factor 21 antibody (FGF21) and anti-aging gene Klotho. A pancreatic acinar cell injury model was successfully established with cerana (100 nmol / L); galactose (25, 50, 100 mmol/L) In a concentration-dependent manner, the inhibitory effect of ceriferin on AR42J injury was inhibited at an optimal concentration of 50 mmol / L. Compared with the ceriferin group, the apoptosis rate of AR42J cells in the galactose group was significantly reduced. table Significantly increased, Bcl-2, FGF21 and Klotho protein expression was significantly increased, Bax protein was significantly decreased; the FGF21 inhibitor can be significantly reduced on galactose these caerulein-induced AR42J cells. Galactose can inhibit the apoptosis and autophagy of pancreatic acinar cells induced by cerana, and its potential mechanism is to up-regulate FGF21 and Klotho, providing a new potential drug for the treatment of acute pancreatitis.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Cell Line; Ceruletide; Fibroblast Growth Factors; Galactose; Glucuronidase; Klotho Proteins; Pancreas, Exocrine; Pancreatitis; Protective Agents; Rats; Signal Transduction

Vagus nerve stimulation (VNS) is effective in reducing inflammation in various diseases, such as rheumatoid arthritis, colitis and acute kidney injury. The anti-inflammatory effect of vagus nerve in these diseases necessitates the interactions of neural activation and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. In this study, we aimed to investigate the effect of VNS on severity in experimental acute pancreatitis (AP).. Two independent AP models were used, which induced in ICR mice with caerulein or pancreatic duct ligation (PDL). Thirty minutes after modeling, the left cervical carotid sheath containing the vagus nerve was electrically stimulated for 2 min. Plasma lipase and amylase activities, TNF-α levels and pancreas histologic damage were evaluated. In caerulein mice, the percentages of α7nAChR. VNS reduced plasma lipase and amylase activities, blunted the concentrations of TNF-α and protected against pancreas histologic damage in two AP models. Survival rates were improved in the PDL model after VNS. In caerulein AP mice, VNS increased the percentages of α7nAChR. VNS reduces disease severity and attenuates inflammation in AP mice. This effect is independent of spleen and is probably related to α7nAChR on macrophage.

Topics: Acute Disease; Adoptive Transfer; Animals; Biomarkers; Ceruletide; Disease Models, Animal; Evoked Potentials; Immunohistochemistry; Male; Mice; Pancreatitis; Severity of Illness Index; Survival Rate; Vagus Nerve Stimulation

Activation of the constitutive nuclear and mitochondrial enzyme poly (ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of cell dysfunction, inflammation, and organ failure in various forms of critical illness. The objective of our study was to evaluate the efficacy and safety of the clinically approved PARP inhibitor olaparib in an experimental model of pancreatitis in vivo and in a pancreatic cell line subjected to oxidative stress in vitro. The preclinical studies were complemented with analysis of clinical samples to detect PARP activation in pancreatitis.. Mice were subjected to cerulein-induced pancreatitis; circulating mediators and circulating organ injury markers; pancreatic myeloperoxidase and malondialdehyde levels were measured and histology of the pancreas was assessed. In human pancreatic duct epithelial cells (HPDE) subjected to oxidative stress, PARP activation was measured by PAR Western blotting and cell viability and DNA integrity were quantified. In clinical samples, PARP activation was assessed by PAR (the enzymatic product of PARP) immunohistochemistry.. In male mice subjected to pancreatitis, olaparib (3 mg/kg i.p.) improved pancreatic function: it reduced pancreatic myeloperoxidase and malondialdehyde levels, attenuated the plasma amylase levels, and improved the histological picture of the pancreas. It also attenuated the plasma levels of pro-inflammatory mediators (TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12, IP-10, KC) but not MCP-1, RANTES, or the anti-inflammatory cytokine IL-10. Finally, it prevented the slight, but significant increase in plasma blood urea nitrogen level, suggesting improved renal function. The protective effect of olaparib was also confirmed in female mice. In HPDE cells subjected to oxidative stress olaparib (1 μM) inhibited PARP activity, protected against the loss of cell viability, and prevented the loss of cellular NAD levels. Olaparib, at 1μM to 30 μM did not have any adverse effects on DNA integrity. In human pancreatic samples from patients who died of pancreatitis, increased accumulation of PAR was demonstrated.. Olaparib improves organ function and tempers the hyperinflammatory response in pancreatitis. It also protects against pancreatic cell injury in vitro without adversely affecting DNA integrity. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of pancreatitis.

Topics: Animals; Cell Culture Techniques; Cell Line; Ceruletide; Disease Models, Animal; Epithelial Cells; Female; Humans; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Pancreatic Ducts; Pancreatitis; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors

Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12 h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60 min and serum amylase within 2 h, but fluorescent autophagosome formation only by 4 h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60 min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15 min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile-Pro-Arg-AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease.

Topics: Animals; Autophagosomes; Autophagy; Ceruletide; Endosomes; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Secretory Vesicles; Trypsin; Trypsinogen

During pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.. We examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.. Pancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.. Our findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.

Topics: Acute Disease; Animals; Anticholesteremic Agents; Autophagy; Ceruletide; Lysosomes; Male; Membrane Fusion; Mice, Inbred C57BL; Pancreatitis; Phagosomes; Simvastatin

Acute pancreatitis (AP) is a severe inflammatory disease. Caerulin induces significant pro-inflammatory responses in macrophages, causing serve damage to pancreatic acinar cells. The potential role of Rab GTPase 21 (Rab21) in this process was tested in this study. In murine bone marrow-derived macrophages (BMDMs), caerulin induced Rab21-TRAF3-MKK3 complex association. Rab21 silencing (by targeted shRNAs) or knockout (by CRISPR/Cas9 method) largely inhibited caerulin-induced MKK3-TRAF3 association, downstream MKK3-p38 activation and production of several pro-inflammatory cytokines (IL-1β, TNF-α and IL-17). Conversely, ectopic Rab21 overexpression in BMDMs potentiated caerulin-induced MKK3-TRAF3 association and pro-inflammatory cytokines production. The cytotoxicity of caerulin-activated BMDMs to co-cultured pancreatic acinar cells was alleviated by Rab21 knockdown or knockout, but exacerbated with Rab21 overexpression. In vivo, administration of Rab21 shRNA lentivirus significantly attenuated pancreatic and systemic inflammations in caerulin-injected AP mice. Collectively, our results suggest that Rab21 mediates caerulin-induced MKK3-p38 activation and pro-inflammatory responses.

Topics: Acinar Cells; Acute Disease; Animals; Cell Death; Ceruletide; Cytokines; Enzyme Activation; Inflammation; Inflammation Mediators; Macrophages; MAP Kinase Kinase 3; Mice, Inbred C57BL; Mice, Knockout; p38 Mitogen-Activated Protein Kinases; Pancreas; Pancreatitis; rab GTP-Binding Proteins; RNA, Small Interfering; TNF Receptor-Associated Factor 3

Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Icariin (ICA), a flavonoid glycoside, has been reported to have several pharmacological effects; however, the anti‑inflammatory effects of ICA against AP require further study. Therefore, we aimed to investigate the effect of ICA on cerulein‑induced AP. In the present study, AP was induced by intraperitoneally administering a supramaximal concentration of cerulein (50 µg/kg/h) for 6 h. ICA was also administered intraperitoneally, and mice were sacrificed 6 h after the final cerulein injection. Blood samples were collected to determine serum amylase and lipase levels. The pancreas and lung were rapidly removed for histological examination, and the analysis of myeloperoxidase activity. In addition, reverse transcription‑quantitative polymerase chain reaction was conducted to analyze the expression of inflammatory cytokines in pancreatic tissues. Our results revealed that the administration of ICA prevented an increase in the pancreas weight/body weight ratio of mice and serum digestive enzyme levels. ICA treatment also inhibited cerulein‑induced histological injury and neutrophil infiltration of the pancreas and lung. In addition, ICA suppressed the production of pro‑inflammatory cytokines, including interleukin (IL)‑1β, IL‑6 and tumor necrosis factor‑α in the pancreas. Furthermore, ICA administration was observed to inhibit p38 activation during cerulein‑induced AP. Inhibition of p38 activation resulted in alleviated pancreatitis. Collectively, our results suggested that ICA exhibits anti‑inflammatory effects in cerulein‑induced AP via the inhibition of p38.

Topics: Amylases; Animals; Biomarkers; Ceruletide; Disease Models, Animal; Female; Flavonoids; Lipase; Mice; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pancreatitis; Severity of Illness Index

In the current study, we explored the role of extracellular ATP (eATP) in promoting systemic inflammation during development of acute pancreatitis (AP). Release of extracellular (e)ATP was evaluated in plasma and bronchoalveolar lavage fluid (BALF) of mice with experimental acute pancreatitis (AP). Prophylactic intervention using apyrase or suramin was used to understand the role and contribution of eATP in pancreatitis-associated systemic injury. AP of varying severity was induced in C57BL/6 mice using 1-day or 2-day caerulein, caerulein + LPS and l-arginine models. eATP was measured in plasma and BALF. Mice were treated with suramin or apyrase in the caerulein and l-arginine models of AP. Plasma cytokines, lung, and pancreatic myeloperoxidase, and morphometric analysis of pancreatic and lung histology, were used to assess the severity of pancreatitis. Plasma eATP and purinergic 2 (P2) receptors in the pancreas and lungs were significantly elevated in the experimental models of AP. Blocking the effect of eATP by suramin led to reduced levels of plasma IL-6 and TNFα as well as reduced lung, and pancreatic injury. Neutralizing eATP with apyrase reduced systemic injury but did not ameliorate local injury. The results of this study support the role of eATP and P2 receptors in promoting systemic inflammation during AP. Modulating purinergic signaling during AP can be an important therapeutic strategy in controlling systemic inflammation and, thus, systemic inflammatory response syndrome during AP.

Topics: Acute Disease; Adenosine Triphosphate; Animals; Apyrase; Arginine; Bronchoalveolar Lavage Fluid; Ceruletide; Cytokines; Inflammation; Lung; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peroxidase; Receptors, Purinergic; Signal Transduction; Suramin

Pancreatitis is characterized by increased influx of Ca. We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf. Pancreatic levels of Ca. In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca

Topics: Acinar Cells; Animals; Calcium; Ceruletide; Disease Models, Animal; HEK293 Cells; Humans; Intracellular Calcium-Sensing Proteins; Membrane Proteins; Mice; Mice, Knockout; Neoplasm Proteins; Pancreas; Pancreatitis; Severity of Illness Index; Stromal Interaction Molecule 1

BACKGROUND Acute pancreatitis (AP) has a high mortality rate and often has serious complications. The Hippo-YAP signaling pathway is mainly involved in cell proliferation and stem cell self-renewal. Recent studies have reported that YAP1 plays a crucial role in pancreatic cancer initiation and acute and chronic pancreatitis (CP). However, the role of YAP1 in AP still needs to be clarified. MATERIAL AND METHODS To assess the role of YAP1 in the progression of AP, we established a cell model of AP in AR42J cells. AR42J, a rat pancreatic acinar cell line, was stimulated with caerulein to mimic AP-like acinar cell injury. Levels of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-alpha) were measured by ELISA to investigate the role of YAP1 in the progression of AP. RESULTS The results showed that YAP1 and MALAT1 were the targets of miR-194 and were upregulated in caerulein-treated AR42J cells. Overexpression of MALAT1 or YAP1 can increase the levels of IL-6 and TNF-alpha secreted by AR42J cells, while miR-194 dramatically counteracts this enhancement effect. CONCLUSIONS Our results demonstrated a regulation loop among MATAL1, miR-194, and YAP1, which dynamically regulates the progression of AP, providing a new therapeutic target for treatment of this disease.

Topics: Acute Disease; Animals; Apoptosis Regulatory Proteins; Base Sequence; Cell Line; Ceruletide; Cytoprotection; Disease Progression; Down-Regulation; Feedback, Physiological; MicroRNAs; Pancreatitis; Protein Binding; Rats; RNA, Long Noncoding; Signal Transduction; Up-Regulation; YAP-Signaling Proteins

Acute pancreatitis (AP) is a severe inflammatory condition of the pancreas, with no specific treatment available. We have previously reported that Nardostachys jatamansi (NJ) ameliorates cerulein-induced AP. However, the specific compound responsible for this inhibitory effect has not been identified. Therefore, in the present study, we focused on a single compound, 8α-hydroxypinoresinol (HP), from NJ. The aim of this study was to determine the effect of HP on the development of pancreatitis in mice and to explore the underlying mechanism(s). AP was induced by the injection of cerulein (50 μg/kg/h) for 6 h. HP (0.5, 5 or 10 mg/kg, i.p.) was administered 1 h prior to and 1, 3 or 5 h after the first cerulein injection, with vehicle- and DMSO-treated groups as controls. Blood samples were collected to determine serum levels of amylase, lipase, and cytokines. The pancreas was removed for morphological examination, myeloperoxidase (MPO) assays, cytokine assays, and assessment of nuclear factor (NF)-κB activation. The lungs were removed for morphological examination and MPO assays. Administration of HP dramatically improved pancreatic damage and pancreatitis-associated lung damage and also reduced amylase and lipase activities in serum. Moreover, administration of HP reduced the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the pancreas and serum during AP. In addition, the administration of HP inhibited degradation of inhibitory κ-Bα (Iκ-Bα), NF-κB p65 translocation into nucleus and NF-κB binding activity in the pancreas. Our results suggest that HP exerted therapeutic effects on pancreatitis and these beneficial effects may be due to the inhibition of NF-κB activation.

Topics: Animals; Ceruletide; Cytokines; Female; Furans; Inflammation; Lignans; Lung; Mice; Mice, Inbred C57BL; Nardostachys; Pancreas; Pancreatitis; Signal Transduction; Tumor Necrosis Factor-alpha

BACKGROUND This study aimed to investigate the effects of maresin-1 (MaR1) in a mouse model of caerulein-induced acute pancreatitis (AP). MATERIAL AND METHODS Fifty C57BL/6 mice with caerulein-induced AP were divided into the untreated control group (N=10), the untreated AP model group (N=10), the MaR1-treated (low-dose, 0.1 μg) AP model group (N=10), the MaR1-treated (middle-dose, 0.5 μg) AP model group (N=10), and the MaR1-treated (high-dose, 1 μg) AP model group (N=10). Enzyme-linked immunoassay (ELISA) measured serum levels of amylase, lipase, tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß (IL-1ß), and IL-6 and mRNA was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Malondialdehyde (MDA), protein carbonyls, superoxide dismutase (SOD), and the ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) were measured. Histology of the pancreas included measurement of acinar cell apoptosis using the terminal-deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay. Western blot measured Toll-like receptor 4 (TLR4), MyD88, and phospho-NF-kappaB p65, and apoptosis-associated proteins Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9. RESULTS Following treatment with MaR1, serum levels of amylase, lipase, TNF-alpha, IL-1ß, and IL-6 decreased, MDA and protein carbonyl levels decreased, SOD and the GSH/GSSG ratio increased in a dose-dependent manner. In the MaR1-treated AP mice, inflammation of the pancreas and the expression of inflammatory cytokines, pancreatic acinar cell apoptosis, Bcl-2 expression, and expression of TLR4, MyD88, and p-NF-kappaB p65 were reduced, but Bax, cleaved caspase-3, and cleaved caspase-9 expression increased. CONCLUSIONS In a mouse model of caerulein-induced AP, treatment with MaR1 reduced oxidative stress and inflammation and reduced apoptosis.

Topics: Animals; Apoptosis; Caspase 3; Ceruletide; Cytokines; Disease Models, Animal; Docosahexaenoic Acids; Inflammation; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Autophagy plays key roles in the development of acute pancreatitis (AP) and the regulation of impaired autophagy has therapeutic potential. The objective of the present study was to investigate whether pharmacological inhibition of autophagy could ameliorate AP in mice and examine the underlying mechanisms. In current study, by imaging-based high-throughput screening, a novel spautin-1 derivative spautin-A41 was identified as a potent autophagy inhibitor. Mice treated with spautin-A41 were resistant to the cerulein-induced elevation of serum pancreatic enzyme activities and pancreatic apoptosis. Mechanistically, spautin-A41 effectively reduced the expression levels of Class III phosphatidylinositol 3 (PI3) kinase complexes and subsequently ameliorated pancreatitis by inhibiting the formation of autophagosome. Therefore, pharmacological inhibition of autophagy by spautin-A41 may serve as new target for treating or lessening the severity of AP.

Topics: Animals; Apoptosis; Autophagy; Benzylamines; Cell Line; Ceruletide; Male; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Phosphatidylinositol 3-Kinases; Quinazolines; Rats

Acute pancreatitis is characterized by an early intracellular protease activation and invasion of leukocytes into the pancreas. Cathepsins constitute a large group of lysosomal enzymes, that have been shown to modulate trypsinogen activation and neutrophil infiltration. Cathepsin G (CTSG) is a neutrophil serine protease of the chymotrypsin C family known to degrade extracellular matrix components and to have regulatory functions in inflammatory disorders. The aim of this study was to investigate the role of CTSG in pancreatitis. Isolated acinar cells were exposed to recombinant CTSG and supramaximal cholezystokinin stimulation. In CTSG

Topics: Acinar Cells; Animals; Cathepsin G; Cells, Cultured; Ceruletide; Disease Models, Animal; Gene Knockout Techniques; Granulocytes; Male; Mice; Neutrophil Infiltration; Neutrophils; Pancreatitis; Trypsinogen

Acute pancreatitis (AP) is a common clinical critical disease with high mortality and the exact pathogenesis is not fully elucidated. The present study aimed to uncover the function of miR-135a in the proliferation, apoptosis, and inflammatory characteristics of diseased pancreatic cells and the potential molecular mechanisms. The expression patterns of miR-135a and family with sequence similarity 129 member A (FAM129A) in patients with AP were analyzed on the basis of the GEO database. The transfection efficiency and expression level of miR-135a in AR42J cells were determined by qRT-PCR. The biological characteristics of AR42J cells treated with cerulein were detected by cell counting kit-8 (CCK-8), flow cytometry, and western blot assays. The potential interaction between miR-135a and FAM129A was confirmed by bioinformatics prediction softwares and luciferase reporter assay. MiR-135a inhibitor and pcDNA3.1-FAM129A were co-transfected to determine the regulation of miR-135a/FAM129A on inflammatory AR42J cell injury. We observed that miR-135a was highly expressed in AP samples. Depletion of miR-135a could alleviate the condition so that the AR42J cells proliferation increased, apoptosis decreased, and the expression of inflammatory cytokines enhanced. In addition, mRNA and protein expression of FAM129A were negatively regulated by miR-135a, and over-expression of FAM129A could strengthen the relief effect of miR-135a inhibitor in AP induced by cerulein. In summary, our data demonstrates that silencing miR-135a reduces AR42J cells injury and inflammatory response in AP induced by cerulein through targeting FAM129A.

Topics: Acute Disease; Animals; Apoptosis; Biomarkers, Tumor; Cell Line; Cell Line, Tumor; Cell Proliferation; Ceruletide; Cytokines; HEK293 Cells; Humans; Inflammation; MicroRNAs; Neoplasm Proteins; Pancreatitis; Rats; RNA, Messenger; Transfection

Nesfatin-1, a recently discovered peptide, was shown to have anti-inflammatory effects. Acute pancreatitis (AP) is a life-threatening condition caused by various reasons. Although the etiology of AP is well-known, its pathogenesis is not clear. The aim of this study is to investigate the possible anti-inflammatory role of nesfatin-1 and its probable protective underlying mechanisms in an acute pancreatitis model. Caerulein was applied intraperitoneally to induce acute pancreatitis in Sprague-Dawley female rats. Nesfatin-1 was administered 5 minutes before the application of caerulein to determine its potential anti-inflammatory role on AP. Five minutes before nesfatin-1 injection, in order to investigate the underlying mechanism, oxytocin receptor antagonist (atosiban), melanocortin receptor antagonist (HS024), or ghrelin receptor antagonist (cortistatin) were administered. Five minutes after nesfatin-1 administration, two doses of caerulein were applied one hour apart. The rats were sacrified 12 hours after the first caerulein dose for serum and pancreatic tissue sampling. Microscopic damage scoring, malondialdehyde and glutathione levels, myeloperoxidase activity, luminol and lucigenin chemiluminescence levels in pancreatic tissue and amylase, lipase, trypsinogen-2 levels in serum were evaluated. Oxidative damage was decreased with nesfatin-1 treatment in the acute pancreatitis model (P < 0.05 - 0.001). The administration of HS024 reversed the effect of nesfatin-1, via increasing lipase, amylase, trypsinogen-2, malondialdehyde (MDA), myeloperoxidase (MPO) and lucigenin levels (P < 0.05 - 0.01). Atosiban pre-treatment elevated MPO activity, luminol and lucigenin chemiluminescence levels (P < 0.01 - 0.001) and cortistatin increased lucigenin and luminol chemiluminescence (P < 0.05 - 0.01). Although receptor antagonists reversed the effect of nesfatin-1 on related biochemical parameters, no significant difference was found in histological scoring. Our results indicated that nesfatin-1 had an anti-inflammatory effect on acute pancreatitis via mainly effecting melanocortin receptors.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Ceruletide; Disease Models, Animal; Female; Nucleobindins; Pancreatitis; Rats; Rats, Sprague-Dawley; Receptors, Melanocortin

Pancreatitis is a disease for which there are numerous etiologies but no effective treatments. Although the expression of the pancreatitis-associated protein-1 (PAP-1) serves as a marker for the disease, its biological function is unknown. The present study was carried out to determine if PAP-1 performs a protective role against oxidative stress-induced pancreatic cell death. For this purpose, we used cerulein-stimulated pancreatic acinar AR42J cells as an experimental model of acute pancreatitis. First, we demonstrated that PAP-1 gene expression is increased by cerulein in a dose- and time-dependent manner. In parallel, the level of active nuclear factor kappaB (NF-κB) was found to be increased in cells treated with cerulein. To test whether activation of the oxidant-sensitive transcription factor NF-κB is mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the primary source of reactive oxygen species, cerulein-stimulated NADPH oxidase activity was suppressed by using the NADPH oxidase inhibitor diphenyleneiodonium and, separately, by anti-sense oligonucleotides directed against NADPH oxidase subunits p22

Topics: Acinar Cells; Acute Disease; Animals; Apoptosis; Cell Survival; Cells, Cultured; Ceruletide; Gene Expression Regulation; NADPH Oxidases; NF-kappa B; Oxidative Stress; Pancreatitis; Pancreatitis-Associated Proteins; Rats; Reactive Oxygen Species

β-Arrestins (β-arrs) are regulators and mediators of G protein-coupled receptor signaling that are functionally involved in inflammation. Nuclear factor-κB p65 (NF-κBp65) activation has been observed early in the onset of pancreatitis. However, the effect of β-arrs in acute pancreatitis (AP) is unclear. The aim of this study is to investigate whether β-arrs are involved in AP through activation of NF-κBp65.. Acute pancreatitis was induced by either caerulein injection or choline-deficient supplemented with ethionine diet (CDE). β-arr1 wild-type and β-arr1 knockout mice were used in the experiment. The survival rate was calculated in the CDE model mice. Histological and western blot analyses were performed in the caerulein model. Inflammatory mediators were detected by real-time polymerase chain reaction in the caerulein-induced AP mice. Furthermore, AR42J and PANC-1 cell lines were used to further study the effects of β-arr1 in caerulein-induced pancreatic cells.. β-Arr1 but not β-arr2 is significantly downregulated in caerulein-induced AP in mice. Targeted deletion of β-arr1 notably upregulated expression of the pancreatic inflammatory mediators including tumor necrosis factor α and interleukin 1β as well as interleukin 6 and aggravated AP in caerulein-induced mice. β-Arr1 deficiency increased mortality in mice with CDE-induced AP. Further, β-arr1 deficiency enhanced caerulein-induced phosphorylation of NF-κBp65 both in vivo and in vitro.. β-Arr1 alleviates AP via repression of NF-κBp65 activation, and it is a potentially therapeutic target for AP.

Topics: Acute Disease; Animals; beta-Arrestin 1; Cell Line, Tumor; Ceruletide; Choline Deficiency; Disease Models, Animal; Down-Regulation; Ethionine; Female; Humans; Interleukin-1beta; Interleukin-6; Mice, Knockout; Pancreatitis; Phosphorylation; Survival Rate; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Our study aimed to probe the effects of rosiglitazone treatment on a severe acute pancreatitis (SAP) model induced by caerulein and investigate the underlying mechanism.. Differentially expressed messenger RNAs (mRNAs) in the mice of a SAP group were screened out by microarray analysis. The inflammatory response pathway was obtained from the online website DAVID Bioinformatics Resources 6.8. The interactions of caerulein and its target proteins were shown by search tool for interactions of chemicals (STITCH). Functional interactions of the genes associated with pancreatitis and the target proteins of caerulein were obtained with search tool for interactions of chemicals (STRING). SAP mice were established by hourly intraperitoneal injection of caerulein. Rosiglitazone was used as treatment drug, and pancreatic inflammation was assessed. The expression of Socs3 was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of interleukin (IL)-6, IL-1b, and Egr1 were studied by RT-PCR and Western blot analysis.. The GSE77983 data were analyzed, and the results showed that Socs3 was overexpressed in SAP tissues. The inflammation response pathway in pancreas was selected by DAVID, STITCH, and STRING. After injection of rosiglitazone in mice, the serum levels of amylase and lipase were decreased. Furthermore, the mRNA and protein levels of Socs3 and inflammatory cytokines in pancreatic tissues were downregulated.. Rosiglitazone could protect mice with SAP from injury by downregulating Socs3 and inhibiting the inflammatory response pathway.

Topics: Animals; Ceruletide; Disease Models, Animal; Early Growth Response Protein 1; Female; Inflammation; Injections, Intraperitoneal; Interleukin-1beta; Interleukin-6; Mice; Mice, Inbred ICR; Pancreatitis; Protective Agents; RNA, Messenger; Rosiglitazone; Severity of Illness Index; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein

Obesity is associated with local and systemic complications in acute pancreatitis. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator and master regulator of mitochondrial biogenesis that exhibits dysregulation in obese subjects. Our aims were: (1) to study PGC-1α levels in pancreas from lean or obese rats and mice with acute pancreatitis; and (2) to determine the role of PGC-1α in the inflammatory response during acute pancreatitis elucidating the signaling pathways regulated by PGC-1α. Lean and obese Zucker rats and lean and obese C57BL6 mice were used first; subsequently, wild-type and PGC-1α knockout (KO) mice with cerulein-induced pancreatitis were used to assess the inflammatory response and expression of target genes. Ppargc1a mRNA and protein levels were markedly downregulated in pancreas of obese rats and mice versus lean animals. PGC-1α protein levels increased in pancreas of lean mice with acute pancreatitis, but not in obese mice with pancreatitis. Interleukin-6 (Il6) mRNA levels were dramatically upregulated in pancreas of PGC-1α KO mice after cerulein-induced pancreatitis in comparison with wild-type mice with pancreatitis. Edema and the inflammatory infiltrate were more intense in pancreas from PGC-1α KO mice than in wild-type mice. The lack of PGC-1α markedly enhanced nuclear translocation of phospho-p65 and recruitment of p65 to Il6 promoter. PGC-1α bound phospho-p65 in pancreas during pancreatitis in wild-type mice. Glutathione depletion in cerulein-induced pancreatitis was more severe in KO mice than in wild-type mice. PGC-1α KO mice with pancreatitis, but not wild-type mice, exhibited increased myeloperoxidase activity in the lungs, together with alveolar wall thickening and collapse, which were abrogated by blockade of the IL-6 receptor glycoprotein 130 with LMT-28. In conclusion, obese rodents exhibit PGC-1α deficiency in the pancreas. PGC-1α acts as selective repressor of nuclear factor-κB (NF-κB) towards IL-6 in pancreas. PGC-1α deficiency markedly enhanced NF-κB-mediated upregulation of Il6 in pancreas in pancreatitis, leading to a severe inflammatory response. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Animals; Ceruletide; Disease Models, Animal; Interleukin-6; Male; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Obesity; Pancreas; Pancreatitis; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphorylation; Rats, Zucker; Signal Transduction; Taurocholic Acid; Transcription Factor RelA; Up-Regulation

The goal of this study was to clarify the protective role of the Wnt/β-catenin pathway agonist SKL2001 in a rat model of Caerulein-induced acute pancreatitis. AR42J cells and rats were divided into 4 groups: control, Caerulein, SKL2001 + Caerulein, and SKL2001 + control. Cell apoptosis was examined using flow cytometry. Hematoxylin-eosin staining was performed to observe pathological changes in pancreatic and small intestinal tissues. Inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA), while genes related to the Wnt/β-catenin pathway were quantified using quantitative real-time PCR. In vitro results showed that Caerulein promoted cell necrosis, inhibited the Wnt/β-catenin pathway, and increased the level of inflammatory cytokines. However, SKL2001 reduced cell necrosis and inflammatory cytokines and activated the Wnt/β-catenin pathway. Additionally, in vivo results demonstrated the accumulation of fluid (i.e., edema), hemorrhage, inflammation and necrosis of the pancreatic acini occurred 6 h after the final Caerulein induction, with the damage reaching a maximal level 12 h after the final Caerulein induction; meanwhile, the Wnt/β-catenin pathway was evidently inhibited with an enhanced level of inflammatory cytokines. The aforementioned damage was further aggravated 12 h later. Nevertheless, the pancreatic and small intestinal tissue damages were alleviated in Caerulein-induced rats treated with SKL2001. In conclusion, activation of the Wnt/β-catenin pathway could inhibit Caerulein-induced cell apoptosis and inflammatory cytokine release, thus improving pancreatic and intestinal damage in rats with acute pancreatitis.

Topics: Acute Disease; Animals; beta Catenin; Ceruletide; Female; Imidazoles; Isoxazoles; Male; Pancreatitis; Rats; Rats, Sprague-Dawley; Wnt Signaling Pathway

Pancreatitis is a major risk factor for the development of pancreatic cancer. In genetically engineered mouse models, induction of pancreatic inflammation dramatically accelerates oncogenic KRas-induced fibrosis, precancerous PanIN formation, and tumorigenesis. Here we describe simple methods of secretagogue-induced experimental acute and chronic pancreatitis, the most commonly used pancreatitis models, and their applications in pancreatic cancer research. Additionally, the preparation of primary pancreatic acinar cells is introduced. Primary acinar cells can be used to study the early events of pancreatic inflammation and pancreatic acinar-to-ductal (ADM) metaplasia.

Topics: Acinar Cells; Animals; Carcinogenesis; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Humans; Metaplasia; Mice; Mice, Transgenic; Mutation; Pancreas; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Primary Cell Culture; Proto-Oncogene Proteins p21(ras); Tumor Cells, Cultured

Acinar-to-ductal metaplasia (ADM) of the pancreas is a process that pancreatic acinar cells differentiate into ductal-like cells with ductal cell traits. The metaplasia of pancreatic acinar cells manifests their ability to adapt to the genetic and environmental pressure they encounter. However, with oncogenic genetic insults and/or sustained environmental stress, ADM may lead to pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic cancer. Understanding the intermediate states of ADM and important molecules that regulate ADM formation may help the development of novel preventive strategies that could be translated to the clinic to benefit the people with high risk of pancreatic cancer. Mouse model is widely used in both in vivo and ex vivo studies of ADM. In this chapter, we describe detailed protocols of injury models of the adult mouse pancreas that can function as a tool to study mechanisms of ADM formation.

Topics: Acinar Cells; Animals; Carcinoma, Pancreatic Ductal; Cell Transdifferentiation; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Humans; Metaplasia; Mice; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Primary Cell Culture; Tumor Cells, Cultured

We aimed to investigate the association of macrophage inflammatory protein (MIP)-1α (CCL3) expression with the severity of acute pancreatitis (AP).. The patients with AP were selected and divided into mild AP (MAP), moderately severe AP (MSAP), and severe AP (SAP) groups according to the severity of AP. The pancreatic acinar cell line Ar42 j was treated with cerulein to induce in vitro cell AP model. The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and the activation of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signaling pathway in stimulated or transfected Ar42 j cells were detected.. We detected that the upregulation of MIP-1α was associated with the severity of AP. Patients with SAP showed the highest MIP-1α contents, followed by MSAP, and, lastly, MAP. In cerulein-stimulated Ar42 j cells, the upregulation of MIP-1α, CCR5, TNF-α, and IL-6 was time dependent. In addition, in human recombinant MIP-1α treated Ar42 j cells, the upregulation of TNF-α and IL-6 was MIP-1α-dose-dependent. In contrast, we detected the inhibition of TNF-α and IL-6 in MIP-1α small interfering RNA (siRNA)-treated cells. Also, the activation of the JNK/p38 MAPK signaling pathway was induced and inhibited by human recombinant MIP-1α and MIP-1α siRNA, respectively.. These results suggested that MIP-1α might be used as a biomarker for the prognosis of AP severity. The MIP-1α-induced inflammatory responses in AP were mediated by TNF-α and IL-6, which were associated with the activation of the JNK/p38 MAPK signaling pathway.

Topics: Adult; Aged; Cell Line; Ceruletide; Chemokine CCL3; Female; Humans; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Middle Aged; Models, Biological; Pancreatitis; Receptors, CCR1; Receptors, CCR5; Severity of Illness Index; Tumor Necrosis Factor-alpha; Up-Regulation; Young Adult

Acute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown.. To uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function.. After the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice.. TLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Ceruletide; Gene Expression Regulation; Humans; Mice, Knockout; Mice, Transgenic; Pancreatitis; Toll-Like Receptor 3

Acute pancreatitis is one of the first pathological processes where autophagy has been described in a human tissue. Autophagy, autodigestion, and cell death are early cellular events in acute pancreatitis. Recent advances in understanding autophagy highlight its importance in pathological conditions. However, methods for monitoring autophagic activity during complex diseases, involving highly differentiated secretory cells, are complicated, and the results are sometimes misinterpreted. Here, we describe methods used to identify autophagic structures and to measure autophagic flux in cultured cell models and animal models of pancreatitis. We also briefly describe the pancreas specific autophagy mouse model that was useful to understand the actual role of autophagy in pancreatitis and to identify a novel selective autophagy pathway named zymophagy. Lastly, we describe the immunomagnetic isolation of autophagosomes and the detection of autophagy in pancreatic tissue samples derived from humans.

Topics: Acinar Cells; Animals; Autophagosomes; Autophagy; Cell Culture Techniques; Cell Line; Ceruletide; Disease Models, Animal; Enzyme Precursors; Humans; Lysosomes; Male; Mice; Microscopy, Electron; Microscopy, Fluorescence; Pancreas; Pancreatectomy; Pancreatitis; Rats; Secretory Vesicles

Syntrophins are a family of proteins forming membrane-anchored scaffolds and serving as adaptors for various transmembrane and intracellular signaling molecules. To understand the physiological roles of β1 syntrophin, one of the least characterized members, we generated mouse models to eliminate β1 syntrophin specifically in the endocrine or exocrine pancreas. β1 syntrophin is dispensable for the morphology and function of insulin-producing β cells. However, mice with β1 syntrophin deletion in exocrine acinar cells exhibit increased severity of cerulein-induced acute pancreatitis. Reduced expression of cystic fibrosis transmembrane conductance regulator and dilation of acinar lumen are potential predisposition factors. During the disease progression, a relative lack of autophagy is associated with deficiencies in both actin assembly and endoplasmic reticulum nucleation. Our findings reveal, for the first time, that β1 syntrophin is a critical regulator of actin cytoskeleton and autophagy in pancreatic acinar cells and is potently protective against cerulein-induced acute pancreatitis.

Topics: Acinar Cells; Animals; Autophagy; Ceruletide; Cystic Fibrosis Transmembrane Conductance Regulator; Dystrophin-Associated Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Protective Agents

Glycosylation of phenolic compounds has been reported to increase water-solubility, reduce toxicity, and sometimes give improved or novel pharmacological activities. Present study was aimed to evaluate and compare the beneficial effects of quercetin aglycone (Quer) and its glycosylated derivative, quercetin 3-O-xyloside (Quer-Xyl), against acute pancreatitis (AP).. The cellular acute pancreatitis model was established by treating the rat pancreatic acinar cells (AR42J) with lipopolysaccharide (10 µg/ml) and cerulein (10. While Quer increased the mRNA expressions of AP marker enzymes, amylase and lipase, Quer-Xyl dose-dependently reversed their expressions. Quer-Xyl suppressed intracellular ROS production and both mRNA and protein levels of GRP78 and PERK, which were significantly elevated in cerulein and LPS-treated AR42J cells. Further, RT-PCR and fluorescence assay revealed that Quer-Xyl dose-dependently augmented the mRNA expressions and activities of caspase-3 and -9.. These results showed that Quer-Xyl, but not Quer, has a significant anti-pancreatitis activity through attenuating intracellular ROS production and ER stress response and enhancing apoptotic cell death, suggesting that it might be useful as a potent functional ingredient in health-beneficial foods or as a therapeutic agent to prevent or treat AP.

Topics: Acinar Cells; Amylases; Animals; Apoptosis; Cell Line; Ceruletide; Dose-Response Relationship, Drug; eIF-2 Kinase; Endoplasmic Reticulum Stress; Glycosides; Heat-Shock Proteins; Lipase; Lipopolysaccharides; Male; Pancreatitis; Quercetin; Rats

We aim to study the protective effect of menadione on caerulein-induced acute pancreatitis (AP) and associated lung injury and to explore the possible mechanism.. Male Swiss mice randomized into control and different experimental groups. AP was induced in mice by six hourly intraperitoneal (i.p) injections of caerulein (50 μg/kg at 1 h interval). Menadione (10 mg/kg) was administered one hour (i.p, 10 mg/kg) after the first caerulein injection and control animals were given hourly intraperitoneal (i.p) injection of isotonic sodium chloride solution for 6 hours.. Administration of menadione attenuated the severity of AP and associated lung injury as shown by the histopathology, reduced MPO and serum amylase activity. Further, the anti-inflammatory effect of menadione was associated with a reduction of pancreatic and pulmonary proinflammatory cytokine interleukin 1β (IL-1β) and hydrogen sulfide (H. The present findings show for the first time that in AP, menadione may exhibit an anti-inflammatory effect by down-regulating substance-P and H

Topics: Amylases; Animals; Ceruletide; Gene Expression Regulation; Hydrogen Sulfide; Interleukin-1beta; Lung Injury; Male; Mice; NF-kappa B; Pancreatitis; Peroxidase; Random Allocation; Substance P; Vitamin K 3

Despite melatonin treatment diminishes inflammatory mediator production and improves organ injury after acute pancreatitis (AP), the mechanisms remain unknown. This study explores whether melatonin improves liver damage after AP through protein kinase B (Akt)-dependent peroxisome proliferator activated receptor (PPAR)-γ pathway.. Male Sprague-Dawley rats were subjected to cerulein-induced AP. Animals were treated with vehicle, melatonin, and melatonin plus phosphoinositide 3-kinase (PI3K)/Akt inhibitor wortmannin 1 h following the onset of AP. Various indicators and targeted proteins were checked at 8 h in the sham and AP groups.. At 8 h after AP, serum alanine aminotransferase/aspartate aminotransferase levels, histopathology score of hepatic injury, liver myeloperoxidase activity, and proinflammatory cytokine production were significantly increased and liver tissue adenosine triphosphate concentration was lower compared with shams. AP resulted in a marked decrease in liver Akt phosphorylation and PPAR-γ expression in comparison with the shams (relative density, 0.442 ± 0.037 versus. 1.098 ± 0.069 and 0.390 ± 0.041 versus ± 1.080 0.063, respectively). Melatonin normalized AP-induced reduction in liver tissue Akt activation (1.098 ± 0.054) and PPAR-γ expression (1.145 ± 0.083) as well as attenuated the increase in liver injury markers and proinflammatory mediator levels, which was abolished by coadministration of wortmannin.. Collectively, our findings suggest that melatonin improves AP-induced liver damage in rats, at least in part, via Akt-dependent PPAR-γ pathway.

Topics: Administration, Intravenous; Animals; Ceruletide; Disease Models, Animal; Humans; Inflammation Mediators; Liver; Liver Failure; Liver Function Tests; Male; Melatonin; Pancreatitis; Phosphorylation; PPAR gamma; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Treatment Outcome; Wortmannin

Proliferation of pancreatic acinar cells is a critical process in the pathophysiology of pancreatic diseases, because limited or defective proliferation is associated with organ dysfunction and patient morbidity. In this context, elucidating the signalling pathways that trigger and sustain acinar proliferation is pivotal to develop therapeutic interventions promoting the regenerative process of the organ. In this study we used genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones to elucidate their role in acinar proliferation following caerulein-mediated acute pancreatitis in mice. In addition, molecular mechanisms mediating the effects of thyroid hormones were identified by genetic and pharmacological inactivation of selected signalling pathways.In this study we demonstrated that levels of the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) transiently increased in the pancreas during acute pancreatitis. Moreover, by using genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones, we showed that T3 was required to promote proliferation of pancreatic acinar cells, without affecting the extent of tissue damage or inflammatory infiltration.Finally, upon genetic and pharmacological inactivation of selected signalling pathways, we demonstrated that T3 exerted its mitogenic effect on acinar cells via a tightly controlled action on different molecular effectors, including histone deacetylase, AKT, and TGFβ signalling.In conclusion, our data suggest that local availability of T3 in the pancreas is required to promote acinar cell proliferation and provide the rationale to exploit thyroid hormone signalling to enhance pancreatic regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Acinar Cells; Animals; Cell Proliferation; Ceruletide; Disease Models, Animal; Histone Deacetylases; Hyperthyroidism; Iodide Peroxidase; Male; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatitis; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Thyroxine; Transforming Growth Factor beta; Triiodothyronine; Up-Regulation

Inflammasomes promote the production of pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18, which are the representative mediators of inflammation. Abnormal activation of inflammasomes leads to the development of inflammatory diseases such as acute pancreatitis (AP). In this study, we demonstrate the inhibitory effects of a new natural compound fraxinellone on inflammasome formation and examine the role of inflammasomes in a mouse model of AP. AP was induced with hourly intraperitoneal injections of supramaximal concentrations of the stable cholecystokinin analogue cerulein (50 μg/kg) for 6 h. Mice were sacrificed 6 h after the final cerulein injection. Blood and pancreas samples were obtained for further experiments. Intraperitoneal injection of fraxinellone significantly inhibited the pancreatic activation of multiple inflammasome molecules such as NACHT, LRR and PYD domains-containing protein 3 (NLRP3), PY-CARD, caspase-1, IL-18, and IL-1β during AP. In addition, fraxinellone treatment inhibited pancreatic injury, elevation in serum amylase and lipase activities, and infiltration of inflammatory cells such as neutrophils and macrophages but had no effect on pancreatic edema. To investigate whether inflammasome activation leads to the infiltration of inflammatory cells, we used parthenolide, a well-known natural inhibitor, and IL-1 receptor antagonist mice. The inhibition of inflammasome activation by pharmacological/or genetic modification restricted the infiltration of inflammatory cells, but not edema, consistent with the results observed with fraxinellone. Taken together, our study highlights fraxinellone as a natural inhibitor of inflammasomes and that inflammasome inhibition may lead to the suppression of inflammatory cells during AP.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Benzofurans; Cell Movement; Ceruletide; Disease Models, Animal; Female; Humans; Inflammasomes; Inflammation; Macrophages; Male; Mice; Mice, Inbred C57BL; Neutrophils; Pancreatitis

Acute pancreatitis is a life-threatening disease accompanied by systemic inflammatory response. NF-κB and p38 signal pathways are activated in AP induced by cerulein. And PAKs are multifunctional effectors of Rho GTPases with kinase activity. In the present study, the function of P21-activated kinase 1 (PAK1) in AP was investigated, and found that PAK1 was up-regulated in pancreas of AP mice model, and led to NF-κB and p38 pathway activation. PAK1 inhibition by shRNA or small molecule inhibitor FRAX597 decreased NF-κB and p38 activity, also alleviated the pathological damage in the pancreas of AP mice model, including decreasing the amylase and lipase levels in serum, decreasing the levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β in AP. These results suggested that PAK1 inhibition protects against AP by inhibiting NF-κB and p38 pathways, and indicated that PAK1 is a potential therapy to alleviate AP patients in clinic, and these need to be explored further.

Topics: Acute Disease; Animals; Antineoplastic Agents; Ceruletide; Cytokines; Disease Models, Animal; Humans; Mice, Inbred C57BL; NF-kappa B; p21-Activated Kinases; p38 Mitogen-Activated Protein Kinases; Pancreatitis; Pyridones; Pyrimidines; RNA Interference; Signal Transduction

Heme oxygenase-1 (HO-1) has an anti-inflammatory action in acute pancreatitis (AP). However, its mechanism of action and natural compounds/drugs to induce HO-1 in pancreas are not well understood. In this study, we investigated the regulatory mechanisms of HO-1 during AP using desoxo-narchinol-A (DN), the natural compound inducing HO-1 in the pancreas. Female C57/BL6 Mice were intraperitoneally injected with supramaximal concentrations of cerulein (50 μg/kg) hourly for 6 h to induce AP. DMSO or DN was administered intraperitoneally, then mice were sacrificed 6 h after the final cerulein injection. Administration of DN increased pancreatic HO-1 expression through activation of activating protein-1, mediated by mitogen-activated protein kinases. Furthermore, DN treatment reduced the pancreatic weight-to-body weight ratio as well as production of digestive enzymes and pro-inflammatory cytokines. Inhibition of HO-1 by tin protoporphyrin IX abolished the protective effects of DN on pancreatic damage. Additionally, DN treatment inhibited neutrophil infiltration into the pancreas via regulation of chemokine (C-X-C motif) ligand 2 (CXCL2) by HO-1. Our results suggest that DN is an effective inducer of HO-1 in the pancreas, and that HO-1 regulates neutrophil infiltration in AP via CXCL2 inhibition.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Chemokine CXCL2; Cytokines; Disease Models, Animal; Female; Heme Oxygenase-1; Humans; Inflammation Mediators; Mice; Mice, Inbred C57BL; Naphthols; Neutrophil Infiltration; Neutrophils; Pancreas; Pancreatitis

The Wilms' tumor suppressor gene (Wt1) encodes a zinc finger transcription factor that plays an essential role in the development of kidneys, gonads, spleen, adrenals and heart. Recent findings suggest that WT1 could also be playing physiological roles in adults. Systemic deletion of WT1 in mice provokes a severe deterioration of the exocrine pancreas, with mesothelial disruption, E-cadherin downregulation, disorganization of acinar architecture and accumulation of ascitic transudate. Despite this extensive damage, pancreatic stellate cells do not become activated and lose their canonical markers. We observed that pharmacological induction of pancreatitis in normal mice provokes de novo expression of WT1 in pancreatic stellate cells, concomitant with their activation. When pancreatitis was induced in mice after WT1 ablation, pancreatic stellate cells expressed WT1 and became activated, leading to a partial rescue of the acinar structure and the quiescent pancreatic stellate cell population after recovery from pancreatitis. We propose that WT1 modulates through the RALDH2/retinoic acid axis the restabilization of a part of the pancreatic stellate cell population and, indirectly, the repair of the pancreatic architecture, since quiescent pancreatic stellate cells are required for pancreas stability and repair. Thus, we suggest that WT1 plays novel and essential roles for the homeostasis of the adult pancreas and, through its upregulation in pancreatic stellate cells after a damage, for pancreatic regeneration. Due to the growing importance of the pancreatic stellate cells in physiological and pathophysiological conditions, these novel roles can be of translational relevance.

Topics: Aldehyde Oxidoreductases; Animals; Cell Lineage; Ceruletide; Disease Models, Animal; Epithelium; Gene Expression; Genes, Wilms Tumor; Homeostasis; Humans; Mice; Mice, Knockout; Mice, Transgenic; Pancreas; Pancreatic Stellate Cells; Pancreatitis; Regeneration; Repressor Proteins; Tissue Distribution; Translational Research, Biomedical; Tretinoin; WT1 Proteins

Acute pancreatitis (AP) is an exocrine dysfunction of the pancreas where oxidative stress and inflammatory cytokines play a key role in induction and progression of the disease. Studies have demonstrated that antioxidant phytochemicals have been effective in improving pancreatitis condition, but there are no clinically approved drugs till date. Our study aims to assess the preventive activity of visnagin, a novel phytochemical isolated from Ammi visnaga against cerulein induced AP. Male Swiss albino mice were divided into six groups (n = 6, each group) comprising of normal control, cerulein control, seven day pre-treatment with visnagin at three dose levels; visnagin low dose (10 mg/kg), visnagin mid dose (30 mg/kg), visnagin high dose (60 mg/kg) and visnagin control (60 mg/kg). AP was induced by six injections of cerulein (50 μg/kg, i.p.) on the 7

Topics: Acute Disease; Ammi; Amylases; Animals; Anti-Inflammatory Agents; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Khellin; Lipase; Male; Mice; Multiple Organ Failure; NF-E2-Related Factor 2; NF-kappa B; Pancreatitis; Signal Transduction

Chemerin, an adipokine, works as the chemoattractant for the immune cells. The role of chemerin in the inflammatory reaction is controversial. Chemerin has been shown to aggravate the inflammatory response, but other studies demonstrated its anti-inflammatory influence. This study assessed the effects of chemerin on acute pancreatitis (AP) in vivo and in vitro.. For in vivo experiments male Wistar rats were used. For in vitro study rat pancreatic AR42J cells were employed. Chemerin (1, 5 or 10 μg/kg) was given to the rats prior to the induction of AP by subcutaneous caerulein infusion (25 μg/kg). For in vitro studies cells were subjected to caerulein (10 nM) with or without chemerin (100 nM). Serum amylase activity was measured by enzymatic method, serum TNFα concentration - by ELISA kit. Western-blot was used to examine cellular proteins.. AP was confirmed by histological examination. Chemerin given to AP rats decreased histological manifestations of AP, reduced serum amylase activity and TNFα concentration. In AR42J cells subjected to caerulein with addition of chemerin signal for TNFα was reduced comparing to the cultures treated with caerulein alone. Analysis of the dynamics of nuclear translocation for p50, p65 and Bcl-3 points out to NF-κB attenuation as a mechanism of observed anti-inflammatory action of chemerin.. Chemerin significantly alleviated severity of AP in the rat, this is possibly due to the inhibition of pro-inflammatory signaling in the pancreatic cells.

Topics: Animals; Cell Line; Ceruletide; Chemokines; Dose-Response Relationship, Drug; Gene Expression Regulation; Intercellular Signaling Peptides and Proteins; Male; NF-kappa B; Pancreas; Pancreatitis; Rats; Rats, Wistar

Oxidative stress plays a major role in acute pancreatitis (AP), leading to massive macrophage infiltration. Nanoyttria (NY) possesses potent free radical scavenging activity. As reactive oxygen species and inflammation play major role in AP, we hypothesized that NY may alleviate cerulein induced AP. NY ameliorated LPS induced oxidative stress in vitro. It reduced ROS, superoxide radical generation and restored the mitochondrial membrane potential in macrophages. Interestingly, NY reduced plasma amylase and lipase levels and attenuated the mitochondrial stress and inflammatory markers. NY suppressed the recruitment of inflammatory cells around the damaged pancreatic acinar cells. Furthermore, NY intervention perturbed the course of AP via reduction of endoplasmic reticulum (ER) stress markers (BiP, IRE1 and Ero1-Lα), and molecular chaperones (Hsp27 and Hsp70). We, to the best of our knowledge, report for first time that NY can attenuate experimental AP by restoration of mitochondrial and ER homeostasis through Nrf2/NFκB pathway modulation.

Topics: Acute Disease; Animals; Antioxidants; Biomarkers; Ceruletide; Endoplasmic Reticulum Stress; Inflammation; Lipid Peroxidation; Lipopolysaccharides; Macrophages; Male; Membrane Potential, Mitochondrial; Mice; Nanoparticles; Neutrophils; Nitrosation; Oxidation-Reduction; Oxidative Stress; Pancreas; Pancreatitis; RAW 264.7 Cells; Reactive Oxygen Species; Severity of Illness Index; Transcription Factor RelA; Yttrium

This study was performed to study the in vitro and in vivo efficacy of hydroalcoholic extract of curry leaf (CLE) rich in carbazole alkaloids, against LPS-induced inflammation in Raw 264.7 macrophages and cerulein-induced acute pancreatitis, respectively. CLE was characterized by Fourier-transform infrared (FTIR) and liquid chromatography-mass spectrometry. Raw 264.7 cells were stimulated with LPS (2 μg/ml) and treated with CLE. The animals were treated with two doses of CLE (100 and 300 mg/kg). Plasma biochemistry, tissue lipid peroxidation, cytokines, and histological examination were evaluated. CLE was found to decently scavenge the activity of DPPH radical. It dose dependently suppressed nitrite production and oxidative stress in macrophages. CLE alleviated LPS-induced inflammation in macrophages as evident from the results of various inflammatory cytokines (IL-1β, IL-6, and TNF-α). In vivo, CLE reduced cerulein-induced pancreatic edema. CLE significantly abrogated the cerulein-induced lipid peroxidation, nitrite, MPO, and GSH levels. The inflammatory cytokines and p65-NFκB activity were significantly reduced by CLE. Mechanistically, CLE reduced the expression of NT, MPO, IL-1β, ICAM-1, and COX-2, and increased the expression of Nrf2. It reduced distant organ damage markers as well. We report for the first time that CLE holds substantial potential for the prevention of acute pancreatitis.

Topics: Animals; Ceruletide; Inflammation; Male; Mice; Murraya; Oxidative Stress; Pancreatitis; Plant Extracts; Plant Leaves

Topics: Acinar Cells; Animals; Autophagosomes; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Nucleus; Ceruletide; Disease Models, Animal; Humans; Inflammation; Lysosomes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pancreas; Pancreatitis; Phosphorylation; Proteasome Endopeptidase Complex; TOR Serine-Threonine Kinases

Acute pancreatitis (AP) is a common digestive disease characterized by inflammation of the pancreas. MiR-155 plays a role in promoting inflammation and inhibiting the activation of anti-inflammatory pathways. Impaired autophagy could promote zymogen activation, abnormal acinar cell secretion, cell death, and the inflammatory response to aggravate AP. The aim of this study was to ascertain the effect of silencing miR-155 on AP through its effects on inflammation and impaired autophagy in vivo. In this study, AAV(adeno-associated virus)-mediated miR-155 and miR-155 sponge were injected through the tail vein of mice. After 3 weeks, AP was induced by intraperitoneal (IP) injections of cerulein. Pancreatic and pulmonary tissues were analyzed after 24 h. Silencing of miR-155 ameliorated pancreas and lung damage in three AP models of mice by preventing accumulation of autophagosomes that are unable to fuse with lysosomes and decreasing pancreatic inflammation by targeting TAB2. 3-MA could reduce the aberrant accumulation of autophagosomes, which alleviates the pancreas damage that was aggravated by increasing miR-155 levels. These findings demonstrate that the inhibition of miR-155 holds promise for limiting pancreatitis.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagosomes; Autophagy; Ceruletide; Disease Models, Animal; Inflammation; Lung; Male; Mice; MicroRNAs; Pancreas; Pancreatitis; Prognosis

Acute pancreatitis (AP) is a multifactorial disease characterized by necroinflammatory changes of the pancreas. Our study is the first study which evaluated the relationship between the free radical production, enzymatic and nonenzymatic antioxidants, oxidative damage, and secretory function of the salivary glands of AP rats. Male Wistar rats were divided equally into 2 groups: control (

Topics: Acute Disease; Animals; Antioxidants; Ceruletide; Male; Oxidative Stress; Pancreatitis; Rats; Rats, Wistar; Salivary Glands

Dietary fat overload (typical to obesity) increases the risk of pancreatic pathologies through mechanisms yet to be defined. We previously showed that saturated dietary fat induces pancreatic acinar lipotoxicity and cellular stress. The endoplasmic reticulum (ER) of exocrine pancreas cells is highly developed and thus predisposed to stress. We studied the combination of saturated and unsaturated FAs in metabolic and pancreatitis like cerulein (CER)-induced stress states on cellular ER stress.Exocrine pancreas AR42J and rat primary exocrine acinar cells underwent acute (24 h) challenge with different FAs (saturated, monounsaturated) at different concentrations (250 and 500 µM) and in combination with acute CER-induced stress, and were analyzed for fat accumulation, ER stress unfolded protein response (UPR) and immune and enzyme markers. Acute exposure of AR42J and pancreatic acinar cells to different FAs and their combinations increased triglyceride accumulation. Palmitic acid significantly dose-dependently enhanced the UPR, immune factors and pancreatic lipase (PL) levels, as demonstrated by XBP1 splicing and elevation in UPR transcripts and protein levels (

Topics: Acinar Cells; Animals; Cell Line; Cells, Cultured; Ceruletide; Dietary Fats; Endoplasmic Reticulum Stress; Inflammation; Oleic Acid; Palmitic Acid; Pancreas, Exocrine; Pancreatitis; Rats; Rats, Sprague-Dawley

Acute pancreatitis (AP) is a common acute abdominal disease with local or systemic inflammatory response, caused by abnormal activation of digestive enzymes. Baicalein has been shown to exert anti-inflammatory effects and to attenuate the pathological changes of AP. The aim of the research was to investigate the effects of baicalein on caerulein induced pancreatitis, and to elucidate the putative underlying mechanism. In this study, the therapeutic potential of baicalein and its mechanism were investigated in a caerulein-induced AP in vivo and in vitro model. The results indicate that baicalein treatment alleviates the caerulein-induced pathological damage in the pancreas. Baicalein decreased the expression level of pro-inflammatory cytokines and chemokines of the pancreas in caerulein treated mice and of isolated pancreatic acinar cells. Moreover, baicalein inhibited the expression of NF-κB p65 and the phosphorylation of p38 MAPK, ERK (extracellular signal-regulated kinase) as well as STAT 3, which indicates that baicalein exerts its anti-inflammatory effects via dampening the NF-κB, MAPK and STAT 3 signaling pathways. Together, this study provides experimental evidence for the clinical application of Scutellaria baicalensis Georgi or baicalein and indicates that baicalein may be a promising candidate for treatment of AP patients in the future.

Topics: Acinar Cells; alpha-Amylases; Animals; Anti-Inflammatory Agents; Cell Survival; Ceruletide; Cytokines; Flavanones; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; NF-kappa B; Pancreas; Pancreatitis; Phytotherapy; RAW 264.7 Cells; STAT3 Transcription Factor

Endoplasmic reticulum (ER) stress in the pancreas is closely associated with the development of acute pancreatitis. However, the role of the protein kinase RNA-like ER kinase (PERK) in this disease is not fully understood. We investigated whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, could improve murine experimental pancreatitis through the amelioration of ER stress.. Acute pancreatitis was induced by the intraperitoneal administration of cerulein (50 μg/kg) six times at 1-h intervals followed by lipopolysaccharide (10 mg/kg). Salubrinal was administered intraperitoneally immediately after lipopolysaccharide injection and 3 h later. Mice were sacrificed 24 h after the first injection of cerulein, and serum amylase and proinflammatory cytokines were measured. The severity of pancreatitis was evaluated histologically using a scoring system. The expression levels of ER stress-related proteins were evaluated by Western blotting.. The administration of salubrinal significantly attenuated the increase in serum amylase levels and improved histologically assessed pancreatitis. The serum levels of proinflammatory cytokines were significantly suppressed in salubrinal-treated mice, as was the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein, and cleaved caspase-3.. The amelioration of ER stress through augmentation of the PERK-signaling pathway may be a therapeutic target for the treatment of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Ceruletide; Cinnamates; Cytokines; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Injections, Intraperitoneal; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Phosphorylation; Thiourea

The purpose of the present study was to investigate the effect of carbon monoxide (CO) released from CO‑releasing molecule 2 (CORM‑2) on mice with acute pancreatitis (AP). To perform the investigation, a mouse AP model was established using caerulein. The mice were treated with or without CORM‑2. The survival rate of the mice in the different groups was analyzed, and serum amylase and lipase levels were measured to assess the degree of pancreatic injury. The severity of AP was also evaluated by histological examination, and histopathological scoring of the pancreatic damage was performed. Pancreatic cell apoptosis was analyzed using a terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labelling assay. The function of the lung and liver was also assessed in the present study. Furthermore, the role of CORM‑2 on oxidative stress, intercellular adhesion molecule 1 (ICAM‑1) and vascular cell adhesion molecule 1 (VCAM‑1) expression, pro‑inflammatory cytokine production, and nuclear factor (NF)‑κB activation in the pancreas of AP mice was determined. The results demonstrated that CORM‑2 reduced the mortality, pancreatic damage, and lung and liver injury of AP mice. CORM‑2 administration also reduced systemic and localized inflammatory cell factors. Furthermore, treatment with CORM‑2 inhibited the expression of ICAM‑1 and VCAM‑1, and the activation of NF‑κB and phosphorylated inhibitor of NF‑κB subunit α, in the pancreas of AP mice. These results indicated that CO released from CORM‑2 exerted protective effects on AP mice, and the beneficial effects were likely due to inhibition of NF‑κB pathway activation.

Topics: Acute Disease; Amylases; Animals; Carbon Monoxide; Ceruletide; Cytokines; Disease Models, Animal; Intercellular Adhesion Molecule-1; Lipase; Lung; Male; Malondialdehyde; Mice; Mice, Inbred C57BL; NF-kappa B; Organometallic Compounds; Oxidative Stress; Pancreas; Pancreatitis; Peroxidase

Acute pancreatitis is a severe systemic disease triggered by a sterile inflammation and initial local tissue damage of the pancreas. Immune cells infiltrating into the pancreas are main mediators of acute pancreatitis pathogenesis. In addition to their antimicrobial potency, macrolides possess anti-inflammatory and immunomodulatory properties which are routinely used in patients with chronic airway infections and might also beneficial in the treatment of acute lung injury. We here tested the hypothesis that the macrolide antibiotic azithromycin can improve the course of acute experimental pancreatitis via ameliorating the damage imposed by sterile inflammation, and could be used as a disease specific therapy. However, our data show that azithromycin does not have influence on caerulein induced acute pancreatitis in terms of reduction of organ damage, and disease severity. Furthermore Infiltration of immune cells into the pancreas or the lungs was not attenuated by azithromycin as compared to controls or ampicillin treated animals with acute experimental pancreatitis. We conclude that in the chosen model, azithromycin does not have any beneficial effects and that its immunomodulatory properties cannot be used to decrease disease severity in the model of caerulein-induced pancreatitis in mice.

Topics: Animals; Anti-Bacterial Agents; Azithromycin; Ceruletide; Disease Models, Animal; Female; Lung; Mice; Pancreas; Pancreatitis; Severity of Illness Index; Treatment Outcome

Because little is known about the role of corticotropin-releasing factor (CRF) agonists in regulating responses in pancreatitis, we evaluated the effects of urocortin 2 (UCN2) and stressin1 in caerulein-induced acute pancreatitis (AP) model in rats. Male rats were pretreated with UCN2 or stressin1 for 30 min followed by induction of AP with supraphysiologic doses of caerulein. Serum amylase and lipase activity, pancreatic tissue necrosis, immune cell infiltrate, nuclear factor (NF)-κB activity, trypsin levels, and intracellular Ca2+ ([Ca2+]i) were ascertained. UCN2, but not stressin1 attenuated the severity of AP in rats. UCN2, but not stressin1, reduced serum amylase and lipase activity, cell necrosis and inflammatory cell infiltration in AP. NF-κB activity in pancreatic nuclear extracts increased in AP and UCN2 treatment reduced caerulein-induced increases in NF-κB activity by 42%. UCN2 treatment prevented caerulein-induced degradation of IκB-α in the cytosolic fraction as well as increased levels of p65 subunit of NF-κB in the cytosolic fraction. Pancreatic UCN2 levels decreased in AP compared with saline. UCN2 evoked [Ca2+]i responses in primary acinar cells and abolished caerulein-evoked [Ca2+]i responses at 0.1nM, and decreased by ~50% at 1.0nM caerulein. UCN2 stimulation resulted in redistribution of a portion of F-actin from the apical to the basolateral pole. UCN2 prevented the massive redistribution of F-actin observed with supraphysiologic doses of caerulein. UCN2, but not stressin1 attenuated severity of an experimental pancreatitis model. The protective effects of UCN2, including anti-inflammatory and anti-necrotic effects involve activation of the CRF2 receptor, [Ca2+]i signaling, and inhibition of NF-κB activity.

Topics: Acinar Cells; Animals; Ceruletide; Corticotropin-Releasing Hormone; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Pancreatitis; Rats; Rats, Sprague-Dawley; Signal Transduction; Urocortins

To investigate the therapeutic effects of resolvin D1 (RvD1) on cerulein and lipopolysaccharide (LPS)-induced severe acute pancreatitis in mice.. The model of severe acute pancreatitis was induced by cerulein combined with LPS in mice. Mice were treated with RvD1 at a dose of 150 mg/kg for 4 h after the last injection of cerulein. Pathological changes and scores were assessed by HE staining, serum amylase and lipase levels were detected by ELISA, serum tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) levels were determined by Luminex Assay.. Cerulein combined with LPS successfully induced severe acute pancreatitis model in mice. RvD1 reduced the pathological changes of pancreas in severe acute pancreatitis mice, decreased the serum levels of amylase and lipase, as well as attenuated the serum levels of TNF-α and IL-6.. RvD1 can reduce the severity of severe acute pancreatitis induced by cerulein and LPS in mice.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Docosahexaenoic Acids; Interleukin-6; Lipase; Lipopolysaccharides; Mice; Pancreas; Pancreatitis; Tumor Necrosis Factor-alpha

To investigate the apoptosis and inflammatory response of microRNA-27a-5p (miR-27a-5p) in pancreatic acinar cells of acute pancreatitis (AP) and its related mechanisms. Rat pancreatic acinar cell line AR42J was treated with caerulein (10nmol/L) to construct an acute pancreatitis cell model. Quantitative real-time polymerase chain reaction was performed to measure the expression of miR-27a-5p; The miR-27a-5p mimic was transfected into cell, and the apoptosis rate of the cells was detected by flow cytometry; The levels of TNF-α, IL-1, and IL-6 in the culture supernatant were determined by enzyme-linked immunosorbent assay; TargetScans database predicted and dual luciferase reporter gene assay verified the relationship between miR-27a-5p and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN); The recovery experiment explored the apoptosis and the effects of inflammatory responses. The expression of miR-27a-5p decreased gradually (P < 0.05) and the expression of PTEN increased gradually (P < 0.05) with the prolongation of acting time. Upregulation of miR-27a-5p significantly promoted cell apoptosis (P < 0.05) and inhibited inflammatory response (P < 0.05); The TargetScans database predicted that the 3'UTR of PTEN contains a base complementary to the miR-27a-5p seed region. Cotransfection of wild-type vector (PTEN-WT) with miR-27a-5p mimic or miR-27a-5p inhibitor significantly affected the relative activity of luciferase (P < 0.05), and no significant impact was observed in mutant PTEN-MUT. Compared with miR-27a-5p + pcDNA group, transfection of miR-27a-5p mimic and pcDNA-PTEN significantly increased the expression of PTEN (P < 0.05), decreased the apoptotic rate (P < 0.05), and increased the inflammatory response (P < 0.05). miR-27a-5p induced apoptosis and inhibited the inflammatory response of pancreatic acinar cells in AP by targeting PTEN.

Topics: Acinar Cells; Animals; Apoptosis; Cell Line; Cell Survival; Ceruletide; Gene Expression Regulation; Interleukin-1; Interleukin-6; MicroRNAs; Pancreatitis; PTEN Phosphohydrolase; Rats; Tumor Necrosis Factor-alpha; Up-Regulation

Acute pancreatitis (AP) is a type of sterile inflammation of the pancreas, potentially leading to systemic inflammatory response syndrome or multiple organ failure. An emerging evidence that dysfunction of miRNA expression may alter pivotal physiological functions and lead to inflammation infiltration and complication of multiple diseases, including AP. Here, the AP model was successfully replicated using cerulein in vitro and in vivo. RT-qPCR was used to detect low expression of miR-148a in AP. This study verified that IL-6 was a direct target of miR-148a. Over-expression of miR-148a decreased the mRNA and protein levels of IL-6 by RT-qPCR and Elisa. Moreover, over-expression of miR-148a improved the pathological state of AP through H&E and MPO staining and transmission electron microscopy. After over-expressing miR-148a, Western blot and immunohistochemical method were used to confirm the reduction of autophagosomes and autolysosomes, blockade of the levels of p-STAT3, LC3-II, ATG7, ATG4c, Beclin1 and the increased p62 expression in AP. The expression of LAMP-2 was not significantly different. In addition, IL-6 and AG490, the IL-6/STAT3 signaling inhibitor, were used to verify the role of IL-6/STAT3 signaling in the regulation of miR-148a on autophagy in cerulein-induced AP in vitro and in vivo. Taken together, our findings indicate that miR-148a suppresses autophagy via regulating IL-6/STAT3 signaling in cerulein-induced AP in vitro and in vivo. The miR-148a appears to be a promising candidate for the gene therapy of AP.

Topics: Acute Disease; Animals; Autophagy; Cell Line; Ceruletide; Down-Regulation; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Interleukin-6; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Pancreatitis; Rats; Signal Transduction; STAT3 Transcription Factor

BACKGROUND Acute pancreatitis is an inflammatory disease of the pancreas associated with high patient morbidity. Lycium barbarum polysaccharide (LBP), a traditional Chinese medicine with an active component extracted from the goji berry, has previously been reported to have anti-inflammatory effects. This study aimed to investigate the effects of LBP in a mouse model of cerulein-induced acute pancreatitis. MATERIAL AND METHODS Acute pancreatitis was induced by intraperitoneal injection of cerulein in C57BL/6 wild-type mice or nuclear factor erythroid-2-related factor 2 (NRF2) gene knockout mice. LBP or normal saline was administrated by gavage once daily for one week before the induction of acute pancreatitis. At 12 hours after the first intraperitoneal injection of cerulein, the mice were euthanized. Blood and pancreatic tissue were sampled for histology and for the measurement of pro-inflammatory cytokines, serum amylase, and lipase. RESULTS In the untreated mouse model of cerulein-induced acute pancreatitis, amylase and lipase levels were increased, and these levels were reduced by LBP treatment when compared with vehicle treatment. In the untreated mouse model, histology of the pancreas showed edema and inflammation, which were reduced in the LBP-treated mice. In the untreated mouse model, increased levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were found, which were reduced in the LBP-treated mice. NRF2 gene knockout mice with cerulein-induced acute pancreatitis showed reduced anti-inflammatory effects of LBP treatment. LBP increased the expression of NRF2 and heme oxygenase-1 (HO-1). CONCLUSIONS In a mouse model of cerulein-induced acute pancreatitis, LBP reduced inflammation by upregulating NRF2 and HO-1.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Cytokines; Disease Models, Animal; Drugs, Chinese Herbal; Heme Oxygenase-1; Interleukin-6; Lipase; Medicine, Chinese Traditional; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; NF-kappa B; Pancreas; Pancreatitis; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) is a common and devastating inflammatory disorder of the pancreas. However, there are still no effective treatments available for the disease. Therefore, it is important to discover new therapeutic targets and strategies for better treatment and prognosis of AP patients. Toll-like receptor 3 (TLR3) ligand polyI:C is a double-stranded RNA mimic that can be used as an immune stimulant. Our current study indicates that polyI:C exerted excellent anti-inflammatory effects in a caerulein-induced AP mouse model and taurocholate-induced pancreatic acinar cell line injury model. We found that polyI:C triggers type I interferon (IFN) production and downstream IFN-α/β receptor (IFNAR)-dependent signaling, which play key roles in protecting the pancreas from inflammatory injury. Knockout of IFN-β and IFNAR in mice abolished the preventive effects of polyI:C on caerulein-induced AP symptoms, which include pancreatic edema, neutrophil infiltration, the accumulation of reactive oxygen species (ROS), and inflammatory gene expression. Treating pancreatic acinar 266-6 cells with an IFNAR inhibitor, which blocks the interaction between type I IFN and IFNAR, diminishes the downregulation of oxidative stress by polyI:C. Additionally, a subsequent transcriptome analysis on the role of polyI:C in treating pancreatitis suggested that chemotaxis of neutrophils and the production of ROS were inhibited by polyI:C in the pancreases damaged by caerulein injection. Thus, polyI:C may act as a type I IFN inducer to alleviate AP, and it has the potential to be a promising therapeutic agent used at the early stages of AP.

Topics: Animals; Cell Line; Ceruletide; Disease Models, Animal; Interferon-beta; Ligands; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Oxidative Stress; Pancreas; Pancreatitis; Poly I-C; Reactive Oxygen Species; Receptors, Interferon; Signal Transduction; Toll-Like Receptor 3

Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast to trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC due to a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the novel Ctrc+ strain the severity of cerulein-induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. Taken together with prior human genetic and biochemical studies, the observations provide conclusive evidence for the protective role of CTRC against pancreatitis.

Topics: Animals; Ceruletide; Chymotrypsin; Disease Models, Animal; Exons; Genetic Predisposition to Disease; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Pancreatitis; Secretagogues; Severity of Illness Index; Species Specificity

Intracellular Ca

Topics: Acinar Cells; Animals; Calcium Signaling; Cell Line; Ceruletide; Docosahexaenoic Acids; Gene Expression Profiling; Gene Expression Regulation; Inositol 1,4,5-Trisphosphate Receptors; Pancreas, Exocrine; Pancreatitis; Proto-Oncogene Proteins c-fos; Rats; Ryanodine Receptor Calcium Release Channel; Sequence Analysis, RNA; Transcription Factor RelB

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cerium; Ceruletide; Inflammation; Macrophages; Male; Mice; Oxidative Stress; Pancreatitis; RAW 264.7 Cells; Superoxide Dismutase

Acute pancreatitis (AP) is a common acute abdominal condition, frequently associated with intestinal barrier dysfunction, which aggravates AP retroactively. Butyrate exhibits anti-inflammatory effects in a variety of inflammatory diseases. However, its potential beneficial effect on AP and the underlying mechanisms have not been investigated.. Experimental AP was induced by caerulein hyperstimulation in wild-type and GPR109A. Butyrate prophylaxis attenuated AP as shown by reduced serum amylase and lipase levels, pancreatic oedema, myeloperoxidase activity, and improved pancreatic morphology. Amelioration of pancreatic damage by butyrate was associated with reduced levels of TNF-α, IL-6, and CCL2 and suppressed activation of the NLRP3 inflammasome in both pancreas and colon. Further, butyrate ameliorated pancreatic inflammation by suppressing interactions between histone deacetylase 1 (HDAC1) and AP1 and STAT1 with increased histone acetylation at H3K9, H3K14, H3K18, and H3K27 loci, resulting in suppression of NLRP3 inflammasome activation and modulation of immune cell infiltration in pancreas. Additionally, butyrate mediated STAT1/AP1-NLRP3 inflammasome suppression via HDAC1 inhibition was demonstrated in peritoneal macrophage. In colon, butyrate inhibited NLRP3 inflammasome activation via GPR109A. Accordingly, the modulatory effects of butyrate on AP, AP-associated gut dysfunction, and NLRP3 inflammasome activation were diminished in GPR109A. Our study dissected tissue-specific anti-inflammatory mechanisms of butyrate during AP, suggesting that increased colonic levels of butyrate may be a strategy to protect against AP.

Topics: Acute Disease; Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Butyrates; Ceruletide; Female; Intestinal Diseases; Intestine, Small; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis

Nonsteroidal anti-inflammatory drugs (NSAIDs) are administered to manage the pain typically found in patients suffering from pancreatitis. NSAIDs also display anti-proliferative activity against cancer cells; however, their effects on normal, untransformed cells are poorly understood. Here, we evaluated whether NSAIDs inhibit the proliferation of pancreatic acinar cells during the development of acute pancreatitis.. The NSAIDs ibuprofen and diclofenac were administered to C57BL/6 mice after induction of pancreatitis with serial injections of cerulein. In addition, ibuprofen was administered concomitantly with 3,5,3-L-tri-iodothyronine (T3), which induces acinar cell proliferation in the absence of tissue inflammation. The development of pancreatic inflammation, acinar de-differentiation into metaplastic lesions and acinar proliferation were quantified by histochemical, biochemical and RT-PCR approaches.. Therapeutic ibuprofen treatment selectively reduced pancreatic infiltration of activated macrophages in vivo, and M1 macrophage polarization and pro-inflammatory cytokine expression both in vivo and in vitro. Reduced macrophage activation was accompanied by reduced acinar de-differentiation into acinar-to-ductal metaplasia. Acinar proliferation was significantly impaired in the presence of ibuprofen and diclofenac, as demonstrated at both the level of proliferation markers and expression of cell cycle regulators. Ibuprofen also reduced acinar cell proliferation induced by mitogenic stimulation with T3, a treatment that does not elicit pancreatic inflammation.. Our study provides evidence that the NSAIDs ibuprofen and diclofenac inhibit pancreatic acinar cell division. This suggests that prolonged treatment with these NSAIDs may negatively affect the regeneration of the pancreas and further studies are needed to confirm these findings in a clinical setting.. This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Topics: Acinar Cells; Animals; Cell Differentiation; Cell Proliferation; Ceruletide; Cytokines; Diclofenac; Ibuprofen; Inflammation; Male; Mice; Mitogens; Neutrophil Infiltration; Pancreas; Pancreatitis; Triiodothyronine

Opioids such as morphine are widely used for the management of pain associated with acute pancreatitis. Interestingly, opioids are also known to affect the immune system and modulate inflammatory pathways in non-pancreatic diseases. However, the impact of morphine on the progression of acute pancreatitis has never been evaluated. In the current study, we evaluated the impact of morphine on the progression and severity of acute pancreatitis.. Effect of morphine treatment on acute pancreatitis in caerulein, L-arginine and ethanol-palmitoleic acid models was evaluated after induction of the disease. Inflammatory response, gut permeability and bacterial translocation were compared. Experiments were repeated in mu (µ) opioid receptor knockout mice (MORKO) and in wild-type mice in the presence of opioid receptor antagonist naltrexone to evaluate the role of µ-opioid receptors in morphine's effect on acute pancreatitis. Effect of morphine treatment on pathways activated during pancreatic regeneration like sonic Hedgehog and activation of embryonic transcription factors like pdx-1 and ptf-1 were measured by immunofluorescence and quantitative PCR.. Histological data show that treatment with morphine after induction of acute pancreatitis exacerbates the disease with increased pancreatic neutrophilic infiltration and necrosis in all three models of acute pancreatitis. Morphine also exacerbated acute pancreatitis-induced gut permeabilisation and bacteraemia. These effects were antagonised in the MORKO mice or in the presence of naltrexone suggesting that morphine's effect on severity of acute pancreatitis are mediated through the µ-opioid receptors. Morphine treatment delayed macrophage infiltration, sonic Hedgehog pathway activation and expression of pdx-1 and ptf-1.. Morphine treatment worsens the severity of acute pancreatitis and delays resolution and regeneration. Considering our results, the safety of morphine for analgesia during acute pancreatitis should be re-evaluated in future human studies.

Topics: Acute Disease; Analgesics, Opioid; Animals; Arginine; Ceruletide; Disease Models, Animal; Disease Progression; Fatty Acids, Monounsaturated; Mice; Mice, Knockout; Morphine; Pancreas; Pancreatitis; Severity of Illness Index; Time Factors

Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca. Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence.. Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca. In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.

Topics: Acute Disease; Animals; Arginine; Autophagy; Bile Acids and Salts; Calcium Signaling; Ceruletide; Choline Deficiency; Cyclophilins; Disease Models, Animal; Endoplasmic Reticulum Stress; Ethionine; Genetic Predisposition to Disease; Humans; Lipid Metabolism; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Proton-Translocating ATPases; Pancreas; Pancreatitis; Peptidyl-Prolyl Isomerase F; Phenotype; Rats; Time Factors; Trehalose

Heat shock factor 1 (HSF1), an important transcriptional molecule in the heat shock process, can regulate the expression of a lot of inflammatory mediators in addition to heat shock proteins. This study evaluated the inhibitive function of HSF1 on the expression of suppressor of cytokine signaling 3 in cerulein-induced acute pancreatitis.. After HSF1 mice, HSF1 mice, and AR42J cells were treated with cerulein, histopathological score, expression of SOCS3 mRNA, and protein levels were analyzed by using RT-PCR, quantitative real-time RT-PCR, and western blotting, respectively. DNA binding and transcription activity of HSF1 to the SOCS3 promoter were detected by chromatin immunoprecipitation and luciferase reporter assays.. The histopathological scores of the pancreas decreased significantly in the cerulein-induced HSF1 mice compared with the cerulein-induced HSF1 mice. SOCS3 mRNA and protein level decreased in the pancreas of the unstimulated HSF1 and HSF1 mice, whereas increased in the pancreas of the cerulein-induced HSF1 and HSF1 mice, with higher in the pancreas of cerulein-induced HSF1mice. In the pcDNA3.1-transfected AR42J cells, SOCS3 protein decreased and was upregulated after the cerulein stimulation, whereas HSF1 overexpression inhibited the upregulation. In the scramble-transfected AR42J cells, SOCS3 protein decreased and was upregulated after the cerulein stimulation, whereas HSF1-RNAi further promoted the upregulation. EMSA and chromatin immunoprecipition showed that HSF1 could directly bind to SOCS3 promoter region. Reporter assays showed that HSF1 could inhibit the transcriptional activity on SOCS3 promoter.. HSF1 can protect AR42J cells from cerulein-induced pancreatitis through inhibiting the expression of SOCS3.

Topics: Animals; Blotting, Western; Ceruletide; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Heat Shock Transcription Factors; Heat-Shock Proteins; Mice; Mice, Mutant Strains; Pancreas; Pancreatitis; Plasmids; Promoter Regions, Genetic; Protein Binding; Real-Time Polymerase Chain Reaction; Suppressor of Cytokine Signaling 3 Protein

Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP).. Changes in mitochondrial membrane potential (Δψm), cytosolic Ca ([Ca]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology.. TRO40303 prevented loss of Δψm and necrosis induced by 100 μM palmitoleic acid ethyl ester or 500 μM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP.. TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP.

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Esters; Fatty Acids; Humans; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria; Necrosis; Oximes; Pancreatitis; Pancreatitis, Alcoholic; Secosteroids; Taurolithocholic Acid

Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18c's functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c

Topics: Aged; Animals; Ceruletide; Cholecystokinin; Endoplasmic Reticulum; Exocytosis; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Munc18 Proteins; Pancreas; Pancreatitis; SNARE Proteins

Clinical studies have revealed that some patients will develop glucose tolerance dysfunction after recovering from acute pancreatitis (AP), which indicated the importance of investigating the potential therapies for restoration of islet β cell function. Cytokeratin 5 (Krt5)-positive cells are considered to function as stem or progenitor cells in the regeneration of lung and salivary gland following injury. In the present study, AP was induced by six hourly intraperitoneal injections of 100 μg/kg cerulein for 4 consecutive days in adult mice, in order to determine the role of Krt5-positive cells in pancreatic regeneration, especially in the restoration of β cell function and the underlying mechanisms. Results showed that glucose homeostasis were deteriorated partly during the recovery process after AP. Furthermore, clusters of Krt5-positive cells were significantly increased in the damaged pancreas marked by inflammatory cells infiltration and acinar cell eradication. In addition, cells co-labelling insulin and Krt5 were found in the injured region after cerulein administration, part of these cells were immunopositive for GLUT2. Taken together, our data demonstrated that Krt5-expressing cells could be involved in the natural pancreas self-healing process and the renewal of β cells after AP in adult mice. It is promising that promoting conversion of Krt5-expressing cells into functional β cells may be a novel method to mitigate the development of diabetes mellitus after AP in vivo.

Topics: Animals; Cell Differentiation; Cells, Cultured; Ceruletide; Female; Insulin-Secreting Cells; Keratin-5; Male; Mice; Mice, Inbred C57BL; Pancreatitis

A various of pharmacological effects of astaxanthin has been confirmed. However, the mechanism underlying protective effect of astaxanthin on acute pancreatitis (AP) induced by cerulein still unclear. The present study is to investigate the mechanism underlying the effect of astaxanthin on autophagy and apoptosis via the JAK/STAT3 pathway.. Intraperitoneal injection of cerulein at hourly intervals followed by lipopolysaccharide injection were used in Balb/C mice. Vehicle or astaxanthin, which intraperitoneal injected in two doses (20 mg/kg and 40 mg/kg), were injected in mice 1 h before the first cerulein injection. At 3 h after the last injection, when the pathological changes were most severe, pancreatic tissue was analyzed by pathologically scored and hematoxylin and eosin (H&E) staining. The severity of AP was assessed by histological grading, proinflammatory cytokine levels, biochemistry, myeloperoxidase (MPO) activity, and analysis of JAK/STAT3 activity.. Astaxanthin administration markedly reduced serum digestive enzyme activities, pancreatic histological scores, proinflammatory cytokine levels (tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), and Interleukin-6 (IL-6)), MPO and JAK/STAT3 activity.. Collectively, these results indicate that astaxanthin inhibits pancreatic injury in AP by targeting JAK/STAT3-mediated apoptosis and autophagy.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Apoptosis; Autophagy; Ceruletide; Cytokines; Disease Models, Animal; Humans; Inflammation Mediators; Janus Kinases; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Pancreas; Pancreatitis; Peroxidase; STAT3 Transcription Factor; Xanthophylls

Acute pancreatitis is a multifactorial disease associated with profound changes of the pancreas induced by release of digestive enzymes that lead to increase in proinflammatory cytokine production, excessive tissue necrosis, edema, and bleeding. Elevated levels of hepatocyte growth factor (HGF) and its receptor c-Met have been observed in different chronic and acute pancreatic diseases including experimental models of acute pancreatitis. In the present study, we investigated the protective effects induced by the recombinant human HGF in a mouse model of cerulein-induced acute pancreatitis. Pancreatitis was induced by 8 hourly administrations of supramaximal cerulein injections (50 µg/kg, ip). HGF treatment (20 µg/kg, iv), significantly attenuated lipase content and amylase activity in serum as well as the degree inflammation and edema overall leading to less severe histologic changes such as necrosis, induced by cerulein. Protective effects of HGF were associated with activation of pro-survival pathways such as Akt, Erk1/2, and Nrf2 and increase in executor survival-related proteins and decrease in pro-apoptotic proteins. In addition, ROS content and lipid peroxidation were diminished, and glutathione synthesis increased in pancreas. Systemic protection was observed by lung histology. In conclusion, our data indicate that HGF exerts an Nrf2 and glutathione-mediated protective effect on acute pancreatitis reflected by a reduction in inflammation, edema, and oxidative stress.

Topics: Animals; Antioxidants; Apoptosis; Ceruletide; Disease Models, Animal; Glutathione; Hepatocyte Growth Factor; Humans; Male; Mice; Oxidative Stress; Pancreatitis; Protective Agents; Proto-Oncogene Proteins c-met; Recombinant Proteins; Signal Transduction; Survival Analysis

Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy.. We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-μm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2. Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen.. In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

Topics: Acinar Cells; Animals; Autophagy; Cell Membrane; Ceruletide; Exocytosis; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatic Neoplasms; Pancreatitis; Secretory Vesicles; Syntaxin 1; Trypsinogen

It is well known that pancreatic recovery after a single episode of injury such as an isolated bout of pancreatitis occurs rapidly. It is unclear, however, what changes are inflicted in such conditions to the molecular landscape of the pancreas. In the caerulein hyperstimulation model of pancreatitis, the murine pancreas has the ability to recover within one week based on histological appearance. In this study, we sought to characterize by RNA-sequencing (RNA-seq) the transcriptional profile of the recovering pancreas up to two weeks post-injury. We found that one week after injury there were 319 differentially expressed genes (DEGs) compared with baseline and that after two weeks there were 53 DEGs. Forty (12.5%) of the DEGs persisted from week one to week two, and another 13 DEGs newly emerged in the second week. Amongst the top up-regulated DEGs were several trypsinogen genes (trypsinogen 4, 5, 12, 15, and 16). To our knowledge, this is the first characterization of the transcriptome during pancreatic recovery by deep sequencing, and it reveals on a molecular basis that there is an ongoing recovery of the pancreas even after apparent histological resolution. The findings also raise the possibility of an emerging novel transcriptome upon pancreatic recovery.

Topics: Animals; Ceruletide; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation; Gene Regulatory Networks; Humans; Mice; Pancreatitis; Regeneration; Sequence Analysis, RNA

We investigated whether intrapancreatic coagulation, with deposition of the fibrinogen-γ dimer (Fib-γD) and hypoxia, affect the severity of acute pancreatitis (AP) in mice. Pancreata of mice with AP induced by administration of cerulein or by L-arginine, or from patients with pancreatitis, had increased deposition of Fib-γD compared with control pancreata. Heparin administration protected mice from cerulein-induced AP and prevented Fib-γD formation. Cerulein administration resulted in activation and stabilization of hypoxia-inducible factor-1α (HIF1α) in pancreata of oxygen-dependent degradation domain-luciferase HIF1α reporter mice. Cerulein also led to induction of genes regulated by HIF1α, including Vegfa and Ero1a, before evidence of Fib-γD deposition or histologic features of AP. Expression of tissue factor, which is regulated by vascular endothelial growth factor, also increased following cerulein administration. Mice with acinar cell-specific disruption of Hif1a (Hif1a

Topics: Acinar Cells; Acute Disease; Animals; Arginine; Ceruletide; Disease Models, Animal; Homeostasis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Pancreas; Pancreas, Exocrine; Pancreatitis; Vascular Endothelial Growth Factor A

Acute pancreatitis (AP) is characterized by severe inflammation and acinar cell death. Transmembrane protein 173 (TMEM173 or STING) is a DNA sensor adaptor protein on immune cells that recognizes cytosolic nucleic acids and transmits signals that activate production of interferons and the innate immune response. We investigated whether leukocyte STING signaling mediates inflammation in mice with AP.. We induced AP in C57BL/6J mice (control) and C57BL/6J-Tmem173gt/J mice (STING-knockout mice) by injection of cerulein or placement on choline-deficient DL-ethionine supplemented diet. In some mice, STING signaling was induced by administration of a pharmacologic agonist. AP was also induced in C57BL/6J mice with bone marrow transplants from control or STING-knockout mice and in mice with disruption of the cyclic GMP-AMP synthase (Cgas) gene. Pancreata were collected, analyzed by histology, and acini were isolated and analyzed by flow cytometry, quantitative polymerase chain reaction, immunoblots, and enzyme-linked immunosorbent assay. Bone-marrow-derived macrophages were collected from mice and tested for their ability to detect DNA from dying acinar cells in the presence and absence of deoxyribonuclease (DNaseI).. STING signaling was activated in pancreata from mice with AP but not mice without AP. STING-knockout mice developed less severe AP (less edema, inflammation, and markers of pancreatic injury) than control mice, whereas mice given a STING agonist developed more severe AP than controls. In immune cells collected from pancreata, STING was expressed predominantly in macrophages. Levels of cGAS were increased in mice with vs without AP, and cGAS-knockout mice had decreased edema, inflammation, and other markers of pancreatic injury upon induction of AP than control mice. Wild-type mice given bone marrow transplants from STING-knockout mice had less pancreatic injury and lower serum levels of lipase and pancreatic trypsin activity following induction of AP than mice given wild-type bone marrow. DNA from dying acinar cells activated STING signaling in macrophages, which was inhibited by addition of DNaseI.. In mice with AP, STING senses acinar cell death (by detecting DNA from dying acinar cells) and activates a signaling pathway that promotes inflammation. Macrophages express STING and activate pancreatic inflammation in AP.

Topics: Acinar Cells; Acute Disease; Animals; Cell Death; Ceruletide; Disease Models, Animal; Inflammation; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nucleotides, Cyclic; Pancreas; Pancreatitis; Signal Transduction

Changes in the protein expression occurring within the initiation phase of acute pancreatitis (AP) might be vital in the development of this complex disease. However, the exact mechanisms involved in the onset of AP remains elusive and most of our knowledge about the pathobiology of AP comes from animal models. We performed in a rat pancreatitic model a high-throughput shotgun proteomic profiling of the soluble and whole membrane fractions from the pancreas during the early phase of cerulein (Cer)-induced AP. We identified 997 proteins, of which 353 were significantly different (22, 276 or 55 in both, the soluble or the membrane fractions, respectively). Gene Ontology and KEGG PATHWAY analyses revealed that these proteins were implicated in molecular mechanisms relevant to AP pathogenesis, including vesicle-mediated and protein transport, lysosomal and mitochondrial impairment or proteolysis. Numerous metabolic processes were downregulated apparently to reduce energy consumption, and a remarkable increase in inflammatory and stress responses was also highlighted. The proteomic data were verified by immunoblotting of 11 and 7 different soluble or membrane-associated proteins, either novel (VPS29 and MCTS1) or known factors in AP. Also, our first observation of the imbalance of some COP proteins during AP early phase deserves further characterization.. AP is one of the most important pathological inflammatory states of the exocrine pancreas but its pathophysiology remains incompletely understood, especially the early acinar events. Proteomic analysis of pancreatic subcellular fractions simplifies protein maps and helps in the identification of new protein alterations and biomarkers characterizing pancreatic tissue damage. Our shotgun approach has not been previously used to profile the early proteomic alterations of the disease, which are considered crucial for its development and for the founding of clinical procedures. Furthermore, our subcellular fractionation protocol allowed us to detect changes in membrane proteins so far overlooked in the proteomic study of AP. Accordingly, using TMT proteomics and bioinformatic tools, we were able to detect significant changes in protein expression related to many pathobiological pathways of acute pancreatitis as from the early phase of the disease. To our knowledge, some of these changes, such as the imbalance of some COP proteins, have never been described in this disease.

Topics: Acute Disease; Animals; Ceruletide; Lysosomes; Male; Mitochondria; Mitochondrial Proteins; Pancreatitis; Proteome; Proteomics; Rats; Rats, Wistar

Acute pancreatitis (AP) is one common clinical acute abdominal disease, for which specific pharmacological or nutritional therapies remain elusive. Lactose, a macronutrient and an inducer of host innate immune responses, possesses immune modulatory functions. The current study aimed to investigate potential modulatory effects of lactose and the interplay between the nutrient and pancreatic immunity during experimentally induced AP in mice. We found that either prophylactic or therapeutic treatment of lactose time-dependently reduced the severity of AP, as evidenced by reduced pancreatic edema, serum amylase levels, and pancreatic myeloperoxidase activities, as well as by histological examination of pancreatic damage. Overall, lactose promoted a regulatory cytokine milieu in the pancreas and reduced infiltration of inflammatory neutrophils and macrophages. On acinar cells, lactose was able to suppress caerulein-induced inflammatory signaling pathways and to suppress chemoattractant tumor necrosis factor (TNF)-α and monocyte chemotactic protein-1 production. Additionally, lactose acted on pancreas-infiltrated macrophages, increasing interleukin-10 and decreasing tumor necrosis factor alpha production. Notably, lactose treatment reversed AP-associated infiltration of activated neutrophils. Last, the effect of lactose on neutrophil infiltration was mimicked by a galectin-3 antagonist, suggesting a potential endogenous target of lactose. Together, the current study demonstrates an immune regulatory effect of lactose to alleviate AP and suggests its potential as a convenient, value-added therapeutic macronutrient to control AP, and lower the risk of its systemic complications.

Topics: Acute Disease; Animals; Ceruletide; Cytokines; Female; Immunologic Factors; Lactose; Macrophages; Mice, Inbred BALB C; Neutrophil Infiltration; Neutrophils; Pancreas; Pancreatitis; Phenotype

BACKGROUND Steroid receptor coactivator-interacting protein (SIP) inhibits the activation of nuclear factor-kappa B (NF-κB) by interacting with p65. The occurrence of acute pancreatitis (AP) is closely associated with pro-inflammatory response. The present study aimed to investigate the role of SIP on myocardial injury caused by AP. MATERIAL AND METHODS Rat pancreatic acinar tumor cell line AR42J cells were treated with caerulein to establish AP cell models. The levels of TNF-α, IL-6, cTnI, CK-MB, and LDH1 were detected by ELISA assay. The mRNA and protein expression levels of SIP, p-p65, and p65 were detected by qRT-PCR and western blot analysis, respectively. Next, the AP cell models were non-transfected or transfected with SIP plasmids or SIP siRNA. ELISA assay was also performed to test the levels of TNF-α, IL-6, cTnI, CK-MB, and LDH1. Moreover, qRT-PCR and western blot analysis were performed to measure the mRNA and protein expression levels of SIP, p-p65, and p65, respectively. RESULTS Caerulein upregulated the levels of TNF-α, IL-6, cTnI, CK-MB, and LDH1. These upregulations were reduced by SIP plasmids and promoted by SIP siRNA, respectively. Caerulein also increased the mRNA and protein expression levels of p-p65. However, the increases were attenuated by SIP plasmids and enhanced by SIP siRNA, respectively. CONCLUSIONS In conclusion, the results suggested that SIP may inhibit the inflammatory response by deactivating p65, thus reducing the myocardial damage caused by AP.

Topics: Acute Disease; Animals; Cell Line, Tumor; Ceruletide; Intracellular Signaling Peptides and Proteins; Models, Biological; Myocardium; Pancreatitis; Phosphorylation; Plasmids; Rats; RNA, Small Interfering; Transcription Factor RelA

Naringenin (Nar) is a type of flavonoid and has been shown to have anti-inflammatory and antioxidative properties. However, the effects of Nar on acute pancreatitis (AP) have not been well studied. In this study, we aimed to investigate the function of Nar in a mouse model of AP.. Mild acute pancreatitis (MAP) was induced by caerulein (Cae), and severe acute pancreatitis (SAP) was induced by L-arginine in mice. Nar was administered intraperitoneally at doses of 25, 50, or 100 mg/kg following MAP induction and at a dose of 100 mg/kg following SAP induction. The serum levels of cytokines, lipase, and amylase were determined, and pancreatic and pulmonary tissues were harvested.. The serum levels of amylase, lipase, and cytokines were significantly decreased in both MAP and SAP models after Nar treatment. The malondialdehyde (MDA) levels of the pancreatic tissue was significantly reduced in both MAP and SAP after Nar treatment. In contrast, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), total sulfhydryl (T-SH), and non-proteinsulthydryl (NP-SH) were markedly increased in both MAP and SAP after Nar treatment. The injury in pancreatic and pulmonary tissues was markedly improved as evidenced by the inhibited expression of myeloperoxidase, nod-like receptor protein 3, and interleukin 1 beta as well as the enhanced expression of nuclear factor erythroid 2-related factor 2/heme oxygenase-1 in pancreatic tissues.. Nar exerted protective effects on Cae-induced MAP and L-arginine-induced SAP in mice, suggesting that Nar may be a potential therapeutic intervention for AP.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Cytokines; Flavanones; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Heme Oxygenase-1; Lipase; Male; Membrane Proteins; Mice; NF-E2-Related Factor 2; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis

Bile acids receptor TGR5 and its agonist INT-777, which has been found to be involved in the NLRP3 inflammasome pathway, play an important role in inflammatory diseases. However, the role of INT-777 in acute pancreatitis (AP) has not been reported. In this present study, we found that TGR5 was expressed in pancreatic tissue and increased after AP onset induced by caerulein and further evaluated the impact of INT-777 on the severity of AP. The results showed that INT-777 could reduce the severity of AP in mice, which was manifested as decreased pancreatic tissue damage as well as the decrease of serum enzymes (amylase and lipase), pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and the expression of necrosis related proteins (RIP3 and p-MLKL). Furthermore, we found that INT-777 reduced the reactive oxygen species (ROS) production in pancreatic acinar cells and inhibited the activation of NLRP3 inflammasome pathway. In conclusion, our data showed that INT-777 could protect pancreatic acinar cell against necrosis and reduce the severity of AP, which may be mediated by inhibiting ROS/NLRP3 inflammasome pathway.

Topics: Acinar Cells; Animals; Ceruletide; Cholic Acids; Disease Models, Animal; Inflammasomes; Male; Mice; Mice, Inbred ICR; Necrosis; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas, Exocrine; Pancreatitis; Protective Agents; Reactive Oxygen Species; Receptors, G-Protein-Coupled

Acute pancreatitis (AP) is a common acute abdominal disease accompanied by systemic inflammatory response syndrome, and could even be complicated by multiple-organ damage. This study aimed to examine whether calycosin, an isoflavone isolated from Radix astragali with antioxidant and anti-inflammatory activity, could protect against AP induced by cerulein. To this end, Balb/C mice were injected with cerulein (50 μg/kg) to establish the animal model of AP. Calycosin (25 and 50 mg/kg, p.o.) was administered 1 h prior to the first cerulein injection. After the last injection of cerulein, the mice were sacrificed and blood was obtained for cytokine analysis. The pancreas was removed for morphological examination, myeloperoxidase (MPO) and malondialdehyde (MDA) analyses, immunohistochemistry, and western blot analysis. Calycosin treatment reversed the increased serum levels of amylase and lipase, alleviated the pathological damage in the pancreas, and decreased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in mice with AP. Additionally, calycosin significantly reduced cerulein-induced pancreatic edema, inhibited MPO activity and increased superoxide dismutase (SOD) activity, and inhibited the expression of NF-κB/p65 and phosphorylation of the inhibitor of NF-κB (IκBα) and p38 MAPK. These results suggested that calycosin protects against AP by exerting anti-inflammatory and anti-oxidative stress effects via the p38 MAPK and NF-κB signal pathways. Calycosin's benefits for AP patients need to be explored further.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Astragalus propinquus; Ceruletide; Cytokines; Disease Models, Animal; Drugs, Chinese Herbal; Isoflavones; Male; Mice, Inbred BALB C; NF-kappa B; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Pancreatitis; Signal Transduction

The objective of this study is to explore the effect of melatonin on endoplasmic reticulum stress in acute pancreatitis (AP) and the molecular mechanism.. Acute pancreatitis was induced in vivo in Sprague-Dawley rats by the retrograde injection of 5% taurocholate into the biliopancreatic duct and in vitro by treating AR42J cells with cerulein (10 nmol/L) plus lipopolysaccharide (LPS) (10 mg/L). The rats and cells were treated with melatonin (50 mg/kg in rats and 0.5, 1, and 2 mmol/L in AR42J cells) 30 minutes before AP was induced. After 9 hours, the cells and rat pancreas tissue were collected for Western blot, reverse transcription polymerase chain reaction, histological examination, immunohistochemistry, and immunofluorescence analysis.. Inositol-requiring 1α (IRE1α)-mediated Jun N-terminal kinase (JNK)/nuclear factor-kappa B (NF-κB) pathway were activated early in AR42J cells and rat AP models. Melatonin significantly inhibited the expression of proinflammatory cytokines. Western blot and immunohistochemical results all indicated that melatonin regulated apoptosis-related protein expression. In addition, melatonin treatment resulted in significantly reduced pancreatic tissue injury, as revealed by histological changes and pathological scores. Furthermore, melatonin treatment significantly reduced the activation of IRE1α-mediated JNK/NF-κB pathway-related proteins.. These findings suggest that melatonin protects AR42J cells and Sprague-Dawley rats against AP-associated injury, probably through downregulation of IRE1α-mediated JNK/NF-κB pathways.

Topics: Acute Disease; Animals; Antioxidants; Apoptosis; Cell Line; Ceruletide; Cytokines; Endoplasmic Reticulum Stress; Gene Expression; Lipopolysaccharides; Male; Melatonin; Pancreas; Pancreatitis; Rats, Sprague-Dawley; Signal Transduction; Taurocholic Acid

Pancreatitis is an inflammatory disease of the exocrine pancreas and ranks among the most common gastrointestinal disorders. Inflamed tissues frequently experience conditions of insufficient oxygen availability, or hypoxia. Here, we demonstrate that hypoxia and consequent stabilization of the hypoxia-inducible factor 1α (HIF1α) transcription factor occur in murine and human pancreatitis. Mice lacking pancreas-specific HIF1α expression display markedly impaired pancreatic regeneration following cerulein-induced pancreatitis, which is associated with excessive intrapancreatic B cell accumulation. Notably, B cell depletion in mice with established pancreatitis significantly enhances tissue regeneration. Our study reveals a crosstalk between pancreatic HIF1α expression and B cell trafficking that regulates tissue regeneration, and identifies plausible molecular targets for treating pancreatitis patients.

Topics: Animals; B-Lymphocytes; Ceruletide; Gene Deletion; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphocyte Depletion; Mice, Inbred C57BL; Pancreas; Pancreatitis; Protein Stability; Regeneration

Acute pancreatitis (AP) is a common and severe gastrointestinal inflammatory disease with poorly understood pathogenesis. We adopted cerulein-induced pancreatitis, a well-established rat model shearing similarities with human AP, to determine the disease background. Special interest was placed on sphingolipids, because their signaling pathways are involved in many pathological states including hepatic steatosis, heart infarction, or pancreatic origin type 1 diabetes.. Sphingolipid levels in the blood and pancreas were determined by the means of chromatography (thin-layer and high-performance liquid chromatography).. We found that AP leads to activation of ceramide de novo synthesis pathway, as evidenced by a significant increment in sphinganine, that is, ceramide synthesis precursor, content (+3.8-fold). Surprisingly, despite the reported growth in sphinganine concentration, we observed a reduced (-38%) ceramide level in the pancreas of rats with AP. The results could be explained by subsequent hydrolysis of ceramide to other secondary messengers, that is, sphingosine (+4-fold) or sphingosine-1-phosphate (+3-fold).. Because it is known that sphingosine-1-phosphate and some of its analogs could have a protective role against AP complications, our findings may contribute to elaboration of new therapeutic strategies in the management of this severe medical condition.

Topics: Acute Disease; Animals; Ceramides; Ceruletide; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Disease Models, Animal; Humans; Pancreas; Pancreatitis; Rats, Wistar; Signal Transduction; Sphingomyelins; Sphingosine

In the present study, we have elaborated the anti-inflammatory mechanism of MSM through homing of CD34. Male Swiss mice were treated with hourly intraperitoneal injections of caerulein (50 μg/kg) for 6 h. MSM (500 mg/kg) was administered intraperitoneally 1 h after the first caerulein injection (therapeutic). The serum amylase activity and myeloperoxidase (MPO) activity in lung and pancreas were measured. The levels of H. Methylsulfonylmethane significantly ameliorated pancreas and lung histopathological changes, decreased serum amylase, MPO activity and inhibited caerulein-induced IL-1β expression. Furthermore, MSM reduced caerulein-induced H. These findings indicate that MSM can effectively reduce inflammatory responses and induce the homing of CD34

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Ceruletide; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Inflammation Mediators; Male; Mice; Pancreatitis; Random Allocation; Sulfones

Inflammatory response is a determinant in the pathological progression of acute pancreatitis (AP). Previous studies have shown that the activation of hedgehog (Hh) signaling is a remarkable change in caerulein-induced AP. However, the relationship between Hh signaling and inflammation is largely ambiguous.. The AP mouse model was induced by injection of cerulein, and histological staining and serum enzymology assays were used to evaluate the establishment of AP. Western blot assay was used to determine the protein levels, cleavage of apoptotic proteins, and activation of the NF-κB signaling pathway. Cytokine array was used to screen inflammatory cytokines, and target cytokines' transcriptional expression and serum levels were examined by real-time PCR and enzyme-linked immunosorbent assay, respectively.. The key transcriptional factor in Hh signaling, Gli2, was upregulated in the pancreas and other tissues during the process of AP, and it seems to be a characteristic feature of local inflammation in pancreatic tissue and systemic inflammatory response in multiple organs. The inflammatory NF-κB pathway is required for the activation of Hh signaling, as blockade of the NF-κB pathway by pyrrolidine dithiocarbamate impaired the Gli2 upregulation. Manipulation of Gli2 expression altered the activation of the NF-κB pathway correspondingly, as well as the cell apoptosis in cerulein-induced AP. Moreover, Gli2 upregulation changed the cytokine expression profile in mouse pancreatic acinar cells, mainly decreasing the pro-inflammatory cytokines interleukin (IL)-6, interferon-γ, and FasL. The anti-inflammatory cytokine IL-10 was upregulated by Gli2 overexpression. Interdiction of Gli2 by the Gli-specific inhibitor GANT61 exacerbated AP in mice and altered the balance of inflammatory cytokines.. This study indicates that Hh activation during AP development is a negative feedback of the inflammatory response, restricting inflammatory injury to the pancreas and other tissues. Thus, manipulation of Hh signaling should shed light on limiting inflammation and alleviating AP damage.

Topics: Acinar Cells; Acute Disease; Animals; Cell Line; Ceruletide; Cytokines; Disease Models, Animal; Hedgehog Proteins; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreatitis; Pyrrolidines; Signal Transduction; Thiocarbamates; Up-Regulation; Zinc Finger Protein Gli2

Acute pancreatitis (AP) is a serious inflammatory disorder of the pancreas with considerable mortality. The clinical therapy is hampered due to lack of any approved drug for AP. In this study, we developed curcumin (cur)-loaded poly (lactic-co-glycolic acid) cur microparticles (CuMPs) for sustained release. CuMPs were prepared by emulsion solvent evaporation method and characterized for shape, size, compatibility, and entrapment efficiency. The in vitro drug release and in vivo pharmacokinetic studies confirmed sustained release pattern of cur from CuMPs. The pharmacodynamic study was conducted in cerulein induced AP model. Prophylactic treatment was planned with single dose of CuMPs (equivalent to 7.5 mg/kg of cur) and compared with free cur given orally (100 mg/kg) and intraperitoneally (7.5 mg/kg) daily for 7 days. Interestingly, the effects of CuMPs were superior compared to the free drug administered either orally or intraperitoneally through repeated administrations. CuMPs showed significant decrease of serum amylase and lipase levels, oxidative and nitrosative stress was also significantly decreased. Moreover, CuMPs impressively decreased inflammatory cytokines. Our results may pave a way to propose similar strategy for many of promising natural products to combat several oxidative stress-mediated disorders via sustained release microparticle approaches.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Ceruletide; Curcumin; Cytokines; Delayed-Action Preparations; Drug Liberation; Male; Mice; Oxidative Stress; Pancreas; Pancreatitis; Polylactic Acid-Polyglycolic Acid Copolymer; Rats, Sprague-Dawley

Acute pancreatitis (AP) is a common inflammatory disease in gastrointestinal tract. Our previous study has shown that caerulin induces TNF receptor-associated factor 3 (TRAF3)-p38 signaling activation and pro-inflammatory response in macrophages, causing damage to co-cultured pancreatic acinar cells. Dihydromyricetin (DHM) is a flavonoid extracted from Ampelopsis grossedentata, which has displayed anti-inflammation and anti-oxidant functions. Our results here show that DHM potently inhibited caerulin-induced expression and productions of multiple pro-inflammatory cytokines (IL-1β, TNF-α and IL-17) in murine bone marrow-derived macrophages (BMDMs). DHM significantly inhibited caerulin-induced TRAF3 protein stabilization, TRAF3-mitogen-activated protein kinase kinase 3 (MKK3) association and following MKK3-p38 activation in BMDMs. Significantly, DHM was ineffective against caerulin in TRAF3-silenced BMDMs. Importantly, DHM supplement attenuated the cytotoxicity of caerulin-activated BMDMs to co-cultured pancreatic acinar cells, resulting in significantly decreased acinar cell death and apoptosis. In vivo, DHM co-administration largely attenuated pancreatic and systemic inflammation in caerulin-injected AP mice. Together, DHM inhibits caerulin-induced TRAF3-p38 signaling activation and AP response. DHM could be further studied as a potential anti-AP agent.

Topics: Acute Disease; Animals; Cell Death; Cell Survival; Cells, Cultured; Ceruletide; Cytokines; Flavonols; Macrophages; Mice; Mice, Inbred C57BL; p38 Mitogen-Activated Protein Kinases; Pancreatitis; Signal Transduction; TNF Receptor-Associated Factor 3

Intrapancreatic activation of the digestive proteases trypsin and chymotrypsin is an early event in the development of pancreatitis. Human genetic studies indicate that chymotrypsin controls trypsin activity via degradation, but there is no evidence of this from animal models. We used CRISPR-Cas9 to disrupt the chymotrypsinogen B1 gene (Ctrb1) in C57BL/6N mice and induced pancreatitis in CTRB1-deficient and C57BL/6N (control) mice by administration of cerulein. CTRB1-deficient mice given cerulein had significant increases in intrapancreatic trypsin activity and developed more severe pancreatitis compared with control mice. CTRB1 therefore protects against secretagogue-induced pancreatitis by reducing trypsin activity. Protease inhibitors developed for treatment of pancreatitis should be designed to target trypsin but not chymotrypsin.

Topics: Animals; Arginine; Ceruletide; Chymotrypsin; Disease Models, Animal; Enzyme Activation; Enzyme Stability; Female; Male; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Proteolysis; Severity of Illness Index; Trypsin

This study aimed to investigate and characterize the anti-inflammatory and anti-hypernociceptive effects of the total polysaccharides of X. americana (TPL-Xa) bark in a mouse model of acute pancreatitis-induced by caerulein and the potential involvement of cannabinoid receptors.. TPL-Xa, containing a heteropolysaccharide composed of glucose, galactose, arabinose, rhamnose, fucose and galacturonic acid, reduced amylase and lipase levels, MPO activity, acinar cell necrosis, edema and neutrophil infiltration. TPL-Xa increased the threshold of visceral hypernociception, an effect reversed by AM630, an antagonist of cannabinoid receptor type 2 (CB2). In addition, TPL-Xa did not alter the animals' motor coordination.. TPL-Xa contains heteropolysaccharides that inhibit inflammation and hypernociception in the experimental model of caerulein-induced AP, by a mechanism involving type CB2 receptors.

Topics: Analgesics; Animals; Anti-Inflammatory Agents; Cannabinoid Receptor Agonists; Carbon-13 Magnetic Resonance Spectroscopy; Ceruletide; Disease Models, Animal; Enzymes; Inflammation Mediators; Male; Mice; Motor Activity; Nociceptive Pain; Olacaceae; Pain Threshold; Pancreas; Pancreatitis; Phytotherapy; Plant Extracts; Plants, Medicinal; Polysaccharides; Proton Magnetic Resonance Spectroscopy; Receptor, Cannabinoid, CB2; Signal Transduction; Time Factors

To date, there still is a lack of specific acute pancreatitis markers and specifically an early marker that can reliably predict disease severity. The inflammatory response in acute pancreatitis is mediated in part through oxidative stress and calcineurin-NFAT (Nuclear Factor of Activated T-cells) signaling, which is inducing its own negative regulator, regulator of calcineurin 1 (RCAN1). Caerulein induction is a commonly used in vivo model of experimental acute pancreatitis. Caerulein induces CN-NFAT signaling, reactive oxygen species and inflammation.. To screen for potential markers of acute pancreatitis, we used the caerulein model of experimental acute pancreatitis (AP) in C57Bl/6 J mice. Pancreata from treated and control mice were used for expression profiling. Promising gene candidates were validated in cell culture experiments using primary murine acinar cells and rat AR42J cells. These candidates were then further tested for their usefulness as biomarkers in mouse and human plasma.. We identified a number of novel genes, including Regulator of calcineurin 1 (Rcan1) and Sestrin 2 (Sesn2) and demonstrated that they are induced by oxidative stress, by stimulation with H. We demonstrated that Rcan1 is regulated by oxidative stress and identified RCAN1 as a potential diagnostic marker of AP.

Topics: Acute Disease; Animals; Biomarkers; Calcium-Binding Proteins; Ceruletide; Gene Expression Profiling; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Muscle Proteins; Oxidative Stress; Pancreatitis; RNA, Messenger

Severe acute pancreatitis is a highly lethal disease caused by systemic inflammatory response syndrome, leading to multiple organ failure. We recently showed that histidine-rich glycoprotein (HRG) supplemental therapy ameliorated septic acute respiratory distress syndrome due to unnecessary neutrophil activation and immunothrombosis formation. Here, we evaluated the effect of HRG on lung inflammation followed by pancreatitis in a severe acute pancreatitis mouse model.. Mice received intraperitoneal injections of cerulein 7 times (100 μg/kg each) at 1-hour intervals to induce acute pancreatitis. Immediately after the first cerulein injection, phosphate-buffered saline, human serum albumin (20 mg/kg), or HRG (20 mg/kg) was intravenously injected. One hour after the last cerulein injection, phosphate-buffered saline or lipopolysaccharide (5 mg/kg) was intravenously injected into the tail vein. We evaluated lung inflammatory level after pancreatitis.. We observed significantly decreased plasma HRG levels in an acute pancreatitis mouse model. Histidine-rich glycoprotein treatment inhibited lung edema and the accumulation of neutrophil in severe acute pancreatitis, but HRG did not directly affect pancreatitis. Moreover, HRG suppressed tumor necrosis factor α, inducible nitric oxide synthase, interleukin 6, and neutrophil elastase mRNA expression and myeloperoxidase activity in the lung.. These data suggested that HRG ameliorated lung inflammation secondary to pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Edema; Gene Expression; Humans; Interleukin-6; Leukocyte Elastase; Lung; Neutrophils; Nitric Oxide Synthase Type II; Pancreatitis; Proteins; Severity of Illness Index; Tumor Necrosis Factor-alpha

The proteasome is involved in the activation of NF-κB and can regulate the progression of inflammatory diseases. However, the role of proteasome in acute pancreatitis (AP) has not been demonstrated. In this study, we first observed that the protein level and activity of proteasome 20S were increased significantly in pancreatic injury tissues after caerulein-induced mild acute pancreatitis (MAP) induction, which was in consistent with the expression of the NF-κB nucleoprotein and positively correlated with the severity of AP. Then, bortezomib, a classical proteasome inhibitor, was used to intervene the progression of MAP in mice. The results showed that bortezomib administration reduced the serum amylase and lipase levels and mitigated histopathological manifestation of pancreatic injury in mice. Meanwhile, bortezomib decreased the expression of NF-κB p65 nucleoprotein as well as total proteasome 20S protein, and inhibited the activity of 20S in pancreatic tissues. In addition, we found that bortezomib could protect pancreatic acinar cell against necrosis and mitigate the severity of AP in a severe acute pancreatitis model induced by sodium taurocholate hydrate. Taken together, our study for the first time confirmed that the proteasome participated in the pathogenesis of AP and its inhibitor bortezomib could protect against AP in mice.

Topics: Acinar Cells; Acute Disease; Animals; Bortezomib; Ceruletide; Disease Progression; Male; Mice, Inbred ICR; Necrosis; Pancreas; Pancreatitis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protective Agents; Taurocholic Acid; Transcription Factor RelA

Tissue nonspecific alkaline phosphatase (TNAP) has a well established role in bone homeostasis and in hepatic/biliary conditions. In addition, TNAP is expressed in the inflamed intestine and is relevant to T and B lymphocyte function. TNAP KO mice are only viable for a few days, but TNAP

Topics: Acinar Cells; Alkaline Phosphatase; Animals; Ceruletide; Disease Models, Animal; Humans; Levamisole; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neutrophils; Pancreas; Pancreatitis; RNA, Messenger; Up-Regulation

Acute pancreatitis is an inflammatory disorder of the pancreas that may precipitate due to various reasons such as chronic alcoholism, gall stone obstruction, and life style. Current treatment options offer limited efficacy, as they provide only symptomatic relief. This study is an attempt to study the effects of Withaferin A (WFA) against Cerulein-induced acute pancreatitis in mice. Animals were pretreated with WFA via intraperitoneal route, for 7 days. Plasma amylase and lipase, tissue malondialdehyde (MDA), and glutathione were evaluated for all groups. Western blot analysis; haematoxylin and eosin staining of the liver, lung, and pancreas; immunohistochemistry for nitrotyrosine; and myeloperoxidase activity were performed. Haematoxylin and eosin stained sections significantly revealed the altered architecture and thereby damage in the pancreas, lungs, and liver that has been low in treatment groups. Increased myeloperoxidase and nitrotyrosine have also been reduced upon treatment with WFA. Increased levels of MDA, NO, and expression of myeloperoxidase and nitrotyrosine in the parameters estimated add evidence to the role of oxidative stress and inflammation in acute pancreatitis. WFA evidently altered these conditions upon pretreatment. Our study shows that this novel steroidal compound has potent anti-inflammatory property. Natural compounds can therefore be good remedies against many diseases if incorporated in routine diet as dietary supplement.

Topics: Acute Disease; Animals; Ceruletide; Inflammation; Male; Mice; Oxidative Stress; Panax; Pancreatitis; Withania; Withanolides

Chronic or repeated episodes of acute pancreatic inflammation, or pancreatitis, are risk factors for the development of pancreatic cancer. Pancreatic cancer is characterized by a strong fibro-inflammatory tumor microenvironment. In pancreatitis, the same fibro-inflammatory reaction is observed concurrently with a loss of normal pancreatic cells. Mouse models are commonly employed to study the progression of pancreatitis and pancreatic cancer, with genetic and pharmacological tools used to elucidate cellular and acellular interactions within pancreatic tumors. Described in this article is a protocol for using Kras

Topics: Adenocarcinoma; Animals; Ceruletide; Disease Models, Animal; Pancreas; Pancreatic Neoplasms; Pancreatitis; Point Mutation; Proto-Oncogene Proteins p21(ras)

Background Chronic pancreatitis (CP) is a persistent inflammation of the pancreas clinically presented with severe abdominal pain, progressive fibrosis, and loss of exocrine and endocrine functions. Inflammasomes, cytosolic multiprotein complexes which regulate the formation of proinflammatory cytokines, are influenced by various factors including heat shock proteins (HSPs). Morus alba L., or white mulberry root bark is a valued traditional Asian medicine with a diverse array of phytochemicals. The aim of this investigation was to define the modulatory action of methanolic extract of Morus alba root bark (MEMARB) on NLRP3 inflammasome, and HSPs in pancreas subjected to inflammatory insult. Methods Pancreatitis was induced in male albino Wistar rats by ethanol (0-36%) and cerulein (20 µg/kg b.wt., i.p.) for 5 weeks with or without MEMARB administration. Serum lipase/amylase (L/A) ratio, oxidative stress index (OSI) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in the pancreas were evaluated. Levels of serum HSP70 was quantified by ELISA. NF-kappa B, NLRP3-ASC, caspase-1, IL-1β, IL-18, and HSP70 gene expression was quantified by quantitative real-time polymerase chain reaction (qPCR). Results L/A ratio and oxidative stress determined in terms of OSI and GSH/GSSG ratio were elevated in pancreatitis-induced rats. The levels were restored in MEMARB co-administered animals. Serum level of HSP70 was increased in pancreatitis-induced animals and dropped significantly in MEMARB co-administrated rats. Pancreatitis-induced group showed increased expression of NF-kappa B, IL-1β, IL-18, caspase-1, NLRP3-ASC and HSP70 mRNA than in MEMARB treated group. Conclusions It can be concluded that the M. alba root extract modulates the expression of HSP70 and NLRP3-ASC which might be attributed to its pancreato-protective effect.

Topics: Animals; Biomarkers; Ceruletide; Ethanol; HSP70 Heat-Shock Proteins; Humans; Inflammasomes; Interleukin-18; Interleukin-1beta; Male; Morus; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis; Plant Bark; Plant Extracts; Plant Roots; Rats; Rats, Wistar

Mice lacking Sirt2 spontaneously develop tumors in multiple organs, as well as when expressed in combination with oncogenic Kras

Topics: Animals; Ceruletide; Disease Models, Animal; Disease Susceptibility; Female; Genetic Predisposition to Disease; Immunohistochemistry; Male; Mice; Mice, Knockout; Mutation; Pancreatitis; Proto-Oncogene Proteins p21(ras); Regeneration; Sirtuin 2

Acute pancreatitis (AP), a common abdominal inflammatory disorder, is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. Although the mechanism remains to be fully understood, inflammation is the main cause of pancreatic damage in AP. A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2-ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)], derived from danshensu, exhibits anti-inflammatory and anti-apoptotic properties

Topics: Acute Disease; Animals; Antioxidants; Biological Products; Cells, Cultured; Ceruletide; Disease Models, Animal; Female; Heme Oxygenase-1; Inflammasomes; Inflammation; Lactates; Macrophages; Mice; Mice, Inbred BALB C; Neutrophils; NF-E2-Related Factor 2; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatitis; Phenylacetates; Signal Transduction; STAT3 Transcription Factor

Our previous study demonstrated that a deficiency of microRNA 21 (miR-21) protects mice from acute pancreatitis, yet the underlying molecular networks associated with miR-21 in pancreatitis and pancreatitis-associated lung injury remain unexplored.. We used next generation sequencing to analyze gene expression profiles of pancreatic tissues from wild-type (WT) and miR-21 knockout (KO) mice treated with caerulein by using a 1-day treatment protocol. The Database for Annotation, Visualization, and Integrated Discovery gene annotation tool and Ingenuity Pathway Analysis were used to analyze the molecular pathways, while quantitative real-time PCR, western blotting, and immunohistochemistry were used to explore the molecular mechanisms.. We identified 152 differentially expressed genes (DEGs) in pancreata between WT and KO mice treated with caerulein. Cellular biogenesis and metabolism were the major pathways affected between WT and KO mice, whereas cell death and inflammatory response discriminated between WT and KO mice under acute pancreatitis. We validated 16 DEGs, consisting of 6 upregulated genes and 10 downregulated genes, involved in pancreatic injury. In particular, the upregulation of Pias3 and downregulation of Hmgb1 in KO pancreata coincided with a reduced severity of pancreatitis. In addition, we found Hmgb1 stimulation resulted in the overexpression of miR-21 in peripheral blood mononuclear cells, and deletion of miR-21 led to a reduction of caerulein-induced acute pancreatitis-associated lung injury by repressing Hmgb1 expression.. Our data support the hypothesis that miR-21 modulates the inflammatory response during acute pancreatitis through the upregulation of Pias3 and downregulation of Hmgb1. Our findings further underscore a role for miR-21 in the promotion of acute pancreatitis.

Topics: Acute Disease; Animals; Cells, Cultured; Ceruletide; Female; Gene Deletion; Gene Expression Regulation; HMGB1 Protein; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Molecular Sequence Annotation; Pancreatitis; Protein Inhibitors of Activated STAT

Pancreatic acinar cell necrosis and inflammatory responses are two key pathologic processes in acute pancreatitis (AP), which determines the severity and outcome of the disease. Recent studies suggest that necroptosis, a programed form of necrosis, is involved in the pathogenesis of AP, but the underlying mechanisms remain unknown. We investigated the expression of necrosome components, including receptor-interacting protein (RIP) 1, RIP3, and mixed lineage kinase domain-like (MLKL), and the molecular mechanisms in pancreatitis-associated necroptosis. We found that RIP3 and phosphorylated MLKL expression was positively related to the degree of necrosis, whereas RIP1 expression was negatively related to the degree of necrosis. Pharmacologic inhibition of RIP1 kinase activity exerted no protection against caerulein/cholecystokinin-8-induced AP, but knockdown of RIP1 with siRNA increased acinar cell necrosis and inhibition of NF-κB activation. RIP1 inhibition led to enhanced RIP3 expression. RIP3 and MLKL inhibition decreased acinar cell necrosis, in which the inhibition of RIP3 reduced the phosphorylation level of MLKL. RIP3 inhibition had no effect on trypsinogen activation but partly inhibited inflammasome activation. Our study strongly suggests that the imbalance between RIP1 and RIP3 shifts the cell death to necrosis, which unravels a new molecular pathogenesis of mechanism of AP and may provide insight into the development of novel therapeutic agent for other necrosis-related diseases.

Topics: Acinar Cells; Acute Disease; Animals; Apoptosis; Ceruletide; Cholecystokinin; Irritants; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Necrosis; Pancreatitis; Peptide Fragments; Phosphorylation; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases

Acute pancreatitis (AP) is one of the most common diseases in gastroenterology. However, neither the etiology nor the pathophysiology of the disease is fully understood and no specific or effective treatment has been developed. Heparanase is an endoglycosidase that cleaves heparan sulfate (HS) side chains of HS sulfate proteoglycans into shorter oligosaccharides, activity that is highly implicated in cellular invasion associated with cancer metastasis and inflammation. Given that AP involves a strong inflammatory aspect, we examined whether heparanase plays a role in AP. Here, we provide evidence that pancreatic heparanase expression and activity are significantly increased following cerulein treatment. Moreover, pancreas edema and inflammation, as well as the induction of cytokines and signaling molecules following cerulein treatment were attenuated markedly by heparanase inhibitors, implying that heparanase plays a significant role in AP. Notably, all the above features appear even more pronounced in transgenic mice over expressing heparanase, suggesting that these mice can be utilized as a sensitive model system to reveal the molecular mechanism by which heparanase functions in AP. Heparanase, therefore, emerges as a potential new target in AP, and heparanase inhibitors, now in phase I/II clinical trials in cancer patients, are hoped to prove beneficial also in AP.

Topics: Acute Disease; Animals; Cathepsin L; Ceruletide; Disease Models, Animal; Disease Susceptibility; Enzyme Activation; Gene Expression; Glucuronidase; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Neutrophil Infiltration; Neutrophils; NF-kappa B; Pancreas; Pancreatitis; STAT3 Transcription Factor

RIPK3 mediates cell death and regulates inflammatory responses. Although genetic studies have suggested that RIPK3-MLKL-mediated necroptosis leads to embryonic lethality in Fadd or Caspase-8-deficient mice, the exact mechanisms are not fully understood. Here, we generated Ripk3 mutant mice by altering the RIPK3 kinase domain (Ripk3

Topics: Animals; Apoptosis; Caspase 8; Ceruletide; Chemokines; Cytokines; Embryo, Mammalian; Embryonic Development; Fas-Associated Death Domain Protein; HEK293 Cells; Humans; Inflammation; Lipopolysaccharides; Macrophages; Mice; Mice, Knockout; Mutagenesis; Necrosis; Oligopeptides; Pancreatitis; Phosphorylation; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases

The present study evaluates the effect of. Pancreatitis was induced pancreatitis by cerulean (50μg/kg, i.p.) five times at an interval of 1 h without any pretreatment of drug. Rats were treated with SS (100 and 200 mg/kg, p. o.) and heparin (150 U/kg, i.p.) alone and in combination for the duration of a week. Later pancreatic weight and blood flow was estimated and different biochemical parameters like concentration of D-dimer and Interleukin 1β (IL-Ιβ) and activity of amylase and lipase were determined in blood of pancreatitis rats. Moreover effect of drug treatment on DNA synthesis and histopathology was also estimated on cerulean induced pancreatitis rats.. Results of this study suggest that treatment with SS alone and in combination with heparin significantly increase in prothrombin time and pancreatic blood flow than negative control group. There was significant decrease in concentration of IL-Ιβ and D-dimer and activity of amylase and lipase in SS and heparin treated group than negative control group. Pancreatic DNA synthesis was also found to be reduced in SS and heparin alone and in combination treated group. Histopathology study also reveals that treatment with SS and heparin alone and in combination reduces edema, hemorrhages, leukocyte infiltration in the TS of pancreatic tissues.. Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway and potentiates the effect of heparin in the management of pancreatitis.

Topics: Amylases; Animals; Anticoagulants; Ceruletide; Drug Therapy, Combination; Fabaceae; Fibrin Fibrinogen Degradation Products; Heparin; Interleukin-1beta; Lipase; Male; Nucleic Acid Synthesis Inhibitors; Pancreas; Pancreatitis; Phytotherapy; Plant Extracts; Plant Stems; Protective Agents; Prothrombin Time; Rats; Rats, Wistar

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras

Topics: Acinar Cells; Animals; Carcinoma in Situ; Cell Proliferation; Cell Transdifferentiation; Cell Transformation, Neoplastic; Cells, Cultured; Ceruletide; Disease Models, Animal; Disease Progression; Genetic Predisposition to Disease; Glycogen Synthase Kinase 3 beta; Homeodomain Proteins; Male; Metaplasia; Mice, Knockout; Pancreas, Exocrine; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Phenotype; Proto-Oncogene Proteins p21(ras); Ribosomal Protein S6 Kinases; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Trans-Activators; Tumor Necrosis Factor-alpha

Ghrelin was shown to exhibit protective and therapeutic effect in the gut. Aim of the study was to investigate the role of sensory nerves (SN) in the protective effect of ghrelin in acute pancreatitis (AP). Studies were performed on male Wistar rats or isolated pancreatic acinar cells. After capsaicin deactivation of sensory nerves (CDSN) or treatment with saline, rats were pretreated intraperitoneally with ghrelin or saline. In those rats, AP was induced by cerulein or pancreases were used for isolation of pancreatic acinar cells. Pancreatic acinar cells were incubated in cerulein-free or cerulein containing solution. In rats with intact SN, pretreatment with ghrelin led to a reversal of the cerulein-induced increase in pancreatic weight, plasma activity of lipase and plasma concentration of tumor necrosis factor-α (TNF-α). These effects were associated with an increase in plasma interleukin-4 concentration and reduction in histological signs of pancreatic damage. CDSN tended to increase the severity of AP and abolished the protective effect of ghrelin. Exposure of pancreatic acinar cells to cerulein led to increase in cellular expression of mRNA for TNF-α and cellular synthesis of this cytokine. Pretreatment with ghrelin reduced this alteration, but this effect was only observed in acinar cells obtained from rats with intact SN. Moreover, CDSN inhibited the cerulein- and ghrelin-induced increase in gene expression and synthesis of heat shock protein 70 (HSP70) in those cells. Ghrelin exhibits the protective effect in cerulein-induced AP on the organ and pancreatic acinar cell level. Sensory nerves ablation abolishes this effect.

Topics: Animals; Capsaicin; Ceruletide; Cytokines; Ghrelin; HSP70 Heat-Shock Proteins; Interleukin-4; Male; Pancreatitis; Rats; Rats, Wistar; Sensory Receptor Cells; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) causes morbidity and mortality. The aim of the present study was to investigate the protective effect of tropisetron against AP induced by cerulein. Cerulein (50μg/kg, 5 doses) was used to induce AP in mice. Six hours after final cerulein injection, animals were decapitated. Hepatic/pancreatic enzymes in the serum, pancreatic content of malondialdehyde (MDA), pro-inflammatory cytokines and myeloperoxidase (MPO) activity were measured. Tropisetron significantly attenuated pancreatic injury markers and decreased the amount of elevated serum amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), MPO activities and pro-inflammatory cytokines levels caused by AP in mice. Tropisetron didn't affect the pancreatic levels of MDA. Our results suggest that tropisetron could attenuate cerulein-induced AP by combating inflammatory signaling. Further clinical studies are needed to confirm its efficacy in patients with AP.

Topics: Acute Disease; Animals; Ceruletide; Cytokines; Disease Models, Animal; Indoles; Lipase; Male; Malondialdehyde; Mice; NF-kappa B; Pancreas; Pancreatitis; Peroxidase; Protective Agents; Signal Transduction; Tropisetron

Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result,

Topics: Acinar Cells; Activating Transcription Factor 3; Animals; Carcinoma, Pancreatic Ductal; Cell Differentiation; Ceruletide; Down-Regulation; Male; Mice; Mice, Knockout; Pancreatic Neoplasms; Pancreatitis; Phenotype

Oxidative stress is an important regulator in the pathogenesis of acute pancreatitis (AP). Reactive oxygen species induce activation of inflammatory cascades, inflammatory cell recruitment, and tissue damage. NF-κB regulates inflammatory cytokine gene expression, which induces an acute, edematous form of pancreatitis. Protein kinase C δ (PKCδ) activates NF-κB as shown in a mouse model of cerulein-induced AP. Docosahexaenoic acid (DHA), an ω-3 fatty acid, exerts anti-inflammatory and antioxidant effects in various cells and tissues. This study investigated whether DHA inhibits cerulein-induced AP in rats by assessing pancreatic edema, myeloperoxidase activity, levels of lipid peroxide and IL-6, activation of NF-κB and PKCδ, and by histologic observation. AP was induced by intraperitoneal injection (i.p.) of cerulein (50 μg/kg) every hour for 7 h. DHA (13 mg/kg) was administered i.p. for three days before AP induction. Pretreatment with DHA reduced cerulein-induced activation of NF-κB, PKCδ, and IL-6 in pancreatic tissues of rats. DHA suppressed pancreatic edema and decreased the abundance of lipid peroxide, myeloperoxidase activity, and inflammatory cell infiltration into the pancreatic tissues of cerulein-stimulated rats. Therefore, DHA may help prevent the development of pancreatitis by suppressing the activation of NF-κB and PKCδ, expression of IL-6, and oxidative damage to the pancreas.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Disease Models, Animal; Docosahexaenoic Acids; Inflammation; Interleukin-6; Lipid Peroxides; Male; NF-kappa B; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Bacillopeptidase is a serine peptidase, known for its fibrinolytic activity. However, a very little information is known about its in vivo inflammatory and/or anti-inflammatory properties. Thus, to understand whether bacillopeptidase incorporation can regulate pancreatitis or not, the cerulein-induced pancreatitis model was used, and the role of bacillopeptidase on pancreatitis was studied. In this study, 46 kDa protein was purified from Bacillus subtilis and identified as bacillopeptidase CFR5 (BPC) through MS/MS analysis. The nutritional prophylactic group was orally fed with two doses of BPC (100 μg/Kg/BW of rat) 6 h before cerulein administration and analyzed for its effect on intestine and pancreas inflammation, cytokines, and pancreatitis marker gene expression. BPC administration significantly reduced the severity of pancreatitis by decreasing serum amylase, lipase, pancreatic edema and myeloperoxidase activity. The pretreatment with BPC suppressed the pancreatic pro-inflammatory and inflammatory cytokines production including IL-6, IL-1β, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-13 in both pancreas and serum samples. Moreover, BPC supplementation restored pancreatitis mediated disruption of intestinal barrier integrity by upregulating tight junction proteins (ZO-1, occludin), antimicrobial peptides (DEFB1, CRAMP), MUC-2, TFF3 expression and by enhancing SCFA's production. Pretreatment with BPC suppressed the intestinal inflammation with reduced cytokines production in the colon and ileal region of cerulein-induced pancreatitis. Thus, BPC based pretreatment protocol is a novel intervention to prevent acute pancreatitis.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antimicrobial Cationic Peptides; Bacillus subtilis; Bacterial Proteins; Cathelicidins; Ceruletide; Cytokines; Defensins; Edema; Gene Expression Regulation; Male; Mucin-2; Occludin; Pancreas; Pancreatitis; Rats; Rats, Wistar; Serine Endopeptidases; Trefoil Factor-3; Zonula Occludens-1 Protein

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease involving intracellular activation of digestive enzymes and pancreatic acinar cell injury. The present study was performed to investigate whether methane-rich saline (MS) was involved in the regulation of AP.. MS (16ml/kg) was administered at different dosing frequencies on mice with cerulein-induced AP. Serum amylase, lipase and histopathological changes in the pancreas tissue were measured. Serum cytokine TNFα, IL-6, IFNγ and IL-10 were detected by ELISA. The mRNA levels of these inflammatory cytokines in the pancreas were detected by real time-PCR. Myeloperoxidase (MPO) and superoxide dismutase (SOD) were determined using commercial kits. Apoptosis was assessed by immunohistochemistry and Western blot.. MS treatment reversed the increased serum level of amylase and lipase, alleviated the pathological damage in the pancreas, and decreased the expression of TNFα, IL-6, IFNγ and IL-10 in cerulean-induced AP mice. In addition, MPO was down-regulated and SOD was up-regulated in the MS treated pancreas, indicating that MS had an anti-oxidant effect against AP. Furthermore, MS protected pancreatic cells against cerulean-induced apoptosis and abolished cleaved caspase-3.. MS exerted anti-inflammatory, anti-oxidant and anti-apoptotic effects on cerulein-induced AP in mice and may proved to be a promising therapeutic agent for the clinical treatment of pancreatitis.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Ceruletide; Cytokines; Humans; Inflammation Mediators; Lipase; Male; Methane; Mice; Mice, Inbred C57BL; Oxidative Stress; Pancreatitis; Saline Waters

Acute pancreatitis is a disease associated with inflammation and tissue damage. One protein that protects against acute injury, including ischemic injury to both the kidney and heart, is renalase, which is secreted into the blood by the kidney and other tissues. However, whether renalase reduces acute injury associated with pancreatitis is unknown. Here, we used both

Topics: Acinar Cells; Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomarkers; Calcium Signaling; Carbachol; Cell Line; Ceruletide; Enzyme Activation; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Enzymologic; Humans; Hypertension; Ligands; Membrane Transport Modulators; Mice; Mice, Knockout; Monoamine Oxidase; Pancreas; Pancreatitis; Plasma Membrane Calcium-Transporting ATPases; Recombinant Fusion Proteins; Taurolithocholic Acid

To create acute pancreatitis condition experimentally in rats using cerulein, and to reveal histopathological effects in pancreatic tissue with erdosteine.. An experimental study.. Department of General Surgery, Duzce University, Turkey, from June to October 2014.. Thirty male Wistar albino rats were divided into three groups. No procedures were applied to Group 1. The rats in Group 2 and Group 3 were injected cerulein, to establish an experimental pancreatitis model and the blood amylase and lipase values were examined. The rats in Group 3 were given 10 mg/kg erdosteine. This treatment was continued for another 2 days and the rats were sacrificed. The pancreatic tissues were examined histopathologically for edema, inflammation, acinar necrosis, fat necrosis, and vacuolization.. The lipase and amylase values and the histopathological examination of pancreatic tissues evidenced that the experimental acute pancreatitis model was established and edema, inflammation, acinar necrosis, fat necrosis, and vacuolization were observed in the pancreatic tissues. The statistical results suggest that erdosteine can decrease the edema, inflammation, acinar necrosis, fat necrosis and vacuolization scores in the tissues.. The severity of acute pancreatitis, induced by cerulein in rats, is reduced with the use of erdosteine.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Disease Models, Animal; Edema; Expectorants; Lipase; Male; Necrosis; Pancreas; Pancreatitis; Rats; Rats, Wistar; Thioglycolates; Thiophenes; Treatment Outcome

Acute pancreatitis (AP) is a disease that can cause local and systemic complications that may have high morbidity and mortality. Currently, there is not any specific treatment for AP. In this study, we created an experimental model of AP in rats, and we aimed to demonstrate the histological effectiveness of tocilizumab treatment that antagonizes interleukin-6 (IL-6), one of the key cytokines in the development of AP.. Forty-eight rats were divided into six groups for this study. AP model was created by subcutaneous injections of cerulein (20 μg/kg) four times at 1-h intervals. Tocilizumab 4 mg/kg was administered to one of the treatment groups and 8 mg/kg to the other treatment group intraperitoneally. The effects of tocilizumab were revealed by examining pancreatic tissue of the rats histopathologically according to the Schonberg scoring system.. A comparison between tocilizumab treatment group and AP control group provides statistically significant improvement in AP (p<0.0001). Furthermore, the dose of 8 mg/kg is shown to be more effective than 4 mg/kg (p=0.004).. Our study points out that tocilizumab may be an effective agent for pancreatitis treatment.

Topics: Acute Disease; Animals; Antibodies, Monoclonal, Humanized; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Gastrointestinal Agents; Pancreas; Pancreatitis; Rats

BACKGROUND Aberrant regulation of nuclear factor-κB (NF-κB) and the signaling pathways that regulate its activity have been found to be involved in various pathologies, particularly cancers, as well as inflammatory and autoimmune diseases. Acute pancreatitis (AP) is a complex pathological process, depending on autodigestion caused by premature activation of zymogens. This study aimed to investigate the effect of high expression of TNIP2 gene on AP and AP-induced myocardial injury. MATERIAL AND METHODS To investigate the effect of TNIP2 on AP and AP-induced myocardial injury, we established an AP cell model and rat model. HE staining was applied for histological examination. ELISA was used to determine the level of pro-inflammatory cytokines (TNF-α and IL-6) and myocardial injury markers (LDH and CK-MB). QRT-PCR and Western blot analysis were performed to determine the mRNA and protein level of related genes, respectively. RESULTS We found that the protein level of TNIP2 was relatively higher in the normal AR42J cells. At 4 h after stimulating with cerulein, the protein level of TNIP2 decreased, reached a minimum at 8 h, and then gradually increased. We also found that TNIP2 was correlated with the activation of NF-κB in cerulein-stimulated AR42J cells, and TNIP2 over-expression inhibited the inflammatory response caused by cerulein. Moreover, our results suggest that TNIP2 over-expression relieved the cerulein-triggered inflammatory response and AP-induced myocardial injury in mice. CONCLUSIONS TNIP2 was shown to exert a protective effect on AP and AP-induced myocardial injury.

Topics: Acute Disease; Adaptor Proteins, Signal Transducing; Animals; Ceruletide; Cytokines; Disease Models, Animal; Female; Mice; Mice, Inbred ICR; Myocardial Infarction; NF-kappa B; Pancreatitis; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

Studies have implied the positive association of JAK2/STAT3 signaling with the onset and severity of acute pancreatitis (AP). However, definitive functional study of JAK2/STAT3 signaling in the pathogenesis of acute pancreatitis in vivo is missing and its potential as a therapeutic target and the underlying mechanisms remain to be determined.. The aim of this study was to explore the role of JAK2/STAT3 signaling in the pathogenesis of hyperlipidemia-intensified caerulin-induced AP and its potential as a therapeutic target.. Using the caerulin-induced acute pancreatitis rat model, we showed that JAK2/STAT3 signaling was activated in pancreas and systemic inflammation was increased during AP. Pharmacological suppression of JAK2 by its inhibitor AG490 robustly protected against tissue damage, attenuated JAK2/STAT3 signaling and inflammatory responses. Local pancreatic tissue damage and phosphor- JAK2 in the pancreatic tissue were enhanced in animals fed with high fat diet compared to chow-diet fed animals. Interestingly, JAK2 inhibitor AG490 significantly inhibited pancreas necrosis and systemic inflammation in animals fed with high fat or chow-diet, but did not affect STAT3 signaling.. These results establish that JAK2 activation plays a significant role in the pathogenesis of caerulin-induced AP in animals on both chow and high-fat diets by regulating necrosis and systemic inflammation. Thus, our results not only clarify novel signaling mechanisms in AP but also suggest that JAK2 might constitute a target in the management of hyperlipidemia-intensified caerulin-induced AP.

Topics: Acute Disease; Animals; Ceruletide; Dietary Fats; Hyperlipidemias; Janus Kinase 2; Male; Pancreatitis; Rats; Rats, Sprague-Dawley; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Inhibitory κB kinase (IKK)/nuclear factor κB (NF-κB) signalling has been implicated in the pathogenesis of pancreatitis, but its precise function has remained controversial. Here, we analyse the contribution of IKK/NF-κB signalling in epithelial cells to the pathogenesis of pancreatitis by targeting the IKK subunit NF-κB essential modulator (NEMO) (IKKγ), which is essential for canonical NF-κB activation.. Mice with a targeted deletion of NEMO in the pancreas were subjected to caerulein pancreatitis. Pancreata were examined at several time points and analysed for inflammation, fibrosis, cell death, cell proliferation, as well as cellular differentiation. Human samples were used to corroborate findings established in mice.. In acute pancreatitis, NEMO deletion in the pancreatic parenchyma resulted in minor changes during the early phase but led to the persistence of inflammatory and fibrotic foci in the recovery phase. In chronic pancreatitis, NEMO deletion aggravated inflammation and fibrosis, inhibited compensatory acinar cell proliferation, and enhanced acinar atrophy and acinar-ductal metaplasia. Gene expression analysis revealed sustained activation of profibrogenic genes and the CXCL12/CXCR4 axis in the absence of epithelial NEMO. In human chronic pancreatitis samples, the CXCL12/CXCR4 axis was activated as well, with CXCR4 expression correlating with the degree of fibrosis. The aggravating effects of NEMO deletion were attenuated by the administration of the CXCR4 antagonist AMD3100.. Our results suggest that NEMO in epithelial cells exerts a protective effect during pancreatitis by limiting inflammation and fibrosis and improving acinar cell regeneration. The CXCL12/CXCR4 axis is an important mediator of that effect and may also be of importance in human chronic pancreatitis.

Topics: Acute Disease; Animals; Biomarkers; Ceruletide; Chemokine CXCL12; Chronic Disease; Disease Progression; Epithelial Cells; Fibrosis; Humans; Intracellular Signaling Peptides and Proteins; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Pancreas; Pancreatitis; Receptors, CXCR4; Regeneration

Pancreas-enriched microRNAs have been experimentally investigated in rodents as candidate serum biomarkers of pancreatic injury with several different acute pancreatic injury models. In the present study, temporal and magnitude responses of exocrine pancreas-enriched miR-216a, miR-216b, and miR-217 and endocrine-enriched miR-375 and miR-148a were measured by droplet digital PCR in serum in a caerulein model of pancreatic injury in the dog. All 5 microRNAs followed a similar time course that mirrored the responses of the conventional serum pancreatic injury biomarkers, amylase and lipase. Detection was improved through the use of assays designed against microRNA isomers (isomirs) identified by sequencing. Serum biomarker increases were concordant with histopathology defined acinar cell injury. Minimal islet cell changes were noted. The pancreas-enriched microRNAs demonstrated similar or greater sensitivity, a larger range of response, and a higher correlation to acinar cell injury compared to amylase and lipase. Our results further support the translational potential of pancreas-enriched microRNAs as sensitive biomarkers of acinar cell injury with evidence from an additional non-clinical model system.

Topics: Animals; Biomarkers; Ceruletide; Disease Models, Animal; Dogs; Male; MicroRNAs; Pancreatitis

Reseda odorata L. has long been used in traditional Asian medicine for the treatment of diseases associated with oxidative injury and acute inflammation, such as endotoxemia, acute lung injury, acute myocardial infarction and hepatitis. Luteolin, the main component of Reseda odorata L., which is also widely found in many natural herbs and vege-tables, has been shown to induce heme oxygenase-1 (HO-1) expression to exert anti-inflammatory and antioxidant effects. In this study, we aimed to examine the effects of luteolin on mice with severe acute pancreatitis (SAP), and to explore the underlying mechanisms. Cerulein and lipopolysaccharide were used to induce SAP in male Institute of Cancer Research (ICR) mice in the SAP group. The SAP group was divided into 4 subgroups, as follows: the vehicle, luteolin, zinc protoporphyrin (ZnPP) only, and luteolin (Lut) + ZnPP (luteolin plus zinc protoporphyrin treatment) groups. The wet/dry weight ratios, hematoxylin and eosin staining and pathological scores of pancreatic tissues were assessed and compared to those of the control mice. Amylase, lipase, nuclear factor-κB (NF-κB) and myeloperoxidase activities, and malondialdehyde, tumor necrosis factor α (TNFα), interleukin (IL)-6, IL-10 and HO-1 levels, as well as the expression of HO-1 were determined in serum and/or pancreatic tissue samples. SAP was successfully induced in male mice compared to normal control mice. The wet/dry weight ratios, pathological scores, and amylase and lipase activity, as well as the levels of TNFα and IL-6 were significantly reduced in the pancreatic tissues of the mice in the Lut group compared with those of the mice in the vehicle group. The Lut group exhibited a significant increase in HO-1 expression in the pancreas and enhanced serum HO-1 and IL-10 levels compared with the vehicle group. The suppression of HO-1 activity in the ZnPP group significantly abolished the protective effects of luteolin. NF-κB expression in the pancreatic tissues from the mice in the Lut + ZnPP group was significantly increased following the suppression of HO-1 activity. On the whole, our findings demonstrate that luteolin protects mice from SAP by inducing HO-1-mediated anti-inflammatory and antioxidant activities, in association with the suppression of the activation of the NF-κB pathway.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Chromatography, High Pressure Liquid; Cytokines; Heme Oxygenase-1; Inflammation Mediators; Lipopolysaccharides; Luteolin; Male; Mice, Inbred ICR; Models, Biological; Necrosis; NF-kappa B; Pancreas; Pancreatitis; Protective Agents; Protoporphyrins

Acute pancreatitis (AP) is a common clinical acute abdominal disease. The intestinal injury associated with AP will aggravate the condition retroactively. This study investigates whether the low-methoxyl pectin (LMP) isolated from lemon could attenuate AP and associated intestinal injury.. Experimental AP was induced in BALB/c mice by caerulien (CAE) hyperstimulation. Nutritional prophylactic group was pre-fed with 5% LMP supplemented forage 3 days before AP induction. We found that LMP supplementation attenuated the severity of AP as evidenced by reduced serum amylase and lipase levels, pancreatic edema and myeloperoxidase activity. The protective effect was also confirmed by histological examination of pancreatic damage. LMP suppressed the production of pancreatic proinflammatory cytokines including TNF-α, IL-1β, and IL-6. Moreover, LMP supplementation restored AP-associated disruption of intestinal barrier integrity as evidenced by upregulation of tight junction modulatory proteins occludin, zonula occludens (ZO)-1, antimicrobial peptides β-defensin-1 (DEFB1) and CRAMP as well as increase in SCFAs production. LMP supplemented mice with AP exhibited suppressed intestinal inflammation as shown by decreased ileal and colon cytokine production compared with CAE group.. Our results support dietary LMP supplementation as an effective nutritional intervention for AP and associated intestinal injury.

Topics: Amylases; Animals; beta-Defensins; Ceruletide; Citrus; Lipase; Male; Mice; Mice, Inbred BALB C; Pancreatitis; Pectins; Tight Junctions

Acute pancreatitis has several underlying etiologies, and results in consequences ranging from mild to complex multi-organ failure. The wide range of pathology suggests a genetic predisposition for progression. We compared the susceptibility to acute pancreatitis in BALB/c and FVB/N mice, coupled with proteomic analysis, in order to identify potential protein associations with pancreatitis progression.. Pancreatitis was induced in BALB/c and FVB/N mice by administration of cerulein or feeding a choline-deficient, ethionine-supplemented (CDE) diet. Histology and changes in serum amylase were examined. Proteome profiling in cerulein-treated mice was performed using 2-dimensional differential in gel electrophoresis (2D-DIGE) followed by mass spectrometry analysis and biochemical validation.. Male and female FVB/N mice manifested more severe cerulein-induced pancreatitis as compared with BALB/c mice, but both strains were similarly susceptible to CDE-induced pancreatitis. Few of the 2D-DIGE alterations were validated by immunoblotting. Clusterin was markedly up-regulated after cerulein-induced pancreatitis in FVB/N but less-so in BALB/c mice. Pyrroline-5-carboxylate reductase (Pycr1), an enzyme involved in proline biosynthesis, had higher basal levels in FVB/N male and female mouse pancreata compared with BALB/c pancreata, and was relatively more resistant to degradation in FVB/N pancreata. However, serum and pancreas tissue proline levels were similar in the two strains.. FVB/N is more susceptible than BALB/c mice to cerulein-induced but not CDE-induced pancreatitis. Most of the 2D-DIGE alterations in the two strains likely relate to posttranslational modifications rather than protein level differences. Clusterin levels increase dramatically in association with pancreatitis severity, while Pycr1 is higher in FVB/N versus BALB/c pancreata basally and after induction of pancreatitis. Changes in proline metabolism may represent a novel potential genetic modifier in the context of pancreatitis.

Topics: Amylases; Animals; Ceruletide; Choline; Clusterin; delta-1-Pyrroline-5-Carboxylate Reductase; Disease Models, Animal; Ethionine; Female; Gene Expression Regulation; Genetic Predisposition to Disease; Male; Mice; Mice, Inbred BALB C; Pancreatitis; Proline; Protein Processing, Post-Translational; Proteome; Pyrroline Carboxylate Reductases; Species Specificity

Resistin, an adipocytokine secreted by fat tissues, has been shown to be associated with increased local and systemic complications in acute pancreatitis (AP). However, the mechanism underlying the effect of resistin in the aggravation of AP remains to be elucidated. The aim of the present study was to investigate the functional consequences of exposing rat pancreatic acinar cells to resistin and to determine whether it amplifies proinflammatory signaling in an in vitro AP model. AR42J cells pretreated with recombinant resistin were activated by cerulein as an in vitro model of AP. The secretion of amylase was measured to evaluate the cytotoxic effect. The mRNA expression levels of tumor necrosis factor (TNF)‑α and interleukin (IL)‑6 were determined using reverse transcription‑quantitative polymerase chain reaction analysis. The nuclear protein expression levels of the nuclear factor (NF)‑κB p65 subunit were determined using western blot analysis. Resistin treatment significantly increased the secretion of amylase, and the mRNA expression levels of TNF‑α and IL‑6 in the cerulein‑induced in vitro AP model. High protein levels of the NF‑κB p65 subunit were observed in the nuclei of cells in the resistin‑treated AP model, compared with the untreated AP model. Pretreatment of the in vitro resistin‑treated AP model with the NF‑κB inhibitor, pyrrolidine dithiocarbamate decreased the protein expression of the NF‑κB p65 subunit in nuclei, and significantly attenuated the increased mRNA expression levels of TNF‑α and IL‑6 induced by resistin. The results of the present study showed that resistin increased the production of the TNF‑α and IL‑6 proinflammatory cytokines via the NF‑κB‑dependent pathway during AP. Thus, the overproduction of obesity‑associated resistin and the associated amplification of the inflammatory response may result in the aggravation of AP severity.

Topics: Acinar Cells; Amylases; Animals; Cell Line; Ceruletide; Cytokines; Interleukin-6; NF-kappa B; Pancreas; Pancreatitis; Rats; Resistin; Tumor Necrosis Factor-alpha

This study was conducted to assess the preventive/therapeutic effects of combined administration of resveratrol and guggulsterone on cerulein-induced acute pancreatitis in mice.. Acute pancreatitis was induced by intraperitoneal injection of cerulein in mice. Serum amylase assay and histology were performed to measure the severity of pancreatitis. Western blotting and multiplex cytokine/chemokine analysis were conducted to understand the action mechanisms of the reagents.. Serum amylase assay and histology revealed that the severity of acute pancreatitis was reduced by the combinatory treatment with resveratrol and guggulsterone, but the ratio of the band intensity implied that reduced nuclear factor-κB activation is primarily responsible for the effect. The reduced amounts of keratinocyte chemoattractant (chemokine [C-X-C motif] ligand 1), interferon gamma-induced protein 10 (C-X-C motif chemokine 10) and interleukin 6 expression in the sera could be involved in attenuated immune cell migration and reduced inflammation by these reagents.. Combinatory treatment with resveratrol and guggulsterone marginally reduced cerulein-induced mild acute pancreatitis in mice.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Ceruletide; Chemokines; Cytokines; Female; Mice, Inbred C57BL; NF-kappa B; Pancreatitis; Pregnenediones; Resveratrol; STAT3 Transcription Factor; Stilbenes

The ever-increasing problem of pancreatitis due to alcohol abuse demands evaluation of novel drugs of plant origin. This study explores the therapeutic effects of the methanolic extract of Brassica oleraceae (MEBO) on ethanol and cerulein induced pancreatitis in rats. The MEBO was subjected to GC-MS and HPLC analysis. Male albino Wistar rats were divided into various groups, fed with alcohol (36% of total calories for 5 weeks) and cerulein (20 μg/kg b.wt i.p, weekly thrice for last three weeks) with or without MEBO (40 mg/kg b.wt). Serum lipase, amylase, IL-1β, IL-18, caspase-1, lipid peroxides, oxidative stress index and antioxidant status were assessed in pancreas. Six compounds were identified in GC-MS analysis. Co-administration of MEBO reduced the pancreatic marker enzymes in serum, IL-1β, IL-18 and caspase-1 and increased the antioxidant status of pancreas. The pancreato-protective effect of Brassica oleraceae may be attributed to well-known anti-inflammatory flavonoids, luteolin, quercetin and myricetin.

Topics: Amylases; Animals; Anti-Inflammatory Agents; Antioxidants; Biomarkers; Brassica; Ceruletide; Chromatography, High Pressure Liquid; Ethanol; Gas Chromatography-Mass Spectrometry; Male; Methanol; Oxidative Stress; Pancreas; Pancreatitis; Plant Extracts; Protective Agents; Rats, Wistar

Topics: Acute Disease; Ceruletide; Humans; Pancreas; Pancreatitis; S100A12 Protein

Previous studies have focused on the effects of propylene glycol alginate sodium sulfate  (PSS)  against  thrombosis,  but  the  anti-inflammatory  potential  is  unknown.  Therefore,  we  specifically focused on the protective effects of PSS on cerulein-induced acute pancreatitis (AP)  using a mouse model, and investigated the mechanism of PSS on autophagy and apoptosis via the  Mitogen-activated  protein  kinase  (MEK)/extracellular  signal-regulated  kinase  (ERK)  pathway.  Cerulein (100 ug/kg) was used to induce AP by ten intraperitoneal injections at hourly intervals in  Balb/C mice. Pretreatment with vehicle or PSS was carried out 1 h before the first cerulein injection  and two doses (25 mg/kg and 50 mg/kg) of PSS were injected intraperitoneally. The severity of AP was  assessed by pathological score, biochemistry, pro-inflammatory cytokine levels, myeloperoxidase  (MPO) activity and MEK/ERK activity. Furthermore, pancreatic histological scores, serum amylase  and lipase activities, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β interleukin (IL)-6 levels, and  MPO activity were significantly reduced by PSS via up-regulated MEK/ERK activity. The representative  molecules of apoptosis and autophagy, such as Bcl-2, Bax, Lc-3, Beclin-1, P62, were remarkably reduced.  Taken together, these results indicate that PSS attenuates pancreas injury by inhibiting autophagy and  apoptosis through a mechanism involving the MEK/ERK signaling pathway.

Topics: Alginates; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Autophagy; Ceruletide; Disease Models, Animal; Humans; Interleukin-1beta; Interleukin-6; Lipase; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Pancreas; Pancreatitis; Peroxidase; Tumor Necrosis Factor-alpha

Transient receptor potential vanilloid-1 (TRPV1) expressed in nociceptors is directly phosphorylated and activated by protein kinase C, and involved in the signaling of pancreatic pain. On the other hand, Cav3.2 T-type Ca2+ channels expressed in nociceptors are functionally upregulated by phosphorylation with protein kinase A and also play a role in pancreatitis-related pain. Calcineurin, a phosphatase, negatively regulates various channel functions including TRPV1, and calcineurin inhibitor-induced pain syndrome by tacrolimus, a calcineurin inhibitor, used as an immunosuppressant, has been a clinical problem. We thus examined the effect of tacrolimus on pancreatitis-related pain in mice. Repeated treatment with cerulein caused referred hyperalgesia accompanying acute pancreatitis, which was unaffected by tacrolimus. Pancreatitis-related symptoms disappeared in 24 h, whereas the referred hyperalgesia recurred following the administration of tacrolimus, which was abolished by the blockers of TRPV1 but not T-type Ca2+ channels. Thus, tacrolimus appears to cause the TRPV1-dependent relapse of pancreatitis-related pain, suggesting the involvement of calcineurin in the termination of pancreatic pain.

Topics: Anilides; Animals; Benzimidazoles; Ceruletide; Cinnamates; Cyclopropanes; Hyperalgesia; Male; Mice; Naphthalenes; Pain; Pancreatitis; Recurrence; Tacrolimus; TRPV Cation Channels

Bromodomain inhibitors (JQ1 and I-BET 762) are a new generation of selective, small molecule inhibitors that target BET (bromodomain and extra terminal) proteins. By impairing their ability to bind to acetylated lysines on histones, bromodomain inhibitors interfere with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell cycle, apoptosis and inflammation. In this study, JQ1 and I-BET 762 decreased c-Myc and p-Erk 1/2 protein levels and inhibited proliferation in pancreatic cancer cells. The tumor microenvironment is known to play an important role in pancreatic cancer, and these drugs suppressed the production of nitric oxide and a variety of inflammatory cytokines, including IL-6, CCL2, and GM-CSF, in both immune and pancreatic cancer cells in vitro. Notably, the bromodomain inhibitors also reduced protein levels of p-Erk 1/2 and p-STAT3 in mouse models of pancreatic cancer. All of these proteins are essential for tumor promotion, progression and metastasis. In conclusion, the bromodomain inhibitors JQ1 and I-BET 762 targeted and suppressed multiple pathways in pancreatic cancer. I-BET 762 and a number of other bromodomain inhibitors are currently being tested in several clinical trials, making them potentially promising drugs for the treatment of pancreatic cancer, an often-fatal disease.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Azepines; Benzodiazepines; Cell Line, Tumor; Cell Proliferation; Ceruletide; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Humans; Inflammation Mediators; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; Pancreatic Neoplasms; Pancreatitis; Phosphorylation; Proto-Oncogene Proteins c-myc; RAW 264.7 Cells; Signal Transduction; Triazoles

Severe acute pancreatitis (SAP) still remains a clinical challenge, not only for its high mortality but the uncontrolled inflammatory progression from acute pancreatitis (AP) to SAP. Cell death, including apoptosis and necrosis are critical pathology of AP, since the severity of pancreatitis correlates directly with necrosis and inversely with apoptosis Therefore, regulation of cell death from necrosis to apoptosis may have practicably therapeutic value. X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the inhibitor of apoptosis proteins (IAP) family, but its function in AP remains unclear. In the present study, we investigated the potential role of XIAP in regulation of cell death and inflammation during acute pancreatitis. The in vivo pancreatitis model was induced by the administration of cerulein with or without lipopolysaccharide (LPS) or by the administration of l-arginine in wild-type or XIAP-deficient mice, and ex vivo model was induced by the administration of cerulein+LPS in AR42J cell line following XIAP inhibition. The severity of acute pancreatitis was determined by serum amylase activity and histological grading. XIAP deletion on cell apoptosis, necrosis and inflammatory response were examined. Caspases activities, nuclear factor-κB (NF-κB) activation and receptor-interacting protein kinase1 (RIP1) degradation were assessed by western blot. Deletion of XIAP resulted in the reduction of amylase activity, decrease of NF-κB activation and less release of TNF-α and IL-6, together with increased caspases activities and RIP1 degradation, leading to enhanced apoptosis and reduced necrosis in pancreatic acinar cells and ameliorated the severity of acute pancreatitis. Our results indicate that deletion of XIAP switches cell death away from necrosis to apoptosis and decreases the inflammatory response, effectively attenuating the severity of AP/SAP. The critical role of XIAP in cell death and inflammation suggests that inhibition of XIAP represents a potential therapeutic strategy for the treatment of acute pancreatitis.

Topics: Animals; Apoptosis; Arginine; Caspases; Cell Death; Cell Line; Ceruletide; Inflammation; Inhibitor of Apoptosis Proteins; Interleukin-6; Mice; Mice, Inbred C57BL; Necrosis; NF-kappa B; Pancreas; Pancreatitis; Receptor-Interacting Protein Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; X-Linked Inhibitor of Apoptosis Protein

To detect the expression ofsignal transducer and activator of transcription 3 (STAT3) in pancreatic tissue of the mouse model of pancreatitis, and to explore its role in the evolution of acute pancreatitis.. Forty-eight healthy male balb/c mice were randomly divided into 3 groups (. Compared with control group, serum amylase activity, pancreatic wet weight ratio and lung MPO activity were significantlyincreased (. The expression of p-STAT3 in MAP and SAP groups are significantly different from that in control group, which indicates that STAT3 isclosely related in acute pancreatitis. Inhibition of STAT3 activity is a potential target to alleviate acute pancreatitis progression.

Topics: Acute Disease; Animals; Ceruletide; Male; Mice; Mice, Inbred BALB C; Pancreas; Pancreatitis; Signal Transduction; STAT3 Transcription Factor

Animal models are essential to understand the pathogenesis of acute pancreatitis (AP) and to develop new therapeutic strategies. Although it has been shown that cerulein-induced AP is associated with pain in experimental animals, most experiments are carried out without any pain-relieving treatment because researchers are apprehensive of an interference of the analgetic agent with AP-associated inflammation. In light of the growing ethical concerns and the legal tightening regarding animal welfare during experiments, this attitude should be changed.. Acute pancreatitis was induced by cerulein in the C57BL/6J and FVB/N mouse inbred strains. One group received vehicle only, and the other was treated with metamizol as analgetic agent. Pain sensation and parameters of AP were analyzed as well as the effect of metamizol in the pancreas and its actions in the brain.. We report that oral administration of metamizol protects cerulein-treated mice from abdominal pain without influencing the clinical and histopathological course of the disease. In addition, it could be shown that metamizol reduces the central pain response.. This study reveals that oral administered metamizol has no influence on the cerulein-induced AP and can be given as an analgesic to increase animal welfare in experiments with induced AP.

Topics: Abdominal Pain; Acute Disease; Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Brain; Ceruletide; Cyclooxygenase 2; Dinoprostone; Dipyrone; Disease Models, Animal; Humans; Hypothalamus; Male; Mice, Inbred C57BL; Mice, Inbred Strains; Pancreatitis; Proto-Oncogene Proteins c-fos; Thalamus

The objective of this study was to determine the mechanism by which activation of peroxisome proliferator-activated receptor-γ promotes apoptosis of acinar cells in pancreatitis.. AR42j cells pretreated with the peroxisome proliferator-activated receptor-γ agonist pioglitazone were activated by cerulein as an in vitro model of acute pancreatitis. Inflammatory cytokines and amylase were detected by enzyme-linked immunosorbent assay. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was measured by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Activity of caspases was determined. Bax and Bcl-2 levels were assayed by Western blot.. Cytokines, amylase, and cellular proliferation decreased in pioglitazone-pretreated cells. Pioglitazone increased the activity of caspases 3, 8, and 9 in cerulein-activated AR42j cells as well as in the pancreas of rats 3 hours after induction of severe acute pancreatitis. Acinar cell apoptosis was induced by reducing the mitochondrial membrane potential in the pioglitazone group. Pioglitazone increased expression of proapoptotic Bax proteins and decreased antiapoptotic Bcl-2 in cerulein-induced AR42j cells and decreased Bcl-2 levels in pancreatic tissue of severe acute pancreatitis rats 1 and 3 hours after induction.. Pioglitazone may promote apoptosis of acinar cells through both intrinsic and extrinsic apoptotic pathways in acute pancreatitis.

Topics: Acinar Cells; Acute Disease; Amylases; Anilides; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cell Survival; Ceruletide; Cytokines; Enzyme-Linked Immunosorbent Assay; Humans; Male; Membrane Potential, Mitochondrial; Pancreatitis; Pioglitazone; PPAR gamma; Proto-Oncogene Proteins c-bcl-2; Rats, Sprague-Dawley; Severity of Illness Index; Thiazolidinediones

Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.

Topics: Acinar Cells; Amylases; Animals; Carcinoma, Pancreatic Ductal; Cell Cycle Checkpoints; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Ceruletide; Epithelial Cells; Fibrosis; Irritants; Lipase; Male; Metaplasia; Mice, Knockout; Mice, Transgenic; Pancreas; Pancreatic Neoplasms; Pancreatitis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta

Activation of the transcription factor NF-κB and expression of pro-inflammatory mediators have been considered as major events of acute pancreatitis (AP). Karyopherin alpha 2 (KPNA2), a member of the importin α family, reportedly modulates p65 subcellular localization.. This study aimed to investigate the expression and possible functions of KPNA2 in the AP cell and animal model, focusing on its association with NF-κB activation.. An AP cell model was established with the cerulein-stimulated AR42J and isolated rat pancreatic acinar cells. The AP rat model was induced by the intraperitoneal injection of cerulein. The secretion of TNF-α, IL-6, and LDH was detected by ELISA kits and the production of NO using nitric oxide kit. Expression of KPNA2 was measured by RT-PCR and Western blot. Expression levels of IKKα, phosphorylation of p65, and total p65 were detected by Western blot. Co-localization of KPNA2 with p65 was observed by immunofluorescence assay. To determine the biological functions of KPNA2 in cerulein-induced inflammatory response, RNA interference was employed to knockdown KPNA2 expression in AR42J and isolated pancreatic acini cells.. Cerulein stimulated KPNA2 expression and IL-6, TNF-α, NO, and LDH production in rat pancreatic acinar cells. Cerulein triggered the phosphorylation and nuclear translocation of NF-κB p65 subunit, indicating the NF-κB activation. The co-localization and nuclear accumulation of KPNA2 and p65 were detected in cerulein-treated cells. Knocking down KPNA2 hindered cerulein-induced nuclear transportation of p65 and alleviated the subsequent inflammatory response in rat pancreatic acinar cells. Additionally, KPNA2 expression was significantly up-regulated in cerulein-induced AP rat model.. KPNA2-facilitated p65 nuclear translocation promotes NF-κB activation and inflammation in acute pancreatitis.

Topics: Acinar Cells; Acute Disease; alpha Karyopherins; Animals; Blotting, Western; Cell Line; Ceruletide; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Gene Expression Regulation; Gene Knockdown Techniques; I-kappa B Kinase; Inflammation; Interleukin-6; Lactate Dehydrogenases; Male; NF-kappa B; Nitric Oxide; Pancreas; Pancreatitis; Phosphorylation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Up-Regulation

Under conditions of inflammation in the absence of micro-organisms (sterile inflammation), necrotic cells release damage-associated molecular patterns that bind to Toll-like receptors on immune cells to activate a signaling pathway that involves activation of IκB kinase and nuclear factor κB (NF-κB). Little is known about the mechanisms that control NF-κB activity during sterile inflammation. We analyzed the contribution of B-cell CLL/lymphoma 3 (BCL3), a transcription factor that associates with NF-κB, in control of sterile inflammation in the pancreas and biliary system of mice.. Acute pancreatitis (AP) was induced in C57BL/6 (control) and Bcl3(-/-) mice by intraperitoneal injection of cerulein or pancreatic infusion of sodium taurocholate. We also studied Mdr2(-/-) mice, which develop spontaneous biliary inflammation, as well as Bcl3(-/-)Mdr2(-/-) mice. We performed immunohistochemical analyses of inflamed and noninflamed regions of pancreatic tissue from patients with AP or primary sclerosing cholangitis (PSC), as well as from mice. Immune cells were characterized by fluorescence-activated cell sorting analysis. Control or Bcl3(-/-) mice were irradiated, injected with bone marrow from Bcl3(-/-) or control mice, and AP was induced.. Pancreatic or biliary tissues from patients with AP or PSC had higher levels of BCL3 and phosphorylated RelA and IκBα in inflamed vs noninflamed regions. Levels of BCL3 were higher in pancreata from control mice given cerulein than from mice without AP, and were higher in biliary tissues from Mdr2(-/-) mice than from control mice. Bcl3(-/-) mice developed more severe AP after administration of cerulein or sodium taurocholate than control mice; pancreata from the Bcl3(-/-) mice with AP had greater numbers of macrophages, myeloid-derived suppressor cells, dendritic cells, and granulocytes than control mice with AP. Activation of NF-κB was significantly prolonged in Bcl3(-/-) mice with AP, compared with control mice with AP. Bcl3(-/-)Mdr2(-/-) mice developed more severe cholestasis and had increased markers of liver injury and increased proliferation of biliary epithelial cells and hepatocytes than Mdr2(-/-) mice. In experiments with bone marrow chimeras, expression of BCL3 by acinar cells, but not myeloid cells, was required for reduction of inflammation during development of AP. BCL3 inhibited ubiquitination and proteasome-mediated degradation of p50 homodimers, which prolonged binding of NF-κB heterodimers to DNA.. BCL3 is up-regulated in inflamed pancreatic or biliary tissues from mice and patients with AP or cholangitis. Its production appears to reduce the inflammatory response in these tissues via blocking ubiquitination and proteasome-mediated degradation of p50 homodimers.

Topics: Acute Disease; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; B-Cell Lymphoma 3 Protein; Bile Ducts; Bone Marrow Transplantation; Ceruletide; Cholangitis, Sclerosing; Humans; I-kappa B Proteins; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Pancreas; Pancreatitis; Phosphorylation; Proteasome Endopeptidase Complex; Protein Multimerization; Proteolysis; Proto-Oncogene Proteins; Signal Transduction; Taurocholic Acid; Time Factors; Transcription Factor RelA; Transcription Factors; Ubiquitination

The extracellular matrix molecule periostin (POSTN, encoded by POSTN), which is secreted by activated pancreatic stellate cells, has important functions in chronic pancreatitis and pancreatic cancer. However, the role of POSTN in acute pancreatitis and subsequent regeneration processes has not been addressed so far. We analyzed the function of POSTN in pancreatic exocrine regeneration after the induction of a severe acute pancreatitis. Postn-deficient mice and wild-type control animals received repetitive cerulein injections, and a detailed histologic analysis of pancreatic tissues was performed. Although there was no difference in pancreatitis severity in the acute inflammatory phase, the recovery of the exocrine pancreas was massively impaired in Postn-deficient mice. Loss of Postn expression was accompanied by strong pancreatic atrophy and acinar-to-adipocyte differentiation, which was also reflected in gene expression patterns. Our data suggest that POSTN is a crucial factor for proper exocrine lineage-specific regeneration after severe acute pancreatitis.

Topics: Animals; Atrophy; Cell Adhesion Molecules; Ceruletide; Disease Models, Animal; Immunohistochemistry; Mice; Mice, Knockout; Pancreas, Exocrine; Pancreatitis; Real-Time Polymerase Chain Reaction; Regeneration

To date, it remains unclear whether mild form of acute pancreatitis (AP) may cause myocardial damage which may be asymptomatic for a long time. Pathogenesis of AP-related cardiac injury may be attributed in part to ROS/RNS overproduction. The aim of the present study was to evaluate the oxidative stress changes in both the pancreas and the heart and to estimate the protective effects of 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (tempol) at the early phase of AP. Cerulein-induced AP led to the development of acute edematous pancreatitis with a significant decrease in the level of sulfhydryl (-SH) groups (oxidation marker) both in heart and in pancreatic tissues as well as a substantial increase in plasma creatine kinase isoenzyme (CK-MB) activity (marker of the heart muscle lesion) which confirmed the role of oxidative stress in the pathogenesis of cardiac damage. The tempol treatment significantly reduced the intensity of inflammation and oxidative damage and decreased the morphological evidence of pancreas injury at early AP stages. Moreover, it markedly attenuated AP-induced cardiac damage revealed by normalization of the -SH group levels and CK-MB activity. On the basis of these studies, it is possible to conclude that tempol has a profound protective effect against cardiac and pancreatic damage induced by AP.

Topics: Amylases; Animals; Anti-Inflammatory Agents; Antioxidants; Cardiotonic Agents; Cell Membrane Permeability; Ceruletide; Creatine Kinase, MB Form; Cyclic N-Oxides; Disease Models, Animal; Free Radical Scavengers; Male; Oxidative Stress; Pancreas; Pancreatitis; Rats, Wistar; Spin Labels; Water

Lysosomal integral membrane protein-2 (LIMP-2) is an important protein in lysosomal biogenesis and function and also plays a role in the tissue inflammatory response. It is known that lysosomes play a central role in acute pancreatitis, with inflammatory cell infiltration triggering the disease early on. In this study we report increases in pancreatic LIMP-2 protein and mRNA levels as early events that occur during the development of cerulein (Cer)-induced acute pancreatitis (AP) in rats. GdCl3, a macrophage inhibitor, but not FK506, a T lymphocyte inhibitor, was able to reverse the increase in LIMP-2 expression after Cer treatment, although such reversion was abolished if the animals were depleted of neutrophils due to a vinblastine sulfate pre-treatment. Immunostaining revealed that the cellular source of LIMP-2 was mainly acinar cells. Additionally, pre-treatments with the MAPKs inhibitors SP600125 and PD98059, inhibitors of JNK and ERK½ activation, respectively, but not of rolipram, a type IV phosphodiesterase inhibitor, suppressed the increase in the expression of LIMP-2 after Cer administration. Together, these results indicate that neutrophils are able to drive a macrophage activation that would regulate the increase in LIMP-2 expression during the early phase of Cer-induced AP, with the stress kinases JNK and ERK½ also playing a coordinated role in the increase of LIMP-2 expression due to Cer.

Topics: Animals; CD36 Antigens; Ceruletide; Cyclic AMP; Gene Expression Regulation; Lysosomal Membrane Proteins; Male; Mitogen-Activated Protein Kinases; Neutrophil Infiltration; Pancreatitis; Rats; Rats, Wistar; Rolipram; Signal Transduction

Acute pancreatitis is an inflammatory process originated in the pancreas; however, it often leads to systemic complications that affect distant organs. Acute respiratory distress syndrome is indeed the predominant cause of death in patients with severe acute pancreatitis. In this study, we aimed to delineate the ameliorative effect of dihydro-resveratrol, a prominent analog of trans-resveratrol, against acute pancreatitis-associated lung injury and the underlying molecular actions. Acute pancreatitis was induced in rats with repetitive injections of cerulein (50 µg/kg/h) and a shot of lipopolysaccharide (7.5 mg/kg). By means of histological examination and biochemical assays, the severity of lung injury was assessed in the aspects of tissue damages, myeloperoxidase activity, and levels of pro-inflammatory cytokines. When treated with dihydro-resveratrol, pulmonary architectural distortion, hemorrhage, interstitial edema, and alveolar thickening were significantly reduced in rats with acute pancreatitis. In addition, the production of pro-inflammatory cytokines and the activity of myeloperoxidase in pulmonary tissues were notably repressed. Importantly, nuclear factor-kappaB (NF-κB) activation was attenuated. This study is the first to report the oral administration of dihydro-resveratrol ameliorated acute pancreatitis-associated lung injury via an inhibitory modulation of pro-inflammatory response, which was associated with a suppression of the NF-κB signaling pathway.

Topics: alpha-Amylases; Animals; Ceruletide; Cytokines; Lung; Lung Diseases; NF-kappa B; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley; Resveratrol; Signal Transduction; Stilbenes

To explore the effect of B7-H3 monoclonal antibody (mAb) on cerulein-induced acute pancreatitis (AP).. Mice were randomly divided into three groups: control group, AP group and B7-H3 mAb treatment group. AP was induced in mice by intraperitoneal injections of cerulein. B7-H3 mAb was administered to the mice by subcutaneous injection 1 hour before the injections of cerulein. The blood, pancreas and lung tissues of the mice were collected 6, 12 and 24 hours after cerulein induction. Expression of B7-H3 protein was detected in the pancreas tissues of the control and AP groups by Western blotting and immunohistochemistry. Serum activities of amylase and lipase were tested by VITROS 5600 Integrated System. The pancreas wet-dry mass ratio was used to value the edema of pancreas. Pathological changes of pancreas and lung tissues were evaluated by HE staining. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were detected by ELISA in all groups.. The level of B7-H3 protein increased in the pancreas tissues of the AP group after successful induction of cerulein, and reached the peak at 12 hours. Serum activities of amylase and lipase in the AP group were significantly higher than those in the control group, while decreased obviously after the intervention of B7-H3 mAb. H&E staining showed that evident inflammation appeared in pancreas and lung tissues of the AP group, and the inflammation and wet-dry mass ratio were markedly reduced in the treatment group. The levels of proinflammatory factors TNF-α, IL-6 and IL-1β in the AP group showed a time-dependent increase, and peaked at 12 hours, while in the treatment group were relatively lower.. B7-H3 is over-expressed in cerulein-induced AP. Anti-B7-H3 mAb can attenuate the inflammation and alleviate the injury of pancreas and lung tissues.

Topics: Acute Disease; Amylases; Animals; Antibodies, Monoclonal; B7 Antigens; Blotting, Western; Ceruletide; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Interleukin-1beta; Interleukin-6; Lipase; Lung; Male; Mice; Pancreas; Pancreatitis; Time Factors; Tumor Necrosis Factor-alpha

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an exuberant inflammatory desmoplastic response. The PDAC microenvironment is complex, containing both pro- and antitumorigenic elements, and remains to be fully characterized. Here, we show that sensory neurons, an under-studied cohort of the pancreas tumor stroma, play a significant role in the initiation and progression of the early stages of PDAC. Using a well-established autochthonous model of PDAC (PKC), we show that inflammation and neuronal damage in the peripheral and central nervous system (CNS) occurs as early as the pancreatic intraepithelial neoplasia (PanIN) 2 stage. Also at the PanIN2 stage, pancreas acinar-derived cells frequently invade along sensory neurons into the spinal cord and migrate caudally to the lower thoracic and upper lumbar regions. Sensory neuron ablation by neonatal capsaicin injection prevented perineural invasion (PNI), astrocyte activation, and neuronal damage, suggesting that sensory neurons convey inflammatory signals from Kras-induced pancreatic neoplasia to the CNS. Neuron ablation in PKC mice also significantly delayed PanIN formation and ultimately prolonged survival compared with vehicle-treated controls (median survival, 7.8 vs. 4.5 mo; P = 0.001). These data establish a reciprocal signaling loop between the pancreas and nervous system, including the CNS, that supports inflammation associated with oncogenic Kras-induced neoplasia. Thus, pancreatic sensory neurons comprise an important stromal cell population that supports the initiation and progression of PDAC and may represent a potential target for prevention in high-risk populations.

Topics: Adenocarcinoma in Situ; Afferent Pathways; Animals; Animals, Newborn; Capsaicin; Carcinoma, Pancreatic Ductal; Ceruletide; Denervation; Disease Progression; Female; Ganglia, Sympathetic; Genes, ras; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelitis; Neoplasm Invasiveness; Pancreas; Pancreatic Neoplasms; Pancreatitis; Precancerous Conditions; Sensory Receptor Cells; Spinal Cord; Spinothalamic Tracts; Thoracic Vertebrae

Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts.

Topics: Animals; Ceruletide; Chromatin; Cytokines; Disease Models, Animal; Extracellular Traps; Flow Cytometry; Humans; Hydrolases; Immunohistochemistry; Interleukin-17; Mice; Neutrophils; Pancreas; Pancreatic Ducts; Pancreatic Juice; Pancreatitis; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Reverse Transcriptase Polymerase Chain Reaction

Trans-resveratrol is a natural stilbenoid possessing multifarious pharmacological benefits; however, when orally consumed, it is rapidly metabolised by colonic microflora and converted to dihydro-resveratrol. Thus, this microbial metabolite is of great therapeutic relevance. In the present study, upon the oral administration of dihydro-resveratrol (10-50 mg/kg), the severity of acute pancreatitis in the cerulein-treated rats was significantly ameliorated as evidenced by decreased α-amylase activities in the plasma and lessened oedema formation in the pancreatic parenchyma. In addition, the generation of intracellular reactive oxidative products, including malondialdehyde and protein carbonyls, was accordingly reduced, so as the production of pro-inflammatory cytokines. While inhibiting the activities of NADPH oxidase and myeloperoxidase, the depletion of glutathione was considerably restored. Importantly, the attenuation of pancreatic oxidative damage by dihydro-resveratrol was associated with a down-regulation of the nuclear factor-kappaB and phosphatidylinositol 3'-kinase-serine/threonine kinase signalling pathways. Furthermore, we demonstrated that the solubility of dihydro-resveratrol was at least 5 times higher than trans-resveratrol whilst exhibiting a much lower cytotoxicity. Collectively, the current findings accentuate new mechanistic insight of dihydro-resveratrol in pancreatic oxidative damage, and advocate its therapeutic potential for the management of acute pancreatitis, particularly for patients unresponsive to trans-resveratrol due to the lack of proper microbial strains.

Topics: Acute Disease; alpha-Amylases; Animals; Antioxidants; Blotting, Western; Ceruletide; Cytokines; Inflammation Mediators; Malondialdehyde; Microscopy, Fluorescence; Molecular Structure; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Phosphatidylinositol 3-Kinases; Protein Carbonylation; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Resveratrol; Signal Transduction; Stilbenes

Systemic damage in acute pancreatitis (AP) can be characterized by oxidative stress and the release of pro-inflammatory cytokines. Roflumilast has been shown to be a potent anti-inflammatory and antioxidant agent. In the present study, we aimed to investigate the effect of roflumilast in cerulein-induced AP.. Thirty-two male rats were divided into four groups: group 1 (sham), group 2 (Roflumilast), group 3 (AP), and group 4 (AP + Roflumilast). AP was induced by injecting 4 × 75 μg/kg of body weight at an interval of 1 h. Rats were killed after 12 h following the last cerulein administration. AP was confirmed by measuring the serum amylase level and inflammatory features.. Morphological changes were observed in the pancreas. Amylase levels were higher in the AP and AP + Roflumilast groups than the sham and Roflumilast groups. The serum levels of TNF-α, IL-1β, and IL-6 increased in the AP group, whereas they decreased in the Roflumilast group. The total oxidant activity (TOA) was higher and the total antioxidant capacity (TAC) was lower in the AP group. The administration of roflumilast decreased the TOA and increased the TAC in comparison with the AP group (p < 0.05 for both).. Roflumilast significantly decreases oxidative stress and inflammatory mediators in the plasma, pancreas, and lung in cerulein-induced AP rats.

Topics: Acute Disease; Aminopyridines; Amylases; Animals; Benzamides; Ceruletide; Cyclopropanes; Disease Models, Animal; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Lung; Male; Oxidative Stress; Pancreas; Pancreatitis; Rats, Wistar; Tumor Necrosis Factor-alpha

Proper regulation of cytosolic Ca(2+) is critical for pancreatic acinar cell function. Disruptions in normal Ca(2+) concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca(2+) homeostasis by promoting rapid Ca(2+) movement. Determining how expression of Ca(2+) modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca(2+) ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca(2+) . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca(2+) levels. Combined, these results suggest SPCA2C affects Ca(2+) homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca(2+) ATPases. J. Cell. Physiol. 231: 2768-2778, 2016. © 2016 Wiley Periodicals, Inc.

Topics: Acinar Cells; Amino Acid Sequence; Animals; Base Sequence; Calcium; Calcium-Transporting ATPases; Ceruletide; Epigenesis, Genetic; Exons; Female; Genome; HEK293 Cells; Histones; Humans; Male; Mice, Inbred C57BL; Mice, Transgenic; Pancreas; Pancreatitis; Promoter Regions, Genetic; RNA, Messenger; Transcription Initiation Site; Transcription, Genetic

Severe acute pancreatitis (AP) is responsible for significant human morbidity and mortality worldwide. Currently, no specific treatments for AP exist, primarily due to the lack of a mechanistic understanding of sterile inflammation and the resultant multisystem organ dysfunction, the pathologic response of AP linked to early death. In this study, we demonstrate that the class III major histocompatibility region III receptor for advanced glycation end products (RAGE) contributes to AP by modulating inflammasome activation in macrophages. RAGE mediated nucleosome-induced absent in melanoma 2 (but not NLRP3) inflammasome activation by modulating dsRNA-dependent protein kinase phosphorylation in macrophages. Pharmacological and genetic inhibition of the RAGE-dsRNA-dependent protein kinase pathway attenuated the release of inflammasome-dependent exosomal leaderless cytokines (e.g., IL-1β and high-mobility group box 1) in vitro. RAGE or absent in melanoma 2 depletion in mice limited tissue injury, reduced systemic inflammation, and protected against AP induced by l-arginine or cerulein in experimental animal models. These findings define a novel role for RAGE in the propagation of the innate immune response with activation of the nucleosome-mediated inflammasome and will help guide future development of therapeutic strategies to treat AP.

Topics: Animals; Arginine; Ceruletide; DNA-Binding Proteins; HMGB1 Protein; Immunity, Innate; Inflammasomes; Interleukin-1beta; Kaplan-Meier Estimate; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; NLR Family, Pyrin Domain-Containing 3 Protein; Nucleosomes; Pancreatitis; Receptor for Advanced Glycation End Products; Signal Transduction; Toll-Like Receptor 4

Autophagy promotes tumor progression downstream of oncogenic KRAS, yet also restrains inflammation and dysplasia through mechanisms that remain incompletely characterized. Understanding the basis of this paradox has important implications for the optimal targeting of autophagy in cancer. Using a mouse model of cerulein-induced pancreatitis, we found that loss of autophagy by deletion of Atg5 enhanced activation of the IκB kinase (IKK)-related kinase TBK1 in vivo, associated with increased neutrophil and T-cell infiltration and PD-L1 upregulation. Consistent with this observation, pharmacologic or genetic inhibition of autophagy in pancreatic ductal adenocarcinoma cells, including suppression of the autophagy receptors NDP52 or p62, prolonged TBK1 activation and increased expression of CCL5, IL6, and several other T-cell and neutrophil chemotactic cytokines in vitro Defective autophagy also promoted PD-L1 upregulation, which is particularly pronounced downstream of IFNγ signaling and involves JAK pathway activation. Treatment with the TBK1/IKKε/JAK inhibitor CYT387 (also known as momelotinib) not only inhibits autophagy, but also suppresses this feedback inflammation and reduces PD-L1 expression, limiting KRAS-driven pancreatic dysplasia. These findings could contribute to the dual role of autophagy in oncogenesis and have important consequences for its therapeutic targeting. Cancer Immunol Res; 4(6); 520-30. ©2016 AACR.

Topics: Acute Disease; Adenocarcinoma; Animals; Autophagy; Autophagy-Related Protein 5; B7-H1 Antigen; Benzamides; Cell Transformation, Neoplastic; Ceruletide; Chemokine CCL5; Cytokines; Enzyme Activation; Gene Deletion; Mice; Pancreatic Neoplasms; Pancreatitis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Pyrimidines; Signal Transduction; Tumor Cells, Cultured

B10 cells are specific B cell subsets with the capacity of producing IL-10 to inhibit immune responses. Several studies have demonstrated that B10 cells are correlated with some immune and inflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CA), colitis and contact hypersensitivity. However, its role in severe acute pancreatitis (SAP) has not been clearly demonstrated yet.. In this study, we show that B10 cells can inhibit inflammation of severe acute pancreatitis (SAP).. Blood from 17 patients with SAP and 22 age-matched healthy volunteers were collected to detect the proportion of B10 cells. CD19-/- mice were used as B10 cell-deficient mice. Amylase and lipase levels, pancreatic edema and HE staining were tested to assess the severity of SAP.. CD19-/- mice, which lack B10 cells, suffered a more severe inflammation in pancreas compared with wild-type mice after caerulein injection. The frequency of B10 cells was decreased both in SAP patients and SAP animal models. Adoptive transfer of B10 cells ameliorates inflammatory injury of pancreatitis in CD19-/- mice.. Thus, we identified B10 cells as a protective factor for SAP and provided a novel target for SAP treatment.

Topics: Adolescent; Adoptive Transfer; Adult; Aged; Animals; Antigens, CD19; B-Lymphocyte Subsets; Ceruletide; Female; Humans; Male; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Pancreas; Pancreatitis; Spleen; Young Adult

Chronic endoplasmic reticulum (ER) stress in pancreatic acinar cells has emerged as a major contributor to the recovery of acute pancreatitis (AP). However, the molecular mechanisms linking AP and ER stress remain not fully understood. In this study, we employed caerulein to induce AP-like inflammation in the AR42J rat pancreatic acinar cells to mimic the AP-like acinar cell injury. Caerulein can activate ER stress in AR42J cells, but the molecular link between AP and ER stress remains to be identified. We here reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident multispanning membrane protein, was involved in the onset of AP-like injury on AR42J cells. TRAM1 was significantly elevated in caerulein-treated AR42J cells. Furthermore, we showed that knockdown of TRAM1 led to hyperactivation of 78 kDa glucose-regulated protein precursor (GRP78) and C/EBP homologous protein (CHOP) and the activation of downstream apoptosis pathway. Given the fact that the activation of ER stress played a protection role in AP, the pro-inflammatory mediators TNF-α and IL-6 and the marker of cell injury LDH were also analyzed. We found that depletion of TRAM1 markedly increased the secretion of TNF-α, IL-6, and LDH in the cells. Moreover, flow cytometry indicated that treatment with caerulein induced a significant decrease of apoptotic index and increase of necrosis index in TRAM1-siRNA cells, compared with control groups, as indicated by downregulated expression of cleaved caspase-3, caspase-8, and caspase-9 mRNA expression activity in TRAM1-siRNA cells. These data implicated that TRAM1 might protect AR42J cells against caerulein-induced AP in AR42J cells through alleviating ER stress.

Topics: Acinar Cells; Animals; Apoptosis; Cell Line; Ceruletide; Endoplasmic Reticulum Stress; Gene Knockdown Techniques; Membrane Glycoproteins; Membrane Transport Proteins; NF-kappa B; Pancreatitis; Rats; RNA Interference

The aim of this study was to establish a pathogenetic mechanism of pancreatitis in celiac disease and IgG4-related disease using gluten-sensitive human leukocyte antigen (HLA)-DQ8 transgenic mice.. Transgenic mice expressing HLA-DQ8 genes were utilized. Control mice were not sensitized but were fed gliadin-free rice cereal. Experimental groups consisted of gliadin-sensitized and gliadin-challenged mice; nonsensitized mice with cerulein hyperstimulation; and gliadin-sensitized and gliadinchallenged mice with cerulein hyperstimulation.. Gliadin-sensitized and gliadin-challenged mice with cerulein hyperstimulation showed significant inflammatory cell infiltrates, fibrosis and acinar atrophy compared with the control mice and the other experimental groups. The immunohistochemical analysis showed greater IgG1-positive plasma cells in the inflammatory infiltrates of gliadin-sensitized and gliadin-challenged mice with cerulein hyperstimulation compared with the control mice and the other experimental groups. Gliadin-sensitized and gliadin-challenged mice with cerulein hyperstimulation or gliadin-sensitized and gliadinchallenged mice showed IgG1-stained inflammatory cell infiltrates in the extrapancreatic organs, including the bile ducts, salivary glands, kidneys, and lungs.. Gliadinsensitization and cerulein hyperstimulation of gluten-sensitive HLA-DQ8 transgenic mice resulted in pancreatitis and extrapancreatic inflammation. This animal model suggests that chronic gliadin ingestion in a susceptible individual with the HLA-DQ8 molecule may be associated with pancreatitis and extrapancreatic inflammation.

Topics: Animals; Autoimmune Diseases; Ceruletide; Disease Models, Animal; Gliadin; HLA-DQ Antigens; Hypersensitivity; Immunoglobulin G; Inflammation; Mice; Mice, Transgenic; Pancreatitis

Acute pancreatitis (AP) is an inflammatory disease mediated by damage to acinar cells and pancreatic inflammation. In patients with AP, subsequent systemic inflammatory responses and multiple organs dysfunction commonly occur. Interactions between cytokines and oxidative stress greatly contribute to the amplification of uncontrolled inflammatory responses. Molecular hydrogen (H2) is a potent free radical scavenger that not only ameliorates oxidative stress but also lowers cytokine levels. The aim of the present study was to investigate the protective effects of H2 gas on AP both in vitro and in vivo. For the in vitro assessment, AR42J cells were treated with cerulein and then incubated in H2-rich or normal medium for 24 h, and for the in vivo experiment, AP was induced through a retrograde infusion of 5% sodium taurocholate into the pancreatobiliary duct (0.1 mL/100 g body weight). Wistar rats were treated with inhaled air or 2% H2 gas and sacrificed 12 h following the induction of pancreatitis. Specimens were collected and processed to measure the amylase and lipase activity levels; the myeloperoxidase activity and production levels; the cytokine mRNA expression levels; the 8-hydroxydeoxyguanosine, malondialdehyde, and glutathione levels; and the cell survival rate. Histological examinations and immunohistochemical analyses were then conducted. The results revealed significant reductions in inflammation and oxidative stress both in vitro and in vivo. Furthermore, the beneficial effects of H2 gas were associated with reductions in AR42J cell and pancreatic tissue damage. In conclusion, our results suggest that H2 gas is capable of ameliorating damage to the pancreas and AR42J cells and that H2 exerts protective effects both in vitro and in vivo on subjects with AP. Thus, the results obtained indicate that this gas may represent a novel therapy agent in the management of AP.

Topics: Amylases; Animals; Cell Line; Cell Survival; Ceruletide; Cytokines; Disease Models, Animal; Gene Expression Regulation; Hydrogen; Lipase; Male; Mice; Oxidative Stress; Pancreatitis; Rats; Taurocholic Acid

To evaluate the effects of certolizumab, a pegylated monoclonal antibody to tumor necrosis factor α (TNF-α), on experimentally induced acute pancreatitis.. Healthy Wistar Albino male rats (n = 36) were randomly divided into 4 groups (9 rats in each group): group 1, control group; group 2, certolizumab group; group 3, cerulein group; and group 4, cerulein + certolizumab group. Acute edematous pancreatitis was induced via intraperitoneal injection of 80-μg/kg cerulein (20 μg/kg, 4 times at 1-hour intervals) in groups 3 and 4. Certolizumab (10 μg) was intraperitoneally administered in groups 2 and 4. Serum levels of amylase, lipase, TNF-α, and lactate dehydrogenase were evaluated. Histopathology and immunohistochemistry of the pancreatic tissue for assessing the activities of malondialdehyde, myeloperoxidase, TNF-α, and caspase-3 were also performed after 72 hours.. Certolizumab treatment significantly decreased the serum levels of amylase, lipase, and lactate dehydrogenase. Histopathological edema, hemorrhage, parenchymal necrosis, and infiltration scores were also decreased, along with a decrease in malondialdehyde, myeloperoxidase, TNF-α, and caspase-3 activities.. This study suggests that certolizumab is a beneficial treatment mode for reducing the severity of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Caspase 3; Ceruletide; Male; Pancreatitis; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia-reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia-reperfusion injury model, the high-dose TNF model, and in A20(-/-) mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia-reperfusion injury model and no benefit in A20(-/-) mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

Topics: Animals; Apoptosis; Ceruletide; Colitis; Dextran Sulfate; Disease Models, Animal; Female; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Reperfusion Injury; Sepsis; Systemic Inflammatory Response Syndrome; Tumor Necrosis Factor alpha-Induced Protein 3

Surface CD24 has previously been described, together with CD44 and ESA, for the characterization of putative cancer stem cells in pancreatic ductal adenocarcinoma (PDAC), the most fatal of all solid tumors. CD24 has a variety of biological functions including the regulation of invasiveness and cell proliferation, depending on the tumor entity and subcellular localization. Genetically engineered mouse models (GEMM) expressing oncogenic KrasG12D recapitulate the human disease and develop PDAC. In this study we investigate the function of CD24 using GEMM of endogenous PDAC and a model of cerulein-induced acute pancreatitis. We found that (i) CD24 expression was upregulated in murine and human PDAC and during acute pancreatitis (ii) CD24 was expressed exclusively in differentiated PDAC, whereas CD24 absence was associated with undifferentiated tumors and (iii) membranous CD24 expression determines tumor subpopulations with an epithelial phenotype in grafted models. In addition, we show that CD24 protein is stabilized in response to WNT activation and that overexpression of CD24 in pancreatic cancer cells upregulated β-catenin expression augmenting an epithelial, non-metastatic signature. Our results support a positive feedback model according to which (i) WNT activation and subsequent β-catenin dephosphorylation stabilize CD24 protein expression, and (ii) sustained CD24 expression upregulates β-catenin expression. Eventually, membranous CD24 augments the epithelial phenotype of pancreatic tumors. Thus we link the WNT/β-catenin pathway with the regulation of CD24 in the context of PDAC differentiation.

Topics: Animals; Carcinoma, Pancreatic Ductal; CD24 Antigen; Cell Differentiation; Cell Membrane; Cell Proliferation; Ceruletide; Epithelial-Mesenchymal Transition; Epithelium; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Knockout; Mice, SCID; Neoplasm Transplantation; Pancreatic Neoplasms; Pancreatitis; Phenotype; Phosphorylation; Proto-Oncogene Proteins p21(ras); Up-Regulation

Chronic pancreatitis is a significant risk factor for pancreatic cancer. Previously, we demonstrated that the pancreatic cancer cells show enhanced expression of collapsin response mediator protein 4 (CRMP4) that strongly correlates with severe venous invasion, liver metastasis, and poor prognosis. However, involvement of CRMP4 in acute or chronic pancreatitis remains unknown.. Acute and chronic pancreatitis mice models were developed by periodic injection of caerulein. The expression levels of CRMP4 and its phosphorylation were examined.. Elevated CRMP4 levels were observed in the infiltrated lymphocytes as well as in the pancreas parenchyma of both acute and chronic pancreatitis. The expression pattern of phosphorylated CRMP4 was similar to that of CRMP4. Cdk5 partially co-localized with the phosphorylated CRMP4.. Pancreatitis induces CRMP4 expression in the pancreas parenchyma and in the infiltrated lymphocytes. Overlapping expression of CRMP4 and Cdk5 may suggest that the Cdk5 is at least, in part, responsible for the phosphorylation of CRMP4. The results suggest that CRMP4 is involved in the inflammatory response in pancreatitis. Understanding the mechanisms of CRMP4 would help us to develop novel therapeutic strategies against acute or chronic pancreatitis, and pancreatic cancer.

Topics: Acute Disease; Animals; Biopsy, Needle; Cell Transformation, Neoplastic; Ceruletide; Chronic Disease; Cyclin-Dependent Kinase 5; Disease Models, Animal; Gene Expression Regulation; Humans; Immunohistochemistry; Mice; Nerve Tissue Proteins; Pancreatic Neoplasms; Pancreatitis; Phosphorylation; Precancerous Conditions; RNA, Small Interfering

Background. Chronic pancreatitis is one of the main risk factors for pancreatic cancer. In acute and chronic pancreatitis, oxidative stress is thought to play a key role. In this respect, the recently described mitochondria-targeted antioxidant SkQ1 effectively scavenges reactive oxygen species at nanomolar concentrations. Therefore, we aimed to characterize the influence of SkQ1 on tissue injury and pain in acute and chronic pancreatitis. Methods. Both acute and chronic pancreatitis were induced in C57BL/6 mice by intraperitoneal cerulein injections and treatment with SkQ1 was carried out by peroral applications. Hyperalgesia was assessed by behavioral observation and measurement of abdominal mechanical sensitivity. Blood serum and pancreatic tissue were harvested for analysis of lipase and histology. Results. SkQ1 did not influence pain, serological, or histological parameters of tissue injury in acute pancreatitis. In chronic pancreatitis, a highly significant reduction of pain-related behavior (p < 0.0001) was evident, but histological grading revealed increased tissue injury in SkQ1-treated animals (p = 0.03). Conclusion. After SkQ1 treatment, tissue injury is not ameliorated in acute pancreatitis and increased in chronic pancreatitis. However, we show an analgesic effect in chronic pancreatitis. Further studies will need to elucidate the risks and benefits of mitochondria-targeted antioxidants as an analgesic.

Topics: Acute Disease; Analgesics; Animals; Antioxidants; Behavior, Animal; Biomarkers; Ceruletide; Disease Models, Animal; Female; Hyperalgesia; Lipase; Mice, Inbred C57BL; Mitochondria; Motor Activity; Pain Perception; Pancreas; Pancreatitis; Pancreatitis, Chronic; Plastoquinone; Risk Factors

The hamster has been shown to share a variety of metabolic similarities with humans. To replicate human acute pancreatitis with hamsters, we comparatively studied the efficacy of common methods, such as the peritoneal injections of caerulein, L-arginine, the retrograde infusion of sodium taurocholate, and another novel model with concomitant administration of ethanol and fatty acid. The severity of pancreatitis was evaluated by serum amylase activity, pathological scores, myeloperoxidase activity, and the expression of inflammation factors in pancreas. The results support that the severity of pathological injury is consistent with the pancreatitis induced in mice and rat using the same methods. Specifically, caerulein induced mild edematous pancreatitis accompanied by minimal lung injury, while L-arginine induced extremely severe pancreatic injury including necrosis and neutrophil infiltration. Infusion of Na-taurocholate into the pancreatic duct induced necrotizing pancreatitis in the head of pancreas and lighter inflammation in the distal region. The severity of acute pancreatitis induced by combination of ethanol and fatty acids was between the extent of caerulein and L-arginine induction, with obvious inflammatory cells infiltration. In view of the advantages in lipid metabolism features, hamster models are ideally suited for the studies of pancreatitis associated with altered metabolism in humans.

Topics: Amylases; Animals; Arginine; Ceruletide; Disease Models, Animal; Ethanol; Fatty Acids; Humans; Injections; Mesocricetus; Pancreatitis; Peroxidase; Severity of Illness Index; Taurocholic Acid

Acute pancreatitis is an inflammatory process that frequently involves peripancreatic tissues and remote organ systems. It has high morbidity and mortality rates in both human and veterinary patients. The severity of pancreatitis is generally determined by events that occur after acinar cell injury in the pancreas, resulting in elevated levels of various proinflammatory mediators, such as interleukin (IL) 1β and 6, as well as tumor necrosis factor alpha (TNF-α). When these mediators are excessively released into the systemic circulation, severe pancreatitis occurs with systemic complications. This pathophysiological process is similar to that of sepsis; thus, there are many striking clinical similarities between patients with septic shock and those with severe acute pancreatitis. We induced acute pancreatitis using caerulein in dogs and measured the change in the gene expression of proinflammatory cytokines. The levels of TNF-α and IL-6 mRNA peaked at 3 h, at twice the baseline levels, and the serum concentrations of amylase and lipase also increased. Histopathological examination revealed severe hyperemia of the pancreas and hyperemia in the duodenal villi and the hepatic sinusoid. Thus, pancreatitis can be considered an appropriate model to better understand the development of naturally occurring sepsis and to assist in the effective treatment and management of septic patients.. La pancréatite aigüe est un processus inflammatoire qui implique fréquemment les tissus péri-pancréatiques et des systèmes organiques éloignés. Elle a des taux de morbidité et de mortalité élevés autant chez les humains que chez les animaux. La sévérité de la pancréatite est généralement déterminée par des évènements qui se produisent suite à des dommages aux cellules acinaires dans le pancréas, et qui induisent des niveaux élevés de différents médiateurs pro-inflammatoires, tels que l’interleukine (IL) 1β et 6, ainsi que le facteur nécrosant des tumeurs alpha (TNFα). Lorsque ces médiateurs sont libérés de manière excessive dans la circulation systémique, une pancréatite sévère se produit avec des complications systémiques. Ce processus pathophysiologique est similaire à celui d’un sepsis; donc, il y a plusieurs similarités cliniques entre des patients avec un choc septique et ceux avec une pancréatite aigüe sévère. Nous avons induit une pancréatite aigüe en utilisant de la caeruléine chez des chiens et avons mesuré le changement dans l’expression des gènes des cytokines pro-inflammatoires. Les niveaux d’ARNm de TNFα et d’IL-6 ont culminé après 3 h, atteignant le double des niveaux de base, et les concentrations sériques d’amylase et de lipase augmentèrent également. Un examen histopathologique a révélé une hyperémie sévère du pancréas et une hyperémie dans les villosités duodénales et les sinusoïdes hépatiques. Ainsi, la pancréatite peut être considérée un modèle approprié pour mieux comprendre le développement d’un sepsis naturel et aider dans le traitement efficace et la gestion de patients septiques.(Traduit par Docteur Serge Messier).

Topics: Animals; Ceruletide; Dog Diseases; Dogs; Gene Expression Regulation; Interleukin-6; Pancreatitis; RNA, Messenger; Tumor Necrosis Factor-alpha

BACKGROUND We evaluated the hematological, biochemical, and histopathological effects of Montelukast on pancreatic damage in an experimental acute pancreatitis model created by cerulein in rats before and after the induction of pancreatitis. MATERIAL AND METHODS Forty rats were divided into 4 groups with 10 rats each. The study groups were: the Cerulein (C) group, the Cerulein + early Montelukast (CMe) group, the Cerulein + late Montelukast (CMl) group, and the Control group. The pH, pO2, pCO2, HCO3, leukocyte, hematocrit, pancreatic amylase, and lipase values were measured in the arterial blood samples taken immediately before rats were killed. RESULTS There were statistically significant differences between the C group and the Control group in the values of pancreatic amylase, lipase, blood leukocyte, hematocrit, pH, pO2, pCO2, HCO3, and pancreatic water content, and also in each of the values of edema, inflammation, vacuolization, necrosis, and total histopathological score (P<0.05). When the CMl group and C group were compared, no statistically significant differences were found in any parameter analyzed. When the CMe group was compared with the C group, pancreatic amylase, lipase, pH, PO2, pCO2, HCO3, pancreatic water content, histopathological edema, inflammation, and total histopathological score values were significantly different between the groups (P<0.05). Finally, when the CMe group and the Control group were compared, significant differences were found in all except 2 (leukocyte and pO2) parameters (P<0.05). CONCLUSIONS Leukotriene receptor antagonists used in the late phases of pancreatitis might not result in any benefit; however, when they are given in the early phases or prophylactically, they may decrease pancreatic damage.

Topics: Acetates; Amylases; Animals; Ceruletide; Cyclopropanes; Disease Models, Animal; Edema; Leukotriene Antagonists; Lipase; Male; Pancreatitis; Quinolines; Rats; Rats, Sprague-Dawley; Sulfides

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

Topics: Animals; Ceruletide; Cysteine Endopeptidases; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis

Perturbation of pancreatic acinar cell state can lead to acinar-to-ductal metaplasia (ADM), a precursor lesion to the development of pancreatic ductal adenocarcinoma (PDAC). In the pancreas, Notch signaling is active both during development and in adult cellular differentiation processes. Hes1, a key downstream target of the Notch signaling pathway, is expressed in the centroacinar compartment of the adult pancreas as well as in both preneoplastic and malignant lesions. In this study, we used a murine genetic in vivo approach to ablate Hes1 in pancreatic progenitor cells (Ptf1a

Topics: Acinar Cells; Animals; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Plasticity; Ceruletide; Disease Models, Animal; Female; Humans; Male; Metaplasia; Mice; Pancreas; Pancreas, Exocrine; Pancreatic Neoplasms; Pancreatitis; Regeneration; Signal Transduction; Stem Cells; Transcription Factor HES-1

In the current study, we have characterized the global miRNA expression profile in mouse pancreatic acinar cells and during acute pancreatitis using next-generation RNA sequencing. We identified 324 known and six novel miRNAs that are expressed in mouse pancreatic acinar cells. In the basal state, miR-148a-3p, miR-375-3p, miR-217-5p, and miR-200a-3p were among the most abundantly expressed, whereas miR-24-5p and miR-421-3p were the least abundant. Treatment of acinar cells with caerulein (100 nM) and taurolithocholic acid 3-sulfate [TLC-S (250 μM)] induced numerous changes in miRNA expression profile. In particular, we found significant overexpression of miR-21-3p in acini treated with caerulein and TLC-S. We further looked at the expression of miR-21-3p in caerulein, l-arginine, and caerulein + LPS-induced acute pancreatitis mouse models and found 12-, 21-, and 50-fold increased expression in the pancreas, respectively. In summary, this is the first comprehensive analysis of global miRNA expression profile of mouse pancreatic acinar cells in normal and disease conditions. Our analysis shows that miR-21-3p expression level correlates with the severity of the disease.

Topics: Acinar Cells; Animals; Ceruletide; Gene Expression; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Mice; MicroRNAs; Pancreatitis; Taurolithocholic Acid

Coagulation is recognized as a key player in inflammatory and autoimmune diseases. The aim of the current research was to examine the effect of pretreatment with acenocoumarol on the development of acute pancreatitis (AP) evoked by cerulein.. AP was induced in rats by cerulein administered intraperitoneally. Acenocoumarol (50, 100 or 150 µg/kg/dose/day) or saline were given once daily for seven days before AP induction.. In rats with AP, pretreatment with acenocoumarol administered at the dose of 50 or 100 µg/kg/dose/day improved pancreatic histology, reducing the degree of edema and inflammatory infiltration, and vacuolization of acinar cells. Moreover, pretreatment with acenocoumarol given at the dose of 50 or 100 µg/kg/dose/day reduced the AP-evoked increase in pancreatic weight, serum activity of amylase and lipase, and serum concentration of pro-inflammatory interleukin-1β, as well as ameliorated pancreatic DNA synthesis and pancreatic blood flow. In contrast, acenocoumarol given at the dose of 150 μg/kg/dose did not exhibit any protective effect against cerulein-induced pancreatitis.. Low doses of acenocoumarol, given before induction of AP by cerulein, inhibit the development of that inflammation.

Topics: Acenocoumarol; Animals; Anticoagulants; Ceruletide; Dose-Response Relationship, Drug; Lipase; Male; Organ Size; Pancreas; Pancreatitis; Rats

A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis.

Topics: Animals; Cell Proliferation; Ceruletide; ErbB Receptors; Female; Gene Dosage; Gene Expression Regulation; Hippo Signaling Pathway; Humans; Mice; Mice, Inbred C57BL; Mucoproteins; Oncogene Proteins; Pancreatitis; Phosphorylation; Protein Serine-Threonine Kinases; Protein Transport; Quinazolines; Regeneration; Signal Transduction; Tyrphostins

Understanding the mechanisms by which cells sense and respond to injury is central to developing therapies to enhance tissue regeneration. Previously, we showed that pancreatic injury consisting of acinar cell damage+β-cell ablation led to islet cell transdifferentiation. Here, we report that the molecular mechanism for this requires activating protease-activated receptor-2 (PAR2), a G-protein-coupled receptor. PAR2 modulation was sufficient to induce islet cell transdifferentiation in the absence of β-cells. Its expression was modulated in an islet cell type-specific manner in murine and human type 1 diabetes (T1D). In addition to transdifferentiation, PAR2 regulated β-cell apoptosis in pancreatitis. PAR2's role in regeneration is broad, as mice lacking PAR2 had marked phenotypes in response to injury in the liver and in digit regeneration following amputation. These studies provide a pharmacologically relevant target to induce tissue regeneration in a number of diseases, including T1D.

Topics: Animals; Carbon Tetrachloride; Cell Death; Cell Lineage; Cell Proliferation; Cell Survival; Cell Transdifferentiation; Ceruletide; Diabetes Mellitus, Type 1; Extremities; Gene Expression Regulation; Glucagon; Homeodomain Proteins; Humans; Insulin; Insulin-Secreting Cells; Liver; Mice, Inbred C57BL; Mice, Knockout; Paired Box Transcription Factors; Pancreatitis; Receptor, PAR-2; Regeneration; Transcription Factors

To investigate the role of interferon regulatory factor 5 (IRF5) in reversing polarization of lung macrophages during severe acute pancreatitis (SAP). A mouse SAP model was established by intraperitoneal (ip) injections of 20 μg/kg body weight caerulein. Pathological changes in the lung were observed by hematoxylin and eosin staining. Lung macrophages were isolated from bronchoalveolar lavage fluid. The quantity and purity of lung macrophages were detected by fluorescence-activated cell sorting and evaluated by real-time polymerase chain reaction (RT-PCR). They were treated with IL-4/IRF5 specific siRNA (IRF5 siRNA) to reverse their polarization and were evaluated by detecting markers expression of M1/M2 using RT-PCR.. SAP associated acute lung injury (ALI) was induced successfully by ip injections of caerulein, which was confirmed by histopathology. Lung macrophages expressed high levels of IRF5 as M1 phenotype during the early acute pancreatitis stages. Reduction of IRF5 expression by IRF5 siRNA reversed the action of macrophages from M1 to M2 phenotype. Treatment with IRF5 siRNA can reverse the pancreatitis-induced activation of lung macrophages from M1 phenotype to M2 phenotype in SAP associated with ALI.

Topics: Acute Lung Injury; Animals; Cells, Cultured; Ceruletide; Disease Models, Animal; Female; Interferon Regulatory Factors; Macrophage Activation; Macrophages, Alveolar; Male; Mice, Inbred C57BL; Pancreatitis; Phenotype; RNA Interference; Severity of Illness Index; Signal Transduction; Time Factors; Transfection

Pancreatic cancer is one of the most aggressive cancers and has an extremely poor prognosis. Despite recent progress in both basic and clinical research, most pancreatic cancers are detected at an incurable stage owing to the absence of disease-specific symptoms. Thus, developing novel approaches for detecting pancreatic cancer at an early stage is imperative. Our in silico and immunohistochemical analyses showed that KIAA1199 is specifically expressed in human pancreatic cancer cells and pancreatic intraepithelial neoplasia, the early lesion of pancreatic cancer, in a genetically engineered mouse model and in human patient samples. We also detected secreted KIAA1199 protein in blood samples obtained from pancreatic cancer mouse models, but not in normal mice. Furthermore, we found that assessing KIAA1199 autoantibody increased the sensitivity of detecting pancreatic cancer. These results indicate the potential benefits of using KIAA1199 as a biomarker for early-stage pancreatic cancer.

Topics: Acute Disease; Animals; Autoantibodies; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Ceruletide; Databases, Genetic; Disease Models, Animal; Early Diagnosis; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hyaluronoglucosaminidase; Male; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Pancreatitis; Proteins; Tissue Array Analysis

Acute pancreatitis is basically considered as activation of inactive proenzymes in the pancreas and digestion of the gland itself. This study was performed to determine if prolidase enzyme, which plays a role in collagen metabolism, could be used as a parameter to assess the severity of pancreatitis in experimentally induced mild and severe pancreatitis.. To create experimentally induced acute pancreatitis 0.1 ml of normal saline solution (NSS) was given five times with an interval of one hour to rats in the first group; 50 µg/kg of cerulein five times with an interval of one hour in the second group; 80 µg/kg of cerulein five times with an interval of one hour in the third group, in the form of intraperitoneal injection.. When the serum prolidase values at beginning, 1st, 5th and 24th hours in group II and III were compared among themselves, there was a statistically significant increase(p < 0.05). The evaluation between groups revealed a statistically significant increase in the value of serum prolidase in group II and group III compared with the control group (p < 0.05). In comparisons performed with tissue values, a statistically significant increase determined in the value of serum prolidase in group II and group III compared with the control group was observed (p < 0.05).. The findings obtained in our study showed that prolidase activity increases directly proportionaly with the severity of pancreatitis. This allows us to postulate that prolidase enzyme activities provide guidance about the metabolism of collagen in patients with acute pancreatitis, serious damage occurring in collagen protein and metabolic control is further distorted depending on the duration and intensity of damage but to be able to speak more precisely, there is a need for further, more detailed and extensive researchs (Tab. 8, Fig. 2, Ref. 30).

Topics: Acute Disease; Amylases; Animals; Ceruletide; Collagen; Dipeptidases; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Severity of Illness Index

Acute pancreatitis continues to be associated with significant rates of mortality and morbidity, and therapeutic options are still very limited. We aimed to investigate the efficacy of trimetazidine on cerulein-induced pancreatic apoptosis and histopathological and biochemistrical consequences of acute pancreatitis.. Thirty-two Wistar albino rats were randomized into four groups (group 1: control group; group 2: acute pancreatitis group; group 3: acute pancreatitis and trimetazidine treatment group; group 4: placebo group). Acute edematous pancreatitis was induced by subcutaneous cerulein injection (20 μg/kg) four times at one-hour intervals. Trimetazidine was prepared in suspension form. In group 3, after gas anesthesia, trimetazidine was administrated to rats via a catheter. Serum interleukin (IL)-1β, tumor necrosis factor (TNF)-α, amylase, lipase and leukocyte levels, pancreatic apoptotic status and pancreatic Schoenberg scores were determined for all groups. Results are given as the mean ± SD. A value of P<0.05 was accepted as statistically significant. SPSS for Windows v15.0 was used for statistical analyses.. In the acute pancreatitis group IL-1β, amylase, lipase and leukocyte levels were elevated and pancreatic histopathological evaluation revealed a diagnosis of acute pancreatitis IL-1β amylase and lipase levels and pancreatic inflammation were decreased significantly in the trimetazidine group (P<0.01). White blood cell counts and TNF-α concentrations for the trimetazidine group and the acute pancreatitis group were not significantly different. Trimetazidine significantly reduced apoptosis in pancreatic tissues and Schoenberg scores were also significantly reduced (P<0.05).. In this study, we showed that trimetazidine treatment significantly decreases the levels of IL-1β, amylase and lipase reduces pancreatic apoptosis and ameliorates the histopathological findings of cerulein-induced acute pancreatitis. Trimetazidine could be a new therapeutic option in the early treatment of acute pancreatitis.

Topics: Animals; Apoptosis; Ceruletide; Disease Models, Animal; Male; Pancreatitis; Random Allocation; Rats; Rats, Wistar; Trimetazidine

Pancreatic acinar cell maturation is dependent on the activity of the pancreas transcription factor 1 (PTF1) complex. Induction of pancreatitis leads to MAP kinase activation and transient suppression of the acinar differentiation programme. We investigated the role of MAP kinase-interacting kinase 1 (Mnk1) in mouse exocrine pancreas development and in the response to secretagogue-induced pancreatitis.. Mnk1 expression was analysed using immunohistochemistry, RT-qPCR and western blotting. Ptf1a binding to Mnk1 was assessed by chromatin immunoprecipitation and qPCR. Acute pancreatitis was induced in wild type and Mnk1(-/-) mice by 7 h intraperitoneal injections of caerulein. In vitro amylase secretion and trypsinogen activation were assessed using freshly isolated acinar cells. In vivo secretion was quantified by secretin-stimulated MRI.. Mnk1 is expressed at the highest levels in pancreatic acinar cells and is a direct PTF1 target. Mnk1 is activated upon induction of pancreatitis and is indispensable for eIF4E phosphorylation. The pancreas of Mnk1(-/-) mice is histologically normal. Digestive enzyme content is significantly increased and c-Myc and Ccnd1 levels are reduced in Mnk1(-/-) mice. Upon induction of acute pancreatitis, Mnk1(-/-) mice show impaired eIF4E phosphorylation, activation of c-Myc and downregulation of zymogen content. Acinar cells show defective relocalisation of digestive enzymes, polarity defects and impaired secretory response in vitro and in vivo.. Mnk1 is a novel pancreatic acinar cell-specific stress response kinase that regulates digestive enzyme abundance and eIF4E phosphorylation. It is required for the physiological secretory response of acinar cells and for the homeostatic response to caerulein administration during acute pancreatitis.

Topics: Acinar Cells; Amylases; Animals; Cell Differentiation; Ceruletide; Cholangiopancreatography, Magnetic Resonance; Down-Regulation; Enzyme Activation; Eukaryotic Initiation Factor-4E; Gene Targeting; Heat-Shock Response; Mice; Mitogen-Activated Protein Kinases; Pancreas, Exocrine; Pancreatitis; Phosphorylation; Protein Serine-Threonine Kinases; Transcription Factors; Trypsinogen

We aimed to evaluate the anti-inflammatory and inhibitory effects of Lithospermum erythrorhizon (LE) on cerulein-induced acute pancreatitis (AP) in a mouse model.. Acute pancreatitis was induced via intraperitoneal injection of cerulein (50 μg/kg) every hour for 6 times. In the LE, water extract (100, 250, or 500 mg/kg) was administered intraperitoneally 1 hour before the first injection of cerulein. Six hours after AP, blood, the pancreas, and the lung were harvested for further examination. In addition, pancreatic acinar cells were isolated using a collagenase method, and then, we investigated the acinar cell viability and cytokine productions.. Treatment with LE reduced pancreatic damage and AP-associated lung injury and attenuated the severity of AP, as evidenced by the reduction in neutrophil infiltration, serum amylase and lipase levels, trypsin activity, and proinflammatory cytokine expression. In addition, treatment with LE inhibited high mobility group box 1 expression in the pancreas during AP. In accordance with in vivo data, LE inhibited the cerulein-induced acinar cell death, cytokine productions, and high-mobility group box 1 expression. Furthermore, LE also inhibited the activation of p38 mitogen-activated protein kinases.. These results suggest that LE plays a protective role during the development of AP by inhibiting the activation of p38.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Biomarkers; Cell Survival; Cells, Cultured; Ceruletide; Cytokines; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Female; HMGB1 Protein; Inflammation Mediators; Lithospermum; Mice, Inbred C57BL; Neutrophil Infiltration; p38 Mitogen-Activated Protein Kinases; Pancreas; Pancreatitis; Phytotherapy; Plant Extracts; Plants, Medicinal; Severity of Illness Index; Signal Transduction; Time Factors

Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of β-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration.

Topics: Animals; beta Catenin; Cell Cycle; Cell Differentiation; Cell Proliferation; Cellular Senescence; Ceruletide; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Down-Regulation; In Vitro Techniques; Metaplasia; Mice; Mice, Knockout; Pancreas; Pancreatitis; Regeneration

In this study, we identified the protein kinases that play the most distinct roles in the occurrence of acute pancreatitis (AP).. Gene expression profile data were downloaded from Gene Expression Omnibus database (GSE3644). The sample was from caerulein-induced AP mice. The intersection of the differentially expressed genes in AP mice taken from a protein kinase database was obtained for screening of the protein kinase encoded genes that were differentially expressed. Database for annotation, visualization, and integrated discovery was used for the functional enrichment analysis. Kinase inhibitors that regulated these kinases were retrieved from PubMed through text mining.. Twenty-nine differentially expressed kinase encoded genes were identified through screening. The functional enrichment analysis demonstrated that the functions of these genes were primarily enriched in "mitogen-activated protein kinase signaling pathway," followed by "extracellular regulated protein kinases pathway," "neurotrophin signaling pathway," "adherens junction," and "gap junction." SRC and epidermal growth factor receptor (EGFR) were related to extracellular regulated protein kinases pathway and also related to adherens junction as well as gap junction. On the basis of the regulated kinases, the kinase inhibitors reported in the literature were classified into multiple groups.. EGFR and SRC may be coexpressed in AP. The kinase inhibitors working together in SRC and EGFR may play better efficacy in the treatment of AP.

Topics: Acute Disease; Animals; Ceruletide; Databases, Genetic; Disease Models, Animal; ErbB Receptors; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Regulatory Networks; Oligonucleotide Array Sequence Analysis; Pancreatitis; Protein Interaction Maps; Protein Kinase Inhibitors; RNA, Messenger; Signal Transduction; src-Family Kinases

Hydrogen sulfide (H(2)S), formed by multiple enzymes, including cystathionine-γ-lyase (CSE), targets Ca(v)3.2 T-type Ca(2+) channels (T channels) and transient receptor potential ankyrin-1 (TRPA1), facilitating somatic pain. Pancreatitis-related pain also appears to involve activation of T channels by H(2)S formed by the upregulated CSE. Therefore, this study investigates the roles of the Ca(v)3.2 isoform and/or TRPA1 in pancreatic nociception in the absence and presence of pancreatitis. In anesthetized mice, AP18, a TRPA1 inhibitor, abolished the Fos expression in the spinal dorsal horn caused by injection of a TRPA1 agonist into the pancreatic duct. As did mibefradil, a T-channel inhibitor, in our previous report, AP18 prevented the Fos expression following ductal NaHS, an H(2)S donor. In the mice with cerulein-induced acute pancreatitis, the referred hyperalgesia was suppressed by NNC 55-0396 (NNC), a selective T-channel inhibitor; zinc chloride; or ascorbic acid, known to inhibit Ca(v)3.2 selectively among three T-channel isoforms; and knockdown of Ca(v)3.2. In contrast, AP18 and knockdown of TRPA1 had no significant effect on the cerulein-induced referred hyperalgesia, although they significantly potentiated the antihyperalgesic effect of NNC at a subeffective dose. TRPA1 but not Ca(v)3.2 in the dorsal root ganglia was downregulated at a protein level in mice with cerulein-induced pancreatitis. The data indicate that TRPA1 and Ca(v)3.2 mediate the exogenous H(2)S-induced pancreatic nociception in naïve mice and suggest that, in the mice with pancreatitis, Ca(v)3.2 targeted by H(2)S primarily participates in the pancreatic pain, whereas TRPA1 is downregulated and plays a secondary role in pancreatic nociceptive signaling.

Topics: Analysis of Variance; Animals; Benzimidazoles; Calcium Channel Blockers; Calcium Channels, T-Type; Ceruletide; Cyclopropanes; Disease Models, Animal; Hydrogen Sulfide; Hyperalgesia; Isothiocyanates; Male; Mice; Naphthalenes; Oligodeoxyribonucleotides, Antisense; Pancreatitis; Posterior Horn Cells; Proto-Oncogene Proteins c-fos; Transient Receptor Potential Channels; TRPA1 Cation Channel; Visceral Pain

Pancreatic acinar cell necrosis and subsequent inflammatory response aggravate acute pancreatitis (AP). Tetraspanin CD9 has been reported to mediate inflammatory signaling by regulating molecular organization at the cell surface. This study aimed to investigate the role of CD9 in caerulein-induced AP (CIP) in mice.. The expression of CD9 was detected in CIP in mice in vivo and cholecystokinin (CCK)/recombinant mouse tumor necrosis factor (rmTNF)-α induced pancreatic acinar cell death in vitro by quantitative real-time polymerase chain reaction, Western blot and immunofluorescence. The roles of CD9 in pancreatic acinar cell death and inflammatory response were further studied through the deletion of CD9 expression using small interfering RNA (siRNA).. CD9 was markedly upregulated in pancreatic tissues in mice during the early onset of CIP and was located mainly at the pancreatic acinar cell surface, which was associated with pancreatic damage. Additionally, incubation with CCK or rmTNF-α directly increased the expression of CD9 in isolated mice pancreatic acinar cells in vitro. The deletion of CD9 expression partially reversed both pancreatic acinar cell death induced by CCK and mRNA levels of proinflammatory cytokines produced by damaged acinar cells.. These results indicate that increased CD9 expression may be involved in pancreatic injury, possibly via the promotion of cytokine expressions in CIP in mice.

Topics: Acinar Cells; Acute Disease; Animals; Ceruletide; Cholecystokinin; Cytokines; Female; Gene Expression; Mice; Mice, Inbred BALB C; Pancreas; Pancreatitis; Random Allocation; RNA; RNA, Small Interfering; Signal Transduction; Tetraspanin 29; Tumor Necrosis Factor-alpha; Up-Regulation

Angiotensin-converting enzyme 2 (ACE2), its product angiotensin-(1-7), and its receptor Mas have been shown to moderate the adverse effects of the ACE-angiotensin II-AT1 axis in many diseases. The aim of this study was to determine whether the ACE2-Ang-(1-7)-Mas axis could have similar effects in a cell culture model of pancreatic damage.. AR42J cells were stimulated with 10 nmol/L cerulein to simulate acute pancreatitis. ACE2, Ang-(1-7), Mas receptor, and PI3K/AKT pathway were measured by quantitative real-time polymerase chain reaction and Western blot analysis.. ACE2 and Mas receptor protein levels in AR42J cells were significantly increased (P < 0.05) between 30 minutes and 6 hours postdisease induction compared with the control group. Mas receptor gene expression was significantly increased (P < 0.05) at 2 hours postdisease induction, and Ang-(1-7) was increased at 6 hours. Treatment with Ang-(1-7) in AR42J cells increased IL-10, decreased IL-6 and IL-8, and reduced the damage to pancreatic cells. Levels of IL-6 and IL-8 in AR42J cell culture were increased significantly after treatment with A779. Moreover, Ang-(1-7) increased the concentration of PI3K/AKT pathway and eNOSin AR42J cells.. ACE2-angiotensin-(1-7)-Mas axis significantly inhibits pancreatitis in response to decreased inflammatory factors by the activation of endothelial nitric oxide synthase and NO signaling pathways.

Topics: Angiotensin I; Angiotensin-Converting Enzyme 2; Animals; Anti-Inflammatory Agents; Cell Line; Ceruletide; Cytoprotection; Inflammation Mediators; Interleukin-6; Interleukin-8; Nitric Oxide; Nitric Oxide Synthase Type III; Pancreas, Exocrine; Pancreatitis; Peptide Fragments; Peptidyl-Dipeptidase A; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Receptors, G-Protein-Coupled; Signal Transduction; Time Factors

In this study, generation 5 (G5) polyamidoamine (PAMAM) dendrimers with two different surface groups, G4.5-COOH and G5-OH, were investigated for their protective effects on pancreas injury in a caerulein-induced acute pancreatitis (AP) mouse model. Both dendrimers significantly decreased pathological changes in the pancreas and reduced the inflammatory infiltration of macrophages in pancreatic tissues. In addition, the expression of pro-inflammatory cytokines was significantly inhibited by the two dendrimers, not only in pancreatic tissues from AP mice but also in vitro in mouse peritoneal macrophages with LPS-induced inflammation. G4.5-COOH, which had better in vivo protective effects for AP than G5-OH, led to a significant reduction in the total number of plasma white blood cells (WBCs) and monocytes in AP mice, and its anti-inflammatory mechanism was related to inhibition of the nuclear translocation of NF-κB in macrophages.

Topics: Animals; Ceruletide; Dendrimers; Macrophages; Mice; Mice, Inbred ICR; NF-kappa B; Pancreatitis; Protective Agents

The onset of acute pancreatitis (AP) is characterized by early protease activation followed by inflammation and organ damage, but the mechanisms are poorly understood.. We hypothesized that histone deacetylase (HDAC) inhibition might exert protective effects on AP and investigated the role of HDAC in trypsin activation, inflammation, and tissue damage in severe AP.. Male C57Bl/6 mice were treated i.p. with the HDAC inhibitor trichostatin A (2 mg/kg) prior to retrograde infusion of taurocholic acid (5 %) into the pancreatic duct. Serum levels of amylase and interleukin (IL)-6, pancreatic levels of macrophage inflammatory protein-2 (MIP-2) as well as tissue morphology and myeloperoxidase activity in the pancreas and lung were determined 24 h after taurocholate challenge. Trypsin activation was analyzed in isolated acinar cells. Quantitative RT-PCR was used to examine the expression of pro-inflammatory mediators in the pancreas.. Pretreatment with trichostatin A decreased amylase levels by 70 % and protected against tissue injury in the pancreas. Moreover, HDAC inhibition reduced systemic IL-6 by more than 95 % and pulmonary myeloperoxidase activity by 75 %. Notably, inhibition of HDAC abolished taurocholate-induced gene expression of cyclooxygenase-2, MIP-2, monocyte chemotactic protein-1, IL-6, and IL-1β in the pancreas. In addition, HDAC inhibition reduced cerulein-induced trypsinogen activation in isolated acinar cells.. Our findings show that HDAC regulates trypsin activation, inflammation, and tissue damage in AP. Thus, targeting HDAC could serve as novel therapeutic approach in the management of severe AP.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Ceruletide; Chemokine CXCL2; Cytoprotection; Disease Models, Animal; Enzyme Activation; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Inflammation Mediators; Injections, Intraperitoneal; Interleukin-6; Lung; Male; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peroxidase; Signal Transduction; Taurocholic Acid; Trypsin

Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Ceruletide; Enzyme Inhibitors; Inflammation; Intra-Abdominal Fat; Lactones; Lipase; Lipolysis; Mice; Mice, Obese; Necrosis; Orlistat; Pancreas; Pancreatitis; Triglycerides

Oncogenic mutations in KRAS contribute to the development of pancreatic ductal adenocarcinoma, but are not sufficient to initiate carcinogenesis. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of the SOX9 gene, are required for tumor formation. Herein we sought to identify the mechanisms that link EGFR signaling with activation of SOX9 during acinar-ductal metaplasia, a transdifferentiation process that precedes pancreatic carcinogenesis.. We analyzed pancreatic tissues from Kras(G12D);pdx1-Cre and Kras(G12D);NFATc1(Δ/Δ);pdx1-Cre mice after intraperitoneal administration of caerulein, vs cyclosporin A or dimethyl sulfoxide (controls). Induction of EGFR signaling and its effects on the expression of Nuclear factor of activated T cells c1 (NFATc1) or SOX9 were investigated by quantitative reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemical analyses of mouse and human tissues and acinar cell explants. Interactions between NFATc1 and partner proteins and effects on DNA binding or chromatin modifications were studied using co-immunoprecipitation and chromatin immunoprecipitation assays in acinar cell explants and mouse tissue.. EGFR activation induced expression of NFATc1 in metaplastic areas from patients with chronic pancreatitis and in pancreatic tissue from Kras(G12D) mice. EGFR signaling also promoted formation of a complex between NFATc1 and C-JUN in dedifferentiating mouse acinar cells, leading to activation of Sox9 transcription and induction of acinar-ductal metaplasia. Pharmacologic inhibition of NFATc1 or disruption of the Nfatc1 gene inhibited EGFR-mediated induction of Sox9 transcription and blocked acinar-ductal transdifferentiation and pancreatic cancer initiation in mice.. EGFR signaling induces expression of NFATc1 and Sox9, leading to acinar cell transdifferentiation and initiation of pancreatic cancer. Strategies designed to disrupt this pathway might be developed to prevent pancreatic cancer initiation in high-risk patients with chronic pancreatitis.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line; Cell Transdifferentiation; Cell Transformation, Neoplastic; Ceruletide; Cyclosporine; Disease Models, Animal; ErbB Receptors; Gene Expression Regulation; Humans; Male; Metaplasia; Mice, Inbred C57BL; Mice, Knockout; Mutation; NFATC Transcription Factors; Pancreas, Exocrine; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Precancerous Conditions; Proto-Oncogene Proteins p21(ras); Signal Transduction; SOX9 Transcription Factor; Tissue Culture Techniques; Transcriptional Activation

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the Western world. The disease has the worst prognosis in the gastrointestinal malignancies with an overall 5-year survival rate of less than 5 %. Therefore, in the search for novel therapeutic targets, biomarkers for early detection and particularly adequate methods to develop and validate therapeutic strategies for this disease are still in urgent demand. Although significant progress has been achieved in understanding the genetic and molecular mechanisms, most approaches have not yet translated sufficiently for better outcome of the patients. In part, this situation is due to inappropriate or insufficient methods in modeling PDAC in laboratory settings. In the past several years, there has been an explosion of genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX) that recapitulate both genetic and morphological alterations that lead to the development of PDAC. Both models are increasingly used for characterization and validation of diagnostic and therapeutic strategies. In this chapter we will discuss state-of-the-art models to consider when selecting an appropriate in vivo system to study disease etiology, cell signaling, and drug development.

Topics: Animals; Cell Line, Tumor; Cell Separation; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Female; Humans; Mice; Mice, Transgenic; Pancreatic Neoplasms; Pancreatitis

The mitogen-activated protein kinases (MAPKs) signaling pathway is involved in inflammatory process. However, the mechanism is not clear. The present study was to investigate the role of p38 MAPK in acute pancreatitis in mice.. Mice were divided into 4 groups: saline control; acute pancreatitis induced with repeated injections of cerulein; control plus p38 MAPK inhibitor SB203580; and acute pancreatitis plus SB203580. The pancreatic histology, pancreatic enzymes, cytokines, myeloperoxidase activity, p38 MAPK and heat shock protein (HSP) 60 and 70 were evaluated.. Repeated injections of cerulein resulted in acute pancreatitis in mice, accompanying with the activation of p38 MAPK and overexpression of HSP60 and HSP70 in the pancreatic tissues. Treatment with SB203580 significantly inhibited the activation of p38 MAPK, and furthermore, inhibited the expression of HSP60 and HSP70 in the pancreas, the inflammatory cytokines in the serum, and myeloperoxidase activity in the lung.. The p38 MAPK signaling pathway is involved in the regulation of inflammatory response and the expression of HSP60 and HSP70 in acute pancreatitis.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Biomarkers; Ceruletide; Chaperonin 60; Disease Models, Animal; HSP70 Heat-Shock Proteins; Imidazoles; Inflammation Mediators; Lung; Mice, Inbred C57BL; Mitochondrial Proteins; p38 Mitogen-Activated Protein Kinases; Pancreas; Pancreatitis; Peroxidase; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Signal Transduction

The aim of this study was to develop aprotinin-loaded microemulsion (MA) for intravenous administration and evaluate the biodistribution and therapeutic potential of developed formulation in acute pancreatitis models in rats. Phase diagrams were constructed to identify microemulsion region and the optimal microemulsion was evaluated for physicochemical properties and treatment effect in rats, and comparisons made with the solution of aprotinin (SA). To evaluate the biodistribution of the drug by gamma scintigraphy aprotinin was radiolabeled with (99m)Tc radionuclide. Mild and severe acute pancreatitis was induced in rats by subcutaneous injections of cerulein and introductal infusion of 3% sodium taurocholate into the bile-pancreatic duct, respectively. In addition, serum amylase and pancreatic tissue myeloperoxidase activities were measured to evaluate the pancreatic damage. According to gamma scintigraphy and biodistribution studies, accumulation times and distribution of (99m)Tc-MA and SA were different. While MA was highly uptake by reticuloendothelial system, SA was mostly excreted by kidneys and bladder. Compared with the mild acute pancreatitis group, treatment with MA significantly decreased the serum amylase activity and pancreas myeloperoxidase activity. Furthermore, the protease inhibitor molecule aprotinin has therapeutic potential in acute pancreatitis. Finally, MA may be suggested as a promising alternative for treatment of acute pancreatitis.

Topics: Administration, Intravenous; Amylases; Animals; Aprotinin; Ceruletide; Emulsions; Male; Pancreatitis; Peroxidase; Radionuclide Imaging; Rats; Serine Proteinase Inhibitors; Taurocholic Acid; Tissue Distribution

Guggulsterone (GS), a plant steroid and a compound found at high levels in Commiphora myrrha, exhibits anti-inflammatory, anti-cancer, and cholesterol-lowering effects. However, the potential of GS to ameliorate acute pancreatitis (AP) is unknown. The aim of this study was to evaluate the effects of GS on cerulein-induced AP. AP was induced by intraperitoneally injecting supramaximal concentrations of the stable cholecystokinin analog cerulein (50 μg/kg) hourly for 6 h. In the GS-treated group, GS was administered intraperitoneally (10, 25, or 50mg/kg) 1 h before the first cerulein injection. Mice were sacrificed 6 h after the final cerulein injection. Blood samples were collected to measure serum lipase levels and evaluate cytokine production. The pancreas and lung were rapidly removed for morphologic and histological examinations, flow cytometry analysis, myeloperoxidase (MPO) assay, and real-time reverse transcription-polymerase chain reaction analysis. Pre-treatment with GS attenuated cerulein-induced histological damage, reduced pancreas weight/body weight ratio, decreased serum lipase levels, inhibited infiltrations of macrophages and neutrophils, and suppressed cytokine production. Additionally, GS treatment suppressed the activation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) in the pancreas in cerulein-induced pancreatitis. In conclusion, our results suggest that GS attenuates AP via deactivation of ERK and JNK.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Blotting, Western; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Female; Injections, Intraperitoneal; JNK Mitogen-Activated Protein Kinases; Lipase; Mice, Inbred C57BL; Pancreas; Pancreatitis; Pregnenediones

Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.

Topics: Acinar Cells; Acute Disease; Animals; Antioxidants; Apoptosis; Ceruletide; Cholecystokinin; Disease Models, Animal; Inflammation; Male; Membrane Potential, Mitochondrial; Mice; Mitochondria; Necrosis; Organophosphorus Compounds; Oxidative Stress; Pancreas; Pancreatitis; Reactive Oxygen Species; Taurolithocholic Acid; Ubiquinone

Recent development of genetically engineered mouse models (GEMs) for pancreatic cancer (PC) that recapitulates human disease progression has helped to identify new strategies to delay/inhibit PC development. We first found that expression of the pancreatic tumor-initiating/cancer stem cells (CSC) marker DclK1 occurs in early stage PC and in both early and late pancreatic intraepithelial neoplasia (PanIN) and that it increases as disease progresses in GEM and also in human PC. Genome-wide next generation sequencing of pancreatic ductal adenocarcinoma (PDAC) from GEM mice revealed significantly increased DclK1 along with inflammatory genes. Genetic ablation of cyclo-oxygenase-2 (COX-2) decreased DclK1 in GEM. Induction of inflammation/pancreatitis with cerulein in GEM mice increased DclK1, and the novel dual COX/5-lipoxygenase (5-LOX) inhibitor licofelone reduced it. Dietary licofelone significantly inhibited the incidence of PDAC and carcinoma in situ with significant inhibition of pancreatic CSCs. Licofelone suppressed pancreatic tumor COX-2 and 5-LOX activities and modulated miRNAs characteristic of CSC and inflammation in correlation with PDAC inhibition. These results offer a preclinical proof of concept to target the inflammation initiation to inhibit cancer stem cells early for improving the treatment of pancreatic cancers, with immediate clinical implications for repositioning dual COX/5-LOX inhibitors in human trials for high risk patients.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Arachidonic Acid; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Proliferation; Ceruletide; Cyclooxygenase 2; Disease Models, Animal; Disease Progression; Doublecortin-Like Kinases; Lipoxygenase Inhibitors; Mice; Mice, Knockout; MicroRNAs; Neoplastic Stem Cells; Pancreatic Neoplasms; Pancreatitis; Protein Serine-Threonine Kinases; Pyrroles

Kaempferol (KF) is the most abundant polyphenol in tea, fruits, vegetables, and beans. However, little is known about its in vivo anti-inflammatory efficacy and mechanisms of action. To study these, several acute mouse inflammatory and nociceptive models, including gastritis, pancreatitis, and abdominal pain were employed. Kaempferol was shown to attenuate the expansion of inflammatory lesions seen in ethanol (EtOH)/HCl- and aspirin-induced gastritis, LPS/caerulein (CA) triggered pancreatitis, and acetic acid-induced writhing.

Topics: Abdominal Pain; Acetic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Ceruletide; Disease Models, Animal; Gastritis; Kaempferols; Mice; Nociception; Pancreatitis

Hypertriglyceridemic pancreatitis (HTGP) is often encountered clinically as a common form of recurrent acute pancreatitis (AP). It is important to evaluate the management of severe hypertriglyceridemia (HTG) or anti-inflammation in the prophylaxis of HTGP in the clinic. FTY720 (2-amino-2[2-(4-octylphenyl) ethyl]-1, 3-propanediol) is a new anti-inflammatory agent with low toxicity and reported to ameliorate lung injury with pancreatitis in rat. We evaluated its protective affection on AP induced by seven hourly intraperitoneal injection of cerulein in apolipoprotein CIII transgenic mice with severe HTG. FTY720 at 1.5 mg/kg was administered by gastric lavage daily for 3 days before induction of AP. The effects of FTY720 to protect against HTGP were assessed by serum amylase, pancreatic pathological scores, immunostaining, and the expression of inflammatory cytokine genes. As a result, injection of cerulein resulted in more severe pathological changes of AP and higher monocyte chemoattractant protein 1 expression in the pancreas in transgenic than in nontransgenic mice. FTY720 pretreatment improved the pathological severity of AP and decreased the expression of monocyte chemoattractant protein 1 in the pancreas significantly, especially near fourfold reduction in transgenic mice. However, FTY720 did not affect plasma triglyceride levels, and other inflammatory factors and plasma amylase were not correlated with the extent of pancreatic damage in AP with or without FTY720 administration. In summary, our study in a new model, apolipoprotein CIII transgenic mice, demonstrated that HTG mice are susceptible to induction of AP. Prophylactic treatment of FTY720 can significantly attenuate cerulein-induced AP and hence warrant further investigation of sphingosine-1-phosphate receptors agonist for potential clinical application in recurrent attacks of HTGP.

Topics: Acute Disease; Amylases; Animals; Apolipoprotein C-III; Ceruletide; Chemokine CCL2; Drug Evaluation, Preclinical; Female; Fingolimod Hydrochloride; Hypertriglyceridemia; Immunosuppressive Agents; Lymphocyte Count; Mice, Transgenic; Pancreas; Pancreatitis

Sphingosine-1-phosphate type-1 receptor (S1P1) agonists have the potential to inhibit the egress of lymphocytes, and have been demonstrated to provide protective effects on some acute inflammatory diseases. However, the value of S1P1 agonists on acute pancreatitis (AP) remains unclear. The aim of this study was to explore the effect of SEW2871, a S1P1-selective agonist, on caerulein-induced AP in mice. AP was induced by giving eight intraperitoneal injections of caerulein (50 µg/kg/h) at hourly intervals. SEW2871 was administered by gavage, at a dose of 20 mg/kg, at 0 h and 12 h after the first intraperitoneal injection of caerulein. The mice were sacrificed at 24 h. Severity of AP, serum amylase and lipase activity, levels of serum cytokines, pancreatic myeloperoxidase (MPO) activity, CD45+CD4+ T lymphocytes in blood, CD4+ T cell infiltration in the pancreas, and proinflammatory cytokine production were assessed. Furthermore, the expression of signal transducer and activator of transcription (STAT) 3 and phospho-STAT3 (p-STAT3) in the pancreas was also evaluated. The results revealed that the administration of SEW2871 ameliorated the severity of AP, by a reduction of serum pancreatic enzyme activity and levels of cytokines, decreased pancreatic MPO activity, depletion of CD4+CD45+ T lymphocytes in the blood and a reduction of CD4+ T cell infiltration in the pancreas. Furthermore, the expression of proinflammatory cytokines mRNA and p-STAT3 were also suppressed by SEW2871 treatment. These results suggest that SEW2871 treatment attenuates the severity of caerulein-induced AP in mice, which may provide a new therapeutic approach for AP therapy.

Topics: Amylases; Animals; Ceruletide; Cytokines; Lipase; Male; Mice, Inbred ICR; Oxadiazoles; Pancreas; Pancreatitis; Peroxidase; RNA, Messenger; STAT3 Transcription Factor; T-Lymphocytes; Thiophenes

Pancreatitis is a significant clinical problem and the lack of effective therapeutic options means that treatment is often palliative rather than curative. A deeper understanding of the pathogenesis of both acute and chronic pancreatitis is necessary to develop new therapies. Pathological changes in pancreatitis are dependent on innate immune cell recruitment to the site of initial tissue damage, and on the coordination of downstream inflammatory pathways. The chemokine receptor CXCR2 drives neutrophil recruitment during inflammation, and to investigate its role in pancreatic inflammation, we induced acute and chronic pancreatitis in wild-type and Cxcr2(-/-) mice. Strikingly, Cxcr2(-/-) mice were strongly protected from tissue damage in models of acute pancreatitis, and this could be recapitulated by neutrophil depletion or by the specific deletion of Cxcr2 from myeloid cells. The pancreata of Cxcr2(-/-) mice were also substantially protected from damage during chronic pancreatitis. Neutrophil depletion was less effective in this model, suggesting that CXCR2 on non-neutrophils contributes to the development of chronic pancreatitis. Importantly, pharmacological inhibition of CXCR2 in wild-type mice replicated the protection seen in Cxcr2(-/-) mice in acute and chronic models of pancreatitis. Moreover, acute pancreatic inflammation was reversible by inhibition of CXCR2. Thus, CXCR2 is critically involved in the development of acute and chronic pancreatitis in mice, and its inhibition or loss protects against pancreatic damage. CXCR2 may therefore be a viable therapeutic target in the treatment of pancreatitis.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Ceruletide; Cytoprotection; Disease Models, Animal; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Neutrophils; Pancreas; Pancreatitis; Pancreatitis, Chronic; Peptides; Receptors, Interleukin-8B; Signal Transduction; Time Factors

Beta-site alpha-amyloid protein cleaving enzyme1 (BACE1) plays a key role in the pathogenesis of Alzheimer's disease. Additional to its moderate expression in the brain, high levels of BACE1 mRNA were found in the pancreas. Murine Bace1 has been immunohistochemicaly detected at the apical pole of acinar cells within the exocrine pancreas of mice and Bace1 activity was observed in pancreatic juice. In vitro experiments revealed enteropeptidase as a putative substrate for Bace1 suggesting a role in acute pancreatitis.. The aim of this study was to address a protective mechanism of Bace1 in acute experimental pancreatitis in mice.. Acute experimental pancreatitis was induced by intraperitoneal injection of caerulein in homozygote Bace1-/- mice and wild type mice. Serum and tissue analyses were carried out after 4 h, 8 h and 24 h. Measurement of plasma amylase and lipase was performed to confirm pancreatitis induction. In order to assess the severity of pancreatitis H&E stained pancreatic sections were examined regarding edema, inflammation and apoptosis. Immunohistochemical detection of myeloperoxidase (MPO) positive cells was carried out to further quantify the extent of inflammation. Expression of Bace2 within the pancreas was analyzed by immunohistochemistry and RT-qPCR.. We demonstrate that total loss of Bace1 in mice leads to no alterations in the course of acute experimental caerulein-pancreatitis. Bace1-/- mice develop a moderate pancreatitis that is comparable in histomorphological and serological features with those seen in wild type mice.. We discuss the results in the context of the applied caerulein induced edematous pancreatitis model and possible compensatory mechanisms via Bace2 that might be responsible for the observed results.

Topics: Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Ceruletide; Mice; Mice, Inbred C57BL; Pancreatitis

The current study aimed to isolate key transcription factors (TFs) in caerulein-induced pancreatitis, and to identify the difference between wild type and Mist1 knockout (KO) mice, in order to elucidate the contribution of Mist1 to pancreatitis. The gene profile of GSE3644 was downloaded from the Gene Expression Omnibus database then analyzed using the t-test. The isolated differentially expressed genes (DEGs) were mapped into a transcriptional regulatory network derived from the Integrated Transcription Factor Platform database and in the network, the interaction pairs involving at least one DEG were screened. Fisher's exact test was used to analyze the functional enrichment of the target genes. A total of 1,555 and 3,057 DEGs were identified in the wild type and Mist1KO mice treated with caerulein, respectively. DEGs screened in Mist1KO mice were predominantly enriched in apoptosis, mitogen-activated protein kinase signaling and other cancer-associated pathways. A total of 188 and 51 TFs associated with pathopoiesis were isolated in Mist1KO and wild type mice, respectively. Out of the top 10 TFs (ranked by P-value), 7 TFs, including S-phase kinase-associated protein 2 (Skp2); minichromosome maintenance complex component 3 (Mcm3); cell division cycle 6 (Cdc6); cyclin B1 (Ccnb1); mutS homolog 6 (Msh6); cyclin A2 (Ccna2); and cyclin B2 (Ccnb2), were expressed in the two types of mouse. These TFs were predominantly involved in phosphorylation, DNA replication, cell division and DNA mismatch repair. In addition, specific TFs, including minichromosome maintenance complex component 7 (Mcm7); lymphoid-specific helicase (Hells); and minichromosome maintenance complex component 6 (Mcm6), that function in the unwinding of DNA were identified to participate in Mist1KO pancreatitis. The DEGs, including Cdc6, Mcm6, Msh6 and Wdr1 are closely associated with the regulation of caerulein-induced pancreatitis. Furthermore, other identified TFs were also involved in this type of regulation.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Division; Ceruletide; Databases, Genetic; DNA Mismatch Repair; DNA Replication; Gene Expression Profiling; Gene Expression Regulation; Gene Regulatory Networks; Mice; Mice, Knockout; Molecular Sequence Annotation; Pancreatitis; Signal Transduction; Transcription Factors

MicroRNAs (miRNAs) regulate apoptosis, yet their role in regulated necrosis remains unknown. miR-21 is overexpressed in nearly all human cancer types and its role as an oncogene is suggested to largely depend on its anti-apoptotic action. Here we show that miR-21 is overexpressed in a murine model of acute pancreatitis, a pathologic condition involving RIP3-dependent regulated necrosis (necroptosis). Therefore, we investigate the role of miR-21 in acute pancreatitis injury and necroptosis. miR-21 deficiency protects against caerulein- or L-arginine-induced acute pancreatitis in mice. miR-21 inhibition using locked-nucleic-acid-modified oligonucleotide effectively reduces pancreatitis severity. miR-21 deletion is also protective in tumour necrosis factor-induced systemic inflammatory response syndrome. These data suggest that miRNAs are critical participants in necroptosis and miR-21 enhances cellular necrosis by negatively regulating tumour suppressor genes associated with the death-receptor-mediated intrinsic apoptosis pathway, and could be a therapeutic target for preventing pathologic necrosis.

Topics: Animals; Apoptosis; Arginine; Bone Marrow Transplantation; Caspases; Ceruletide; Fas Ligand Protein; fas Receptor; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Imidazoles; Indoles; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Necrosis; Oligonucleotides; Pancreatitis; Receptor-Interacting Protein Serine-Threonine Kinases; RNA Interference; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) is an inflammatory disease, and is one of the most common gastrointestinal disorders worldwide. Soluble epoxide hydrolase (sEH; encoded by Ephx2) deficiency and pharmacological inhibition have beneficial effects in inflammatory diseases. Ephx2 whole-body deficiency mitigates experimental AP in mice, but the suitability of sEH pharmacological inhibition for treating AP remains to be determined. We investigated the effects of sEH pharmacological inhibition on cerulein- and arginine-induced AP using the selective sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered before and after induction of pancreatitis. Serum amylase and lipase levels were lower in TPPU-treated mice compared with controls. In addition, circulating levels and pancreatic mRNA of the inflammatory cytokines tumor necrosis factor-α, interleukin Il-1β, and Il-6 were reduced in TPPU-treated mice. Moreover, sEH pharmacological inhibition before and after induction of pancreatitis was associated with decreased cerulein- and arginine-induced nuclear factor-κB inflammatory response, endoplasmic reticulum stress, and cell death. sEH pharmacological inhibition before and after induction of pancreatitis mitigated cerulein- and arginine-induced AP. This work suggests that sEH pharmacological inhibition may be of therapeutic value in acute pancreatitis.

Topics: Amylases; Animals; Arginine; Ceruletide; Disease Models, Animal; Epoxide Hydrolases; Gene Expression Regulation; Interleukin-1beta; Interleukin-6; Lipase; MAP Kinase Signaling System; Mice; Pancreatitis; Phenylurea Compounds; Piperidines; Tumor Necrosis Factor-alpha

We describe the first mouse model of pancreatic intraepithelial neoplasia (PanIN) lesions induced by alcohol in the presence and absence of chronic pancreatitis.. Pdx1-Cre;LSL-K-ras mice were exposed to Lieber-DeCarli alcohol diet for 6 weeks with cerulein injections. The PanIN lesions and markers of fibrosis, inflammation, histone deacetylation, epithelial-to-mesenchymal transition (EMT), and cancer stemness were measured by immunohistochemistry and Western.. Exposure of Pdx1-Cre;LSL-K-ras mice to an alcohol diet significantly stimulated fibrosis and slightly but not significantly increased the level of PanIN lesions associated with an increase in tumor-promoting M2 macrophages. Importantly, the alcohol diet did not increase activation of stellate cells. Alcohol diet and cerulein injections resulted in synergistic and additive effects on PanIN lesion and M2 macrophage phenotype induction, respectively. Cerulein pancreatitis caused stellate cell activation, EMT, and cancer stemness in the pancreas. Pancreatitis caused histone deacetylation, which was promoted by the alcohol diet. Pancreatitis increased EMT and cancer stemness markers, which were not further affected by the alcohol diet.. The results suggest that alcohol has independent effects on promotion of PDAC associated with fibrosis formed through a stellate cell-independent mechanism and that it further promotes early PDAC and M2 macrophage induction in the context of chronic pancreatitis.

Topics: Acetylation; Acute Disease; Animals; Carcinoma in Situ; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Epithelial-Mesenchymal Transition; Ethanol; Fibrosis; Histones; Macrophages; Mice, Transgenic; Neoplastic Stem Cells; Pancreas; Pancreatic Neoplasms; Pancreatic Stellate Cells; Pancreatitis; Pancreatitis, Alcoholic; Pancreatitis, Chronic; Time Factors

Lupeol is a triterpenoid commonly found in fruits and vegetables and is known to exhibit a wide range of biological activities, including antiinflammatory and anti-cancer effects. However, the effects of lupeol on acute pancreatitis specifically have not been well characterized. Here, we investigated the effects of lupeol on cerulein-induced acute pancreatitis in mice. Acute pancreatitis was induced via an intraperitoneal injection of cerulein (50 µg/kg). In the lupeol treatment group, lupeol was administered intraperitoneally (10, 25, or 50 mg/kg) 1 h before the first cerulein injection. Blood samples were taken to determine serum cytokine and amylase levels. The pancreas was rapidly removed for morphological examination and used in the myeloperoxidase assay, trypsin activity assay, and real-time reverse transcription polymerase chain reaction. In addition, we isolated pancreatic acinar cells using a collagenase method to examine the acinar cell viability. Lupeol administration significantly attenuated the severity of pancreatitis, as was shown by reduced pancreatic edema, and neutrophil infiltration. In addition, lupeol inhibited elevation of digestive enzymes and cytokine levels, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interleukin (IL)-6. Furthermore, lupeol inhibited the cerulein-induced acinar cell death. In conclusion, these results suggest that lupeol exhibits protective effects on cerulein-induced acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Cell Survival; Ceruletide; Cytokines; Injections, Intraperitoneal; Lipase; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Pancreas; Pancreatitis; Pentacyclic Triterpenes; Peroxidase; Plant Extracts; Protective Agents; Tumor Necrosis Factor-alpha

Visceral pain represents a major clinical challenge in the management of many gastrointestinal disorders, eg, pancreatitis. However, cerebral neurobiological mechanisms underlying visceral nociception are poorly understood. As a representative model of visceral nociception, we applied cerulein hyperstimulation in C57BL6 mice to induce acute pancreatitis and performed a behavioral test battery and c-Fos staining of brains. We observed a specific pain phenotype and a significant increase in c-Fos immunoreactivity in the paraventricular nucleus of the thalamus (PVT), the periaqueductal gray, and the medial prefrontal cortex (mPFC). Using neuronal tracing, we observed projections of the PVT to cortical layers of the mPFC with contacts to inhibitory GABAergic neurons. These inhibitory neurons showed more activation after cerulein treatment suggesting thalamocortical "feedforward inhibition" in visceral nociception. The activity of neurons in pancreatitis-related pain centers was pharmacogenetically modulated by designer receptors exclusively activated by designer drugs, selectively and cell type specifically expressed in target neurons using adeno-associated virus-mediated gene transfer. Pharmacogenetic inhibition of PVT but not periaqueductal gray neurons attenuated visceral pain and induced an activation of the descending inhibitory pain pathway. Activation of glutamatergic principle neurons in the mPFC, but not inhibitory neurons, also reversed visceral nociception. These data reveal novel insights into central pain processing that underlies visceral nociception and may trigger the development of novel, potent centrally acting analgesic drugs.

Topics: Affect; Animals; Behavior, Animal; Ceruletide; Dependovirus; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Midline Thalamic Nuclei; Neural Pathways; Neuronal Tract-Tracers; Neurons; Nociception; Pancreatitis; Periaqueductal Gray; Prefrontal Cortex; Proto-Oncogene Proteins c-fos; Visceral Pain

Extracellular histones are rapidly cleared by the liver and rarely detectable in the circulation unless there is extensive cell death, as in severe trauma and sepsis. This study investigated whether circulating histones are elevated in experimental acute pancreatitis models and correlate to disease severity.. Acute pancreatitis was induced in mice by: (1) 4 or (2) 12 intraperitoneal injections of cerulein (50 μg/kg) at 1 hour apart; (3) retrograde infusion of 3.5% sodium taurocholate into the biliopancreatic duct. Mice were sacrificed at various time points to collect blood and tissues. Severity of pancreatitis was assessed by biochemical markers and histopathology. Circulating histones were determined by Western blotting.. Four cerulein injections induced edematous pancreatitis, whereas 12 cerulein injections and ductal taurocholate infusion caused necrotizing pancreatitis. Circulating histones were barely detectable in the blood of animals with edematous pancreatitis but significantly increased in necrotizing pancreatitis. The levels of circulating histones were strongly correlated to histopathological scores of necrosis of the pancreas.. Circulating histones increased significantly in necrotizing pancreatitis due to extensive pancreatic acinar cell death. Levels of circulating histones may have translational potential as a biomarker of disease severity.

Topics: Acute Disease; Analysis of Variance; Animals; Biomarkers; Blotting, Western; Ceruletide; Disease Models, Animal; Histones; Humans; Male; Mice, Inbred C57BL; Pancreatitis; Pancreatitis, Acute Necrotizing; Severity of Illness Index; Taurocholic Acid

Activation of the transcription factor κB (NF-κB) and secretion of pro-inflammatory mediators are major events in acute pancreatitis (AP). Recently, O-linked-N-acetylglucosamine (O-GlcNAc) modification, one type of posttranslational modifications, reportedly attunes NF-κB function. However, the expression of O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation of proteins, in AP, and the possible contribution of OGT-mediated O-GlcNAcylation to the NF-κB inflammatory activation in pancreatic acinar cells and to the AP progression have not been understood. This study focused on the effects and mechanisms of OGT-mediated O-GlcNAcylation during AP.. An AP cell model was established with the caerulein-stimulated AR42 J rat pancreatic acinar cells. The secretion of pro-inflammatory cytokines TNF-α was detected by ELISA kits, and the production of NO was determined using the colorimetric Griess reaction. Expression of OGT was measured by RT-PCR and Western blot. Expression levels of RL2, phosphorylation of p65, total p65, IKKα were detected by Western blot. The NF-κB activity was evaluated by luciferase reporter gene assay. To determine the biological functions of OGT in caerulein-induced inflammatory response, RNA interference and PUGNAc, the inhibitor of O-GlcNAcase (OGA) was employed to regulate OGT expression in AR42 J cells.. Caerulein significantly up-regulated the expression of OGT, and increased the global protein O-GlcNAcylation level in AR42 J cells. Reduction of OGT by small interfering RNA (siRNA) inhibited caerulein-triggered inflammation, assessed by the production of pro-inflammatory mediators (TNF-α and NO). We also demonstrated that O-GlcNAcylation directly modified the NF-κB p65 subunit and its upstream activating kinases IKKα in AR42 J cells. Lowering O-GlcNAcylation by OGT knockdown attenuated p65 activating phosphorylation, nuclear translocation, NF-κB transcriptional activity and levels of NF-κB transcriptional targets TNF-α and NO; on the contrary, elevating O-GlcNAc through PUGNAc increased IKKα and p65 O-GlcNAcylation accompanied by increased p65 phosphorylation, activity and levels of TNF-α and NO in caerulein-treated cells.. Our results demonstrate for the first time that OGT-mediated O-GlcNAcylation promotes NF-κB signaling activation and inflammation in pancreatic acinar cells, which might promote the progression of AP.

Topics: Acinar Cells; Acute Disease; Acylation; Animals; Cell Line; Ceruletide; Cytokines; Immunoprecipitation; Inflammation; N-Acetylglucosaminyltransferases; NF-kappa B; Nitric Oxide; Pancreatitis; Rats; RNA Interference; RNA, Small Interfering; Tumor Necrosis Factor-alpha

The mechanisms by which drugs induce pancreatitis are unknown. A definite cause of pancreatitis is due to the antiepileptic drug valproic acid (VPA). On the basis of three crucial observations-that VPA inhibits histone deacetylases (HDACs), HDACs mediate pancreas development, and aspects of pancreas development are recapitulated during recovery of the pancreas after injury-we hypothesized that VPA does not cause injury on its own, but it predisposes patients to pancreatitis by inhibiting HDACs and provoking an imbalance in pancreatic recovery. In an experimental model of pancreatic injury, we found that VPA delayed recovery of the pancreas and reduced acinar cell proliferation. In addition, pancreatic expression of class I HDACs (which are the primary VPA targets) increased in the midphase of pancreatic recovery. VPA administration inhibited pancreatic HDAC activity and led to the persistence of acinar-to-ductal metaplastic complexes, with prolonged Sox9 expression and sustained β-catenin nuclear activation, findings that characterize a delay in regenerative reprogramming. These effects were not observed with valpromide, an analog of VPA that lacks HDAC inhibition. This is the first report, to our knowledge, that VPA shifts the balance toward pancreatic injury and pancreatitis through HDAC inhibition. The work also identifies a new paradigm for therapies that could exploit epigenetic reprogramming to enhance pancreatic recovery and disorders of pancreatic injury.

Topics: Acinar Cells; Animals; Anticonvulsants; Cell Differentiation; Cell Proliferation; Ceruletide; Histone Deacetylase Inhibitors; Histone Deacetylases; Male; Mice; Pancreas; Pancreatitis; Regeneration; Up-Regulation; Valproic Acid

Nr5a2 participates in biliary acid metabolism and is a major regulator of the pancreatic exocrine programme. Single nucleotide polymorphisms in the vicinity of NR5A2 are associated with the risk of pancreatic ductal adenocarcinoma (PDAC).. To determine the role of Nr5a2 in pancreatic homeostasis, damage-induced regeneration and mutant KRas-driven pancreatic tumourigenesis.. Nr5a2+/- and KRas(G12V);Ptf1a-Cre;Nr5a2+/- mice were used to investigate whether a full dose of Nr5a2 is required for normal pancreas development, recovery from caerulein-induced pancreatitis, and protection from tumour development.. Adult Nr5a2+/- mice did not display histological abnormalities in the pancreas but showed a more severe acute pancreatitis, increased acino-ductal metaplasia and impaired recovery from damage. This was accompanied by increased myeloid cell infiltration and proinflammatory cytokine gene expression, and hyperactivation of nuclear factor κb and signal transducer and activator of transcription 3 signalling pathways. Induction of multiple episodes of acute pancreatitis was associated with more severe damage and delayed regeneration. Inactivation of one Nr5a2 allele selectively in pancreatic epithelial cells was sufficient to cause impaired recovery from pancreatitis. In comparison with Nr5a2+/+ mice, KRas(G12V);Ptf1a(Cre/+);Nr5a2+/- mice showed a non-statistically significant increase in the area affected by preneoplastic lesions. However, a single episode of acute pancreatitis cooperated with loss of one Nr5a2 allele to accelerate KRas(G12V)-driven development of preneoplastic lesions.. A full Nr5a2 dose is required to restore pancreatic homeostasis upon damage and to suppress the KRas(G12V)-driven mouse pancreatic intraepithelial neoplasia progression, indicating that Nr5a2 is a novel pancreatic tumour suppressor. Nr5a2 could contribute to PDAC through a role in the recovery from pancreatitis-induced damage.

Topics: Animals; Blotting, Western; Carcinoma, Pancreatic Ductal; Ceruletide; Heterozygote; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; NF-kappa B; Pancreatic Neoplasms; Pancreatitis; Polymerase Chain Reaction; Proto-Oncogene Proteins p21(ras); Receptors, Cytoplasmic and Nuclear; Signal Transduction; STAT3 Transcription Factor

Drug-induced pancreatitis (DIP) is an underdiagnosed condition that lacks sensitive and specific biomarkers. To better understand the mechanisms of DIP and to identify potential tissue biomarkers, we studied experimental pancreatitis induced in male C57BL/6 mice by intraperitoneal injection of caerulein (10 or 50 μg/kg) at 1-hr intervals for a total of 7 injections. Pancreata from caerulein-treated mice exhibited consistent acinar cell autophagy and apoptosis with infrequent necrosis. Kinetic assays for serum amylase and lipase also showed a dose-dependent increase. Terminal deoxynucleotidyl transferase-mediated biotin-dNTP nick labeling (TUNEL) detected dose-dependent acinar cell apoptosis. By light microscopy, autophagy was characterized by the formation of autophagosomes and autolysosomes (ALs) within the cytoplasm of acinar cells. Immunohistochemical studies with specific antibodies for proteins related to autophagy and pancreatic stress were conducted to evaluate these proteins as potential biomarkers of pancreatitis. Western blots were used to confirm immunohistochemical results using pancreatic lysates from control and treated animals. Autophagy was identified as a contributing process in caerulein-induced pancreatitis and proteins previously associated with autophagy were upregulated following caerulein treatment. Autophagosomes and ALs were found to be a common pathway, in which cathepsins, lysosome-associated membrane protein 2, vacuole membrane protein 1, microtubule-associated protein 1 light chain 3 (LC3), autophagy-related protein 9, Beclin1, and pancreatitis-associated proteins were simultaneously involved in response to caerulein stimulus. Regenerating islet-derived 3 gamma (Reg3γ), a pancreatic acute response protein, was dose-dependently induced in caerulein-treated mice and colocalized with the autophagosomal marker, LC3. This finding supports Reg3γ as a candidate biomarker for pancreatic injury.

Topics: Acinar Cells; Amylases; Animals; Autophagy; Biomarkers; Ceruletide; Immunohistochemistry; Lipase; Male; Mice; Mice, Inbred C57BL; Necrosis; Pancreas; Pancreatitis; Proteins

Emerging evidence from mouse models suggests that mutant Kras can drive the development of pancreatic ductal adenocarcinoma (PDA) precursors from acinar cells by enforcing ductal de-differentiation at the expense of acinar identity. Recently, human genome-wide association studies have identified NR5A2, a key regulator of acinar function, as a susceptibility locus for human PDA. We investigated the role of Nr5a2 in exocrine maintenance, regeneration and Kras driven neoplasia.. To investigate the function of Nr5a2 in the pancreas, we generated mice with conditional pancreatic Nr5a2 deletion (PdxCre(late); Nr5a2(c/c)). Using this model, we evaluated acinar differentiation, regeneration after caerulein pancreatitis and Kras driven pancreatic neoplasia in the setting of Nr5a2 deletion.. We show that Nr5a2 is not required for the development of the pancreatic acinar lineage but is important for maintenance of acinar identity. Nr5a2 deletion leads to destabilisation of the mature acinar differentiation state, acinar to ductal metaplasia and loss of regenerative capacity following acute caerulein pancreatitis. Loss of Nr5a2 also dramatically accelerates the development of oncogenic Kras driven acinar to ductal metaplasia and PDA precursor lesions.. Nr5a2 is a key regulator of acinar plasticity. It is required for maintenance of acinar identity and re-establishing acinar fate during regeneration. Nr5a2 also constrains pancreatic neoplasia driven by oncogenic Kras, providing functional evidence supporting a potential role as a susceptibility gene for human PDA.

Topics: Animals; Carcinoma, Acinar Cell; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Ceruletide; Mice; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); Real-Time Polymerase Chain Reaction; Receptors, Cytoplasmic and Nuclear

The objective of this study was to investigate the potential protective effects of fucoidan, an L- and P-selectin modulator, in 2 murine models of acute pancreatitis.. Acute pancreatitis was induced in mice either by the retrograde infusion of taurolithocholic acid sulfate into the pancreatic duct or by intraperitoneal injections of cerulein (50 μg/kg per hour). The experimental groups received fucoidan (25 mg/kg, intravenously) before pancreatitis induction, whereas control groups received only saline. After 24 hours, serum amylase, lipase, interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and nitrite were measured. In addition, myeloperoxidase (MPO) activity (lung and pancreas) and histological assessment (pancreas) were determined.. Serum amylase, lipase, nitrite, TNF-α, and IL-1β, and pancreatic and lung MPO were increased in both taurolithocholic acid sulfate and cerulein acute pancreatitis compared with the respective control groups. Fucoidan significantly decreased the augmented levels of amylase, lipase, pancreatic and lung MPO, TNF-α, IL-1β, and nitrite in both models. Pancreas histological changes observed in both acute pancreatitis models were significantly attenuated by fucoidan.. Fucoidan reduced the severity of acute pancreatitis in mice by decreasing neutrophil infiltration and systemic inflammation, suggesting that modulation of selectins may constitute a promising therapeutic approach.

Topics: Acute Disease; Amylases; Analysis of Variance; Animals; Anti-Ulcer Agents; Ceruletide; Interleukin-1beta; L-Selectin; Lipase; Lung; Male; Mice; Neutrophil Infiltration; Nitrites; P-Selectin; Pancreas; Pancreatitis; Peroxidase; Polysaccharides; Taurolithocholic Acid; Tumor Necrosis Factor-alpha

The protein tyrosine phosphatases (PTPs) SHP-1, SHP-2 and PTP1B are overexpressed early on during the development of cerulein -induced acute pancreatitis (AP) in rats, and their levels can be modulated by some species of mitogen-activated protein kinases (MAPKs), the intracellular levels of cAMP and by general leukocyte infiltration, the latter at least for SHP-2 and PTP1B. In this study we show that cerulein treatment activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not p38 MAPK during the early phase of cerulein-induced AP (2h after the first injection of cerulein). Therefore, by using the MAPK inhibitors SP600125 (a specific JNK inhibitor) and PD98059 (a specific ERK inhibitor), we have unmasked the particular MAPK that underlies the modulation of the expression levels of these PTPs. JNK would act by preventing SHP-1 protein expression from increasing beyond a certain level. ERK 1/2 was the main MAPK involved in the increase in SHP-2 protein expression due to cerulein. JNK negatively modulated the SH2-domain containing PTPs. Both MAPKs played a role in the increase in PTP1B protein expression due to cerulein. Finally, by using the white blood cell inhibitors vinblastine sulfate, gadolinium chloride and FK506 (tacrolimus), we show that the macrophage activity or T-lymphocytes does not modulate the expression of any of the PTPs, although neutrophil infiltration was found to be a regulator of SHP-2 and PTP1B protein expression due to cerulein.

Topics: Acute Disease; Animals; Anthracenes; Ceruletide; Cyclic AMP; Flavonoids; Immunoblotting; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neutrophil Infiltration; Pancreas; Pancreatitis; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Rats; Rats, Wistar; Time Factors

In the past decades, a greater understanding of acute pancreatitis has led to improvement in mortality rates. Nevertheless, this disease continues to be a health care system problem due to its economical costs. Future strategies such as antioxidant supplementation could be very promising, regarding to beginning and progression of the disease. For this reason, this study was aimed at assessing the effect of exogenous administration of resveratrol during the induction process of acute pancreatitis caused by the cholecystokinin analog cerulein in rats. Resveratrol pretreatment reduced histological damage induced by cerulein treatment, as well as hyperamylasemia and hyperlipidemia. Altered levels of corticosterone, total antioxidant status, and glutathione peroxidase were significantly reverted to control levels by the administration of resveratrol. Lipid peroxidation was also counteracted; nevertheless, superoxide dismutase enzyme was overexpressed due to resveratrol pretreatment. Related to immune response, resveratrol pretreatment reduced pro-inflammatory cytokine IL-1β levels and increased anti-inflammatory cytokine IL-10 levels. In addition, pretreatment with resveratrol in cerulein-induced pancreatitis rats was able to reverse, at least partially, the abnormal calcium signal induced by treatment with cerulein. In conclusion, this study confirms antioxidant and immunomodulatory properties of resveratrol as chemopreventive in cerulein-induced acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Antioxidants; Ceruletide; Corticosterone; Female; Glutathione Peroxidase; Interleukin-10; Interleukin-1beta; Lipase; Lipid Peroxidation; Male; Malondialdehyde; Oxidative Stress; Pancreas; Pancreatitis; Rats; Rats, Wistar; Resveratrol; Stilbenes; Superoxide Dismutase

High calcium concentrations are an established risk factor for pancreatitis. We have investigated whether increasing magnesium concentrations affect pathological calcium signals and premature protease activation in pancreatic acini, and whether dietary or intraperitoneal magnesium administration affects the onset and course of experimental pancreatitis.. Pancreatic acini were incubated with up to 10 mM magnesium; [Ca(2+)](i) (fura-2AM) and intracellular protease activation (fluorogenic substrates) were determined over 60 min. Wistar rats received chow either supplemented or depleted for magnesium (<300 ppm to 30 000 ppm) over two weeks before pancreatitis induction (intravenous caerulein 10 µg/kg/h/4 h); controls received 1 µg/kg/h caerulein or saline. C57BL6/J mice received four intraperitoneal doses of magnesium (NaCl, Mg(2+) 55 192 or 384 mg/kg bodyweight) over 72 h, then pancreatitis was induced by up to eight hourly supramaximal caerulein applications. Pancreatic enzyme activities, protease activation, morphological changes and the immune response were investigated.. Increasing extracellular Mg(2+) concentration significantly reduced [Ca(2+)](i) peaks and frequency of [Ca(2+)](i) oscillations as well as intracellular trypsin and elastase activity. Magnesium administration reduced pancreatic enzyme activities, oedema, tissue necrosis and inflammation and somewhat increased Foxp3-positiv T-cells during experimental pancreatitis. Protease activation was found in animals fed magnesium-deficient chow-even with low caerulein concentrations that normally cause no damage.. Magnesium supplementation significantly reduces premature protease activation and the severity of pancreatitis, and antagonises pathological [Ca(2+)](i) signals. Nutritional magnesium deficiency increases the susceptibility of the pancreas towards pathological stimuli. These data have prompted two clinical trials on the use of magnesium in patients at risk for pancreatitis.

Topics: Acute Disease; Animals; Biomarkers; Calcium; Ceruletide; Dietary Supplements; Disease Progression; Hydrolases; Magnesium; Magnesium Deficiency; Male; Mice; Pancreatitis; Peptide Hydrolases; Rats; Rats, Wistar; Severity of Illness Index; Treatment Outcome

The aim of this study was to evaluate the effects of Opuntia humifusa (OH) on cerulein-induced acute pancreatitis (AP).. Acute pancreatitis was induced via intraperitoneal injection of cholecystokinin analog cerulein (50 μg/kg). In the OH pretreatment group, OH was administered intraperitoneally (100, 250, or 500 mg/kg) 1 hour before first cerulein injection. In the posttreatment group, OH was administered intraperitoneally (500 mg/kg) 1 hour after the first cerulein injection. Furthermore, we isolated the pancreatic acinar cells using collagenase method, then investigated the acinar cell viability, cytokine productions, and the regulating mechanisms.. The both pretreatment and posttreatment of OH treatment attenuated the severity of AP, as shown by the histology of the pancreas and lung, and inhibited neutrophil infiltration; serum amylase and lipase activities; proinflammatory cytokine expression such as interleukin 1, interleukin 6, and tumor necrosis factor α; and cell death including apoptosis and necrosis. Furthermore, OH inhibited the activation of c-Jun N-terminal kinases.. These results suggest that OH reduces the severity of AP by inhibiting acinar cell death through c-Jun N-terminal kinases.

Topics: Acinar Cells; Acute Disease; Amylases; Animals; Apoptosis; Blotting, Western; Cell Survival; Cells, Cultured; Ceruletide; Cytokines; Dose-Response Relationship, Drug; Female; Gene Expression; HMGB1 Protein; Injections, Intraperitoneal; Lipase; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Opuntia; Pancreas; Pancreatitis; Plant Extracts; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis.. We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses.. After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor κB, degradation of inhibitor of κB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor κB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection.. In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage.

Topics: Acute Disease; Animals; Arginine; Cell Death; Ceruletide; Disease Models, Animal; DNA Damage; High Mobility Group Proteins; Histones; Immunity, Innate; Mice; Mice, Inbred C57BL; Mice, Knockout; Nucleosomes; Oxidative Stress; Pancreas; Pancreatitis; Reactive Oxygen Species; Repressor Proteins; Signal Transduction; Time Factors

Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin II (Ang II) have been implicated in the pathogenesis of pancreatitis. Angiotensin-converting enzyme 2 (ACE2) degrades Ang II to angiotensin-(1-7) [Ang-(1-7)] and has recently been described to have an antagonistic effect on ACE signalling. However, the specific underlying role of ACE2 in the pathogenesis of severe acute pancreatitis (SAP) is unclear. In the present study, the local imbalance of ACE and ACE2, as well as Ang II and Ang-(1-7) expression, was compared in wild-type (WT) and ACE2 knock-out (KO) or ACE2 transgenic (TG) mice subjected to cerulein-induced SAP. Serum amylase, tumour necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-10 levels and histological morphometry were used to determine the severity of pancreatitis. In WT mice, pancreatic ACE and Ang II and serum Ang II expression increased (P < 0.05), while pancreatic ACE2 and Ang-(1-7) and serum Ang-(1-7) levels were also significantly elevated (P < 0.05) from 2 to 72 h after the onset of SAP. However, the ratio of pancreatic ACE2 to ACE expression was significantly reduced (from 1.46 ± 0.09 to 0.27 ± 0.05, P < 0.001) and paralleled the severity of pancreatitis. The Ace2 KO mice exhibited increased levels of tumour necrosis factor-α, IL-1β, IL-6, multifocal coagulative necrosis and inflammatory infiltrate, and lower levels of serum IL-10 and pancreatic Ang-(1-7) (4.70 ± 2.13 versus 10.87 ± 2.51, P < 0.001) compared with cerulein-treated WT mice at the same time point. Conversely, Ace2 TG mice with normal ACE expression were more resistant to SAP challenge as evidenced by a decreased inflammatory response, attenuated pathological changes and increased survival rates. These data suggest that the ACE2-ACE imbalance plays an important role in the pathogenesis of SAP and that pancreatic ACE2 is an important factor in determining the severity of SAP.

Topics: Acute Disease; Amylases; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Biomarkers; Ceruletide; Disease Models, Animal; Genotype; Inflammation Mediators; Male; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Pancreas; Pancreatitis; Peptide Fragments; Peptidyl-Dipeptidase A; Phenotype; Severity of Illness Index; Time Factors

Gene expression is affected by modifications to histone core proteins within chromatin. Changes in these modifications, or epigenetic reprogramming, can dictate cell fate and promote susceptibility to disease. The goal of this study was to determine the extent of epigenetic reprogramming in response to chronic stress that occurs following ablation of MIST1 (Mist1(-/-) ), which is repressed in pancreatic disease. Chromatin immunoprecipitation for trimethylation of lysine residue 4 on histone 3 (H3K4Me3) in purified acinar cells from wild type and Mist1(-/-) mice was followed by Next Generation sequencing (ChIP-seq) or ChIP-qPCR. H3K4Me3-enriched genes were assessed for expression by qRT-PCR in pancreatic tissue before and after induction of cerulein-induced pancreatitis. While most of H3K4Me3-enrichment is restricted to transcriptional start sites, >25% of enrichment sites are found within, downstream or between annotated genes. Less than 10% of these sites were altered in Mist1(-/-) acini, with most changes in H3K4Me3 enrichment not reflecting altered gene expression. Ingenuity Pathway Analysis of genes differentially-enriched for H3K4Me3 revealed an association with pancreatitis and pancreatic ductal adenocarcinoma in Mist1(-/-) tissue. Most of these genes were not differentially expressed but several were readily induced by acute experimental pancreatitis, with significantly increased expression in Mist1(-/-) tissue relative to wild type mice. We suggest that the chronic cell stress observed in the absence of MIST1 results in epigenetic reprogramming of genes involved in promoting pancreatitis to a poised state, thereby increasing the sensitivity to events that promote disease.

Topics: Acinar Cells; Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Pancreatic Ductal; Ceruletide; Chromatin; Epigenesis, Genetic; Histones; Male; Metabolic Networks and Pathways; Methylation; Mice; Mice, Knockout; Pancreas; Pancreatic Neoplasms; Pancreatitis

The modulating effect of methanolic extract of Emblica officinalis (MEEO) on ethanol (EtOH)- and cerulein (Cer)-induced pancreatitis in rats was investigated in this study.. Male albino Wistar rats were divided into four groups. Group 1 and 2 rats served as control and fed normal diet. Group 3 and 4 rats were fed isocalorically adjusted diet containing EtOH (36% of total calories) for 5 weeks and also subjected to intraperitoneal injection of Cer 20 µg/kg b.wt. thrice weekly for the last 3 weeks of the experimental period. In addition, group 2 and 4 rats received 200 mg/kg b.wt. of MEEO from 15th day till the experimental period. Serum levels of lipase (L), amylase (A), cytokines IL-1β, IL-18, caspase-1 and oxidative stress index (OSI) were determined. Levels of fecal trypsin, total collagen, caspase-1, myeloperoxidase (MPO), antioxidants and mRNA expression of caspase-1, IL-1β and IL-18 were determined in the pancreas.. HPLC analysis showed the presence of rutin in MEEO. We observed a significant elevation in serum L/A ratio, IL-1β, IL-18, caspase-1, OSI, collagen, MPO activity and the mRNA expression of IL-1β, IL-18 and caspase-1 and significant reduction in fecal trypsin and antioxidant status in EtOH- and Cer-administered rats. The inflammatory markers were found to be reduced and the antioxidant status of pancreas was maintained in MEEO-coadministered rats.. The rutin rich nature of E. officinalis can be claimed for its anti-inflammatory and pancreato protective effects.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Biomarkers; Ceruletide; Chromatography, High Pressure Liquid; Collagen; Cytokines; Ethanol; Fruit; Male; Oxidative Stress; Pancreas; Pancreatitis; Phyllanthus emblica; Phytotherapy; Plant Extracts; Rats, Wistar; Rutin; Trypsin

The aim of this study was to investigate whether BML-111 can exert protective effects on cerulein-induced acute pancreatitis-associated lung injury (APALI) via activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) signaling pathway. Severe acute pancreatitis (SAP) was established by intraperitoneal injection of cerulein (50 μg/kg) seven times at hourly intervals and Escherichia coli lipopolysaccharide (10 mg/kg) once after the last dose of cerulein immediately. BML-111 (1 mg/kg) was administered 1 h before the first injection of cerulein. Samples were taken at 3, 6, 12, and 24 h after the last injection. Pathologic lesions of the pancreas and lung tissues as well as the levels of serum amylase were analyzed; Myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), Nrf2, heme oxygenase-1 (HO-1), and. quinone oxidoreductase-1 (NQO1) of lung tissue were determined. The findings revealed that the injuries of pancreas and lung were typically induced by cerulein. The administration of BML-111 reduced the levels of serum amylase, lung MPO, lung MDA, the wet-to-dry weight ratio, and the pathology injury scores of the lung and pancreas, which increased in the SAP group. The expressions of Nrf2, HO-1, NQO1, and activity of SOD in lung tissue increased in the BML-111 group compared with those in the SAP group. This study indicates that BML-111 may play a critical protective role in APALI induced by cerulein. The underlying mechanisms of protective role may be attributable to its antioxidant effects through the activation of Nrf2/ARE pathway.

Topics: Acute Disease; Acute Lung Injury; Animals; Ceruletide; Disease Models, Animal; Heme Oxygenase-1; Heptanoic Acids; Lipopolysaccharides; Lung; Male; Malondialdehyde; Membrane Proteins; Mice; Mice, Inbred BALB C; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Pancreatitis; Peroxidase; Response Elements; Signal Transduction; Superoxide Dismutase

This study investigated the relationship of Fas and Fas ligand (FasL) expression and apoptosis of lymphocytes in relation to the pathogenic immune response and infectious complications observed in experimental severe acute pancreatitis in mice. Forty male Balb/c mice were randomly divided into control, mild (MAP), and severe acute pancreatitis (SAP) groups. Overexpression of Fas/FasL messenger ribonucleic acid (mRNA) and protein was observed in spleen-derived lymphocytes in SAP (p < 0.01). Apoptosis of these resulted in a depletion of circulating lymphocytes in this group (p < 0.05). A further significant change in the SAP group with infectious complications was observed. A positive relationship was found between the Fas/FasL expression and lymphocyte apoptosis, and negative relationships were observed between Fas/FasL expression and CD4(+) and CD19(+) lymphocytes and the CD4(+)/CD8(+) ratio in SAP mice (p < 0.01). The results suggest that the overexpression of Fas/FasL is associated with infectious complications and severity of experimental severe acute pancreatitis by promoting apoptosis of lymphocytes.

Topics: Animals; Antigens, CD19; Apoptosis; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Ceruletide; Fas Ligand Protein; fas Receptor; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Pancreatitis; RNA, Messenger; Spleen

Three classical rodent models of acute pancreatitis were created in an effort to identify potential pre-clinical models of drug-induced pancreatitis (DIP) and candidate non-invasive biomarkers for improved detection of DIP. Study objectives included designing a lexicon to minimize bias by capturing normal variation and spontaneous and injury-induced changes while maintaining the ability to statistically differentiate degrees of change, defining morphologic anchors for novel pancreatic injury biomarkers, and improved understanding of mechanisms responsible for pancreatitis. Models were created in male Sprague-Dawley rats and C57BL6 mice through: 1) administration of the cholecystokinin analog, caerulein; 2) administration of arginine; 3) surgical ligation of the pancreatic duct. Nine morphologically detectable processes were used in the lexicon; acinar cell hypertrophy; acinar cell autophagy; acinar cell apoptosis; acinar cell necrosis; vascular injury; interstitial edema, inflammation and hemorrhage; fat necrosis; ductal changes; acinar cell atrophy. Criteria were defined for scoring levels (0 = absent, 1 = mild, 2 = moderate, 3 = severe) for each lexicon component. Consistent with previous studies, histopathology scores were significant greater in rats compared to mice at baseline and after treatment. The histopathology scores in caerulein and ligation-treated rats and mice were significantly greater than those of arginine-treated rats and mice. The present study supports a multifaceted pathogenesis for acute pancreatitis in which intra-acinar trypsinogen activation, damage to acinar cells, fat cells, and vascular cells as well as activation/degranulation of mast cells and activated macrophages all contribute to the initiation and/or progression of acute inflammation of the exocrine pancreas.

Topics: Animals; Arginine; Ceruletide; Disease Models, Animal; Ligation; Male; Mice; Mice, Inbred C57BL; Pancreatic Ducts; Pancreatitis; Rats; Rats, Sprague-Dawley

Autophagy is an intracellular degradation system in eukaryotic cells that occurs at a basal level. It can also be induced in response to environmental signals including nutrients, hormones, microbial pathogens, and growth factors, although the mechanism is not known in detail. We previously demonstrated that excessive autophagy is induced within pancreatic acinar cells deficient in Spink3, which is a trypsin inhibitor. SPINK1, the human homolog of murine Spink3, has structural similarity to epidermal growth factor (EGF), and can bind and stimulate the EGF receptor (EGFR). To analyze the role of the EGFR in pancreatic development, in the regulation of autophagy in pancreatic acinar cells, and in cerulein-induced pancreatitis, we generated and examined acinar cell-specific Egfr-deficient (Egfr(-/-)) mice. Egfr(-/-) mice showed no abnormalities in pancreatic development, induction of autophagy, or cerulein-induced pancreatitis, suggesting that Egfr is dispensable for autophagy regulation in pancreatic acinar cells.

Topics: Acinar Cells; Animals; Autophagy; Carrier Proteins; Ceruletide; ErbB Receptors; Female; Glycoproteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatitis; Prostatic Secretory Proteins; Signal Transduction; Trypsin Inhibitor, Kazal Pancreatic

Ulinastatin is a drug used effectively to alleviate symptoms and improve the pathophysiology of various types of pancreatitis. However, the molecular mechanism responsible for its action remains unknown. Therefore, we further explore the therapeutic effects of ulinastatin and investigate possible molecular pathways modulated by this drug in the development of severe acute pancreatitis (SAP).. SAP mouse model was created by administering intraperitoneal injections of cerulein and lipopolysaccharide. Pancreatic injury was assessed by performing biochemical and histological assays and by measuring the inflammatory response of the pancreas. Specifically, we examined changes in the expression of components of the rennin-angiotensin system (RAS), including angiotensin-converting enzyme (ACE)-angiotensin II (Ang II)-angiotensin type 1 receptor (AT-1R), and ACE2-Ang-(1-7)-Mas receptor.. When SAP mouse models were treated with ulinastatin at a dosage of 50,000 U/kg body weight, we found, through biochemical and histopathological analyses, that the pancreatic injury was significantly ameliorated. Administration of ulinastatin to SAP mice led to increased expression of ACE2, Ang-(1-7), and Mas receptor, decreased expression of serum Ang II and pancreatic AT-1R, and no alterations in the expression of pancreatic ACE and Ang II when compared to cerulein-treated control group that did not receive ulinastatin.. This study shows that ulinastatin has differential effects on the two axes of the RAS during SAP. Our results further suggest that upregulation of components of the ACE2-Ang-(1-7)-Mas pathway might be an important mechanism contributing to the therapeutic role of ulinastatin in alleviating pancreatitis-associated symptoms.

Topics: Acute Disease; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Ceruletide; Disease Models, Animal; Gene Expression; Glycoproteins; Lipopolysaccharides; Mice, Inbred C57BL; Molecular Targeted Therapy; Pancreatitis; Peptide Fragments; Peptidyl-Dipeptidase A; Prospective Studies; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; Severity of Illness Index

Acute pancreatitis (AP) is a potentially lethal disease characterized by inflammation and parenchymal cell death; also, the severity of AP correlates directly with necrosis and inversely with apoptosis. However, mechanisms of regulating cell death in AP remain unclear. The endoplasmic reticulum (ER) chaperone protein GRP78 has anti-apoptotic properties, in addition to modulating ER stress responses. This study used RNA interference (RNAi) approach to investigate the potential role of GRP78 in regulating apoptosis during AP. In vitro models of AP were successfully developed by treating AR42J cells with cerulein or cerulein plus lipoplysaccharide (LPS). There was more pancreatic inflammation and less apoptosis with the cerulein plus LPS treatment. Furthermore, knockdown of GRP78 expression markedly promoted apoptosis and reduced necrosis in pancreatic acinar cells. This was accomplished by enhancing the activation of caspases and inhibiting the activity of X-linked inhibitor of apoptosis protein (XIAP), as well as a receptor interacting protein kinase-1(RIPK1), which is a key mediator of necrosis. This attenuated the severity of pancreatic inflammation, especially after cerulein plus LPS treatment. In conclusion, these findings indicate that GRP78 plays an anti-apoptotic role in regulating the cell death response during AP. Therefore, GRP78 is a potential therapeutic target for AP.

Topics: Acinar Cells; Amylases; Animals; Apoptosis; Caspases; Cell Line; Ceruletide; Cytokines; Gene Expression; Gene Knockdown Techniques; Heat-Shock Proteins; Lipase; Lipopolysaccharides; Pancreas, Exocrine; Pancreatitis; Rats; RNA Interference; RNA, Small Interfering; Signal Transduction

The NACHT, LRR, and pyrin domain-containing protein 3 (NLRP3) inflammasome induces inflammation in response to organ injury, but little is known about its regulation. Toll-like receptors (TLRs) provide the first signal required for activation of the inflammasome and stimulate aerobic glycolysis to generate lactate. We examined whether lactate and the lactate receptor, Gi-protein-coupled receptor 81 (GPR81), regulate TLR induction of signal 1 and limit inflammasome activation and organ injury.. Primary mouse macrophages and human monocytes were incubated with TLR4 agonists and lactate and assayed for levels of pro-interleukin (IL)1β, NLRP3, and caspase-1 (CASP1); release of IL1β; and activation of nuclear factor-κB (NF-κB) and caspase-1. Small interfering RNAs were used to reduce levels of GPR81 and arrestin β-2 (ARRB2), and an NF-κB luciferase reporter transgene was transfected in RAW 264.7 cells. Cell lysates were analyzed by immunoprecipitation with an antibody against GPR81. Acute hepatitis was induced in C56BL/6N mice by administration of lipopolysaccharide and D-galactosamine. Acute pancreatitis was induced by administration of lipopolysaccharide and cerulein. Some mice were given intraperitoneal injections of sodium lactate or small interfering RNA against Gpr81. Activation of NF-κB in tissue macrophages was assessed in mice that expressed a reporter transgene.. In macrophages and monocytes, increasing concentrations of lactate reduced TLR4-mediated induction of Il1B, Nlrp3, and Casp1; activation of NF-κB; release of IL1β; and cleavage of CASP1. GPR81 and ARRB2 physically interacted and were required for these effects. The administration of lactate reduced inflammation and organ injury in mice with immune hepatitis; this reduction required Gpr81 dependence in vivo. Lactate also prevented activation of NF-κB in macrophages of mice, and, when given after injury, reduced the severity of acute pancreatitis and acute liver injury.. Lactate negatively regulates TLR induction of the NLRP3 inflammasome and production of IL1β, via ARRB2 and GPR81. Lactate could be a promising immunomodulatory therapy for patients with acute organ injury.

Topics: Animals; Anti-Inflammatory Agents; Arrestins; beta-Arrestin 2; beta-Arrestins; Carrier Proteins; Cell Line; Ceruletide; Chemical and Drug Induced Liver Injury; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Galactosamine; Humans; Immunity, Innate; Inflammasomes; Injections, Intraperitoneal; Interleukin-1beta; Lipopolysaccharides; Liver; Macrophages; Male; Mice; Mice, Inbred C57BL; Monocytes; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatitis; Receptors, G-Protein-Coupled; RNA Interference; RNA, Small Interfering; Signal Transduction; Sodium Lactate; Toll-Like Receptor 4; Toll-Like Receptors; Transfection

Cancer-associated inflammation is a molecular key feature in pancreatic ductal adenocarcinoma. Oncogenic KRAS in conjunction with persistent inflammation is known to accelerate carcinogenesis, although the underlying mechanisms remain poorly understood. Here, we outline a novel pathway whereby the transcription factors NFATc1 and STAT3 cooperate in pancreatic epithelial cells to promote Kras(G12D)-driven carcinogenesis. NFATc1 activation is induced by inflammation and itself accelerates inflammation-induced carcinogenesis in Kras(G12D) mice, whereas genetic or pharmacologic ablation of NFATc1 attenuates this effect. Mechanistically, NFATc1 complexes with STAT3 for enhancer-promoter communications at jointly regulated genes involved in oncogenesis, for example, Cyclin, EGFR and WNT family members. The NFATc1-STAT3 cooperativity is operative in pancreatitis-mediated carcinogenesis as well as in established human pancreatic cancer. Together, these studies unravel new mechanisms of inflammatory-driven pancreatic carcinogenesis and suggest beneficial effects of chemopreventive strategies using drugs that are currently available for targeting these factors in clinical trials.. Our study points to the existence of an oncogenic NFATc1-STAT3 cooperativity that mechanistically links inflammation with pancreatic cancer initiation and progression. Because NFATc1-STAT3 nucleoprotein complexes control the expression of gene networks at the intersection of inflammation and cancer, our study has significant relevance for potentially managing pancreatic cancer and other inflammatory-driven malignancies.

Topics: Animals; Cell Line, Tumor; Ceruletide; Gene Expression Regulation, Neoplastic; Mice, Transgenic; NFATC Transcription Factors; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); STAT3 Transcription Factor

Diabetes and fibrosis can be concurrent processes in several diseases such as cystic fibrosis or chronic pancreatitis. To evaluate whether diabetes can influence fibrosis and thus aggravate the pathological process, the progression of chronic pancreatitis was assessed in diabetic and non diabetic mice. For this purpose, insulin producing beta-cells in C57Bl/6J mice were selectively impaired by administration of streptozotocin. Chronic pancreatitis was then induced by repetitive administration of cerulein in normoglycaemic and hyperglycaemic mice. Diabetes caused enhanced collagen I deposition within three weeks of the onset of chronic pancreatitis and increased the proliferation of interstitial cells. This was accompanied by an increased number of interlobular fibroblasts, which expressed S100A4 (fibroblast-specific protein-1) and stimulation of α-smooth muscle actin expression of pancreatic stellate cells. In addition, the observed aggravation of chronic pancreatitis by diabetes also led to a significantly enhanced atrophy of the pancreas, increased infiltration of inflammatory chloracetate esterase positive cells and enhanced acinar cell death. We conclude that diabetes has a detrimental influence on the progression of chronic pancreatitis by aggravating fibrosis, inflammation and pancreatic atrophy.

Topics: Actins; Animals; Ceruletide; Chronic Disease; Collagen Type I; Diabetes Mellitus, Experimental; Fibrosis; Mice; Pancreas; Pancreatitis; S100 Calcium-Binding Protein A4; S100 Proteins; Streptozocin

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.

Topics: Acinar Cells; Animals; Apoptosis; Ceruletide; Gene Expression; Humans; Immunohistochemistry; Inflammation; Mice, Inbred C57BL; Mice, Transgenic; Organ Specificity; Pancreas; Pancreatitis; Rats; Transgenes; Trypsin

Pancreatic acinar cell necrosis is indicative of severe pancreatitis and the degree of necrosis is an index of its outcome. We studied whether the dose and duration of injury correlates with severity, particularly in terms of necrosis, in caerulein-induced acute pancreatitis (AP) in Swiss albino mice. In addition to control group 1 (G1), groups 2 and 3 received four injections of caerulein every hour but were sacrificed at five hours (G2) and nine hours (G3) respectively, and group 4 received eight injections and was sacrificed at nine hours (G4). The severity of pancreatitis was assessed histopathologically and biochemically. The histopathological scores of pancreatitis in groups 3 and 4 were significantly higher than in groups 1 and 2 (4 vs. 1, 4 vs. 2, 3 vs. 1, 3 vs. 2; P < 0.05). TUNEL-positive apoptotic cells were significantly higher in groups 2 and 3 compared with groups 1 and 4 (P < 0.05). Necrosis was significantly more in group 4 than other groups (37.49% (4.68) vs. 19.97% (1.60) in G2; 20.36% (1.56) in G3; P = 0.006 for G 2 vs. 4 and P = 0.019 for G 3 vs. 4). Electron microscopy revealed numerous autophagosomes in groups 2 and 3 and mitochondrial damage and necrosis in group 4. The pancreatic and pulmonary myeloperoxidase activity in group 4 was significantly higher than that in the other groups (P < 0.01). Hence, severity of pancreatitis is a function of the dose of injurious agent, while inflammation is both dose and duration dependent, which may also explain the wide spectrum of severity of AP seen in clinical practice.

Topics: Acute Disease; Animals; Apoptosis; Behavior, Animal; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Kidney; Lung; Male; Mice; Necrosis; Pancreas; Pancreatitis; Peroxidase; Severity of Illness Index; Time Factors

Severe acute pancreatitis is a life-threatening disease with a high mortality; so far, no causal treatment is known. The aim of this study was to evaluate the therapeutic potential of hydroxyethyl starch (HES) and cell-free hemoglobin in an experimental model.. Thirty-nine pigs were randomly assigned into 3 groups. Severe acute pancreatitis was induced by intraductal injection of glycodeoxycholic acid in combination with intravenous administration of cerulein. All animals were kept in isovolemic conditions by application of Ringer solution, 10% HES, or cell-free hemoglobin. The pancreatic microcirculation was evaluated over 8 hours. Thereafter, the animals were observed for 6 days followed by killing of the animals and histopathologic examination.. The administration of HES and cell-free hemoglobin led to improved microcirculation and tissue oxygenation compared with the Ringer's group. Consequently, the histopathologic damage was reduced (5.5 [3-8.5] vs 9.5 [7.5-11]; P < 0.001). In addition, the mean survival was significantly longer at 121 hours (95% confidence interval, 102-139) versus the Ringer group's 57 hours (95% confidence interval, 32-82; P < 0.001).. The administration of HES and cell-free hemoglobin can improve microcirculation in severe acute porcine pancreatitis, with consequent reduction in histopathologic damage and mortality. Therefore, this might represent an interesting therapeutic option in the treatment of severe acute pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Glycodeoxycholic Acid; Hemoglobins; Hydroxyethyl Starch Derivatives; Isotonic Solutions; Microcirculation; Oxygen Consumption; Pancreas; Pancreatitis; Random Allocation; Ringer's Solution; Severity of Illness Index; Survival Analysis; Swine; Treatment Outcome

Acute pancreatitis (AP) is a complicated disease which is largely undiscovered. Fisetin, a natural flavonoid from fruits and vegetables, has been shown to have anti-inflammatory, antioxidant, and anti-cancer activities in various disease models. However, the effects of fisetin on AP have not been determined. Pre- and post- treatment of mice with fisetin reduced the severity of AP and pancreatitis-associated lung injury and inhibited several biochemical parameters (pancreatic weight to body weight ratio, amylase, lipase, and myeloperoxidase activity) and production of inflammatory cytokines. In pancreatic acinar cells, fisetin also inhibited cell death and production of inflammatory cytokines. In addition, fisetin inhibited activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-κB in vivo and in vitro. In conclusion, these results suggest that fisetin exhibits anti-inflammatory effect on AP and could be a beneficial agent in the treatment of AP and its pulmonary complications.

Topics: Acinar Cells; Acute Disease; Administration, Oral; Animals; Ceruletide; Down-Regulation; Enzyme Activation; Female; Flavonoids; Flavonols; Injections, Intraperitoneal; JNK Mitogen-Activated Protein Kinases; Lung; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreas; Pancreatitis; Signal Transduction

Acute pancreatitis (AP) is defined as an inflammatory disease of the pancreas. The purpose of this study was to examine the effectiveness of Anakinra on cerulein-induced experimental pancreatitis rat model by using the results of biochemical and histopathological findings.. Cerulein was administered to induce AP in rats. Group 1 was the sham group. Subcutancerulein was injected to the rats in group 2 for experimental pancreatitis group. In groups 3 and 4, 100 and 50 mg/kg intraperitoneal Anakinra were injected after the induction of experimental pancreatitis by subcutaneous cerulein in rats, respectively. Lastly, in group 5, rats were injected with intraperitoneal saline and subcutan cerulean for placebo group. The following parameters were evaluated: histopathological score of pancreatitis, apoptotic index, amylase, lipase, TNF-α levels, IL-1β and the leukocyte count.. When the results of serum amylase, lipase, TNF-α and IL-1β levels, the leukocyte count, histopathologic scores and apoptotic indices of control group compared to the results of other groups, the differences exhibited statistical significance (all p < 0.05). On the other hand, when the results of fourth group compared with the results of third group, the data demonstrated statistical insignificance (p > 0.05). However, no any significant differences were found between the results of fourth and fifth groups (p > 0.05).. In the light of these results, cerulein is an appropriate agent for experimental AP rat model and Anakinra has a favorable therapeutic effect on acute experimental pancreatitis model. Moreover, Anakinra significantly decreases cerulein-related pancreatic tissue injury and pancreatic apoptosis.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Ceruletide; Disease Models, Animal; Interleukin 1 Receptor Antagonist Protein; Interleukin-1beta; Leukocyte Count; Lipase; Male; Pancreatitis; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

Diosmetin (3', 5, 7-trihydroxy-4'-methoxyflavone), the aglycone part of the flavonoid glycosides diosmin occurs naturally in citrus fruit, was considered to exhibit anti-inflammatory and antioxidant properties. Our study aimed to investigate the effect of diosmetin in a murine model of cerulein-induced acute pancreatitis (AP). Experimental AP was induced in mice by seven intraperitoneal injection of cerulein (50 ug/kg) at hourly intervals. Diosmetin (100 mg/kg) or vehicle was pretreated 2 h before the first cerulein injection. After 6 h, 9 h, 12 h of the first cerulein injection, the severity of acute pancreatitis was evaluated biochemically and morphologically. Pretreatment with diosmetin significantly reduced serum levels of amylase and lipase; the histological injury; the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6; myeloperoxidase (MPO) activity, trypsinogen activation peptide (TAP) level, the expression of inducible nitric oxide synthase (iNOS); and the nuclear factor (NF)-κB activation in cerulein-induced AP. This study showed that administration of diosmetin demonstrated a beneficial effect on the course of cerulein-induced AP in mice. Therefore, diosmetin may become a new therapeutic agent in future clinical trials for treatment of AP.

Topics: Active Transport, Cell Nucleus; Acute Disease; Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Cytoprotection; Disease Models, Animal; Flavonoids; Inflammation Mediators; Male; Mice, Inbred C57BL; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Severity of Illness Index; Signal Transduction; Time Factors

To investigate the migratory path of stem cells in pancreatic tissues damaged by pancreatitis and to preliminarily identify stem cells that efficiently contribute to the repair of damaged pancreatic tissues.. An animal model of acute pancreatitis was established, in which rats in the experimental group were given intraperitoneal (IP) injections of caerulein. Before the rats were sacrificed, 5-bromo-2'-deoxyuridine (BrdU) was administered by IP injection to label proliferating pancreatic cells. The localization and distribution of the stem cell-specific marker proteins nestin and c-kit in pancreatic tissues were examined using an immunohistochemical approach, and proliferation-specific BrdU incorporation was also analyzed.. (1) The nestin-positive cells first appeared in the pancreatic interlobar vessels, and then, were observed in the pancreatic acinar and islet tissues. (2) C-kit-positive cells were located only in the pancreatic islets. (3) BrdU-positive cells first appeared in the area surrounding the interlobular region, and then were diffusely distributed and filled the pancreatic lobules.. (1) The stem cells, participated in the repair of damaged pancreatic tissue, appear firstly in the pancreatic interlobar vessels, then migrate toward the pancreatic lobules by using the interlobar vessels as channels and penetrate through the vascular endothelium into the pancreatic acinar tissues. A portion of the stem cells eventually penetrate into the islet tissue. (2) Exogenous stem cells, rather than the tissue-resident stem cells, efficiently contribute to the repair of damaged pancreatic tissues.

Topics: Acute Disease; Animals; Biomarkers; Cell Movement; Cell Proliferation; Ceruletide; Disease Models, Animal; Female; Immunohistochemistry; Male; Nestin; Pancreas; Pancreatitis; Proto-Oncogene Proteins c-kit; Rats, Sprague-Dawley; Stem Cells; Time Factors; Wound Healing

A polymorphism in the ATP synthase 8 (ATP8) gene of the murine mitochondrial genome, G-to-T transversion at position 7778, has been suggested to increase susceptibility to multiple autoimmune diseases, including autoimmune pancreatitis (AIP). The polymorphism also induces mitochondrial reactive oxygen species generation, secretory dysfunction and β-cell mass adaptation. Here, we have used two conplastic mouse strains, C57BL/6N-mtAKR/J (B6-mtAKR; nt7778 G; control) and C57BL/6N-mtFVB/N (B6-mtFVB; nt7778 T), to address the question if the polymorphism also affects the course of cerulein-induced acute pancreatitis in mice. Therefore, two age groups of mice (3 and 12-month-old, respectively) were subjected to up to 7 injections of the secretagogue cerulein (50 µg/kg body weight) at hourly intervals. Disease severity was assessed at time points from 3 hours to 7 days based on pancreatic histopathology, serum levels of α-amylase and activities of myeloperoxidase (MPO) in lung tissue. A comparison of cerulein-induced pancreatic tissue damage and increases of α-amylase and MPO activities showed no differences between the age-matched groups of both strains. Interestingly, histological evaluation of pancreatic tissue of both untreated and cerulein-treated B6-mtAKR and B6-mtFVB mice also revealed the presence of infiltrates of immune cells surrounding ducts and vessels; a finding that is compatible with an early stage of AIP. After recovery from cerulein-induced pancreatitis (day 7 after the injections), 12-month-old B6-mtFVB mice but not B6-mtAKR mice displayed aggravated lymphocytic lesions. A comparison of 12-month-old mice with other age groups of both strains revealed that lymphocytic foci were largely absent in 3-month-old mice, while 24-month-old mice were more affected. Together, our data suggest that the mtDNA nt7778 G/T polymorphism does not aggravate cerulein-induced acute pancreatitis. Autoimmune-like lesions, however, may progress faster if additional tissue damage occurs.

Topics: alpha-Amylases; Animals; Ceruletide; DNA, Mitochondrial; Humans; Lymphocytes; Mice; Mitochondria; Pancreatitis; Peroxidase; Polymorphism, Single Nucleotide; Reactive Oxygen Species

Pancreatitis is a necroinflammatory disease with acute and chronic manifestations. Accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic pancreatitis (CP). Pancreatic parathyroid hormone-related protein (PTHrP) levels are elevated in a mouse model of cerulein-induced AP. Here, we show elevated PTHrP levels in mouse models of pancreatitis induced by chronic cerulein administration and pancreatic duct ligation. Because acinar cells play a major role in the pathophysiology of pancreatitis, mice with acinar cell-specific targeted disruption of the Pthrp gene (PTHrP(Δacinar)) were generated to assess the role of acinar cell-secreted PTHrP in pancreatitis. These mice were generated using Cre-LoxP technology and the acinar cell-specific elastase promoter. PTHrP(Δacinar) exerted protective effects in cerulein and pancreatic duct ligation models, evident as decreased edema, histological damage, amylase secretion, pancreatic stellate cell (PSC) activation, and extracellular matrix deposition. Treating acinar cells in vitro with cerulein increased IL-6 expression and NF-κB activity; these effects were attenuated in PTHrP(Δacinar) cells, as were the cerulein- and carbachol-induced elevations in amylase secretion. The cerulein-induced upregulation of procollagen I expression was lost in PSCs from PTHrP(Δacinar) mice. PTHrP immunostaining was elevated in human CP sections. The cerulein-induced upregulation of IL-6 and ICAM-1 (human acinar cells) and procollagen I (human PSCs) was suppressed by pretreatment with the PTH1R antagonist, PTHrP (7-34). These findings establish PTHrP as a novel mediator of inflammation and fibrosis associated with CP. Acinar cell-secreted PTHrP modulates acinar cell function via its effects on proinflammatory cytokine release and functions via a paracrine pathway to activate PSCs.

Topics: Acinar Cells; Amylases; Animals; Carbachol; Cells, Cultured; Ceruletide; Fibrosis; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Mice; Mice, Inbred BALB C; Mice, Knockout; NF-kappa B; Pancreatic Ducts; Pancreatitis; Parathyroid Hormone-Related Protein; Procollagen

Understanding the regulation of death pathways, necrosis and apoptosis, in pancreatitis is important for developing therapies directed to the molecular pathogenesis of the disease. Protein kinase Cε (PKCε) has been previously shown to regulate inflammatory responses and zymogen activation in pancreatitis. Furthermore, we demonstrated that ethanol specifically activated PKCε in pancreatic acinar cells and that PKCε mediated the sensitizing effects of ethanol on inflammatory response in pancreatitis. Here we investigated the role of PKCε in the regulation of death pathways in pancreatitis. We found that genetic deletion of PKCε resulted in decreased necrosis and severity in the in vivo cerulein-induced pancreatitis and that inhibition of PKCε protected the acinar cells from CCK-8 hyperstimulation-induced necrosis and ATP reduction. These findings were associated with upregulation of mitochondrial Bak and Bcl-2/Bcl-xL, proapoptotic and prosurvival members in the Bcl-2 family, respectively, as well as increased mitochondrial cytochrome c release, caspase activation, and apoptosis in pancreatitis in PKCε knockout mice. We further confirmed that cerulein pancreatitis induced a dramatic mitochondrial translocation of PKCε, suggesting that PKCε regulated necrosis in pancreatitis via mechanisms involving mitochondria. Finally, we showed that PKCε deletion downregulated inhibitors of apoptosis proteins, c-IAP2, survivin, and c-FLIPs while promoting cleavage/inactivation of receptor-interacting protein kinase (RIP). Taken together, our findings provide evidence that PKCε activation during pancreatitis promotes necrosis through mechanisms involving mitochondrial proapoptotic and prosurvival Bcl-2 family proteins and upregulation of nonmitochondrial pathways that inhibit caspase activation and RIP cleavage/inactivation. Thus PKCε is a potential target for prevention and/or treatment of acute pancreatitis.

Topics: Acinar Cells; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; CASP8 and FADD-Like Apoptosis Regulating Protein; Ceruletide; Cytochromes c; Ethanol; Gene Deletion; Inhibitor of Apoptosis Proteins; Mice; Mice, Inbred C57BL; Necrosis; Pancreas; Pancreatitis; Protein Kinase C-epsilon; Proto-Oncogene Proteins c-bcl-2; Receptor-Interacting Protein Serine-Threonine Kinases; Sincalide

Mild injury of the exocrine pancreas is often asymptomatic and can be under- or mis-diagnosed. The pancreas-enriched microRNAs miR-216a and miR-217 were evaluated as potential serum biomarkers of exocrine pancreas injury in rodent models of acute pancreatitis induced by caerulein, l-arginine, and pancreatic duct ligation. Both microRNAs showed time- and dose- relevant responses to pancreatic injury and wider dynamic ranges of response than serum amylase or lipase. Pancreas-selective microRNAs were found to be relatively sensitive serum biomarkers of pancreatic injury in rodents with potentially greater specificity than the current standard assays.

Topics: Amylases; Animals; Arginine; Biomarkers; Ceruletide; Male; Mice, Inbred C57BL; MicroRNAs; Pancreas; Pancreatitis; Rats, Sprague-Dawley; ROC Curve

Inflammasomes are protein complexes formed in response to tissue injury and inflammation to regulate the formation of proinflammatory cytokines. Nod-like receptor pyrin domain containing 3 (NLRP3) is one such inflammasome involved in pancreatic inflammation. Caspase activation recruitment domain (CARD) is an interaction motif found in all the major components of NLRP3 inflammasome such as apoptosis associated speck-like CARD containing protein (ASC) and procaspase-1. NLRP3 activates procaspase-1 with the concerted action of CARD domain of ASC. In the present study, the effect of rutin, a natural flavonoid on the expression of ASC of NLRP3, was investigated in rats treated with ethanol (EtOH) and cerulein (Cer). Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats were fed normal diet, whereas groups 3 and 4 rats were fed EtOH (36 % of total calories) containing diet for a total period of 5 weeks and also administered Cer (20 µg/kg body weight i.p.) thrice weekly for the last 3 weeks. In addition, groups 2 and 4 rats received daily 100 mg/kg body weight of rutin from third week. Rutin co-administration significantly decreased the level of pancreatic marker enzymes, oxidative stress markers, inflammatory markers, mRNA expression of caspase-1, cytokines, ASC-NLRP3, and protein expression of caspase-1 and ASC in rats received EtOH-Cer. The results of the study revealed that rutin can reduce inflammation in pancreas probably by influencing the down regulation of ASC-NLRP3 which might result in the reduced activation of caspase-1 and controlled cytokine production.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Apoptosis Regulatory Proteins; Biomarkers; CARD Signaling Adaptor Proteins; Carrier Proteins; Caspase 1; Ceruletide; Disease Models, Animal; Enzymes; Ethanol; Glutathione; Inflammasomes; Male; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Pancreatitis; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Rutin

The gene p8 was initially described in pancreatic tissue during acute experimental pancreatitis, a disease that is characterized by a systemic immune response. Although early reports suggested that p8 affects leukocyte migration during acute pancreatitis (AP), no studies revealing its immune-modulatory effects have been performed.. We investigated the composition of the cellular immune system in naive p8 knockout (p8(−/−)) mice and compared with matched wild-type mice during pancreatitis.. In young mice, there were no relevant differences in the composition of peripheral and splenic CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD11b(+)Gr-1(-), and Gr-1 cells. In mature p8(−/−) mice, increased splenic CD4CD25FoxP3 cells, spleen siderosis, and increased marginal zones in the splenic white pulp were found. During AP, peripheral and splenic CD3(+) and CD3CD4 declined stronger in the p8(−/−) mice. The spleen of the p8(−/−) mice showed severe hypoplasia of the white pulp and mild hyperplasia of the red pulp. This was associated with a significantly increased rate of apoptosis.. We conclude that p8 has no impact on the cellular composition of the adaptive and innate immune systems in noninflammatory conditions. However, it may limit apoptosis and maintain homeostasis of the immune reaction during AP.

Topics: Acute Disease; Animals; Apoptosis; Cell Count; Ceruletide; DNA-Binding Proteins; Female; Hemosiderosis; Lymphocyte Count; Lymphocyte Subsets; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Neoplasm Proteins; Organ Specificity; Pancreatitis; Splenic Diseases; Splenomegaly

Topics: Animals; Antioxidants; Ceruletide; Female; Male; Melatonin; Oxidative Stress; Pancreas; Pancreatitis

Oxidative stress is recognized as a pivotal effector of several pathogenic processes, including acute pancreatitis. Reactive oxygen species not just cause damage on the main cellular components, but also influence the expression of antioxidant system genes. Antioxidant molecules, such as melatonin, could be good candidates for the treatment of this multidimensional disease. The present study was to evaluate the chemopreventive effect of melatonin in a rat model of cerulein-induced acute pancreatitis.. Four subcutaneous injections of cerulein (20 μg/kg body weight) were given to Wistar rats at two hours intervals; melatonin was injected intraperitoneally (25 mg/kg body weight) 30 minutes before each injection of cerulein. Lipid peroxidation, protein oxidation (carbonyl groups), total antioxidant status, and glutathione peroxidase activity were determined in pancreatic tissue using commercial kits.. The chemopreventive administration of melatonin caused a reduction in lipid peroxidation and protein oxidation due to injections of cerulein. Additionally, melatonin treatment was also able to revert glutathione peroxidase activity and total antioxidant status near to control levels, suggesting that melatonin could prevent from oxidative phenomena in the pancreas, such as lipid peroxidation and protein oxidation, and could stimulate, directly or indirectly, the expression of antioxidant enzymes.. Melatonin, a polyvalent antioxidant, protected the pancreatic damage via the decrease of oxidative stress and increase of the activities of antioxidant enzymes in cerulein-induced acute pancreatitis.

Topics: Acute Disease; Animals; Antioxidants; Ceruletide; Cytoprotection; Disease Models, Animal; Female; Glutathione Peroxidase; Lipid Peroxidation; Male; Melatonin; Oxidative Stress; Pancreas; Pancreatitis; Protein Carbonylation; Rats, Wistar

Acute pancreatitis is characterized by inflammatory processes affecting not only the pancreas, but also the lung. Here, we investigated timing of leucocyte infiltration and chemokine expression within lung and pancreas during pancreatitis and whether treatments selectively inhibiting chemokines (using Evasins) could improve organ injury.. C57Bl/6 mice were submitted in vivo to 10-h intraperitoneal injections of cerulein and followed for up to 168 h. Five minutes after the first cerulein injection, a single intraperitoneal injection of 10 μg Evasin-3, 1 μg Evasin-4 or an equal volume of vehicle (PBS) was performed. Leucocytes, reactive oxygen species (ROS), necrosis and chemokine/cytokine mRNA expression were assessed in different organs by immunohistology and real-time RT-PCR, respectively.. In the lung, neutrophil infiltration and macrophage infiltration peaked at 12 h and were accompanied by increased CXCL2 mRNA expression. CCL2, CXCL1 and TNF-alpha significantly increased after 24 h as compared to baseline. No increase in CCL3 and CCL5 was observed. In the pancreas, neutrophil infiltration peaked at 6 h, while macrophages increased only after 72 h. Treatment with Evasin-3 decreased neutrophil infiltration, ROS production and apoptosis in the lung and reduced neutrophils, macrophages apoptosis and necrosis in the pancreas. Evasin-4 only reduced macrophage content in the lung and did not provide any benefit at the pancreas level.. Chemokine production and leucocyte infiltration are timely regulated in lung and pancreas during pancreatitis. CXC chemokine inhibition with Evasin-3 improved neutrophil inflammation and injury, potentially interfering with damages in acute pancreatitis and related pulmonary complications.

Topics: Animals; Anti-Inflammatory Agents; Arthropod Proteins; Ceruletide; Chemokine CXCL1; Chemokine CXCL2; Disease Models, Animal; Leukocytes; Male; Mice, Inbred C57BL; Necrosis; Neutrophil Infiltration; Neutrophils; Oxidative Stress; Pancreas; Pancreatitis; Reactive Oxygen Species; Receptors, CXCR; Salivary Proteins and Peptides

The change in exocrine mass is an important parameter to follow in experimental models of pancreatic injury and regeneration. However, at present, the quantitative assessment of exocrine content by histology is tedious and operator-dependent, requiring manual assessment of acinar area on serial pancreatic sections. In this study, we utilized a novel computer-generated learning algorithm to construct an accurate and rapid method of quantifying acinar content. The algorithm works by learning differences in pixel characteristics from input examples provided by human experts. HE-stained pancreatic sections were obtained in mice recovering from a 2-day, hourly caerulein hyperstimulation model of experimental pancreatitis. For training data, a pathologist carefully outlined discrete regions of acinar and non-acinar tissue in 21 sections at various stages of pancreatic injury and recovery (termed the "ground truth"). After the expert defined the ground truth, the computer was able to develop a prediction rule that was then applied to a unique set of high-resolution images in order to validate the process. For baseline, non-injured pancreatic sections, the software demonstrated close agreement with the ground truth in identifying baseline acinar tissue area with only a difference of 1% ± 0.05% (p = 0.21). Within regions of injured tissue, the software reported a difference of 2.5% ± 0.04% in acinar area compared with the pathologist (p = 0.47). Surprisingly, on detailed morphological examination, the discrepancy was primarily because the software outlined acini and excluded inter-acinar and luminal white space with greater precision. The findings suggest that the software will be of great potential benefit to both clinicians and researchers in quantifying pancreatic acinar cell flux in the injured and recovering pancreas.

Topics: Acinar Cells; Algorithms; Animals; Automation; Ceruletide; Humans; Mice; Pancreatitis

Acute pancreatitis (AP) is a frequent gastrointestinal disorder that causes significant morbidity, and its incidence has been progressively increasing. AP starts as a local inflammation in the pancreas that often leads to systemic inflammatory response and complications. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition in murine models has beneficial effects in inflammatory diseases, but its significance in AP remains unexplored.. To investigate whether sEH may have a causal role in AP we utilized Ephx2 knockout (KO) mice to determine the effects of sEH deficiency on cerulein- and arginine-induced AP. sEH expression increased at the protein and messenger RNA levels, as well as enzymatic activity in the early phase of cerulein- and arginine-induced AP in mice. In addition, amylase and lipase levels were lower in cerulein-treated Ephx2 KO mice compared with controls. Moreover, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B and IL-6 were lower in cerulein-treated Ephx2 KO mice compared with controls. Further, Ephx2 KO mice exhibited decreased cerulein- and arginine-induced NF-κB inflammatory response, MAPKs activation and decreased cell death. Conclusions -These findings demonstrate a novel role for sEH in the progression of cerulein- and arginine-induced AP.

Topics: Acute Disease; Animals; Cell Death; Ceruletide; Disease Models, Animal; Epoxide Hydrolases; Gene Expression; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; NF-kappa B; Pancreatitis

Previously we have investigated the cerulein-induced acute pancreatitis and provided data on its micro-rheological impact in the rat. We hypothesized that non-steroid anti-inflammatory agent flunixin, the xanthine-derivate pentoxifylline and the low molecular weight heparin enoxaparin may have various beneficial effects improving microcirculatory and rheological parameters. In female rats, under general anesthesia, 10 μg/kg cerulein s.c. was administered and 2 hours afterwards microcirculation was tested by laser Doppler flowmetry on the tongue and after performing laparotomy on the small intestine, liver and pancreas prior to terminal blood sampling. From blood samples hematological parameters, blood pH, lactate concentration, erythrocyte deformability, osmoscan parameters and erythrocyte aggregation were tested. Compared to normal control in acute pancreatitis group we found severe deterioration in tissue microcirculation together with impaired erythrocyte deformability and enhanced aggregation, accompanied by acidic pH and increasing lactate concentration. Improvement was found when using flunixin (s.c.), pentoxifylline (i.p.) or enoxaparin (s.c.). These drugs could partly improve the blood flux on the surface of the investigated organs, and the flunixin had the most expressed improving effects on micro-rheological parameters. Surprisingly, the improving effect of pentoxifylline on micro-rheological parameters was not obvious (red blood cell deformability did not improved better than in the other treated groups), however, microcirculatory parameters improved.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticoagulants; Ceruletide; Clonixin; Enoxaparin; Erythrocyte Aggregation; Erythrocyte Deformability; Female; Microcirculation; Pancreatitis; Pentoxifylline; Rats; Vasodilator Agents

Severe acute pancreatitis (SAP) is a serious systemic disease with a sustained high mortality rate. Extensive evidence has shown that gut barrier dysfunction plays a critical role in the pathophysiology of SAP.. Investigating the role of intestinal mucosa oxidative stress in gut barrier dysfunction of SAP.. Twenty-four BALB/c mice were randomly divided into two groups with twelve mice each group. The SAP group mice received six intraperitoneal injections of cerulein (50 µg/kg) at 1-hour intervals, then given one intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS from E. coli) for inducing SAP. Normal saline was given to the mice of control group. The animals of each group were averaged to two batches. Four and eight hours after the final injection, respectively, mice were anesthetized and blood and tissue samples were harvested for examination. The pathological changes of pancreas and gut were observed and scored. The serum levels of diamine oxidase (DAO), amylase and tumor necrosis factor-alpha (TNF-α) were measured. The contents of malondialdehyde (MDA) and reduced glutathione (GSH) and activity of superoxide dismutase (SOD) and xanthine oxidase (XO) in gut mucosa were detected. In gut mucosa, the caspase-3 activity was measured and the cell apoptosis and apoptosis index (AI) were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The data were analyzed by ANOVA and t-test.. At four and eight hours after SAP induction, the SAP group mice had significantly higher pancreatic and gut pathological scores (p < 0.01) and increased serum levels of amylase (p < 0.05), DAO and TNF-α (p < 0.01) and increased MDA contents and XO activity of gut mucosa (p < 0.01) compared with those of control mice. There were significantly lower GSH contents (p < 0.05) and SOD activity (p < 0.01) of gut mucosa in the SAP mice. It was also observed that the gut mucosa cells of SAP mice had significantly higher caspase-3 activity and apoptosis index (p < 0.01).. In SAP, waterfall-style release of inflammatory factors such as TNF-α led to ischemia-reperfusion injury of gut mucosa which resulted in serious oxidative stress and activation of caspase-3 pathway and severe apoptosis of gut mucosa. Therefore, intestinal mucosal oxidative stress may play an important role in the mechanism of gut barrier dysfunction.

Topics: Acute Disease; Analysis of Variance; Animals; Apoptosis; Caspase 3; Ceruletide; Disease Models, Animal; In Situ Nick-End Labeling; Inflammation Mediators; Intestinal Mucosa; Male; Mice; Mice, Inbred BALB C; Oxidative Stress; Pancreatitis; Random Allocation; Reperfusion Injury; Severity of Illness Index; Tumor Necrosis Factor-alpha

To evaluate the inhibitory effects of Scolopendra subspinipes mutilans (SSM) on cerulein-induced acute pancreatitis (AP) in a mouse model.. SSM water extract (0.1, 0.5, or 1 g/kg) was administrated intraperitoneally 1 h prior to the first injection of cerulein. Once AP developed, the stable cholecystokinin analogue, cerulein was injected hourly, over a 6 h period. Blood samples were taken 6 h later to determine serum amylase, lipase, and cytokine levels. The pancreas and lungs were rapidly removed for morphological examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction. To specify the role of SSM in pancreatitis, the pancreatic acinar cells were isolated using collagenase method. Then the cells were pre-treated with SSM, then stimulated with cerulein. The cell viability, cytokine productions and high-mobility group box protein-1 (HMGB-1) were measured. Furthermore, the regulating mechanisms of SSM action were evaluated.. The administration of SSM significantly attenuated the severity of pancreatitis and pancreatitis associated lung injury, as was shown by the reduction in pancreatic edema, neutrophil infiltration, vacuolization and necrosis. SSM treatment also reduced pancreatic weight/body weight ratio, serum amylase, lipase and cytokine levels, and mRNA expression of multiple inflammatory mediators such as tumor necrosis factor-α and interleukin-1β. In addition, treatment with SSM inhibited HMGB-1 expression in the pancreas during AP. In accordance with in vivo data, SSM inhibited the cerulein-induced acinar cell death, cytokine, and HMGB-1 release. SSM also inhibited the activation of c-Jun NH2-terminal kinase, p38 and nuclear factor (NF)-κB.. These results suggest that SSM plays a protective role during the development of AP and pancreatitis associated lung injury via deactivating c-Jun NH2-terminal kinase, p38 and NF-κB.

Topics: Acute Disease; Acute Lung Injury; Amylases; Animals; Anti-Inflammatory Agents; Arthropod Venoms; Cell Survival; Cells, Cultured; Ceruletide; Cytokines; Disease Models, Animal; Enzyme Activation; HMGB1 Protein; Inflammation Mediators; JNK Mitogen-Activated Protein Kinases; Lipase; Mice; Mice, Inbred C57BL; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pancreas; Pancreatitis; Signal Transduction; Time Factors

Pancreatitis is classified into acute pancreatitis (AP) and chronic pancreatitis (CP). Apelin, a small regulatory peptide, is the endogenous ligand for the APJ receptor. Apelin and APJ are expressed in the pancreas. The aims of this study were to examine whether apelin influences the inflammatory and fibrosis responses to pancreatitis in mice and to identify mechanisms behind apelin's activities. Supramaximal cerulein induction of AP or CP caused significant (P < 0.05) elevations in pancreatic apelin and APJ expression. Levels declined during the recovery phases. In apelin gene-knockout mice with pancreatitis, pancreatic neutrophil invasion and myeloperoxidase activity were enhanced significantly, and apelin treatment suppressed both. Apelin exposure reduced CP-induced elevations of extracellular matrix-associated proteins. Apelin inhibited PDGF-simulated connective tissue growth factor production and proliferation of pancreatic stellate cells (PSCs). Serum granulocyte colony-stimulating factor and keratinocyte cytokine levels were higher in apelin gene-knockout than wild-type mice with pancreatitis. Apelin reduced AP- and CP-induced elevations in pancreatic NF-κB activation. Together, these findings imply that the pancreatic apelin-APJ system functions to curb the inflammatory and fibrosis responses during pancreatitis. Furthermore, findings suggest that apelin reduces inflammation and fibrosis by reducing neutrophil recruitment and PSC activity. Inhibition of neutrophil invasion may be mediated by reduced keratinocyte cytokine and granulocyte colony-stimulating factor secretion. Apelin-induced reductions in PSC proliferation and connective tissue growth factor production are putative mechanisms underlying apelin's inhibition of extracellular matrix production. The apelin-associated changes in NF-κB binding may be linked to apelin's regulation of pancreatic inflammatory and fibrosis responses during pancreatitis.

Topics: Adipokines; Animals; Apelin; Apelin Receptors; Ceruletide; Chemokines; Gene Expression Regulation; Granulocyte Colony-Stimulating Factor; Intercellular Signaling Peptides and Proteins; Interleukin-3; Mice; Mice, Knockout; Pancreatitis; Receptors, G-Protein-Coupled; Recombinant Fusion Proteins; RNA, Messenger

Diseases of the exocrine pancreas are often associated with perturbed differentiation of acinar cells. MicroRNAs (miRNAs) regulate pancreas development, yet little is known about their contribution to acinar cell differentiation. We aimed to identify miRNAs that promote and control the maintenance of acinar differentiation.. We studied mice with pancreas- or acinar-specific inactivation of Dicer (Foxa3-Cre/Dicer(loxP/-) mice), combined (or not) with inactivation of hepatocyte nuclear factor (HNF) 6 (Foxa3-Cre/Dicer(loxP/-)/Hnf6-/- mice). The role of specific miRNAs in acinar differentiation was investigated by transfecting cultured cells with miRNA mimics or inhibitors. Pancreatitis-induced metaplasia was investigated in mice after administration of cerulein.. Inhibition of miRNA synthesis in acini by inactivation of Dicer and pancreatitis-induced metaplasia were associated with repression of acinar differentiation and with induction of HNF6 and hepatic genes. The phenotype of Dicer-deficient acini depends on the induction of HNF6; overexpression of this factor in developing acinar cells is sufficient to repress acinar differentiation and to induce hepatic genes. Let-7b and miR-495 repress HNF6 and are expressed in developing acini. Their expression is inhibited in Dicer-deficient acini, as well as in pancreatitis-induced metaplasia. In addition, inhibiting let-7b and miR-495 in acinar cells results in similar effects to those found in Dicer-deficient acini and metaplastic cells, namely induction of HNF6 and hepatic genes and repression of acinar differentiation.. Let-7b, miR-495, and their targets constitute a gene network that is required to establish and maintain pancreatic acinar cell differentiation. Additional studies of this network will increase our understanding of pancreatic diseases.

Topics: Acinar Cells; Animals; Biomarkers; Cell Differentiation; Ceruletide; Flow Cytometry; Gene Expression Regulation; Hepatocyte Nuclear Factor 6; Immunohistochemistry; Metaplasia; Mice; Mice, Knockout; MicroRNAs; Pancreas, Exocrine; Pancreatitis; Real-Time Polymerase Chain Reaction

Acute pancreatitis (AP) is an inflammatory condition wherein pro-inflammatory mediators, oxidative stress, and NF-κB signaling play a key role. Currently, no specific therapy exists and treatment is mainly supportive and targeted to prevent local pancreatic injury and systemic inflammatory complications. This study was aimed to examine whether 1,8-cineole, a plant monoterpene with antioxidant and anti-inflammatory properties could ameliorate cerulein-induced acute pancreatitis.. AP was induced in Swiss mice by six one hourly injections of cerulein (50 μg/kg, i.p.). 1,8-cineole (100, 200 and 400mg/kg, p.o.) was administered 1h prior to first cerulein injection, keeping vehicle and thalidomide treated groups as controls. Blood samples were taken 6-h later to determine serum levels of amylase and lipase, and cytokines. The pancreas was removed for morphological examination, myeloperoxidase (MPO) and malondialdehyde (MDA) assays, reduced glutathione (GSH) levels, and for nuclear factor (NF)-κB immunostaining.. 1,8-cineole effectively reduced the cerulein-induced histological damage, pancreatic edema and NF-κB expression, levels of MPO activity and MDA, and replenished the GSH depletion. Cerulein increased serum levels of amylase and lipase, and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were also decreased by 1,8-cineole pretreatment, similar to thalidomide, a TNF-α inhibitor. The anti-inflammatory IL-10 cytokine level was, however, enhanced by 1,8-cineole.. These findings indicate that 1,8-cineole can attenuate cerulein-induced AP via an anti-inflammatory mechanism and by combating oxidative stress. Further studies are needed to clearly elucidate its benefits in patients on acute pancreatitis.

Topics: Animals; Ceruletide; Cyclohexanols; Cytokines; Eucalyptol; Male; Mice; Monoterpenes; NF-kappa B; Oxidative Stress; Pancreatitis

To investigate the effects of long term pretreatment with low-, medium- and high-dose aspirin (acetylsalicylic acid, ASA) on a model of acute pancreatitis (AP) induced in rats.. Forty male Wistar rats were used. Three experimental groups, each consisting of eight animals, received low- (5 mg/kg per day), medium- (150 mg/kg per day) and high-dose (350 mg/kg per day) ASA in supplemented pellet chow for 100 d. Eight animals, serving as the AP-control group, and another eight, serving as reference value (RV) group, were fed with standard pellet chow for the same period. After pretreatment, AP was induced in the experimental animals by intraperitoneal administration of cerulein (2 × 50 μg/kg), while the RV group received saline in the same way. Twelve hours after the second injection, the animals were sacrificed. Pancreatic tissue and plasma samples were collected. One part of the collected pancreatic tissues was used for histopathological evaluation, and the remaining portion was homogenized. Cytokine levels [tumor necrosis factor, interleukin (IL)-1β, IL-6], hemogram parameters, biochemical parameters (amylase and lipase), nuclear factor-κB, aspirin triggered lipoxins and parameters related to the antioxidant system (malondialdehyde, nitric oxide, hemeoxygenase-1, catalase and superoxide dismutase) were measured.. Cerulein administration induced mild pancreatitis, characterized by interstitial edema (total histopathological score of 5.88 ± 0.44 vs 0.25 ± 0.16, P < 0.001). Subsequent pancreatic tissue damage resulted in an increase in amylase (2829.71 ± 772.48 vs 984.57 ± 49.22 U/L, P = 0.001) and lipase (110.14 ± 75.84 U/L vs 4.71 ± 0.78 U/L, P < 0.001) in plasma, and leucocytes (6.89 ± 0.48 vs 4.36 ± 0.23, P = 0.001) in peripheral blood. Cytokines, IL-1β (18.81 ± 2.55 pg/μg vs 6.65 ± 0.24 pg/μg, P = 0.002) and IL-6 (14.62 ± 1.98 pg/μg vs 9.09 ± 1.36 pg/μg, P = 0.04) in pancreatic tissue also increased. Aspirin pretreatment reduced the increase in the aforementioned parameters to a certain degree and partially improved the histopathological alterations caused by cerulein. No evidence of side effects related to chronic ASA administration (e.g., inflammation or bleeding) was observed in the gastrointestinal tract in macroscopic and histopathological examination.. Long term ASA pretreatment could prevent and/or ameliorate certain hematological, serological and histological alterations caused by cerulein-induced AP.

Topics: Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Aspirin; Biomarkers; Ceruletide; Disease Models, Animal; Drug Administration Schedule; Inflammation Mediators; Lipase; Lipid Peroxidation; Male; Oxidative Stress; Pancreas; Pancreatitis; Rats; Rats, Wistar; Time Factors

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2-deficient (Crhr2-/-) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF2) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2-/- mice. Crhr2-/- mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2-/- male mice during pancreatitis. WT and Crhr2-/- female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2-/- male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2-/- mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2-/- mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.

Topics: Acinar Cells; Amylases; Animals; Cell Line; Ceruletide; Endoplasmic Reticulum Stress; Female; Male; Mice; Mice, Transgenic; Pancreatitis; Receptors, Corticotropin-Releasing Hormone; Sex Factors; Urocortins

Pancreatic ductal adenocarcinoma (PDA) is a leading cause of cancer-related death. Through the process of acinar-to-ductal metaplasia (ADM), pancreatic acinar cells give rise to pancreatic intraepithelial neoplasia (PanIN), the most common precursor of PDA. However, even when Kras is activated in a majority of acinar cells, ADM and subsequent development of PanINs is inefficient in the absence of additional stresses. Numb regulates cell junctions, integrins, and the activity of embryonic signaling pathways; therefore, we investigated its effects on acinar cell dedifferentiation, regeneration, and metaplasia.. We used mouse models of pancreatic regeneration and PDA as well as mice with loss-of-function alleles of Numb (p48Cre/p48Cre(ER);Numb(f/f) and p48Cre/p48Cre(ER);Kras(G12D);Numb(f/f) mice) to study the roles of Numb in pancreatic regeneration and ADM.. Loss of Numb resulted in premature dedifferentiation of acinar cells in response to injury due to administration of the cholecystokinin analogue cerulein and interfered with acinar cell regeneration. Numb was found to regulate multiple signaling pathways in acinar cells during cerulein-induced pancreatitis. Disruption of Numb accelerated and destabilized ADM in the context of oncogenic Kras (in p48Cre;Kras(G12D);Numb(f/f) and p48Cre(ER);Kras(G12D);Numb(f/f) mice).. Numb is an important regulator of acinar cell differentiation and viability during metaplasia. In mice with pancreatitis or pancreatic injury, elimination of Numb causes dedifferentiated acinar cells to undergo apoptosis, and this is not mitigated by oncogenic Kras.

Topics: Acinar Cells; Animals; Apoptosis; Cell Dedifferentiation; Cell Survival; Ceruletide; Disease Models, Animal; Membrane Proteins; Metaplasia; Mice; Mice, Inbred Strains; Nerve Tissue Proteins; Pancreas; Pancreatic Ducts; Pancreatitis; Proto-Oncogene Proteins p21(ras); Regeneration; Signal Transduction; Tumor Suppressor Protein p53

We have previously reported that bee venom (BV) has a protective role against acute pancreatitis (AP). However, the effects of apamin, the major compound of BV, on AP have not been determined. The aim of this study was to evaluate the effects of apamin on cerulein-induced AP.. AP was induced via intraperitoneal injection of supramaximal concentrations of the stable cholecystokinin analogue cerulein (50 μg/kg) every hour for 6 times. In the apamin treatment group, apamin was administered subcutaneously (10, 50, or 100 μg/kg) at both 18 and 1 h before the first cerulein injection. The mice were sacrificed at 6 h after the final cerulein injection. Blood samples were obtained to determine serum amylase and lipase levels, as well as cytokine production. The pancreas and lung were rapidly removed for morphologic and histological examination, myeloperoxidase (MPO) assay, and real-time reverse transcription-polymerase chain reaction. Furthermore, we isolated the pancreatic acinar cells to specify the role of apamin in AP.. Pre-treatment with apamin inhibited histological damage, pancreatic weight/body weight ratio, serum level of amylase and lipase, MPO activity, and cytokine production. In addition, apamin treatment significantly inhibited cerulein-induced pancreatic acinar cell death. Furthermore, apamin treatment inhibited the cerulein-induced activation of c-Jun NH2-terminal kinases (JNK).. These results could suggest that apamin could protect against AP by inhibition of JNK activation.

Topics: Acute Disease; Animals; Apamin; Ceruletide; Cholecystokinin; Cytokines; Disease Models, Animal; Injections, Intraperitoneal; Injections, Subcutaneous; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Pancreas; Pancreatitis

The main goal of this work was to get insight into the mechanism of cerulein-induced reactive oxygen species (ROS) formation and impact of c-Jun NH(2)-terminal kinase (JNK) on this process.. The study was performed on Wistar rats and on a cellular model of acute pancreatitis (AP) using AR42J cell line.. First of all, we observed that during AP, the iron storage protein ferritin in the rat pancreas undergoes degradation accompanied by an increased formation of protein carbonyls. Pancreatic acinar AR42J cells stimulated by cerulein showed increased labile iron pool that was accompanied by a decrease in the cellular ferritin-L level and an increase in the ROS formation. The changes in the ferritin-L level were inversely correlated with the ROS formation. The cells expressing inactive JNK1 mutant were completely resistant to cerulein-induced ferritin degradation.. Our data showed that cerulein-induced AP in rats and on cellular model is accompanied by JNK1-dependent ferritin degradation, increases labile iron pool and ROS formation.

Topics: Animals; Cell Line; Ceruletide; Ferritins; Iron; Mitogen-Activated Protein Kinase 8; Mutant Proteins; Pancreatitis; Rats; Rats, Wistar; Reactive Oxygen Species

Poly(ADP-ribose) polymerase (Parp) 1 is a key regulator of cell death, its inhibition prevented streptozotocin-induced diabetes and attenuated caerulein-induced acute pancreatitis. Reg family proteins are significantly induced by Parp1 inhibitor, experimental diabetes and/or acute pancreatitis. We propose that Reg proteins are involved in the protection of pancreatic cells by Parp1 inhibition. To test this possibility, Parp1-/- and wild-type mice were injected with streptozotocin to induce diabetes. Separately, acute pancreatitis was induced with repeated injections of caerulein. Upon streptozotocin administration, Parp1-/- mice displayed much decreased hyperglycemia and preserved serum insulin level. The treatment induced similar levels of Reg1, -2, -3α and -3β genes in the pancreas of both wild-type and Parp1-/- mice, suggesting that the upregulation of Reg family genes during streptozotocin-induced diabetes was independent of Parp1 ablation. In caerulein-induced pancreatitis, unlike being reported, Parp1 knockout caused no relief on the severity of pancreatitis; the upregulation of pancreatic Reg1, -2, -3α and -3β genes upon caerulein was unaffected by Parp1 deletion. Our results reconfirmed the protective effect of Parp1 gene deletion on islet β-cells but questioned its effect on the acinar cells. In either case, the significant induction of Reg family genes seemed independent of Parp1-mediated cell death.

Topics: Animals; Ceruletide; Diabetes Mellitus, Experimental; Glucose Transporter Type 2; Insulin-Secreting Cells; Lithostathine; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Pancreatitis; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Streptozocin; Transcriptional Activation; Up-Regulation

Hydrogen sulfide (H2S) has been reported to be involved in the signaling of the inflammatory response; however, there are differing views as to whether it is pro- or anti-inflammatory. In this study, we sought to determine whether endogenously synthesized H2S via cystathionine-γ-lyase (CSE) plays a pro- or anti-inflammatory role in caerulein-induced pancreatitis. To investigate this, we used mice genetically deficient in CSE to elucidate the function of CSE in caerulein-induced acute pancreatitis. We compared the inflammatory response and tissue damage of wild-type (WT) and CSE knockout (KO) mice following 10 hourly administrations of 50 μg/kg caerulein or saline control. From this, we found that the CSE KO mice showed significantly less local pancreatic damage as well as acute pancreatitis-associated lung injury compared with the WT mice. There were also lower levels of pancreatic eicosanoid and cytokines, as well as reduced acinar cell NF-κB activation in the CSE KO mice compared with WT mice. Additionally, in WT mice, there was a greater level of pancreatic CSE expression and sulfide-synthesizing activity in caerulein-induced pancreatitis compared with the saline control. When comparing the two saline-treated control groups, we noted that the CSE KO mice showed significantly less pancreatic H2S-synthesizing activity relative to the WT mice. These results indicate that endogenous H2S generated by CSE plays a key proinflammatory role via NF-κB activation in caerulein-induced pancreatitis, and its genetic deletion affords significant protection against acute pancreatitis and associated lung injury.

Topics: Animals; Ceruletide; Cystathionine gamma-Lyase; Gene Expression Regulation; Hydrogen Sulfide; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Pancreatitis; Protein Kinase C

To investigate the pathophysiological role of C/EBP homologous protein (CHOP) in severe acute pancreatitis and associated lung injury.. A severe acute pancreatitis model was induced with 6 injections of cerulein (Cn, 50 μg/kg) at 1-h intervals, then intraperitoneal injection of lipopolysaccharide (LPS, 7.5 mg/kg) in CHOP-deficient (Chop(-/-)) mice and wild-type (WT) mice. Animals were sacrificed under anesthesia, 3 h or 18 h after LPS injection. Serum amylase, lipase, and cytokines [interleukin (IL)-6 and tumor necrosis factor (TNF)-α], pathological changes, acute lung injury, and apoptosis in the pancreas were evaluated. Serum amylase and lipase activities were detected using a medical automatic chemical analyzer. Enzyme-linked immunosorbent assay kits were used to evaluate TNF-α and IL-6 levels in mouse serum and lung tissue homogenates. Apoptotic cells in sections of pancreatic tissues were determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) analysis. The mouse carotid arteries were cannulated and arterial blood samples were collected for PaO2 analysis. The oxygenation index was expressed as PaO2/FiO2.. Administration of Cn and LPS for 9 and 24 h induced severe acute pancreatitis in Chop(-/-) and WT mice. When comparing Chop(-/-) mice and WT mice, we observed that CHOP-deficient mice had greater increases in serum TNF-α (214.40 ± 19.52 pg/mL vs 150.40 ± 16.70 pg/mL; P = 0.037), amylase (4236.40 ± 646.32 U/L vs 2535.30 ± 81.83 U/L; P = 0.041), lipase (1678.20 ± 170.57 U/L vs 1046.21 ± 35.37 U/L; P = 0.008), and IL-6 (2054.44 ± 293.81 pg/mL vs 1316.10 ± 108.74 pg/mL; P = 0.046) than WT mice. The histopathological changes in the pancreases and lungs, decreased PaO2/FiO2 ratio, and increased TNF-α and IL-6 levels in the lungs were greater in Chop(-/-) mice than in WT mice (pancreas: Chop(-/-) vs WT mice, hemorrhage, P = 0.005; edema, P = 0.005; inflammatory cells infiltration, P = 0.005; total scores, P = 0.006; lung: hemorrhage, P = 0.017; edema, P = 0.017; congestion, P = 0.017; neutrophil infiltration, P = 0.005, total scores, P = 0.001; PaO2/FiO2 ratio: 393 ± 17.65 vs 453.8, P = 0.041; TNF-α: P = 0.043; IL-6, P = 0.040). Results from TUNEL analysis indicated increased acinar cell apoptosis in mice following the induction of acute pancreatitis. However, Chop(-/-) mice displayed significantly reduced pancreatic apoptosis compared with the WT mice (201.50 ± 31.43 vs 367.00 ± 47.88, P = 0.016).. These results suggest that CHOP can exert protective effects against acute pancreatitis and limit the spread of inflammatory damage to the lungs.

Topics: Acute Disease; Acute Lung Injury; Amylases; Animals; Apoptosis; Biomarkers; Ceruletide; Disease Models, Animal; Inflammation Mediators; Interleukin-6; Lipase; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Severity of Illness Index; Transcription Factor CHOP; Tumor Necrosis Factor-alpha

To investigate whether miRNA-155 (miR-155) dysregulates apical junctional complex (AJC) protein expression in experimental severe acute pancreatitis (SAP).. Twenty-four male BALB/c mice were randomly assigned to two groups: the SAP group (n = 12) receiving sequential intraperitoneal injection of 50 µg/kg caerulein and 10 mg/kg lipopolysaccharide over 6 h, and the control group (n = 12) receiving intraperitoneal injection of normal saline. Animals were sacrificed 3 h following the last injection for collection of blood samples and pancreas and distal ileal segment specimens. Routine pancreas and intestine histology was used to assess SAP pathology and intestinal epithelial barrier damage. Levels of serum amylase, diamine oxidase (DAO), and tumor necrosis factor (TNF)-α were determined using commercial kits. Total RNA samples were isolated from intestinal epithelial specimens and reversely transcribed into cDNA. miR-155 and RhoA mRNA expression profiles were determined using quantitative real-time polymerase chain reaction. Target genes for miR-155 were predicted using the miRTarBase database, RNA22 and PicTar computational methods. Western blotting was performed to quantitate the protein expression levels of the target gene RhoA, as well as zonula occludens (ZO)-1 and E-cadherin, two AJC component proteins.. Intraperitoneal injection of caerulein and lipopolysaccharide successfully induced experimental acute pancreatic damage (SAP vs control, 10.0 ± 2.0 vs 3.2 ± 1.2, P < 0.01) and intestinal epithelial barrier damage (3.2 ± 0.7 vs 1.4 ± 0.7, P < 0.01). Levels of serum amylase (21.6 ± 5.1 U/mL vs 14.3 ± 4.2 U/mL, P < 0.01), DAO (21.4 ± 4.1 mg/mL vs 2.6 ± 0.8 mg/mL, P < 0.01), and TNF-α (61.0 ± 15.1 ng/mL vs 42.9 ± 13.9 ng/mL, P < 0.01) increased significantly in SAP mice compared to those in control mice. miR-155 was significantly overexpressed in SAP intestinal epithelia (1.94 ± 0.50 fold vs 1.03 ± 0.23 fold, P < 0.01), and RhoA gene containing three miR-155-specific binding sites in the three prime untranslated regions was one of the target genes for miR-155. RhoA (22.7 ± 5.8 folds vs 59.6 ± 11.6 folds, P < 0.01), ZO-1 (46 ± 18 folds vs 68 ± 19 folds, P < 0.01), and E-cadherin proteins (48 ± 15 folds vs 77 ± 18 folds, P < 0.01) were underexpressed in SAP intestinal epithelia although RhoA mRNA expression was not significantly changed in SAP (0.97 ± 0.18 folds vs 1.01 ± 0.17 folds, P > 0.05).. TNF-α-regulated miR-155 overexpression inhibits AJC component protein syntheses of ZO-1, and E-cadherin by downregulating post-transcriptional RhoA expression, and disrupts intestinal epithelial barrier in experimental SAP.

Topics: Acute Disease; Amine Oxidase (Copper-Containing); Amylases; Animals; Cadherins; Ceruletide; Disease Models, Animal; Epithelial Cells; Ileum; Intestinal Mucosa; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Pancreatitis; Permeability; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; RNA, Messenger; Severity of Illness Index; Tight Junctions; Tumor Necrosis Factor-alpha; Up-Regulation; Zonula Occludens-1 Protein

Acute pancreatitis accounts for almost 250.000 hospital admissions annually in the United States. Most promising treatment approaches are preventive; however, little is known about the early factors initiating acute pancreatitis. We aimed to evaluate the preventive effects of enoxaparin and hesperidin in cerulein-induced acute pancreatitis.. We used 70 Wistar albino rats for this study. Rats were divided into 7 groups: control group, and groups that were administered cerulein(Group 2), enoxaparin (Group 3), hesperidin (Group 4), cerulein with enoxaparin (Group 5), cerulein with hesperidin (Group 6), and cerulein with both enoxaparin and hesperidin (Group 7). Edema formation; leukocyte infiltration; measurement of the amylase level, pancreatic tissue weight, and pancreatic tissue oxidative capacity; and chemiluminescence using luminol, lucigenin, and nitric oxide levels as indices of tissue oxidative capacity were used to evaluate pancreatitis.. Acute edematous mild pancreatitis was induced in groups 2, 5, and 6 by cerulein injections. Enoxaparin and hesperidin significantly decreased (p < 0.001) all the tested parameters in these rats. Enoxaparin and hesperidin did not offer complete protection but showed 50% decrease in edema formation. The preventive agents showed no superiority to each other. Further, when enoxaparin and hesperidin were used in combination, no significant additive effects with regard to anti-inflammatory and anti-oxidative actions were present.. We showed that both enoxaparin and hesperidin exerted significant preventive effects in all the parameters related to acute pancreatitis in our experimental rat model.

Topics: Acute Disease; Amylases; Animals; Anticoagulants; Antioxidants; Ceruletide; Edema; Enoxaparin; Hesperidin; Male; Neutrophil Infiltration; Nitric Oxide; Pancreas; Pancreatitis; Rats, Wistar; Reactive Oxygen Species

In acute pancreatitis (AP), inflammatory cells and products disseminated in abdominal lymph and blood induce systemic inflammation. Interruption of abdominal lymph flow, and thereby reduction of lymphatic dissemination, could alter the course of the disease. Therefore, we investigated whether thoracic duct ligation (TDL) in a rat model of cerulein-induced AP results in reduced lung damage as a marker for reduction of systemic dissemination through the lymphatic system. Thirty-four male rats were assigned to TDL (TDL-rats, n=8), AP (AP-rats, n=8), TDL+AP (TDL+AP-rats, n=9) or sham TDL (Ctr-rats, n=9) groups. TDL and sham TDL were established first. Two days later, AP was induced in AP- and TDL+AP-rats by a series of subcutaneous injections of cerulein. Vehicle was injected in the same manner in Ctr- and TDL-rats as controls. Rats were sacrificed six hours after the end of the serial injections. Histological examination showed that AP-induced damage to the pancreas and ileum were similar in AP- and TDL+AP-rats whereas lung damage was less severe in TDL+AP-rats than in AP-rats. Assays demonstrated that: hepatic and pulmonary myeloperoxidase activities were increased in AP-rats but not in the TDL+AP-rats; more Il-6 was found in AP-rat than TDL+AP-rat lungs; and lung-lavage fluid from AP-rats yielded more angiopoietin-2 than TDL+AP-rats. In conclusion, prior TDL in the rat attenuates lung damage in acute pancreatitis.

Topics: Acute Disease; Acute Lung Injury; Animals; Ceruletide; Ligation; Male; Pancreatitis; Rats; Rats, Wistar; Thoracic Duct

The purpose of our study was to evaluate the protective effect of melatonin in a rat model of caerulein-induced acute pancreatitis. For the induction of experimental acute pancreatitis, four subcutaneous injections of caerulein (20 mgkg–1 body weight) were given to Wistar rats at 2-h intervals. Melatonin was injected intraperitoneally (25 mg kg–1 body weight) 30 min before each caerulein injection. After 12 h, rats were sacrificed by decapitation. Blood and pancreas samples were collected and processed for serological and histopathological studies,respectively. Lipase, a-amylase, corticosterone, total antioxidant power and cytokines interleukin (IL)-1b, IL-4 and tumour necrosis factor(TNF)-a were determined using commercial kits. ANOVA and Tukey tests (P<0.05) were performed for the statistical analysis of the results.Results showed that the administration of melatonin reduced histological damage induced by caerulein treatment as well as the hyperamylasemia and hyperlipidemia. Corticosterone and antioxidant total power were also reverted to basal activities. Furthermore, melatonin pre-treatment reduced pro-inflammatory cytokines IL-1b and TNF-a and increased the serum levels of anti-inflammatory cytokine IL-4. In conclusion,the findings suggest that the protective effect of melatonin in caerulein-induced acute pancreatitis is mediated by the anti-inflammatory ability of this indolamine. Thus, melatonin may have a protective effect against acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Antioxidants; Ceruletide; Cytokines; Female; Lipase; Male; Melatonin; Pancreatitis; Rats; Rats, Wistar

Serotonin (5-hydroxytryptamine, 5-HT) is a potent bioactive molecule involved in a variety of physiological processes. In this study, the authors analysed whether 5-HT regulates zymogen secretion in pancreatic acinar cells and the development of pancreatic inflammation, a potentially lethal disease whose pathophysiology is not completely understood.. 5-HT regulation of zymogen secretion was analysed in pancreatic acini isolated from wild-type or tryptophan hydoxylase-1 knock-out (TPH1(-/-)) mice, which lack peripheral 5-HT, and in amylase-secreting pancreatic cell lines. Pancreatitis was induced by cerulein stimulation and biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks.. Absence and reduced intracellular levels of 5-HT inhibited the secretion of zymogen granules both ex vivo and in vitro and altered cytoskeleton dynamics. In addition, absence of 5-HT resulted in attenuated pro-inflammatory response after induction of pancreatitis. TPH1(-/-) mice showed limited zymogen release, reduced expression of the pro-inflammatory chemokine MCP-1 and minimal leucocyte infiltration compared with wild-type animals. Restoration of 5-HT levels in TPH1(-/-) mice recovered the blunted inflammatory processes observed during acute pancreatitis. However, cellular damage, inflammatory and fibrotic processes accelerated in TPH1(-/-) mice during disease progression.. These results identify a 5-HT-mediated regulation of zymogen secretion in pancreatic acinar cells. In addition, they demonstrate that 5-HT is required for the onset but not for the progression of pancreatic inflammation. These findings provide novel insights into the normal physiology of pancreatic acinar cells and into the pathophysiology of pancreatitis, with potential therapeutic implications.

Topics: Acinar Cells; Actin Cytoskeleton; Amylases; Animals; Cell Line; Ceruletide; Chemotaxis, Leukocyte; Disease Progression; Enzyme Precursors; Fibrosis; Immunohistochemistry; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis; Serotonin

The peroxisome proliferator-activated receptor-α (PPAR-α) has attracted considerable attention for its anti-inflammatory properties; however, Toll-like receptor (TLR) pathways have an essential proinflammatory role in acute pancreatitis (AP). This study aimed to evaluate the attenuation of inflammation by PPAR-α and to investigate the interaction between PPAR-α and TLR pathways in AP.. Acute pancreatitis was induced in rats by administration of cerulein. The PPAR-α agonist WY14643 and/or antagonist MK886 was administered. The severity of AP was determined by measuring serum amylase, lipase, Ca(2+), pathological changes, myeloperoxidase activity, serum levels of interleukin (IL)-6, and intercellular adhesion molecule-1 (ICAM-1). The TLR2 and TLR4 messenger RNA (mRNA) and proteins were determined by real-time reverse transcriptase polymerase chain reaction and Western blotting, respectively. The mRNA expressions of target molecules of TLR pathways, including IL-6, IL-10, ICAM-1, and tumor necrosis factor α were also measured.. Treatment with WY14643 significantly decreased amylase, lipase, myeloperoxidase activity, pathological scores, IL-6, and ICAM-1 levels. The TLR2 and TLR4 mRNA and proteins were markedly decreased after treatment with WY14643, along with IL-6, ICAM-1, and tumor necrosis factor α mRNA levels. However, these effects were completely reversed by the coadministration of MK886.. Activation of PPAR-α played a protective role in AP, partially mediated by modulation of TLR pathways.

Topics: Amylases; Animals; Anti-Inflammatory Agents; Biomarkers; Blotting, Western; Calcium; Ceruletide; Cytokines; Disease Models, Animal; Gene Expression Regulation; Indoles; Intercellular Adhesion Molecule-1; Lipase; Male; Neutrophil Infiltration; Pancreas; Pancreatitis; Peroxidase; PPAR alpha; Pyrimidines; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 9; Toll-Like Receptors

The synthetic tripeptide feG (D-Phe-D-Glu-Gly) is a novel pharmacologic agent that decreases neutrophil recruitment, infiltration, and activation in various animal models of inflammatory disease. We aimed to investigate the effect of feG as both a preventive treatment when administered before acute lung injury and as a therapeutic treatment administered following initiation of acute lung injury.. Lung injury was assessed following prophylactic or therapeutic intratracheal feG administration in a “two-hit” rodent model of acute pancreatitis plus intratracheal lipopolysaccharide.. Following both prophylactic and therapeutic feG administration, there were significant improvements in arterial blood oxygenation and respiratory mechanics and decreased lung edema, BAL protein concentration, histologic tissue injury scores, BAL cell infiltration, and lung myeloperoxidase activity. Most indices of lung damage were reduced to baseline control values.. feG reduced leukocyte infiltration, ameliorated the severity of inflammatory damage, and restored lung function when administered either prophylactically or therapeutically in a two-hit rat model of acute pancreatitis plus intratracheal lipopolysaccharide.

Topics: Acute Disease; Acute Lung Injury; Animals; Cell Movement; Ceruletide; Disease Models, Animal; Male; Neutrophils; Oligopeptides; Pancreatitis; Rats; Rats, Sprague-Dawley; Respiratory Mechanics; Severity of Illness Index; Treatment Outcome

The anti-inflammatory effects of O-1602 and cannabidiol (CBD), the ligands of G protein-coupled receptor 55 (GPR55), on experimental acute pancreatitis (AP) were investigated.. Acute pancreatitis was induced in C57BL mice by intraperitoneal injection of 50 μg/kg cerulein hourly, with a total of 6 times. Drugs (O-1602, 10 mg/kg, or CBD, 0.5 mg/kg) were given by intraperitoneal injection 2 times at 30 minutes before the first injection and immediately before the fifth cerulein injection. At 3 hours after the last injection, the blood, the lungs, and the pancreas were harvested for the pancreatic enzyme activity, myeloperoxidase activity, and pro-inflammatory cytokines measurement; and the expressions of GPR55 mRNA and protein in the pancreas were detected.. Cannabidiol or O-1602 treatment significantly improved the pathological changes of mice with AP and decreased the enzyme activities, IL-6 and tumor necrosis factor α; levels, and the myeloperoxidase activities in plasma and in the organ tissues. G protein-coupled receptor 55 mRNA and protein expressed in the pancreatic tissue, and the expressions were decreased in the mice with AP, and either CBD or O-1602 attenuated these changes to a certain extent.. Cannabidiol and O-1602 showed anti-inflammatory effects in mice with AP and improved the expression of GPR55 in the pancreatic tissue as well.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Blotting, Western; Cannabidiol; Ceruletide; Disease Models, Animal; Immunohistochemistry; Inflammation Mediators; Injections, Intraperitoneal; Interleukin-6; Lipase; Lung; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peroxidase; Real-Time Polymerase Chain Reaction; Receptors, Cannabinoid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha

To investigate the role of bone morphogenetic protein (BMP) signaling in acute pancreatitis (AP) by administration of noggin, an endogenous BMP antagonist, in a cerulein-induced AP model.. Acute pancreatitis was induced by 9 hourly intraperitoneal injections of cerulein (50 μg/kg). Control mice received phosphate-buffered saline injections. In a separate group, noggin (0.5 mg/kg) was given intraperitoneally at 1 hour before and 2, 4, and 6 hours after AP induction. The mice were euthanized at 1 hour after completion of AP induction. The blood samples and the pancreas were harvested for analysis. Isolated pancreatic acini from normal mice and AR42J cells were treated with BMP2 and cerulein. AR42J cells were also treated with noggin. Phosphorylation of Smad1/5/8 was measured.. Bone morphogenetic protein signaling was up-regulated in AP mouse pancreas. Bone morphogenetic protein 2 and cerulein-induced phosphorylation of Smad1/5/8 in the acinar cells in vitro, which was blocked by noggin. Noggin administration in vivo attenuated AP induction, decreased vacuole formation in acinar cells, blocked LC3-II levels, and partially restored Beclin-1 and lysosomal-associated membrane protein 2 levels.. Bone morphogenetic protein signaling seems to promote AP induction and autophagy, as suggested by our study showing that noggin ameliorates AP and partially restores autophagic homeostasis.

Topics: Acute Disease; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Bone Morphogenetic Protein 2; Carrier Proteins; Cell Line; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Intraperitoneal; Lysosomal-Associated Membrane Protein 2; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Pancreas; Pancreatitis; Phosphorylation; Rats; Recombinant Proteins; Signal Transduction; Smad1 Protein; Smad5 Protein; Smad8 Protein; Time Factors

To examine the effect of inflexinol on the development of acute pancreatitis (AP) and to investigate the mechanisms responsible for the protective effect against AP.. Acute pancreatitis was induced in mice by intraperitoneal injection of cerulein. Inflexinol was administered intraperitoneally 4 times every 6 hours from 1 hour before the first cerulein injection. Serum amylase activity and histology of the pancreas were measured. Determination of pancreatic nuclear factor-κB (NF-κB) p65 expression was conducted by Western blotting and immunohistochemistry to investigate the mechanisms responsible for the inflexinol effects.. Serum amylase activity in the cerulein group was significantly higher than that in the control group (P < 0.05). Pancreatic histology revealed marked inflammatory changes in the cerulein group such as interstitial edema, vacuolization, necrosis, and infiltration of inflammatory cells; and Western blotting and immunohistochemistry showed marked NF-κB p65 expression. Treatment with inflexinol significantly attenuated the inflammatory changes in pancreatic histology at 24, 48, and 72 hours (P < 0.05). Pancreatic NF-κB p65 expression decreased significantly after inflexinol treatment (P < 0.05).. Inflexinol reduced the severity of cerulein-induced AP by inhibiting NF-κB activation.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Biomarkers; Blotting, Western; Ceruletide; Disease Models, Animal; Diterpenes, Kaurane; Female; Immunohistochemistry; Mice; Mice, Inbred BALB C; Pancreas; Pancreatitis; Severity of Illness Index; Time Factors; Transcription Factor RelA

Nuclear factor-κB (NF-κB) is activated during early stages of pancreatitis. This transcription factor regulates genes that control many cell activities, including inflammation and survival. There is evidence that activation of NF-κB protects against pancreatitis, and, in other cases, that it promotes this disease. We compared the effects of NF-κB in different mouse models of pancreatitis to understand these complications.. To model constitutive activation of NF-κB, we expressed a transgene that encodes its p65 subunit or the inhibitor of κB kinase (IKK)2 in pancreatic acinar cells of mice. We analyzed effects on pancreatic tissues and levels of NF-κB target genes in these mice and compared them with mice that did not express transgenic p65 or IKK2 (controls).. Transgenic expression of p65 led to compensatory expression of the inhibitory subunit IKB-α and, therefore, no clear phenotype. However, p65 transgenic mice given injections of cerulein, to induce acute pancreatitis, had higher levels of NF-κB activity in acinar cells, greater levels of inflammation, and more severe outcomes than control mice. In contrast, constitutive expression of IKK2 directly increased the activity of NF-κB in acinar cells and induced pancreatitis. Prolonged activity of IKK2 (3 months) resulted in activation of stellate cells, loss of acinar cells, and fibrosis, which are characteristics of chronic pancreatitis. Co-expression of IKK2 and p65 greatly increased the expression of inflammatory mediators and the severity of pancreatitis, compared with control mice.. The level of NF-κB activation correlates with the severity of acute pancreatitis in mice. Longer periods of activation (3 months) lead to chronic pancreatitis. These findings indicate that strategies to inactivate NF-κB might be used to treat patients with acute or chronic pancreatitis.

Topics: Acinar Cells; Animals; Ceruletide; Disease Models, Animal; Fibrosis; Gene Expression Regulation; I-kappa B Kinase; I-kappa B Proteins; Mice; Mice, Transgenic; NF-kappa B; NF-KappaB Inhibitor alpha; Pancreatic Stellate Cells; Pancreatitis; Severity of Illness Index; Time Factors; Transcription Factor RelA

The transcription factor nuclear factor-κB (NF-κB) (a heterodimer of NF-κB1p50 and RelA) is activated rapidly in acute pancreatitis (AP). However, it is not clear whether NF-κB promotes or protects against AP. We used the NF-κB inhibitor protein, inhibitor of κB (IκB)α, to study the roles of NF-κB in the development of AP in mice.. IκBα or the combination of IκBα and RelA selectively were deleted from pancreas of mice using the Cre/locus of cross-over P strategy; cerulein or L-arginine were used to induce AP. We performed microarray analyses of the IκBα- and RelA-deficient pancreata. DNA from healthy individuals and patients with acute or chronic pancreatitis were analyzed for variants in coding regions of alpha-1-antichymotrypsin.. Mice with pancreas-specific deletion of IκBα had constitutive activation of RelA and a gene expression profile consistent with NF-κB activation; development of AP in these mice was attenuated and trypsin activation was impaired. However, AP was fully induced in mice with pancreas-specific deletion of IκBα and RelA. By using genome-wide expression analysis, we identified a cluster of NF-κB-regulated genes that might protect against the development of AP. The serine protease inhibitor 2A (Spi2a) was highly up-regulated in IκBα-deficient mice. Lentiviral-mediated expression of Spi2A reduced the development of AP in C57BL/6 and RelA-deficient mice. However, we did not correlate any variants of alpha-1-antichymotrypsin, the human homologue of Spi2a, with acute or chronic pancreatitis.. Pancreas-specific deletion of IκBα results in nuclear translocation of RelA and reduces AP induction and trypsin activation in mice after administration of cerulein or L-arginine. Constitutive activation of RelA up-regulates Spi2A, which protects mice against the development of AP.

Topics: Acinar Cells; alpha 1-Antichymotrypsin; Animals; Arginine; Ceruletide; Cytosol; Disease Models, Animal; Gene Expression Profiling; Genetic Vectors; Genotype; I-kappa B Proteins; Lentivirus; Mice; Mice, Inbred C57BL; Microarray Analysis; NF-kappa B; NF-KappaB Inhibitor alpha; Nuclear Proteins; Pancreas; Pancreatitis; Phosphorylation; Serpins; Signal Transduction; Transcription Factor RelA; Trypsin; Up-Regulation

The endocannabinoid system has been shown to mediate beneficial effects on gastrointestinal inflammation via cannabinoid receptors 1 (CB(1)) and 2 (CB(2)). These receptors have also been reported to activate the MAP kinases p38 and c-Jun NH(2)-terminal kinase (JNK), which are involved in early acinar events leading to acute pancreatitis and induction of proinflammatory cytokines. Our aim was to examine the role of cannabinoid receptor activation in an experimental model of acute pancreatitis and the potential involvement of MAP kinases. Cerulein pancreatitis was induced in wild-type, CB(1)-/-, and MK2-/- mice pretreated with selective cannabinoid receptor agonists or antagonists. Severity of pancreatitis was determined by serum amylase and IL-6 levels, intracellular activation of pancreatic trypsinogen, lung myeloperoxidase activity, pancreatic edema, and histological examinations. Pancreatic lysates were investigated by Western blotting using phospho-specific antibodies against p38 and JNK. Quantitative PCR data, Western blotting experiments, and immunohistochemistry clearly show that CB(1) and CB(2) are expressed in mouse pancreatic acini. During acute pancreatitis, an upregulation especially of CB(2) on apoptotic cells occurred. The unselective CB(1)/CB(2) agonist HU210 ameliorated pancreatitis in wild-type and CB(1)-/- mice, indicating that this effect is mediated by CB(2). Furthermore, blockade of CB(2), not CB(1), with selective antagonists engraved pathology. Stimulation with a selective CB(2) agonist attenuated acute pancreatitis and an increased activation of p38 was observed in the acini. With use of MK2-/- mice, it could be demonstrated that this attenuation is dependent on MK2. Hence, using the MK2-/- mouse model we reveal a novel CB(2)-activated and MAP kinase-dependent pathway that modulates cytokine expression and reduces pancreatic injury and affiliated complications.

Topics: Amylases; Animals; Anti-Inflammatory Agents; Apoptosis; Blotting, Western; Cannabinoids; Ceruletide; Disease Models, Animal; Dronabinol; Edema; Enzyme Activation; Immunohistochemistry; Interleukin-6; Intracellular Signaling Peptides and Proteins; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; p38 Mitogen-Activated Protein Kinases; Pancreas, Exocrine; Pancreatitis; Peroxidase; Phosphorylation; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Trypsinogen

Shikonin, a highly liposoluble naphthoquinone pigment isolated from the traditional medical herbs Lithospermum erythrorhizon (LE), was considered to exhibit an anti-inflammatory property. While the potential of shikonin to ameliorate acute pancreatitis (AP) is unknown. Our aim was to investigate the effects of shikonin in a murine model of cerulein-induced pancreatitis.. AP was induced in mice by six intraperitoneal injection of cerulein (50 μg/kg) at hourly intervals. Vehicle or shikonin (50 mg/kg) was pretreated 2 h before the first cerulein injection. After 6 h, 9 h and 12 h of the first cerulein injection, the severity of acute pancreatitis was assessed by biochemistry, myeloperoxidase activity, histological grading, proinflammatory cytokines levels and nuclear factor kappa B (NF-κB) activity.. Shikonin administration significantly reduced serum amylase and lipase activities, pancreatic histological scores, TNF-α, IL-1β, IL-6 levels, MPO activity and NF-κB activity.. Taken together, these results suggest that shikonin might protect against experimental pancreatitis by reducing release of inflammatory cytokines via inhibition of NF-κB activity. The therapeutic role of shikonin in AP needs further investigation.

Topics: Animals; Anti-Inflammatory Agents; Ceruletide; Cytokines; Male; Mice; Mice, Inbred BALB C; Naphthoquinones; NF-kappa B; Pancreatitis; Peroxidase; Phytotherapy; RNA, Messenger

Our aim in the present study was to investigate the potential roles of the 78-kDa glucose-regulated protein (GRP78) and the X-linked inhibitor of apoptosis protein (XIAP) in the regulation of apoptosis during cerulein-induced acute pancreatitis (CAP). A rat CAP model was induced by injection of cerulein (50 µg/kg), and the severity of CAP was estimated by measuring serum amylase and lipase, pancreatic edema and histological changes. Pancreatic acinar cell apoptosis was determined by terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) assay, and the expression of GRP78, XIAP and the apoptotic genes caspase-3, -7 and -9 were determined by real‑time quantitative PCR and western blotting. After induction with cerulein, increased serum amylase and lipase, pancreatic edema, inflammation and apoptosis were observed in CAP rats. Furthermore, the mRNA and protein levels of GRP78 and XIAP were significantly downregulated in CAP rats, while the mRNA levels of caspase-3, -7 and -9, as well as the cell apoptotic index were markedly increased when compared with control rats (P<0.05). The expression of GRP78 and XIAP was negatively correlated with caspase expression in CAP (P<0.05). This study suggests that the downregulation of GRP78 and XIAP were correlated with apoptosis in pancreatic acinar cells, and that this may occur through the regulation of caspase activation during CAP.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Caspase 3; Caspase 7; Caspase 9; Ceruletide; Disease Models, Animal; Down-Regulation; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Lipase; Male; Pancreatitis; Rats; Rats, Wistar; RNA, Messenger; X-Linked Inhibitor of Apoptosis Protein

Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2(-/-) mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca(2+) concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.

Topics: Acinar Cells; Animals; Bile Acids and Salts; Biliary Tract Diseases; Calcium; Calcium Signaling; Ceruletide; Cholangiopancreatography, Endoscopic Retrograde; Chymotrypsinogen; Coloring Agents; Enzyme Activation; Enzyme Precursors; Gallstones; Indicators and Reagents; Lipopeptides; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis; Propidium; Receptor, PAR-2; Trypsinogen

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25-30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10(-6) M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R.

Topics: Acetophenones; Acinar Cells; Animals; Benzopyrans; Cells, Cultured; Ceruletide; Male; Mice; NF-kappa B; Nitriles; Pancreas, Exocrine; Pancreatitis; Phosphorylation; Protein Precursors; Receptors, Neurokinin-1; Signal Transduction; Substance P; Sulfones; Tachykinins; Up-Regulation

Acute pancreatitis is a potentially fatal disease with no known cure. The initial events in acute pancreatitis may occur within the acinar cells. We examined the effect of sesamol on (i) a cerulein-induced pancreatic acinar cancer cell line, AR42J, and (ii) cerulein-induced experimental acute pancreatitis in rats. Sesamol inhibited amylase activity and increased cell survival. It also inhibited medium lipid peroxidation and 8-hydroxydeoxyguanosine in AR42J cells compared with the cerulein-alone groups. In addition, in cerulein-treated rats, sesamol inhibited serum amylase and lipase levels, pancreatic edema, and lipid peroxidation, but it increased pancreatic glutathione and nitric oxide levels. Thus, we hypothesize that sesamol attenuates cerulein-induced experimental acute pancreatitis by inhibiting the pancreatic acinar cell death associated with oxidative stress in rats.

Topics: Acute Disease; Amylases; Animals; Antioxidants; Benzodioxoles; Cell Line, Tumor; Cell Survival; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Lipid Peroxidation; Male; Mice; Oxidative Stress; Pancreatitis; Phenols; Rats; Rats, Wistar

The purpose of our study was to evaluate the protective effect of the strong antioxidant and anti-inflammatory agent, lycopene, on oxidative stress in a rat model of cerulein-induced acute edematous pancreatitis.. Sprague-Dawley rats were pretreated with lycopene (50 mg/kg, i.p.) or saline 15 min before cerulein was given 20 μg/kg (i.p.) at 1-h intervals within 4 h. Twelve hours after cerulein or saline injections, the animals were killed by decapitation. Blood samples were collected to analyze amylase, lipase, and proinflammatory cytokines (TNF-α and IL-1ß). Pancreatic tissues were taken for the determination of tissue glutathione (GSH) and malondialdehyde (MDA) levels, Na(+)/K(+)-ATPase, and myeloperoxidase (MPO) activities. Tissue samples were also examined histologically.. Acute pancreatitis caused significant decrease in tissue GSH levels and Na(+)/K(+)-ATPase activity, while pancreatic MDA levels and MPO activity were increased. Furthermore, TNF-α, IL-1ß, and amylase lipase levels were also significantly increased. On the other hand, lycopene pretreatment reserved all these biochemical indices as well as histopathologic alterations that were induced by cerulein.. According to the results, lycopene protects the pancreatic tissues from oxidative damage induced by cerulein, and this effect possibly involves the inhibition of neutrophil infiltration and lipid peroxidation. These results suggest that high dietary intake of tomatoes may have protective effects against acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Antioxidants; Carotenoids; Ceruletide; Cytokines; Disease Models, Animal; Female; Glutathione; Lipase; Lipid Peroxidation; Lycopene; Male; Malondialdehyde; Oxidative Stress; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley

Based on the function of chemokine fractalkine (FKN), acting as both adhesion and chemoattractant, FKN plays a role in acute inflammatory response. In this study, we investigated the mechanism of FKN mediated upregulation inflammation in severe acute pancreatitis (SAP) rat models. Western blot, reverse transcriptase-polymerase chain reaction, and immunofluorescence demonstrated that FKN and its receptor CX3CR1 were overexpressed in cerulein-stimulated AR42J cells. AG490 and FKN-siRNA inhibited activation of Janus kinase/signal transducers and activators of transcription (Jak/Stat) in cerulein-stimulated AR42J cells. Following exposure AG490 and FKN-siRNA inhibited tumor necrosis factor-alpha expression by enzyme-linked immunosorbent assay and immunohistochemistry in vivo the SAP rat models. These results showed FKN and CX3CR1 were involved inflammatory response in cerulein-stimulated AR42J cells. FKN upregulates inflammation through CX3CR1 and the Jak/Stat pathway in SAP rat models.

Topics: Animals; Cell Line; Ceruletide; Chemokine CX3CL1; CX3C Chemokine Receptor 1; Inflammation; Inflammation Mediators; Janus Kinases; Pancreatitis; Rats; Rats, Sprague-Dawley; Receptors, Chemokine; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Up-Regulation

Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein- and L-arginine-induced acute pancreatitis in mice.. Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages.. Almost all types of immune cells, except for CD11b(high)CD11c(-) cells, were detected in the pancreas of healthy mice. However, activated CD11b(high)CD11c(-) cells, including Gr-1(low) macrophages and Gr-1(high) cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2(-/-) mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b(high)CD11c(-)Gr-1(low) macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b(high)CD11c(-)Gr-1(low) macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1(low) macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b(high)CD11c(-)Gr-1(low) macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2.. Cerulein induction of pancreatitis in mice involves migration of CD11b(high)CD11c(-)Gr-1(low) macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3-dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis.

Topics: Acute Disease; Animals; Arginine; Biomarkers; CD11b Antigen; CD11c Antigen; Ceruletide; Chemokine CCL2; Chemotaxis; Disease Models, Animal; DNA-Binding Proteins; Enzymes; Immunity, Innate; Lymphocyte Depletion; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Parabiosis; Receptors, CCR2; Receptors, Chemokine; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

Pancreatitis occurs when digestive enzymes are activated in the pancreas. Severe pancreatitis has a 10-30% mortality rate. No specific treatments for pancreatitis exist now. Here, we discovered that interleukin-22 (IL-22) may have therapeutic potential in treating acute and chronic pancreatitis. Wild-type and IL-22 knockout mice were equally susceptible to cerulein-induced acute and chronic pancreatitis, whereas liver-specific IL-22 transgenic mice were completely resistant to cerulein-induced elevation of serum digestive enzymes, pancreatic necrosis and apoptosis, and inflammatory cell infiltration. Treatment of wild-type mice with recombinant IL-22 or adenovirus IL-22 markedly attenuated the severity of cerulein-induced acute and chronic pancreatitis. Mechanistically, we show that the protective effect of IL-22 on pancreatitis was mediated via the induction of Bcl-2 and Bcl-X(L), which bind to Beclin-1 and subsequently inhibit autophagosome formation to ameliorate pancreatitis. In conclusion, IL-22 ameliorates cerulein-induced pancreatitis by inhibiting the autophagic pathway. IL-22 could be a promising therapeutic drug to treat pancreatitis.

Topics: Animals; Autophagy; bcl-X Protein; Ceruletide; Interleukin-22; Interleukins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pancreatitis; Protective Agents; Proto-Oncogene Proteins c-bcl-2

The size of the pancreas is determined by intrinsic factors, such as the number of progenitor cells, and by extrinsic signals that control the fate and proliferation of those progenitors. Both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury. Whether the expansion of these lineages relies on similar mechanisms is unknown. Although we have shown that the Wnt signaling component β-catenin is selectively required in mouse embryos for the generation of exocrine acinar cells, this protein has been ascribed various functions in the postnatal pancreas, including proliferation and regeneration of islet as well as acinar cells. To address whether β-catenin remains important for the maintenance and expansion of mature acinar cells, we have established a system to follow the behavior and fate of β-catenin-deficient cells during postnatal growth and regeneration in mice. We find that β-catenin is continuously required for the establishment and maintenance of acinar cell mass, extending from embryonic specification through juvenile and adult self-renewal and regeneration. This requirement is not shared with islet cells, which proliferate and function normally in the absence of β-catenin. These results make distinct predictions for the relative role of Wnt-β-catenin signaling in the etiology of human endocrine and exocrine disease. We suggest that loss of Wnt-β-catenin activity is unlikely to drive islet dysfunction, as occurs in type 2 diabetes, but that β-catenin is likely to promote human acinar cell proliferation following injury, and might therefore contribute to the resolution of acute or chronic pancreatitis.

Topics: Acinar Cells; Aging; Animals; Animals, Newborn; beta Catenin; Cell Differentiation; Cell Lineage; Cell Proliferation; Ceruletide; Gene Deletion; Homeostasis; Humans; Mice; Mice, Knockout; Pancreas, Exocrine; Pancreatitis; Regeneration

Although microcirculatory disturbances play pivotal role in the pathomechanism of acute pancreatitis (AP), very few papers can be found which had been tested any of hemorheological parameters. The aim of our study was to analyze the hemorheological changes in cerulein-induced experimental acute pancreatitis in rat in two doses (5 and 10 μg/kg, s.c.). Male and female rats were subjected to Control group, or AP with 5 or 10 μg/kg cerulein groups. Blood samplings (lateral caudal vein) were completed before cerulein administration, and 1, 2 and 24 hours later. Hematological parameters, amylase activity, erythrocyte deformability (ektacytometry) and aggregation (light-transmission method) were tested. The presence of AP could be confirmed by amylase testing and histological examination. The earliest impairment of the red blood cell deformability could be observed 1 hour after cerulein administration in 10 μg/kg dosage. Female animals had the worst rheological results with high mortality. In conclusion, subcutaneously administrated cerulein in dosage of 5 and 10 μg/kg resulted in AP in rats, with significant changes in red blood cell deformability and alterations in erythrocyte aggregation. This model seems to be suitable for further comparative studies.

Topics: Acute Disease; Animals; Blood Cell Count; Ceruletide; Erythrocyte Aggregation; Erythrocyte Deformability; Female; Hemorheology; Male; Microcirculation; Pancreatitis; Rats; Rats, Sprague-Dawley; Sex Factors

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1-36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1-36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7-34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.

Topics: Acinar Cells; Animals; Apoptosis; Blotting, Western; Bone Neoplasms; Cells, Cultured; Central Nervous System Depressants; Ceruletide; Collagen Type I; Ethanol; Fibronectins; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Mice; Mice, Inbred C57BL; Osteosarcoma; Pancreatic Stellate Cells; Pancreatitis; Parathyroid Hormone-Related Protein; Rats; Real-Time Polymerase Chain Reaction; RNA, Messenger

It is well established that acute pancreatitis often causes diabetes and that a high blood glucose level associated with pancreatitis is a marker of poor prognosis. The aim of this study was to evaluate if diabetes merely reflects the severity of pancreatitis or whether it can also aggravate the progression of this disease in a vicious circle.. Reversible acute oedematous pancreatitis was induced in untreated and streptozotocin-treated diabetic mice by injection of cerulein. Progression of pancreatitis was studied by immunohistochemistry, ELISA and various other enzyme assays. The production of regenerating islet-derived 3β (REG3β) was determined by western blot and immunohistochemistry.. While cerulein treatment in non-diabetic mice resulted in acute pancreatitis followed by regeneration of the pancreas within 7 days, diabetes aggravated pancreatitis, inhibited the regeneration of the exocrine tissue and led to strong atrophy of the pancreas. The aggravation of pancreatitis by diabetes was characterised by decreased production of the anti-inflammatory protein REG3β, increased inflammation, augmented oedema formation and increased cell death during the acute phase of pancreatitis (p < 0.05). During the regenerative phase, diabetes augmented inflammation, increased cell death, reduced acinar cell expansion and increased the expansion of duct as well as interstitial cells, resulting in the formation of tubular complexes (p < 0.05). Administration of insulin reversed the observed phenotype in diabetic mice.. Diabetes aggravates acute pancreatitis and suppresses regeneration of the exocrine tissue. Thus, diabetes is not just a concomitant phenomenon of pancreatitis, but can have a fundamental influence on the progression of acute pancreatitis.

Topics: Animals; Cell Death; Cell Proliferation; Ceruletide; Diabetes Mellitus, Experimental; Hypoglycemic Agents; Insulin; Male; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Proteins; Regeneration

Endoplasmic reticulum (ER) stress and hypertriglyceridemia (HTG) have been implicated in acute pancreatitis (AP).. For cellular model, rat exocrine acinar cells were preincubated with palmitic acid (0.05 or 0.1 mmol/L, 3 h) and stimulated with a cholecystokinin analog, CCK-8 (100 pmol/L, 30 min). For animal model, rats fed a high-fat diet to cause HTG and AP was induced by injection of caerulein (20 μg/kg). Injury to pancreatic cells was estimated by measuring amylase secretion, intracellular calcium concentration, apoptosis and histological changes. Expression of genes involved in ER stress-induced unfolded protein response (UPR) was monitored by RT-PCR and immunohistology.. In CCK-8 stimulated rat acinar cells, preincubation with PA caused an increased secretion of amylase, a higher and prolonged accumulation of intracellular calcium and increased apoptosis. Rats on high-fat diet had significantly elevated serum triglyceride levels. Induction of AP led to increased apoptosis in pancreatic tissue on high-fat diet than controls. For favoring HTG, expression of UPR components, GRP78/Bip, XBP-1, GADD153/CHOP and caspase-12 was upregulated.. Levels of markers of AP pathogenesis and components of UPR were elevated in the presence of excess fatty acids in pancreatic acinar cells. HTG appears to aggravate ER-stress and pathogenesis of AP.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Calcium; Ceruletide; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Gene Expression Regulation; Hypertriglyceridemia; Immunohistochemistry; Male; Palmitic Acid; Pancreas, Exocrine; Pancreatitis; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Sincalide; Time Factors; Tissue Culture Techniques; Unfolded Protein Response

Hydrogen sulfide (H(2)S), a novel gaseous messenger, is synthesized endogenously from L-cysteine by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). S-propargyl-cysteine (SPRC) is a slow H(2)S releasing drug that provides cysteine, a substrate of CSE. The present study was aimed to investigate the effects of SPRC in an in vivo model of acute pancreatitis (AP) in mice. AP was induced in mice by hourly caerulein injections (50 µg/kg) for 10 hours. Mice were treated with SPRC (10 mg/kg) or vehicle (distilled water). SPRC was administered either 12 h before or 3 h before the induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and processed to measure the plasma amylase, plasma H(2)S, myeloperoxidase (MPO) activities and cytokine levels in pancreas and lung. The results revealed that significant reduction of inflammation, both in pancreas and lung was associated with SPRC given 3 h prior to the induction of AP. Furthermore, the beneficial effects of SPRC were associated with reduction of pancreatic and pulmonary pro-inflammatory cytokines and increase of anti-inflammatory cytokine. SPRC administered 12 h before AP induction did not cause significant improvement in pancreatic and lung inflammation. Plasma H(2)S concentration showed significant difference in H(2)S levels between control, vehicle and SPRC (administered 3 h before AP) treatment groups. In conclusion, these data provide evidence for protective effects of SPRC in AP possibly by virtue of its slow release of endogenous H(2)S.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Ceruletide; Cysteine; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gases; Hydrogen Sulfide; Inflammation; Lung; Male; Mice; Pancreas; Pancreatitis; Peroxidase; Time Factors

Novel and effective drugs against acute pancreatitis are required. Therefore, we examined the changes in the metabolite levels in the serum and pancreatic tissue of mice with cerulein- and arginine-induced pancreatitis using gas-chromatography/mass-spectrometry (GC/MS) and investigated whether these alterations affected the severity of acute pancreatitis. In the cerulein-induced pancreatitis model, 93 and 129 metabolites were detected in the serum and pancreatic tissue, respectively. In the L-arginine-induced acute pancreatitis model, 120 and 133 metabolites were detected in the serum and pancreatic tissue, respectively. Among the metabolites, the concentrations of tricarboxylic acid (TCA) cycle intermediates and amino acids were altered in pancreatitis, and in pancreatic tissue, the levels of the intermediates involved in the initial part of the TCA cycle were increased and those of the intermediates involved in the latter part of the TCA cycle were decreased. Some metabolites exhibited similar changes in both pancreatitis mouse models, e.g., the levels of glutamic acid and O-phosphoethanolamine were significantly decreased in the pancreatic tissue. Supplementation with glutamic acid and O-phosphoethanolamine attenuated the severity of cerulein-induced acute pancreatitis. Our results suggest that GC/MS-based metabolomics is capable of accurately representing the status of acute pancreatitis, leading to the discovery of therapeutic agents for pancreatitis.

Topics: Acute Disease; Amino Acids; Animals; Arginine; Ceruletide; Citric Acid Cycle; Disease Models, Animal; Ethanolamines; Gas Chromatography-Mass Spectrometry; Glutamic Acid; Male; Metabolomics; Mice; Mice, Inbred C57BL; Pancreatitis

Acute pancreatitis is a common disease, which is divided into mild pancreatitis and severe pancreatitis. For the latter, a systemic inflammatory response may occur and lead to distant organ damage and the development of multiple organ dysfunction syndrome (MODS), which accounts for significant morbidity and mortality in humans. Chemokines and their receptors are being believed to play a pivotal role in the pathogenesis of acute pancreatitis. Chemokine receptor CXCR3 is reported to be involved in acute tissue injury, for example acute lung injury induced by cigarette smoking, but its role in acute pancreatitis is not yet known. In this study, two animal models of acute pancreatitis (cerulein- and arginine-induced pancreatitis) were applied in CXCR3⁻/⁻ mice and wild-type mice, in order to explore the role of CXCR3 in acute pancreatitis. Serum amylase, lipase and histological observations revealed that CXCR3 knockout did not affect the severity of acute pancreatitis. However, edema and inflammatory cell infiltrate in the lung tissue were attenuated in CXCR3⁻/⁻ mice when acute pancreatitis was induced. In conclusion, chemokine receptor CXCR3 is not involved in acute pancreatic injury, but has a connection with acute pancreatitis-associated lung injury. Acute pulmonary injury is attenuated in CXCR3 knockout mice in experimental acute pancreatitis.

Topics: Acute Disease; Acute Lung Injury; Amylases; Animals; Arginine; Ceruletide; Disease Models, Animal; Lipase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis; Receptors, CXCR3; Severity of Illness Index

Peroxisome proliferator-activated receptors (PPARs) are ligand activated transcription factors belonging to the nuclear receptor superfamily. PPARs activation has a profound impact on the local immune response with consequences affecting the progression of chronic inflammatory diseases. Relatively little is known on the role of PPAR-β/δ in the regulation of inflammatory responses. The aim of the present study was to evaluate the influence of PPAR-β/δ receptor in a model of edematous pancreatitis induced in mice by administration of cerulein at supramaximal doses, as well as in necrohemorrhagic model induced by intraductal administration of sodium taurocholate (STC).. Mice were treated with cerulein (50 μg/kg) or STC (5%). GW0742 (0.3 mg/kg) was intraperitoneally administered 1 and 6 hours after cerulein injection or was injected 2 hours before STC infusion. The pancreas and exopancreatic organs were carefully removed for microscopic examination. Pancreatic weight, serum amylase, lipase, tumor necrosis factor-α and interleukin-1β levels, as well as cytokines, adhesion molecules, nitrotyrosine, poly (ADP-ribose), inducible nitric oxide, FAS ligand, Bax, Bcl-2 expression by immunohistochemistry, and myeloperoxidase activity of the pancreas were assayed. Moreover, the involvement of nuclear factor-κB pathway was investigated by Western blot analysis.. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by edema, neutrophil infiltration and apoptosis, and elevated serum levels of amylase and lipase. Taurocholate challenge caused a clear increase in serum amylase, neutrophil infiltration, and tissue damage in the pancreas. Tissue and inflammatory changes in the pancreata were significantly less in GW0742 group than in cerulein or STC groups. In addition, the pancreatic water content was reduced in mice treated with PPAR-β/δ agonist. In the mild pancreatitis, GW0742 was also able to decrease the expression of proinflammatory cytokines and enzymes, as well as of proteins involved in apoptosis and nuclear factor-Kappa B pathway.. GW0742 attenuated pancreatic damage in 2 different experimental models of pancreatitis in mice.

Topics: Acute Disease; Amylases; Animals; Cell Adhesion Molecules; Cell Movement; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Lipase; Male; Mice; Mice, Inbred Strains; Neutrophils; Pancreatitis; PPAR delta; PPAR-beta; Taurocholic Acid; Thiazoles; Time Factors

Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.

Topics: ADP-Ribosylation Factor 1; Animals; Autophagy; Blotting, Western; Brefeldin A; Cathepsin B; Ceruletide; Enzyme Precursors; Golgi Apparatus; Mice; Microscopy, Electron; Microscopy, Fluorescence; Pancreatitis; Rats; Real-Time Polymerase Chain Reaction; Trypsinogen

Bmi1 is a member of the Polycomb protein family and represses transcription by modifying chromatin organization at specific promoters. Bmi1 is implicated in the control of stem cell self-renewal and has been shown to regulate cell proliferation, tissue homeostasis, and differentiation. Bmi1 is present in a subpopulation of self-renewing pancreatic acinar cells and is expressed in response to pancreatic damage. We investigated the role of Bmi1 in regeneration of exocrine pancreas.. Acute pancreatitis was induced in Bmi1(-/-) mice with cerulein; pancreatic cell regeneration, differentiation, and apoptosis were assessed. Cultured Bmi1(-/-) and wild-type primary acini were analyzed in vitro to determine acinar-specific consequences of Bmi1 deletion. To investigate cell autonomous versus non-cell autonomous roles for Bmi1 in vivo, pancreatitis was induced in Bmi1(-/-) mice reconstituted with a wild-type hematopoietic system.. Bmi1 expression was up-regulated in the exocrine pancreas during regeneration after cerulein-induced pancreatitis. Exocrine regeneration was impaired following administration of cerulein to Bmi1(-/-) mice. Pancreata of Bmi1(-/-) mice were hypoplastic, and the exocrine pancreas was replaced with ductal metaplasia that had increased apoptosis and decreased cell proliferation compared with that of wild-type mice. Expression of Cdkn2a and p53-dependent apoptotic genes was markedly up-regulated in Bmi1(-/-) pancreas compared with wild-type mice after injury. Furthermore, after transplantation of bone marrow from wild-type to Bmi1(-/-) mice, the chimeric mice had intermediate levels of pancreatic hypoplasia and significant but incomplete rescue of impaired exocrine regeneration after cerulein injury.. Bmi1 contributes to regeneration of the exocrine pancreas after cerulein-induced injury through cell autonomous mechanisms, in part by regulating Cdkn2a expression, and non-cell autonomous mechanisms.

Topics: Acute Disease; Animals; Apoptosis; Bone Marrow Transplantation; Cell Differentiation; Cell Proliferation; Ceruletide; Choline Deficiency; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Ethionine; Female; Gene Expression Regulation; Green Fluorescent Proteins; Mice; Mice, Knockout; Mice, Transgenic; Nuclear Proteins; Pancreas, Exocrine; Pancreatitis; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; Regeneration; Repressor Proteins; Time Factors; Tissue Culture Techniques; Transplantation Chimera; Tumor Suppressor Protein p53

To study the potential role of the 78kDa glucose regulated protein (GRP78) in the pathogenesis of acute pancreatitis (AP) in vitro.. AR42J cells were stimulated by cerulein or cerulein plus lipoplysaccharide (LPS). The severity of pancreatic inflammation was evaluated by amylase, lipase, TNF-a, and IL-6. Apoptosis was determined by flow cytometry; the expressions of apoptotic genes, GRP78 and the downstream molecules were determined by real-time quantitative PCR and Western blot.. After cerulein stimulation, the levels of amylase, lipase, TNF-a and IL-6 were all increased, with a more pronounced increase after cerulein plus LPS stimulation. Apoptosis was different in two cell models, high apoptosis in cerulein group; whereas cerulein plus LPS induced relatively less apoptosis. Apoptotic gene expressions revealed more pronounced increase in the cerulein group than those in cerulein plus LPS group. The expressions of GRP78 and downstream molecules were different in two cell models. GRP78 expression was down-regulated in cerulein group and upregulated in cerulein plus LPS group.. GRP78 expression was associated with apoptosis and the severity of cerulein-induced pancreatic inflammation, indicating that GRP78 might prevent apoptosis in pancreatic acinar cells thereby deteriorating the severity of AP.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Blotting, Western; Cell Line; Ceruletide; DNA-Binding Proteins; Flow Cytometry; Gene Expression Regulation; Heat-Shock Proteins; Interleukin-6; Lipase; Lipopolysaccharides; Necrosis; Pancreas, Exocrine; Pancreatitis; Rats; Real-Time Polymerase Chain Reaction; Regulatory Factor X Transcription Factors; RNA, Messenger; Severity of Illness Index; Signal Transduction; Time Factors; Transcription Factor CHOP; Transcription Factors; Tumor Necrosis Factor-alpha

The purpose of this study was to test the hypothesis that activation of endogenous peroxisome proliferator-activated receptor (PPARγ) inhibits induction of early growth response factor-1 (Egr-1), which is rapidly induced in the pancreas following cerulein intraperitoneal injection. Acute pancreatitis was induced in mice by hourly intraperitoneal injection of cerulein. Pioglitazone was administered prophylactically and pancreatic inflammation was assessed. AR42J cells were stimulated with caerulein 10⁻⁸ M co-incubated in presence of different concentration of pioglitazone. The expression of PPARγ, Egr-1, and the target genes of Egr-1 were studied by real-time reverse transcriptase polymerase chain reaction (PCR), Western blot, and immunohistochemistry. In vitro, a PPAR-γ activator (pioglitazone) strikingly diminished Egr-1 mRNA and protein expression corresponding to Egr-1. In vivo, treatment with pioglitazone prior to the intraperitoneal injection of cerulein induction of Egr-1 and its target genes such as, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1). The inhibitory effect of pioglitazone on Egr-1 expression induced by cerulein was almost fully restored by GW9662. Activation of PPAR-γ suppressed the activation of Egr-1 and its inflammatory gene targets and provided potent protection against pancreas injury. These data suggest a new mechanism in which PPAR-γ activation may decrease tissue inflammation in response to a cerulein insult.

Topics: Acinar Cells; Acute Disease; Anilides; Animals; Cell Line, Tumor; Ceruletide; Chemokine CCL2; Chemokines; Disease Models, Animal; Early Growth Response Protein 1; Hypoglycemic Agents; Male; Pancreatitis; Pioglitazone; PPAR gamma; Rats; Rats, Sprague-Dawley; RNA, Messenger; Severity of Illness Index; Thiazolidinediones; Translational Research, Biomedical

Alterations in protein expression within the initiation phase of acute pancreatitis (AP) might play an important role in the development of this disease, lysosomes being involved in its pathophysiology. The use of pancreatic subcellular fractions in proteomic analysis, simplifies protein maps and helps in the identification of new protein changes and biomarkers characterizing tissue damage. The present study aims to determine the differentially expressed acidic proteins in the pancreatic soluble and lysosomal+mitochondrial (L+M) fractions from rats during the early phase of the experimental model of cerulein (Cer)-induced AP. Subcellular pancreatic extracts from diseased and control rats were analyzed by 2-DE (3-5.6 pH range) and MALDI-TOF/TOF MS. Comparative analysis afforded the conclusive identification of 13 (soluble fraction) and 7 (L+M fraction) proteins or protein fragments occuring in different amounts between diseased and control pancreas, some of them being newly described in AP. In the soluble fraction, we detected changes related to inflammation and apoptosis (α1-inhibitor-3, α-1 antitrypsin, α-1 macroglobulin, haptoglobin, STRAP), oxidative stress and stress response (peroxiredoxin-2, thioredoxin-like 1, GRP94/TRA1, heat shock cognate 71kDa protein), digestive proteases (elastase 3B), serine protease inhibition (serpins B6 and A3L) and translation processes (EF 1-δ). In the L+M fraction, we detected changes mainly related to energy generation or cellular metabolism (ATP synthase β subunit, chymotrypsinogen B, triacylglycerol lipase), cell redox homeostasis (iodothyronine 5´monodeiodinase) and digestive proteases (carboxypeptidase B1). The data should provide valuable information for unraveling the early pathophysiologic mechanisms of Cer-induced AP.

Topics: Acute Disease; Animals; Apoptosis; Biomarkers; Ceruletide; Electrophoresis, Gel, Two-Dimensional; Hydrogen-Ion Concentration; Lysosomes; Male; Mitochondria; Pancreas; Pancreatitis; Proteome; Proteomics; Rats; Rats, Wistar; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions

The aim of the present study was to develop a model of hypertriglyceridemic (HTG) acute pancreatitis and to investigate the effects of probucol in this model.. Hamsters were fed a high-fat diet (HFD) or a normal diet for 3 weeks. Probucol was added at 1% to the HFD in the treated group. Pancreatitis was induced by 7 peritoneal injections of cerulein to the normal and HFD hamster groups. The severity of the pancreatitis and whole body oxidative stress were assessed.. The HFD induced severe HTG (>1000 mg/dL) in the hamsters. A more severe pancreatitis was observed in the HFD group. The HFD did not influence plasma-reduced glutathione level, but there was a significant increase after 1% probucol was provided in the diet. Plasma malonaldehyde levels in the HFD group were significantly higher than the normal chow group, whereas probucol administration significantly decreased plasma hydrogen peroxide and malonaldehyde levels. We also found that probucol significantly reduced levels of amylase and lipase in the plasma and pathological scores in pancreatic tissue.. This study presents a novel model of severe HTG acute pancreatitis, and our results support the potential therapeutic application of probucol in HTG acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Antioxidants; Biomarkers; Ceruletide; Cricetinae; Diet, High-Fat; Disease Models, Animal; Glutathione; Hydrogen Peroxide; Hypertriglyceridemia; Lipase; Malondialdehyde; Oxidative Stress; Pancreas; Pancreatitis; Probucol; Severity of Illness Index

To determine if the fraction of Nardostachys jatamansi (NJ) has the potential to ameliorate the severity of acute pancreatitis (AP).. Mice were administered the biologically active fraction of NJ, i.e., the 4th fraction (NJ4), intraperitoneally, and then injected with the stable cholecystokinin analogue cerulein hourly for 6 h. Six hours after the last cerulein injection, the pancreas, lung, and blood were harvested for morphological examination, measurement of cytokine expression, and examination of neutrophil infiltration.. NJ4 administration attenuated the severity of AP and lung injury associated with AP. It also reduced cytokine production and neutrophil infiltration and resulted in the in vivo up-regulation of heme oxygenase-1 (HO-1). Furthermore, NJ4 and its biologically active fraction, NJ4-2 inhibited the cerulein-induced death of acinar cells by inducing HO-1 in isolated pancreatic acinar cells.. These results suggest that NJ4 may be a candidate fraction offering protection in AP and NJ4 might ameliorate the severity of pancreatitis by inducing HO-1 expression.

Topics: Acute Disease; Animals; Cell Death; Ceruletide; Cytokines; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Enzymes; Female; Heme Oxygenase-1; Inflammation Mediators; Lung; Membrane Proteins; Mice; Mice, Inbred C57BL; Nardostachys; Neutrophil Infiltration; Pancreas; Pancreatitis; Plant Extracts; Plant Roots; Severity of Illness Index; Time Factors; Up-Regulation

Heat shock protein (Hsp) 72 is a molecular chaperone which is upregulated in response to a variety of stress situations and has a general cytoprotective function. Increased Hsp72 levels were implicated in protection from acute pancreatitis; a hypothesis which was not tested in a transgenic mouse model yet.. To analyze the role of Hsp72 during acute pancreatitis, well-characterized transgenic animals overexpressing rat Hsp72 (Hsp72 mice) under the control of the ß-actin promoter were subjected to caerulein- and L-arginine-induced acute pancreatitis. The severity of experimental pancreatitis was determined via serum lipase levels, morphometric evaluation and quantification of pancreatic edema/inflammation.. Hsp72 mice displayed ∼100-times Hsp72 overexpression, but no changes in the remaining chaperones. Robust Hsp72 signal was observed in pancreatic acini, but not in islets or ductal cells. In both models, elevated Hsp72 did not protect from development of acute pancreatitis and the pancreatitis-associated lung injury, but accelerated recovery from caerulein-induced tissue injury (lower lipase levels, edema, inflammation and necrosis 36 h after caerulein administration). The observed protective function of Hsp72 in caerulein-induced pancreatitis is likely due to an attenuated NF-κB signalling.. Hsp72 overexpression accelerates the recovery from acute pancreatitis and may represent a potential treatment strategy.

Topics: Acute Disease; Animals; Arginine; Ceruletide; Disease Models, Animal; Gene Expression; Gene Expression Regulation; HSP72 Heat-Shock Proteins; Mice; Mice, Transgenic; NF-kappa B; Pancreas, Exocrine; Pancreatitis; Recovery of Function; Signal Transduction

Clinical studies indicate that cigarette smoking increases the risk for developing acute pancreatitis. The nicotine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a major cigarette smoke toxin. We hypothesized that NNK could sensitize to pancreatitis and examined its effects in isolated rat pancreatic acini and in vivo. In acini, 100 nM NNK caused three- and fivefold activation of trypsinogen and chymotrypsinogen, respectively, above control. Furthermore, NNK pretreatment in acini enhanced zymogen activation in a cerulein pancreatitis model. The long-term effects of NNK were examined in vivo after intraperitoneal injection of NNK (100 mg/kg body wt) three times weekly for 2 wk. NNK alone caused zymogen activation (6-fold for trypsinogen and 2-fold for chymotrypsinogen vs. control), vacuolization, pyknotic nuclei, and edema. This NNK pretreatment followed by treatment with cerulein (40 μg/kg) for 1 h to induce early pancreatitis responses enhanced trypsinogen and chymotrypsinogen activation, as well as other parameters of pancreatitis, compared with cerulein alone. Potential targets of NNK include nicotinic acetylcholine receptors and β-adrenergic receptors; mRNA for both receptor types was detected in acinar cell preparations. Studies with pharmacological inhibitors of these receptors indicate that NNK can mediate acinar cell responses through an nonneuronal α(7)-nicotinic acetylcholine receptor (α(7)-nAChR). These studies suggest that prolonged exposure to this tobacco toxin can cause pancreatitis and sensitize to disease. Therapies targeting NNK-mediated pathways may prove useful in treatment of smoking-related pancreatitis.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Atropine; Carcinogens; Cells, Cultured; Ceruletide; Edema; Enzyme Precursors; L-Lactate Dehydrogenase; Male; Mecamylamine; Nicotiana; Nitrosamines; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Receptors, Nicotinic; Sincalide

The intracellular sensor NOD1 has important host-defense functions relating to a variety of pathogens. Here, we showed that this molecule also participates in the induction of a noninfectious pancreatitis via its response to commensal organisms. Pancreatitis induced by high-dose cerulein (a cholecystokinin receptor agonist) administration depends on NOD1 stimulation by gut microflora. To analyze this NOD1 activity, we induced pancreatitis by simultaneous administration of a low dose of cerulein (that does not itself induce pancreatitis) and FK156, an activator of NOD1 that mimics the effect of gut bacteria that have breached the mucosal barrier. The pancreatitis was dependent on acinar cell production of the chemokine MCP-1 and the intrapancreatic influx of CCR2(+) inflammatory cells. Moreover, MCP-1 production involved activation of the transcription factors NF-κB and STAT3, each requiring complementary NOD1 and cerulein signaling. These studies indicate that gut commensals enable noninfectious pancreatic inflammation via NOD1 signaling in pancreatic acinar cells.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Acinar Cells; Animals; Bacteria; Ceruletide; Chemokine CCL2; Diaminopimelic Acid; Humans; Immunity, Mucosal; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mucous Membrane; NF-kappa B; Nod1 Signaling Adaptor Protein; Pancreatitis; Receptors, CCR2; Signal Transduction; STAT3 Transcription Factor

Hedgehog signaling plays critical roles in pancreatic oncogenesis and chronic pancreatitis, but its roles in acute pancreatitis (AP) are largely ambiguous. In this study, we provide evidence that Sonic hedgehog (Shh), but neither Desert hedgehog (Dhh) nor Indian hedgehog (Ihh), is the main protein whose expression is activated during the development of cerulein-induced acute pancreatitis in mice, and the Shh serves as an anti-inflammation factor in an autocrine manner. Blocking autocrine Shh signaling with anti-Shh neutralizing antibody aggravates the progression of acute pancreatitis. Mechanistic insight into Shh signaling activation in acute pancreatitis indicates that inflammatory stimulation activates Shh expression and secretion, and subsequently upregulates the expression and secretion of interleukin-10 (IL-10). Moreover, inhibition of Shh signaling with neutralizing antibody abolishes IL-10 production in vivo and in vitro. Molecular biological studies show that autocrine Shh signaling activates the key transcriptional factor Gli1 so that the target gene IL-10 is upregulated, leading to the protective and anti-inflammatory functions in the mouse model of acute pancreatitis. Thus, this study suggests autocrine Shh signaling functions as a protective signaling in the progression of acute pancreatitis.

Topics: Acute Disease; Animals; Autocrine Communication; Cell Line; Ceruletide; Gene Expression; Gene Expression Regulation; Hedgehog Proteins; Interleukin-10; Kruppel-Like Transcription Factors; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Pancreas; Pancreatitis; Signal Transduction; Up-Regulation; Zinc Finger Protein GLI1

Acute pancreatitis (AP) is a complicated inflammatory disease that has an unknown underlying pathogenesis. Because alpha-pinene can modulate inflammation, we examined whether alpha-pinene plays a role in AP.. Alpha-pinene was administered intraperitoneally 1h prior to the first injection of cerulein. Once AP developed, cerulein, a stable cholecystokinin analog, was injected hourly over a 6-h period. Blood samples were taken 6h later to determine serum amylase and lipase levels. The pancreas and lungs were rapidly removed for morphological examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction. We also isolated the pancreatic acinar cells using a collagenase solution. Cell viability, and cytokine productions were measured in pancreatic acini.. Intraperitoneal administration of alpha-pinene reduced the pancreatic weight (PW) to body weight (BW) ratio and the serum levels of amylase and lipase. Alpha-pinene treatment also reduced histological damage and myeloperoxidase activity in the pancreas and lungs. Furthermore, alpha-pinene pretreatment reduced the production of pancreatic tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 during cerulein-induced AP. In vitro, alpha-pinene inhibited cerulein-induced cell death and cytokine production in isolated cerulein-treated pancreatic acinar cells.. These findings suggest that alpha-pinene has an anti-inflammatory effect during cerulein-induced AP.

Topics: Acute Disease; Amylases; Animals; Bicyclic Monoterpenes; Body Weight; Cells, Cultured; Ceruletide; Female; Immunologic Factors; Injections, Intraperitoneal; Lipase; Lung; Mice; Mice, Inbred C57BL; Monoterpenes; Pancreas; Pancreatitis; Peroxidase

We explored the role of the MyD88 signaling pathway. This pathway mediates the recognition of pathogen-associated molecular patterns and damage-associated molecular patterns via Toll-like receptors (TLRs) and/or IL-1/IL-18 via each cytokine receptor in a murine model of acute pancreatitis induced by cerulein administration. Our analysis revealed that: various TLRs and MyD88 molecules were constitutively expressed in the pancreas of cerulein-treated and untreated wild-type (WT) mice. MyD88⁻/⁻ mice administered cerulein developed severe pancreatitis as compared with MyD88⁺/⁺ WT mice. The number of IL-10-expressing CD11b⁺Gr-1(high) cells in cerulein-administered MyD88⁻/⁻ mice was significantly decreased. This was in accordance with a reciprocal increase in the infiltration of CD4⁺ T cells as compared with that in control MyD88⁺/⁺ mice. WT mice pretreated with antibiotics and administered cerulein developed milder pancreatitis as compared with control cerulein-administered mice without antibiotic treatment. The MyD88 signaling pathway contributes to the induction of regulatory IL-10-producing macrophages/myeloid-derived suppressor cells, possibly in response to non-bacterial components in the damaged pancreas. These results provide a new concept for therapeutic strategies against acute pancreatitis.

Topics: Ampicillin; Animals; Anti-Bacterial Agents; CD11b Antigen; CD4-Positive T-Lymphocytes; Ceruletide; Flow Cytometry; Interleukin-10; Macrophage Activation; Macrophages; Metronidazole; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Neomycin; Pancreas; Pancreatitis; Receptors, Cell Surface; Signal Transduction; Toll-Like Receptors; Vancomycin

The type of immune response during development of acute pancreatitis (AP) determines disease severity. Pancreatic epithelial cells express the interleukin (IL)-22 receptor A1 (IL-22RA1). The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates expression of IL-22. We investigated sources and role of IL-22 in the pancreas, along with the effects of AhR activation on IL-22 expression and AP progression in mice.. We analyzed the effects of recombinant IL-22, a monoclonal antibody against IL-22, and agonists and antagonists of AhR in mice with AP (induced with caerulein or a choline-deficient diet supplemented with DL-ethionine) and control mice. We also analyzed transgenic mice with AhR deficiency (AhR(d) and AhR(-/-) mice).. CD4(+) T cells were the main source of IL-22 in pancreatic tissues from healthy mice. During development of AP, numbers of IL-22(+) CD4(+) T cells were reduced, whereas IL-22RA1 was up-regulated. Consistent with high levels of IL-22RA1 expression, pancreatic acinar cells responded to IL-22 signaling via signal transducers and activators of transcription 3; administration of IL-22 reduced AP and associated lung injury in mice. AhR was required for production of IL-22 and protected mice from AP. Mice that did not respond to AhR activation developed AP, but administration of IL-22 reduced AP; blockade of IL-22 reversed the ability of activated AhR to protect against AP.. AhR activation protects mice from AP by inducing expression of IL-22. AhR therefore mediates interactions between pancreatic leukocytes and epithelial cells and might be developed as a therapeutic target.

Topics: Acute Disease; Animals; CD4-Positive T-Lymphocytes; Ceruletide; Choline Deficiency; Disease Models, Animal; Female; Interleukin-22; Interleukins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Pancreas; Pancreatitis; Phosphorylation; Receptors, Aryl Hydrocarbon; Signal Transduction; STAT3 Transcription Factor

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that controls expressions of several genes involved in cell survival, proliferation and differentiation, and tissue inflammation. However, the significance of pancreatic STAT3 in acute pancreatitis remains unclear. We generated conditional STAT3 knockout (stat3(Δ/Δ)) mice by crossing stat3(flox/flox) mice with Pdx1-promoter Cre transgenic mice. Caerulein administration activated pancreatic STAT3 and induced acute pancreatitis as early as 3 hours in wild-type mice, and full recovery from the induced pancreatic injury was observed within 7 days. The levels of serum amylase and lipase and histologic scores of pancreatic necrosis and inflammatory cell infiltration were significantly higher at 3 hours in stat3(Δ/Δ) mice than in stat3(flox/flox) mice. Pancreatic recovery after pancreatitis was significantly delayed in stat3(Δ/Δ) mice compared with stat3(flox/flox) mice. Although stat3(flox/flox) mice had marked production in the pancreas of pancreatitis-associated protein 1 (PAP1), a serum acute phase protein, this induction was completely abrogated in stat3(Δ/Δ) mice. Enforced production of PAP1 by a hydrodynamic procedure in the liver significantly suppressed pancreatic necrosis and inflammation and also promoted pancreatic regeneration and recovery in stat3(Δ/Δ) mice to levels similar to those observed in stat3(flox/flox) mice. In conclusion, pancreatic STAT3 is indispensable for PAP1 production, and this STAT3/PAP1 pathway plays a protective role in caerulein-induced pancreatitis.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Ceruletide; Female; Gene Deletion; Gene Expression; Gene Knockdown Techniques; Lectins, C-Type; Liver; Male; Mice; Mice, Knockout; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; STAT3 Transcription Factor

The role of endothelins in acute pancreatitis remains obscure. To assess the effects of endothelins (ETs) in early (4 h) caerulein-induced acute pancreatitis (AP) in rats, ET-1, ET-2 and ET-3 (0.5 or 1.0 nmol/kg) were applied twice with i.p. caerulein (2×40 μg/kg) at 1h interval. Histological and ultrastructural examinations of pancreases and the assay of trypsinogen activation in whole homogenate were performed. All ETs, especially ET-1 at the higher dose, decreased inflammatory cell infiltration despite an increase in the edema score. The vacuolization and necrosis of acinar cells were slightly increased after the lower dose of ET-1 and ET-2. Ultrastructural changes were generally improved after the higher dose of ETs. Trypsinogen activation increased from 4.8±1.3% in control to 18.4±3.8% in AP (p<0.01). It was attenuated to 6.4±1.3% (p<0.01) by the higher dose of ET-1 and to 8.8±1.5% (p<0.05) by the lower dose of ET-3. In summary, ETs, especially ET-1 at the higher dose, were found to have some beneficial effects on morphological changes and trypsinogen activation in the pancreas in early caerulein-induced AP.

Topics: Animals; Ceruletide; Dose-Response Relationship, Drug; Endothelins; Enzyme Activation; Male; Microscopy, Electron, Transmission; Pancreatitis; Rats; Rats, Wistar; Trypsinogen

In our previous study, we reported that cerulein-induced acute pancreatitis is aggravated in pancreatic β-cell-specific pituitary adenylate cyclase-activating polypeptide (PACAP) transgenic mice, showing that an increase in pancreatic PACAP is a risk factor for progression of acute pancreatitis. Accordingly, in this study, we examined the progression of cerulein-induced acute pancreatitis in PACAP knockout (KO) mice. Unexpectedly, after cerulein, about 60% of the KO mice showed severe hypothermia below 30°C by 12 h and most of them died within 72 h. In contrast, the remaining KO and wild-type mice showed normothermia with no mortality. Thus, KO mice could be classified into two groups as hypothermic (HT-KO) and normothermic (NT-KO) to cerulein. Only HT-KO mice subsequently showed severe mortality, although both HT-KO and NT-KO mice exhibited similar susceptibility of lungs to cerulein toxicity, comparable to that in wild-type mice. Regarding pancreatitis, HT-KO mice showed ameliorated pancreatic damage without any rise in serum enzyme activities, whereas NT-KO mice exhibited a similar degree of pancreatitis to wild-type mice. Taken together, the present results indicate that lack of pancreatic PACAP did not aggravate, but rather ameliorated, cerulein-induced pancreatitis. In addition, about half of KO mice showed a novel phenotype in which cerulein caused rapid and severe hypothermia, followed by death.

Topics: Animals; Body Temperature; Ceruletide; Hypothermia; Lung; Mice; Mice, Knockout; Pancreatitis; Peroxidase; Pituitary Adenylate Cyclase-Activating Polypeptide; Survival Rate

The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) is known to be an endogenous negative feedback or compensatory mechanism that serves to limit pro-inflammatory and chemotactic events in response to injury. The aim of this study is to elucidate whether Akt plays any role in 17β-estradiol (E2)-mediated attenuation of lung injury after acute pancreatitis (AP).. Male Sprague-Dawley rats underwent cerulein-induced AP. Rats were treated with vehicle (cyclodextrin), E2 (1 mg/kg body weight [BW]), or E2 plus PI3K/Akt inhibitor Wortmannin (100 μg/kg BW) 1h after the onset of AP. At 8 h after sham operation or AP, various parameters were measured.. AP led to a significant decrease in lung Akt phosphorylation, which was associated with increased lung tissue myeloperoxidase (MPO) activity, wet-to-dry weight ratios, interleukin (IL)-6, tumor necrosis factor (TNF)-α, cytokine-induced neutrophil chemoattractant (CINC)-1, and CINC-3 levels. Administration of E2 after AP restored the AP-induced decrease in Akt phosphorylation and attenuated the increase in lung injury markers (MPO activity and wet-to dry weight ratios) and pro-inflammatory mediator production. The effects of E2 on the lung were abolished by co-administration of Wortmannin.. These results collectively suggest evidences that the Akt pathway seems to be required for E2-mediated protection of lung injury after AP.

Topics: Androstadienes; Animals; Blotting, Western; Ceruletide; Chemokine CXCL1; Chemokine CXCL2; Cyclodextrins; Estradiol; Interleukin-6; Lung Injury; Male; Pancreatitis; Peroxidase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Wortmannin

The discovery of novel and effective treatment methods would be of great help to patients with acute pancreatitis. The aims of this study were to determine the inhibitory effects of vitamin K3 (VK3) against cerulein-induced acute pancreatitis in mice and to examine the mechanisms behind these effects.. Acute pancreatitis in mice was induced by intraperitoneal injection of cerulein 6 times at hourly intervals. Vitamin K3 was administered once before the first injection of cerulein or twice before and after the first injection of cerulein. The degrees of inflammation and autophagy in the pancreatic tissue were estimated by histological examination, measurement of enzyme activity, confocal microscopy, and Western blotting. The inhibitory effects of VK3 against rapamycin-induced autophagy were also examined using HeLa cells stably expressing green fluorescent protein LC3.. Cerulein-induced acute pancreatitis was markedly attenuated by the administration of VK3. In addition, VK3 led to the inhibition of cerulein-evoked autophagic changes and colocalization of autophagosomes and lysosomes in the pancreatic tissue. Vitamin K3 also reduced rapamycin-induced autophagy in HeLa/green fluorescent protein LC3 cells.. Our data suggest that the administration of VK3 reduces pancreatic inflammation in acute pancreatitis through inhibition of the autophagic pathway. Vitamin K3 may be an effective therapeutic strategy against acute pancreatitis.

Topics: Acute Disease; Animals; Autophagy; Ceruletide; Female; HeLa Cells; Humans; Lysosomes; Mice; Mice, Inbred C57BL; Pancreatitis; Phagosomes; Sirolimus; Vitamin K 2; Vitamin K 3

Platelets not only control thrombosis and haemostasis but may also regulate inflammatory processes. Acute pancreatitis (AP) is characterized by changes in both coagulation and proinflammatory activities. The role of platelets in AP is not yet known.. AP was induced in C57BL/6 mice by repeated caerulein administration (50 µg/kg intraperitoneally). Mice received a platelet-depleting or control antibody before caerulein challenge. Neutrophil infiltration, myeloperoxidase (MPO) and macrophage inflammatory protein (MIP) 2 levels, acinar cell necrosis and haemorrhage in the pancreas, as well as serum amylase activity, were determined 24 h after caerulein injection. In an alternative model of pancreatitis, L-arginine (4 g/kg intraperitoneally) was given twice with an interval of 1 h and tissue samples were taken after 72 h [Correction added after online publication 29 September 2010: in the preceding sentence, 4 mg/kg was corrected to 4 g/kg].. Caerulein administration increased acinar cell necrosis, neutrophil infiltration, focal haemorrhage and serum amylase levels. Platelet depletion reduced acinar cell necrosis, haemorrhage and serum amylase levels in AP. Depletion of platelets decreased caerulein-induced MPO levels and neutrophil recruitment in the pancreas. Platelet depletion abolished caerulein-induced MIP-2 generation in the pancreas and circulation. The effects of platelet depletion on necrosis, neutrophils and MPO levels were confirmed in L-arginine-induced pancreatitis.. Platelets play a crucial role in AP by regulating neutrophil infiltration, most likely mediated by MIP-2 production in the pancreas.

Topics: Amylases; Animals; Antibodies; Arginine; Blood Platelets; Ceruletide; Chemokine CXCL2; Flow Cytometry; Male; Mice; Mice, Inbred C57BL; Neutrophil Activation; Pancreatitis; Peroxidase; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex

Lysosomes play an important role in acute pancreatitis (AP). Here we developed a method for the isolation of lysosome subpopulations from rat pancreas and assessed the stability of lysosomal membranes.. AP was induced by four subcutaneous injections of 20 μg caerulein/kg body weight at hourly intervals. The animals were killed 9h after the first injection. Marker enzymes [N-acetyl-β-D-glucosaminidase (NAG), cathepsin B and succinate dehydrogenase (SDH)] were assayed in subcellular fractions from control pancreas and in pancreatitis. Lysosomal subpopulations were separated by Percoll density gradient centrifugation and observed by electron microscopy. NAG molecular forms were determined by DEAE-cellulose chromatography.. AP was associated with: (i) increases in the specific activity of lysosomal enzymes in the soluble fraction, (ii) changes in the size and alterations in the morphology of the organelles from the lysosomal subpopulations, (iii) the appearance of large vacuoles in the primary and secondary lysosome subpopulations, (iv) the increase in the amount of the NAG form associated with the pancreatic lysosomal membrane as well as its release towards the soluble fraction.. Lysosome subpopulations are separated by a combination of differential and Percoll density gradient centrifugations. Primary lysosome membrane stability decreases in AP.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Lysosomes; Male; Pancreatitis; Rats; Rats, Wistar

Angiotensin II is a vasoactive peptide that controls blood pressure and homeostasis. Emerging evidence shows that locally generated angiotensin II plays a crucial role in normal physiology, as well as pathophysiological conditions such as pancreatitis. We recently reported that angiotensin II activates pancreatic NFκB in obstructive pancreatitis. However, the specific cell type responsible for this activation remains unclear. In this study, we investigated whether pancreatic acinar cells respond to angiotensin II. These cells are the most abundant pancreatic cells and the most vulnerable to pancreatitis. Pancreatic acinar AR42J cells were used as an in vitro model of pancreatic inflammation. Our results demonstrated that treatment with caerulein, a cholecystokinin receptor agonist, induced hypersecretion and NFκB activation, as demonstrated by elevated amylase secretion and degradation of inhibitor of NFκB (IκBβ). Angiotensin II, either alone or in combination with caerulein, augmented IκBβ degradation. Pre-treatment with losartan, an antagonist of the angiotensin type I (AT₁) receptor, abolished NFκB activation by angiotensin II and caerulein in a dose-dependent manner. Treatment with PD123319, a blocker of the angiotensin type II (AT₂) receptor, enhanced the activation of NFκB by angiotensin II and caerulein. Preliminary data further demonstrated that angiotensin II could extend caerulein-induced ERK1/2 activation in acinar cells. These results indicated that inflammation triggered by hyperstimulation of pancreatic acinar cells is enhanced by angiotensin II, via the AT₁ receptor. In contrast, stimulation of the AT₂ receptor protects against caerulein-induced NFκB activation. The differential roles of the AT₁ and AT₂ receptors might be useful in developing potential therapies for pancreatic inflammation.

Topics: alpha-Amylases; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin Receptor Antagonists; Animals; Cell Line; Ceruletide; Enzyme Activation; Imidazoles; Losartan; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Pancreas, Exocrine; Pancreatitis; Pyridines; Rats; Receptor, Angiotensin, Type 1

Substance P (SP) is involved in the pathophysiology of acute pancreatitis (AP) via binding to its high-affinity receptor, neurokinin-1-receptor (NK1R). An up-regulation of SP and NK1R expression was observed in experimental AP and in caerulein-stimulated pancreatic acinar cells. However, the mechanisms that lead to this up-regulation are not fully understood. In this study, we showed the role of protein kinase C (PKC) in caerulein-induced SP and NK1R production in isolated mouse pancreatic acinar cells. Caerulein (10(-7) M) stimulation rapidly activated the conventional PKC-α and novel PKC-δ as observed by the phosphorylation of these molecules. Pre-treatment of pancreatic acinar cells with Gö6976 (1-10 nM) and rottlerin (1-10 μM) inhibited PKC-α and PKC-δ phosphorylation, respectively, but not the other way round. At these concentrations used, PKC-α and PKC-δ inhibition reversed the caerulein-induced up-regulation of SP and NK1R, indicating an important role of PKCs in the modulation of SP and NK1R expression. Further experiments looking into signalling mechanisms showed that treatment of pancreatic acinar cells with both Gö6976 and rottlerin inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Inhibition of PKC-α or PKC-δ also affected caerulein-induced transcription factor activation, as represented by nuclear factor-κB and AP-1 DNA-binding activity. The findings in this study suggested that PKC is upstream of the mitogen-activated protein kinases and transcription factors, which then lead to the up-regulation of SP/NK1R expression in caerulein-treated mouse pancreatic acinar cells.

Topics: Acetophenones; Acinar Cells; Animals; Benzopyrans; Ceruletide; Gene Expression Regulation; MAP Kinase Kinase 4; Mice; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Pancreas; Pancreatitis; Phosphorylation; Protein Kinase C-alpha; Protein Kinase C-delta; Receptors, Neurokinin-1; Signal Transduction; Substance P

Curcuma longa (CL) has been reported to possess a variety of pharmacological activities. However, the effects of CL on acute pancreatitis (AP) have not yet been determined. To this end, we examined the effects of CL on cerulein-induced AP. Cell viability and cytokine productions were measured in pancreatic acini. Mice were divided into 3 groups: i) Normal group, ii) normal saline-treated group, iii) group treated with CL at a dose of 0.05, 0.1, 0.5 and 1 g/kg. CL was administered orally to mice for 7 days. The mice were intraperitoneally injected with the stable cholecystokinin analogue, cerulein (50 μg/kg), every hour for a total of 6 h. The mice were sacrificed 6 h after the completion of the cerulein injections. Blood samples were obtained to determine serum amylase, lipase and cytokine levels. The pancreas was rapidly removed for morphological examination, measurement of tissue myeloperoxidase activity, as well as the level of cytokines and heme oxygenase-1 (HO-1). The CL treatment reduced cerulein-induced cell death and cytokine production in pancreatic acini. The administration of CL significantly ameliorated the severity of pancreatitis and pancreatitis-associated lung injury, as was shown by the reduction in pancreatic edema, neutrophil infiltration, vacuolization, necrosis, serum amylase, lipase and cytokine levels, and mRNA expression of multiple inflammatory mediators such as interleukin (IL)-1ß and -6 and tumor necrosis factor (TNF)-α. In order to identify the regulatory mechanism of CL on cerulein-induced pancreatitis, we examined the level of HO-1 in the pancreas. We found that the administration of CL induced HO-1. Our results suggest that CL plays a protective role in the development of AP and pancreatitis-associated lung injury.

Topics: Acute Disease; Animals; Ceruletide; Curcuma; Cytokines; Female; Heme Oxygenase-1; Lung Injury; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peroxidase; Plant Extracts

Acute lung injury is a common complication of acute pancreatitis (AP) and contributes to the majority of AP-associated deaths. Although some aspects of AP-induced lung inflammation have been demonstrated, investigation of resultant changes in lung function is limited. The aim of this study was to characterize lung injury in caerulein-induced AP. Male Sprague Dawley rats (n = 7-8/group) received 7 injections of caerulein (50 μg/kg) at 12, 24, 48, 72, 96, or 120 hours before measurement of lung impedance mechanics. Bronchoalveolar lavage (BAL), plasma, pancreatic, and lung tissue were collected to determine pancreatic and lung measures of acute inflammation. AP developed between 12 and 24 hours, as indicated by increased plasma amylase activity and pancreatic myeloperoxidase (MPO) activity, edema, and abnormal acinar cells, before beginning to resolve by 48 hours. In the lung, MPO activity peaked at 12 and 96 hours, with BAL cytokine concentrations peaking at 12 hours, followed by lung edema at 24 hours, and BAL cell count at 48 hours. Importantly, no significant changes in BAL protein concentration or arterial blood gas-pH levels were evident over the same period, and only modest changes were observed in respiratory mechanics. Caerulein-induced AP results in minor lung injury, which is not sufficient to allow protein permeability and substantially alter respiratory mechanics.

Topics: Acute Lung Injury; Amylases; Animals; Bronchoalveolar Lavage; Ceruletide; Male; Pancreatitis; Peroxidase; Pneumonia; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Respiratory Mechanics

Pancreatitis represents a life-threatening inflammatory condition where leucocytes, cytokines and vascular endothelium contribute to the development of the inflammatory disease. The glucocorticoid-induced tumour necrosis factor (TNF) receptor family-related protein (GITR) is a costimulatory molecule for T lymphocytes, modulates innate and adaptive immune system and has been found to participate in a variety of immune responses and inflammatory processes. Our purpose was to verify whether inhibition of GITR triggering results in a better outcome in experimental pancreatitis.. In male GITR knock-out (GITR(-/-)) and GITR(+/+) mice on Sv129 background, acute pancreatitis was induced after i.p. administration of cerulein. Other experimental groups of GITR(+/+) mice were also treated with different doses of Fc-GITR fusion protein (up to 6.25 µg·mouse⁻¹), given by implanted mini-osmotic pump. Clinical score and pro-inflammatory parameters were evaluated.. A less acute pancreatitis was found in GITR(-/-) mice than in GITR(+/+) mice, with marked differences in oedema, neutrophil infiltration, pancreatic dysfunction and injury. Co-treatment of GITR(+/+) mice with cerulein and Fc-GITR fusion protein (6.25 µg·mouse⁻¹) decreased the inflammatory response and tissue injury, compared with treatment with cerulein alone. Inhibition of GITR triggering was found to modulate activation of nuclear factor κB as well as the production of TNF-α, interleukin-1β, inducible nitric oxide synthase, nitrotyrosine, poly-ADP-ribose, intercellular adhesion molecule-1 and P-selectin.. The GITR-GITR ligand system is crucial to the development of acute pancreatitis in mice. Our results also suggest that the Fc-GITR fusion protein could be useful in the treatment of acute pancreatitis.

Topics: Animals; Apoptosis; Ceruletide; Edema; Glucocorticoid-Induced TNFR-Related Protein; I-kappa B Proteins; Intercellular Adhesion Molecule-1; Interleukin-1beta; Ligands; Male; Mice; Mice, 129 Strain; Mice, Knockout; Neutrophil Infiltration; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; P-Selectin; Pancreatitis; Poly(ADP-ribose) Polymerases; Receptors, Nerve Growth Factor; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; T-Lymphocytes; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) has a high mortality rate; repetitive AP induces chronic AP and pancreatic adenocarcinoma. Mesenchymal stem cells (MSCs) have immunoregulatory effects and reduce inflammation. We developed a protocol to isolate human bone marrow-derived clonal MSCs (hcMSCs) from bone marrow aspirate and investigated the effects of these cells in rat models of mild and severe AP.. Mild AP was induced in Sprague-Dawley rats by 3 intraperitoneal injections of cerulein (100 μg/kg), given at 2-hour intervals; severe AP was induced by intraparenchymal injection of 3% sodium taurocholate solution. hcMSCs were labeled with CM-1,1'-dioctadecyl-3,3,3'-tetramethylindo-carbocyanine perchloride and administered to rats through the tail vein.. hcMSCs underwent self-renewal and had multipotent differentiation capacities and immunoregulatory functions. Greater numbers of infused hcMSCs were detected in pancreas of rats with mild and severe AP than of control rats. Infused hcMSCs reduced acinar-cell degeneration, pancreatic edema, and inflammatory cell infiltration in each model of pancreatitis. The hcMSCs reduced expression of inflammation mediators and cytokines in rats with mild and severe AP. hcMSCs suppressed the mixed lymphocyte reaction and increased expression of Foxp3(+) (a marker of regulatory T cells) in cultured rat lymph node cells. Rats with mild or severe AP that were given infusions of hcMSCs had reduced numbers of CD3(+) T cells and increased expression of Foxp3(+) in pancreas tissues.. hcMSCs reduced inflammation and damage to pancreatic tissue in a rat model of AP; they reduced levels of cytokines and induced numbers of Foxp3(+) regulatory T cells. hcMSCs might be developed as a cell therapy for pancreatitis.

Topics: Acute Disease; Animals; Biomarkers; Bone Marrow Transplantation; CD3 Complex; Cell Differentiation; Cell Proliferation; Cells, Cultured; Ceruletide; Coculture Techniques; Cytokines; Disease Models, Animal; Forkhead Transcription Factors; Humans; In Situ Hybridization, Fluorescence; Inflammation Mediators; Mesenchymal Stem Cell Transplantation; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Rats, Wistar; Regeneration; Severity of Illness Index; T-Lymphocytes; Taurocholic Acid; Time Factors

Topics: Acute Disease; Animals; Bicarbonates; Ceruletide; Disease Progression; Galanin; Mice; Pancreatitis

Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure. Melanocortin peptides have been successfully used in experimental models of organ failure and shock, and their protective effect occurs through the activation of a vagus nerve-mediated cholinergic anti-inflammatory pathway by acting at brain melanocortin 4 receptors. In the light of these observations, we studied the effects of the selective melanocortin 4 receptor agonist RO27-3225 in an experimental model of cerulein-induced pancreatitis.. Randomized experiment.. Research laboratory at a university hospital.. Experimental pancreatitis in rats.. Acute pancreatitis was induced in male Sprague-Dawley rats by intraperitoneal injections of cerulein (80 μg/kg, four injections at hourly intervals). Before pancreatitis induction, groups of animals were subjected to bilateral cervical vagotomy, pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective melanocortin 4 receptor antagonist HS024, or not pretreated. Thirty minutes after the first cerulein injection, rats were intraperitoneally treated with a nanomolar dose of RO27-3225 or vehicle. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 mins after treatment with RO27-3225 or vehicle, and for a 30-min period.. Serum lipase and amylase activity, tumor necrosis factor-α and interleukin-6 expression, pancreatic myeloperoxidase activity, and histologic damage were evaluated; neural efferent activity of vagal fibers was also assessed. RO27-3225 reduced cerulein-induced serum lipase and amylase activity, blunted the expression of tumor necrosis factor-α and interleukin-6, abated the increase in pancreatic myeloperoxidase activity, and protected against histologic damage. Furthermore, RO27-3225 markedly increased neural efferent activity along the vagus nerve. Vagotomy, chlorisondamine, and HS024 abated these protective effects of RO27-3225.. Our data show that melanocortin 4 receptor agonists reduce pancreatitis severity through the activation of the cholinergic anti-inflammatory pathway. These findings could be of particular interest in the clinical setting.

Topics: Acute Disease; Analysis of Variance; Animals; Blotting, Western; Ceruletide; Cholinergic Agents; Disease Models, Animal; Immunohistochemistry; Inflammation Mediators; Interleukin-6; Male; Pancreatitis; Peptides; Peroxidase; Random Allocation; Rats; Rats, Sprague-Dawley; Receptor, Melanocortin, Type 4; Receptors, Nicotinic; Sensitivity and Specificity; Severity of Illness Index; Signal Transduction; Tumor Necrosis Factor-alpha; Vagus Nerve

This study aimed to determine the effect of hydrogen sulfide (H2S) on Toll-like receptor 4 (TLR4)-mediated innate immune signaling in acute pancreatitis (AP) via substance P.. Male Swiss mice were treated with hourly intraperitoneal injections of cerulein (50 μg/kg) for 10 hours. dl-propargylglycine ([PAG] 100 mg/kg, intraperitoneally), an inhibitor of H2S formation, was administered 1 hour after the induction of AP. Pancreatic acinar cells from male preprotachykinin-A gene-knockout mice (PPTA) and their wild-type counterparts were incubated with or without cerulein (10 M for 60 minutes). To better understand the effect of H2S in inflammation, acinar cells were stimulated with cerulein after addition of H2S donor, sodium hydrosulfide. In addition, cerulein-treated pancreatic acinar cells were pretreated with PAG (30 μM) for 1 hour.. The H2S inhibitor PAG eliminated TLR4, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and nuclear factor-κB (NF-κB) levels in in vitro and in vivo models of cerulein-induced AP. PPTA gene deletion reduced TLR4, myeloid differentiation factor 88, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and NF-κB in cerulein-treated pancreatic acinar cells, whereas administration of sodium hydrosulfide resulted in a further rise in TLR4 and NF-κB levels in cerulein-treated pancreatic acinar cells.. The present findings show for the first time that in AP, H2S may up-regulate the TLR4 pathway and NF-κB via substance P.

Topics: Acute Disease; Animals; Base Sequence; Ceruletide; DNA Primers; Gene Deletion; Hydrogen Sulfide; Immunity, Innate; Interleukin-1 Receptor-Associated Kinases; Male; Mice; Myeloid Differentiation Factor 88; NF-kappa B; Pancreas; Pancreatitis; Protein Precursors; Signal Transduction; Substance P; Tachykinins; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4; Up-Regulation

Acute pancreatitis is an inflammatory process of variable severity. Leucocytes are thought to play a key role in the development of pancreatitis and pancreatitis-associated lung injury. The interactions between inflammatory cells and their mediators are crucial for determining tissue damage. Monocyte chemoattractant protein-1 (or CCL-2), CCR-2 and CCR-4 are chemokines and chemokine receptors involved in leucocyte trafficking. The aim of the study was to evaluate the role of the CCL-2, CCR-2 and CCR-4 chemokine receptors in the pathogenesis of cerulein-induced pancreatitis and pancreatitis-associated lung injury. To address the role of CCL-2, CCR-2 and CCR-4 that attracts leucocytes cells in inflamed tissues, pancreatitis was induced by administering supramaximal doses of cerulein in mice that do not express CCL-2, CCR-2 or CCR-4.. The severity of pancreatitis was measured by serum amylase, pancreatic oedema and acinar cell necrosis. Lung injury was quantitated by evaluating lung microvascular permeability and lung myeloperoxidase activity. Chemokine and chemokine-receptor expression were quantitated by real-time PCR. The nature of inflammatory cells invading the pancreas and lungs was studied by immunostaining.. The authors have found that pancreas CCL-2 and CCR-2 levels rise during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in CCL-2, whereas the deficiency in either CCR-2 or CCR-4 does not reduce the severity of both the pancreatitis and the lung injury. The amounts of neutrophils and monocyte/macrophages (MOMA)-2 cells were significantly lower in mice deficient in CCL-2 compared with their sufficient littermates.. These results suggest that CCL-2 plays a key role in pancreatitis by modulating the infiltration by neutrophils and MOMA-2 cells, and that its deficiency may improve the outcome of the disease.

Topics: Acute Disease; Animals; Ceruletide; Chemokine CCL2; Disease Models, Animal; Immunohistochemistry; Lung Injury; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Knockout; Neutrophil Infiltration; Organ Size; Pancreas; Pancreatitis; Receptors, CCR2; Receptors, CCR4

Acute pancreatitis is a substantial clinical problem accounting for 240,000 hospital admissions yearly in the United States. Obesity is epidemic and is clearly an independent risk factor for increased severity of acute pancreatitis (AP). Adipose tissue is an endocrine organ that secretes a variety of metabolically active substances termed adipokines. However, the role of adipokines in modulating acute pancreatitis severity remains incompletely understood. Dietary fish oil is rich in omega-3 free fatty acids and attenuates adipose tissue-induced inflammation. Therefore, we hypothesized that feeding obese mice diets rich in fish oil would alter the adipokine milieu and attenuate the severity of pancreatitis.. Lean (C57BL/6 J) and obese (LepDb) mice were fed either a soybean oil- or fish oil-rich diet for 4 weeks. AP was induced by six hourly intraperitoneal injections of cerulein (50 μg/kg). Serum adipokine levels were measured, and pancreatitis severity was assessed histologically and by measuring pancreatic concentrations of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), myleoperoxidase (MPO), and monocyte chemoattractant protein-1 (MCP-1).. Obese mice developed more severe pancreatitis than lean mice. Fish oil significantly decreased serum leptin (lean and obese) and increased serum adiponectin (lean only). Fish oil did not alter the baseline pancreatic inflammatory milieu, nor did it change histologic or biochemical pancreatitis severity.. These data demonstrate that a diet rich in fish oil altered the adipokine milieu in lean and congenitally obese mice; however, fish oil did not improve pancreatitis severity induced with cerulein hyperstimulation.

Topics: Adipokines; Adiponectin; Animals; Ceruletide; Chemokine CCL2; Dietary Fats, Unsaturated; Female; Fish Oils; Interleukin-1beta; Interleukin-6; Leptin; Mice; Mice, Inbred C57BL; Obesity; Pancreatitis; Pancrelipase; Peroxidase; Severity of Illness Index; Soybean Oil

Excessive Ca2+ influx mediates many cytotoxic processes, including those associated with autoimmune inflammatory diseases such as acute pancreatitis and Sjögren syndrome. Transient receptor potential (canonical) channel (TRPC) 3 is a major Ca2+ influx channel in pancreatic and salivary gland cells. We investigated whether genetic or pharmacologic inhibition of TRPC3 protects pancreas and salivary glands from Ca2+-dependent damage.. We developed a Ca2+-dependent model of cell damage for salivary gland acini. Acute pancreatitis was induced by injection of cerulein into wild-type and Trpc3-/- mice. Mice were also given the Trpc3-selective inhibitor pyrazole 3 (Pyr3).. Salivary glands and pancreas of Trpc3-/- mice were protected from Ca2+-mediated cell toxicity. Analysis of Ca2+ signaling in wild-type and Trpc3-/- acini showed that Pyr3 is a highly specific inhibitor of Tprc3; it protected salivary glands and pancreas cells from Ca2+-mediated toxicity by inhibiting the Trpc3-mediated component of Ca2+ influx.. TRPC3-mediated Ca2+ influx mediates damage to pancreas and salivary glands. Pharmacologic inhibition of TRPC3 with the highly selective TRPC3 inhibitor Pyr3 might be developed for treatment of patients with acute pancreatitis and Sjögren syndrome.

Topics: Acute Disease; Animals; Calcium Channel Blockers; Calcium Signaling; Ceruletide; Disease Models, Animal; Epithelial Cells; Mice; Mice, Knockout; Pancreas; Pancreatitis; Pyrazoles; Salivary Gland Diseases; Salivary Glands; Severity of Illness Index; Time Factors; TRPC Cation Channels

To evaluate the anti-inflammatory effect of α,β-amyrin, a pentacyclic triterpenoid from Protium heptaphyllum, on cerulein-induced acute pancreatitis in mice.. Acute pancreatitis was induced in Swiss mice by five intraperitoneal injections of cerulein (50 μg/kg), at 1 h intervals. Mice received α,β-amyrin (10, 30 and 100 mg/kg), thalidomide (200 mg/kg), or vehicle (3% Tween 80) orally 1 h before and 12 h after the cerulein challenge. The severity of pancreatitis was evaluated 24 h after cerulein by assessing serum pro-inflammatory cytokines and amylase activity, pancreatic myeloperoxidase (MPO), and thiobarbituric acid-reactive substances (TBARS), as well as by histology.. α,β-Amyrin and thalidomide significantly attenuated the cerulein-induced increase in tumor necrosis factor (TNF)-α, interleukin-6, lipase, amylase, MPO, and TBARS. Moreover, α,β-amyrin greatly suppressed the pancreatic edema, inflammatory cell infiltration, acinar cell necrosis, and expressions of TNFα and inducible nitric oxide synthase.. α,β-Amyrin ameliorates cerulein-induced acute pancreatitis by acting as an anti-inflammatory and antioxidant agent.

Topics: Amylases; Animals; Anti-Inflammatory Agents; Burseraceae; Ceruletide; Disease Models, Animal; Immunosuppressive Agents; Interleukin-6; Male; Mice; Nitric Oxide Synthase Type II; Oleanolic Acid; Pancreatitis; Peroxidase; Random Allocation; Thalidomide; Tumor Necrosis Factor-alpha

Recent data suggest that platelets not only control thrombosis and hemostasis but may also regulate inflammatory processes such as acute pancreatitis. However, the specific role of platelet-derived mediators in the pathophysiology of acute pancreatitis is not known. Herein, we examined the role of CD40 ligand (CD40L, CD154) in different models of acute pancreatitis. Acute pancreatitis was induced by repetitive caerulein administration (50μg/kg, i.p.) or infusion of sodium taurocholate (5%-10μl) into the pancreatic duct in wild-type C57BL/6 and CD40L-deficient mice. Neutrophil infiltration, myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2) levels, acinar cell necrosis, edema and hemorrhage in the pancreas as well as serum amylase activity and lung levels of MPO were quantified 24h after induction of acute pancreatitis. Caerulein and taurocholate challenge caused a clear-cut pancreatic damage characterized by increased acinar cell necrosis, neutrophil infiltration, focal hemorrhage, edema formation as well as increased levels of serum amylase and MIP-2 in the pancreas and lung MPO and histological damage. Notably, CD40L gene-deficient animals exhibited a similar phenotype as wild-type mice after challenge with caerulein and taurocholate. Similarly, administration of an antibody directed against CD40L had no effect against acute pancreatitis. Our data suggest that CD40L does not play a functional role in experimental acute pancreatitis. Thus, other candidates than CD40L needs to be explored in order to identify platelet-derived mediators in the pathophysiology of acute pancreatitis.

Topics: Acute Disease; Animals; CD40 Ligand; Ceruletide; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Taurocholic Acid

Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR(2)), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR(2) and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Injections of a subinflammatory dose of exogenous trypsin increased c-Fos immunoreactivity, indicative of spinal nociceptive activation, but did not cause inflammation, as assessed by measuring serum amylase and myeloperoxidase activity and by histology. The same dose of trypsin IV and P23 increased some inflammatory end points and caused a more robust effect on nociception, which was blocked by melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant trypsin isoforms. To determine the contribution of endogenous activation of trypsin and its minor isoforms, recombinant enterokinase (ENK), which activates trypsins in the duodenum, was administered into the pancreas. Intraductal ENK caused nociception and inflammation that were diminished by polypeptide inhibitors, including soybean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by melagatran. Finally, the secretagogue cerulein induced pancreatic nociceptive activation and nocifensive behavior that were reversed by melagatran. Thus trypsin and its minor isoforms mediate pancreatic pain and inflammation. In particular, the inhibitor-resistant isoforms trypsin IV and P23 may be important in mediating prolonged pancreatic inflammatory pain in pancreatitis. Our results suggest that inhibitors of these isoforms could be novel therapies for pancreatitis pain.

Topics: Abdominal Pain; Acute Disease; Amylases; Analgesics; Animals; Azetidines; Benzylamines; Ceruletide; Disease Models, Animal; Enteropeptidase; Enzyme Activation; Humans; Kinetics; Male; Pain Measurement; Pancreas; Pancreatitis; Peroxidase; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Recombinant Proteins; Signal Transduction; Soybean Proteins; Spinal Cord; Trypsin; Trypsin Inhibitors

An overproduction of proinflammatory mediators in severe acute pancreatitis contributes to the systemic inflammatory response, which may lead to multiorgan damage and even death. Thus, inflammatory cytokines, e.g., tumor necrosis factor (TNF)-α and interleukin (IL)-1β, may be novel targets for the treatment of acute pancreatitis. The aim of this study was to investigate the therapeutic effects of pomalidomide (or CC-4047), a thalidomide analog and immunomodulatory agent, in acute pancreatitis.. Acute pancreatitis was induced in C57BL/6 mice by intraperitoneal administration of cerulein (100 μg/kg/h × 8). Pomalidomide was administered (0.5 mg/kg orally) 1 h before the first or 1 h after the last cerulein administration. The severity of the acute pancreatitis was evaluated biochemically and morphologically.. Pretreatment with pomalidomide significantly reduced the plasma levels of amylase and lipase; the histological injury; and the expression of TNF-α, IL-1β, monocyte chemotactic protein-1 (MCP-1), and inducible nitric oxide synthase (iNOS) in cerulein-induced acute pancreatitis. Post-treatment with pomalidomide also decreased the cerulein-induced elevation of plasma amylase and lipase and decreased the pancreatic damage.. Treatment with pomalidomide ameliorated the severity of cerulein-induced acute pancreatitis in mice. Our data suggest that pomalidomide may become a new therapeutic agent in future clinical trials for the treatment of acute pancreatitis.

Topics: Acute Disease; Administration, Oral; Amylases; Animals; Ceruletide; Disease Models, Animal; Immunologic Factors; Lipase; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Severity of Illness Index; Thalidomide

Acute pancreatitis is characterized by early activation of intracellular proteases followed by acinar cell death and inflammation. Activation of damage-associated molecular pattern (DAMP) receptors and a cytosolic complex termed the inflammasome initiate forms of inflammation. In this study, we examined whether DAMP-receptors and the inflammasome provide the link between cell death and the initiation of inflammation in pancreatitis.. Acute pancreatitis was induced by caerulein stimulation in wild-type mice and mice deficient in components of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain [ASC], NLRP3, caspase-1), Toll-like receptor 9 (TLR9), or the purinergic receptor P2X(7). Resident and infiltrating immune cell populations and pro-interleukin-1β expression were characterized in control and caerulein-treated adult murine pancreas. TLR9 expression was quantified in pancreatic cell populations. Additionally, wild-type mice were pretreated with a TLR9 antagonist before induction of acute pancreatitis by caerulein or retrograde bile duct infusion of taurolithocholic acid 3-sulfate.. Caspase-1, ASC, and NLRP3 were required for inflammation in acute pancreatitis. Genetic deletion of Tlr9 reduced pancreatic edema, inflammation, and pro-IL-1β expression in pancreatitis. TLR9 was expressed in resident immune cells of the pancreas, which are predominantly macrophages. Pretreatment with the TLR9 antagonist IRS954 reduced pancreatic edema, inflammatory infiltrate, and apoptosis. Pretreatment with IRS954 reduced pancreatic necrosis and lung inflammation in taurolithocholic acid 3-sulfate-induced acute pancreatitis.. Components of the inflammasome, ASC, caspase-1, and NLRP3, are required for the development of inflammation in acute pancreatitis. TLR9 and P2X(7) are important DAMP receptors upstream of inflammasome activation, and their antagonism could provide a new therapeutic strategy for treating acute pancreatitis.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Apoptosis; Apoptosis Regulatory Proteins; CARD Signaling Adaptor Proteins; Carrier Proteins; Caspase 1; Ceruletide; Cytoskeletal Proteins; Disease Models, Animal; DNA; Inflammasomes; Interleukin-1; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Neutrophil Infiltration; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatitis; Pneumonia; Protein Precursors; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7; RNA, Messenger; Severity of Illness Index; Signal Transduction; Taurolithocholic Acid; Toll-Like Receptor 9

Acute pancreatitis (AP) is characterized by premature zymogen activation, systemic inflammatory response resulting in inflammatory infiltrates, sustained intracellular calcium, neurogenic inflammation and pain. The inhibitory neurotransmitter and cytoprotective amino acid glycine exerts a direct inhibitory effect on inflammatory cells, inhibits calcium influx and neuronal activation and therefore represents a putative therapeutic agent in AP.. To explore the impact of glycine, mild AP was induced in rats by supramaximal cerulein stimulation (10 μg/kg BW/h) and severe AP by retrograde injection of sodium taurocholate solution (3%) into the common biliopancreatic duct. 100/300 mmol glycine was administered intravenously before induction of AP. To elucidate the effect of glycine on AP, we determined pathomorphology, pancreatic cytokines as well as proteases, serum lipase and amylase, pancreatic and lung MPO activity and pain sensation.. Glycine administration resulted in a noticeable improvement of pathomorphological alterations in AP, such as a reduction of necrosis, inflammatory infiltrates and cytoplasmic vacuoles in cerulein pancreatitis. In taurocholate pancreatitis, glycine additionally diminished pancreatic cytokines and MPO activity, as well as serum lipase and amylase levels.. Glycine reduced the severity of mild and much more of severe AP by attenuating the intrapancreatic and systemic inflammatory response. Therefore, glycine seems to be a promising tool for prophylactic treatment of AP. and IAP.

Topics: Animals; Ceruletide; Chemoprevention; Cytokines; Disease Models, Animal; Enzymes; Glycine; Glycine Agents; Injections, Intravenous; Male; Necrosis; Pain Measurement; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Taurocholic Acid

Although the way for pain management associated with acute pancreatitis has been searched for, there are not enough medications available for it. The aim of the present study was to investigate the role of bradykinin (BK) in pain related to acute pancreatitis. After repeated injections of caerulein (50 μg/kg and 6 times), mice showed edema in the pancreas, and blood concentrations of pancreatic enzymes (amylase and lipase) were clearly elevated. A histopathological study demonstrated that caerulein caused tissue damage characterized by edema, acinar cell necrosis, interstitial hemorrhage, and inflammatory cell infiltrates. Furthermore, the mRNA levels of interleukin-1β and monocyte chemotactic protein (MCP)-1 were significantly increased in the pancreas of caerulein-treated mice. The sensitivity of abdominal organs as measured by abdominal balloon distension was enhanced in caerulein-injected mice, suggesting that caerulein caused pancreatic hyperalgesia. Moreover, repeated treatment with caerulein resulted in cutaneous tactile allodynia of the upper abdominal region as demonstrated by the use of von Frey filaments, indicating that caerulein-treated mice exhibited referred pain. Under this condition, the mRNA levels of bradykinin B1 receptor (BKB1R) and bradykinin B2 receptor (BKB2R) were significantly increased in the dorsal root ganglion (DRG). Finally, we found that des-Arg⁹-(Leu⁸)-bradykinin (BKB1R antagonist) and HOE-140 (BKB2R antagonist) attenuated the acute pancreatitis pain-like state in caerulein-treated mice. These findings suggest that the upregulation of BK receptors in the DRG may, at least in part, contribute to the development of the acute pancreatitis pain-like state in mice.

Topics: Animals; Ceruletide; Ganglia, Spinal; Gene Expression; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Pain; Pancreatitis; Receptors, Bradykinin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

Multiple organ dysfunction is the main cause of death in severe acute pancreatitis. Primary mitochondrial dysfunction plays a central role in the development and progression of organ failure in critical illness. The present study investigated mitochondrial function in seven tissues during early experimental acute pancreatitis.. Twenty-eight male Wistar rats (463 ± 2 g; mean ± SEM) were studied. Group 1 (n= 8), saline control; Group 2 (n= 6), caerulein-induced mild acute pancreatitis; Group 3 (n= 7) sham surgical controls; and Group 4 (n= 7), taurocholate-induced severe acute pancreatitis. Animals were euthanased at 6 h from the induction of acute pancreatitis and mitochondrial function was assessed in the heart, lung, liver, kidney, pancreas, duodenum and jejunum by mitochondrial respirometry.. Significant early mitochondrial dysfunction was present in the pancreas, lung and jejunum in both models of acute pancreatitis, however, the Heart, liver, kidney and duodenal mitochondria were unaffected.. The present study provides the first description of early organ-selective mitochondrial dysfunction in the lung and jejunum during acute pancreatitis. Research is now needed to identify the underlying pathophysiology behind the organ selective mitochondrial dysfunction, and the potential benefits of early mitochondrial-specific therapies in acute pancreatitis.

Topics: Acute Disease; Animals; Biomarkers; Cell Respiration; Ceruletide; Disease Models, Animal; Energy Metabolism; Jejunum; Lung; Male; Mitochondria; Mitochondrial Diseases; Multiple Organ Failure; Pancreas; Pancreatitis; Rats; Rats, Wistar; Severity of Illness Index; Taurocholic Acid; Time Factors

NADPH oxidase is potentially associated with acute pancreatitis by producing reactive oxygen species (ROS). We investigated whether NADPH oxidase mediates the activation of Janus kinase (Jak)2/signal transducers and activators of transcription (Stat)3 and mitogen-activated protein kinases (MAPKs) to induce the expression of transforming growth factor-β1 (TGF-β1) in cerulein-stimulated pancreatic acinar cells.. AR42J cells were treated with an NADPH oxidase inhibitor diphenyleneiodonium (DPI) or a Jak2 inhibitor AG490. Other cells were transfected with antisense or sense oligonucleotides (AS or S ODNs) for NADPH oxidase subunit p22(phox) or p47(phox).. TGF-β1 was determined by enzyme-linked immonosorbent assay. STAT3-DNA binding activity was measured by electrophoretic mobility shift assay. Levels of MAPKs as well as total and phospho-specific forms of Jak1/Stat3 were assessed by Western blot analysis.. Cerulein induced increases in TGF-β1, Stat3-DNA binding activity and the activation of MAPKs in AR42J cells. AG490 suppressed these cerulein-induced changes, similar to inhibition by DPI. Cerulein-induced activation of Jak2/Stat3 and increases in MAPKs and TGF-β1 levels were inhibited in the cells transfected with AS ODN for p22(phox) and p47(phox) compared to S ODN controls.. Inhibition of NADPH oxidase may be beneficial for prevention and treatment of pancreatitis by suppressing Jak2/Stat3 and MAPKs and expression of TGF-β1 in pancreatic acinar cells.

Topics: Animals; Cell Line; Ceruletide; Enzyme Activation; Enzyme Inhibitors; Humans; Janus Kinase 2; Mitogen-Activated Protein Kinases; NADPH Oxidases; Pancreas, Exocrine; Pancreatitis; Rats; Reactive Oxygen Species; STAT3 Transcription Factor; Transforming Growth Factor beta1; Tyrphostins

Stanniocalcin 2 (STC2) is a secreted protein activated by (PKR)-like Endoplasmic Reticulum Kinase (PERK) signalling under conditions of ER stress in vitro. Over-expression of STC2 in mice leads to a growth-restricted phenotype; however, the physiological function for STC2 has remained elusive. Given the relationship of STC2 to PERK signalling, the objective of this study was to examine the role of STC2 in PERK signalling in vivo.. Since PERK signalling has both physiological and pathological roles in the pancreas, STC2 expression was assessed in mouse pancreata before and after induction of injury using a cerulein-induced pancreatitis (CIP) model. Increased Stc2 expression was identified within four hours of initiating pancreatic injury and correlated to increased activation of PERK signalling. To determine the effect of STC2 over-expression on PERK, mice systemically expressing human STC2 (STC2Tg) were examined. STC2Tg pancreatic tissue exhibited normal pancreatic morphology, but altered activation of PERK signalling, including increases in Activating Transcription Factor (ATF) 4 accumulation and autophagy. Upon induction of pancreatic injury, STC2Tg mice exhibited limited increases in circulating amylase levels and increased maintenance of cellular junctions.. This study links STC2 to the pathological activation of PERK in vivo, and suggests involvement of STC2 in responding to pancreatic acinar cell injury.

Topics: Activating Transcription Factor 4; Amylases; Animals; Autophagy; Ceruletide; eIF-2 Kinase; Female; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Pancreatitis; Signal Transduction

To study the expression of X-linked inhibitor of apoptosis protein (XIAP) and cell apoptosis in vitro model of acute pancreatitis (AP), we carried out experiments to stimulate AR42J cell line with caerulein (10(-8) mol/L) for 12 hours, then collected cells at various time points (0 h, 4 h, 8 h, 12 h, and 24 h, respectively). We then observed the morphologic changes of AR42J cells with the stimulation of caerulein with electronic microscope. The gene expression of XIAP, caspase-3 and caspase-9 was detected using real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), and the protein expression of XIAP was assessed by western blot. The activation of nuclear factor-kappa B (NF-kappaB) was measured by flow cytometry (FCM). With the stimulation of caerulein, the expression of XIAP and the NF-kappaB activation could first decrease and then increase, but the change of caspase-3 and caspase-9 expressions were opposite. XIAP may inhibit the cell apoptosis in rat pancreatic acinus AR42J cell lines at first with the stimulation of caerulein, then NF-kappaB can upgrade the expression of XIAP and increase the cell apoptosis.

Topics: Acinar Cells; Animals; Apoptosis; Cell Line; Ceruletide; NF-kappa B; Pancreas; Pancreatitis; Rats; X-Linked Inhibitor of Apoptosis Protein

Obesity is an independent risk factor for severe acute pancreatitis, though the mechanisms underlying this association are unknown. The powerful anti-inflammatory adipokine adiponectin is decreased in obesity. We recently showed that the severity of pancreatitis in obese mice is inversely related to circulating adiponectin levels, and therefore hypothesized that adiponectin upregulation would attenuate the severity of pancreatitis in obese mice.. Forty congenitally obese mice were studied. Seven days prior to study, 20 mice received a single tail vein injection of adenovirus expressing recombinant murine adiponectin (APN; 2 × 10⁸ plaque forming unit (pfu)), and the remainder received a control adenoviral vector expressing β-galactosidase (β-gal; 2 × 10⁸ pfu). Half of the mice in each group had pancreatitis induced by cerulein injection (50 mcg/kg IP hourly for 6 h). The other half received saline on the same schedule. Serum APN concentration and pancreatic tissue concentrations of interleukin (IL)-6, IL-1β, and MCP-1 were measured by ELISA. Histologic pancreatitis score was calculated based on the degree of inflammation (0-4), edema (0-4), and vacuolization (0-4). Data were analyzed by ANOVA and Tukey's tests; p < 0.05 was considered significant.. No difference in body weight was observed between groups. Serum APN was significantly upregulated in the APN group compared with the β-gal group. Pancreatic tissue concentration of IL-6 was significantly decreased in the APN group compared with the β-gal group. No change either in pancreatic tissue concentration of IL-1β and MCP-1 or in the severity of histologic pancreatitis were observed.. Adiponectin upregulation modulates the pancreatic cytokine milieu but does not attenuate pancreatitis in this model of mild acute pancreatitis.

Topics: Adenoviridae; Adiponectin; Analysis of Variance; Animals; Ceruletide; Chemokine CCL2; DNA, Recombinant; Female; Genetic Vectors; Interleukin-1beta; Interleukin-6; Mice; Mice, Obese; Obesity; Pancreas; Pancreatitis; Up-Regulation

Piperine is a phenolic component of black pepper (Piper nigrum) and long pepper (Piper longum), fruits used in traditional Asian medicine. Our previous study showed that piperine inhibits lipopolysaccharide-induced inflammatory responses. In this study, we investigated whether piperine reduces the severity of cerulein-induced acute pancreatitis (AP). Administration of piperine reduced histologic damage and myeloperoxidase (MPO) activity in the pancreas and ameliorated many of the examined laboratory parameters, including the pancreatic weight (PW) to body weight (BW) ratio, as well as serum levels of amylase and lipase and trypsin activity. Furthermore, piperine pretreatment reduced the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 during cerulein-induced AP. In accordance with in vivo results, piperine reduced cell death, amylase and lipase activity, and cytokine production in isolated cerulein-treated pancreatic acinar cells. In addition, piperine inhibited the activation of mitogen-activated protein kinases (MAPKs). These findings suggest that the anti-inflammatory effect of piperine in cerulein-induced AP is mediated by inhibiting the activation of MAPKs. Thus, piperine may have a protective effect against AP.

Topics: Alkaloids; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Benzodioxoles; Ceruletide; Enzyme Activation; Interleukin-1beta; Interleukin-6; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Pancreatitis; Piperidines; Polyunsaturated Alkamides; Tumor Necrosis Factor-alpha

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease without effective chemopreventive or therapeutic approaches. Although the role of oncogenic Kras in initiating development of PDAC is well established, downstream targets of aberrant Ras signaling are poorly understood. Acinar-ductal metaplasia (ADM) appears to be an important prerequisite for development of pancreatic intraepithelial neoplasia (PanIN), a common precursor to PDAC. RAS-related C3 botulinum substrate 1 (Rac1), which controls actin reorganization, can be activated by Ras, is up-regulated in several human cancers, and is required for cerulein-induced morphologic changes in acini. We investigated effects of loss of Rac1 in Kras-induced pancreatic carcinogenesis in mice.. Using a Cre/lox approach, we deleted Rac1 from pancreatic progenitor cells in different mouse models of PDAC and in mice with cerulein-induced acute pancreatitis. Acinar epithelial explants of mutant mice were used to investigate the role of Rac1 in vitro.. Rac1 expression increased in mouse and human pancreatic tumors, particularly in the stroma. Deletion of Rac1 in Kras(G12D)-induced PDAC in mice reduced formation of ADM, PanIN, and tumors and significantly prolonged survival. Pancreatic epithelial metaplasia was accompanied by apical-basolateral redistribution of F-actin, along with basal expression of Rac1. Acinar epithelial explants that lacked Rac1 or that were incubated with inhibitors of actin polymerization had a reduced ability to undergo ADM in 3-dimensional cultures.. In mice, Rac1 is required for early metaplastic changes and neoplasia-associated actin rearrangements in development of pancreatic cancer. Rac1 might be developed as a diagnostic marker or therapeutic target for PDAC.

Topics: Actins; Animals; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Genes, ras; Humans; Kaplan-Meier Estimate; Keratin-19; Metaplasia; Mice; Models, Animal; Pancreas; Pancreatic Neoplasms; Pancreatitis; rac1 GTP-Binding Protein; Signal Transduction; Survival Rate

Pancreatic acinar cells are used to study regulated exocytosis. We investigated the role of interferon regulatory factor-2 (IRF2) in exocytosis in pancreatic acinar cells.. Pancreas tissues from Irf2⁺/⁺, Irf2⁺/⁻), and Irf2⁻/⁻ mice were examined by microscopy, immunohistochemical, and immunoblot analyses; amylase secretion was quantified. We also compared salivary glands and pancreatic islets of Irf2⁻/⁻ mice with those of Irf2⁺/⁻ mice. To examine the effects of increased signaling by type I interferons, we studied pancreatic acini from Irf2⁻/⁻Ifnar1⁻/⁻ mice. The effect of IRF2 on amylase secretion was studied using an acinar cell line and a retroviral system. We studied expression of IRF2 in wild-type mice with cerulein-induced pancreatitis and changes in pancreatic tissue of Irf2⁻/⁻ mice, compared with those of Irf2⁺/⁻ mice.. Irf2⁻/⁻ pancreas was white and opaque; numerous and wide-spread zymogen granules were observed throughout the cytoplasm, along with lack of fusion between zymogen granules and the apical membrane, lack of secretagogue-stimulated amylase secretion, and low serum levels of amylase and elastase-1, indicating altered regulation of exocytosis. The expression pattern of soluble N-ethylmaleimide-sensitive factor attachment protein receptors changed significantly, specifically in pancreatic acini, and was not rescued by disruption of type I interferon signaling. Down-regulation of IRF2 decreased amylase secretion in an acinar cell line. In mice with pancreatitis, levels of IRF2 were reduced. Irf2⁻/⁻ acini were partially resistant to induction of pancreatitis.. IRF2 regulates exocytosis in pancreatic acinar cells; defects in this process might be involved in the early phases of acute pancreatitis.

Topics: Animals; Autophagy; Cell Line; Cells, Cultured; Ceruletide; Disease Models, Animal; Exocytosis; Interferon Regulatory Factor-2; Interferons; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatitis; Signal Transduction; SNARE Proteins

Acute pancreatitis (AP) is an inflammatory disease involving acinar cell injury and rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. 2',4',6'-Tris (methoxymethoxy) chalcone (TMMC), a synthetic chalcone derivative, displays potent anti-inflammatory effects. Therefore, we aimed to investigate whether TMMC might affect the severity of AP and pancreatitis-associated lung injury in mice. We used the cerulein hyperstimulation model of AP. Severity of pancreatitis was determined in cerulein-injected mice by histological analysis and neutrophil sequestration. The pretreatment of mice with TMMC reduced the severity of AP and pancreatitis-associated lung injury and inhibited several biochemical parameters (activity of amylase, lipase, trypsin, trypsinogen, and myeloperoxidase and production of proinflammatory cytokines). In addition, TMMC inhibited pancreatic acinar cell death and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 by inhibiting NF-κB and extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation. Neutralizing antibodies for TNF-α, IL-1β, and IL-6 inhibited cerulein-induced cell death in isolated pancreatic acinar cells. Moreover, pharmacological blockade of NF-κB/ERK1/2 reduced acinar cell death and production of TNF-α, IL-1β, and IL-6 in isolated pancreatic acinar cells. In addition, posttreatment of mice with TMMC showed reduced severity of AP and lung injury. Our results suggest that TMMC may reduce the complications associated with pancreatitis.

Topics: Amylases; Animals; Anti-Inflammatory Agents; Ceruletide; Chalcones; Interleukin-1beta; Interleukin-6; Lipase; Lung Injury; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; NF-kappa B; Pancreatitis; Peroxidase; Tumor Necrosis Factor-alpha

In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.

Topics: Acinar Cells; Animals; Ceruletide; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Inflammation; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Taurochenodeoxycholic Acid; Unfolded Protein Response

The cellular mediators of acute pancreatitis are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis.. Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier method.. Numbers of major histocompatibility complex II(+)CD11c(+) DCs increased 100-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes. Intrapancreatic DCs acquired a distinct immune phenotype in mice with acute pancreatitis; they expressed higher levels of major histocompatibility complex II and CD86 and increased production of interleukin-6, membrane cofactor protein-1, and tumor necrosis factor-α. However, rather than inducing an organ-destructive inflammatory process, DCs were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DCs and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DCs died from acinar cell death within 4 days. Depletion of DCs from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DCs did not require infiltrating neutrophils, activation of nuclear factor-κB, or signaling by mitogen-activated protein kinase or tumor necrosis factor-α.. DCs are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress.

Topics: Acute Disease; Animals; Arginine; Ceruletide; Dendritic Cells; Diphtheria Toxin; Disease Models, Animal; Early Growth Response Protein 1; Interleukin-6; Kaplan-Meier Estimate; Male; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Phenotype; Time Factors; Tissue Survival

Reactive oxygen radicals, pro-inflammatory mediators and cytokines have been implicated in caerulein induced acute pancreatitis. Nordihydroguaiaretic acid (NDGA), a plant lignin, has marked anti-inflammatory properties. The present study aimed to investigate the possible protective effect of NDGA against caerulein induced pancreatitis. Acute pancreatitis was induced by intraperitoneal administration of eight doses of caerulein in male swiss albino mice. NDGA was administered after 9 h of acute pancreatitis induction. Pancreatic damage and the protective effect of NDGA were assessed by oxidative stress parameters and histopathology of pancreas. The mRNA expression of heat shock proteins (DNAJ C15 and HSPD1) was examined by real-time RT-PCR analysis. Expression of HSP 27, NF-κB, TNF-α, p-p38, Bcl-2, p-PP2A, procaspase-3, caspase-3 and histone modifications were examined by western blotting. NDGA attenuated the oxidative stress, led to increased plasma α-amylase and decreased IGF-1 in AP mice. It modulated the mRNA and protein levels of heat shock proteins and reduced the expression of NF-κB, TNF-α and p-p38. It increased the number of TUNEL positive apoptotic cells in the pancreas of AP mice. In addition, NDGA prevented the changes in modifications of histone H3 in acute pancreatitis. To best of our knowledge, this is the first report which suggests that NDGA prevents the progression of acute pancreatitis by involving alteration of histone H3 modifications and modulating the expression of genes involved in inflammatory/apoptotic cascade, which may be responsible for decreased necrosis and increased apoptosis in this model of acute pancreatitis.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Apoptosis; Caspase 3; Ceruletide; Chaperonin 60; Histones; In Situ Nick-End Labeling; Inflammation; Lipoxygenase Inhibitors; Male; Masoprocol; Mice; Mitochondrial Proteins; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Real-Time Polymerase Chain Reaction; Signal Transduction; Tumor Necrosis Factor-alpha

Pigment epithelium-derived factor (PEDF) is important for maintaining the normal extracellular matrix. We hypothesized that the initiation of pancreatic fibrosis is dependent on the loss of PEDF. Pancreatic PEDF expression was assessed in wild-type mice fed either a control or ethanol diet using an intragastric feeding model. Pancreatitis responses were elicited with either a single episode or a repetitive cerulein-induced (50 μg/kg, 6 hourly i.p. injections) protocol in wild-type and PEDF-null mice. Quantitative real-time PCR and immunoblotting were performed to assess fibrogenic responses. In wild-type animals, PEDF expression increased with pancreatitis and was more pronounced in mice fed ethanol. Compared with wild-type mice, α-smooth muscle actin staining and expression levels of fibrogenic markers (eg, transforming growth factor-β1, platelet-derived growth factor, collagen I, and thrombospondin-1) were higher in PEDF-null mice at baseline. Sirius red staining revealed more fibrosis in PEDF-null versus wild-type pancreas 1 week after pancreatitis. Differences in tissue fibrosis resolved with longer recovery periods. PEDF overexpression suppressed thrombospondin-1 levels in vitro. Ethanol feeding and experimental pancreatitis increased PEDF expression in wild-type mice. PEDF-null mice, however, demonstrated enhanced early fibrotic responses compared with wild-type mice with pancreatitis. These findings indicate that PEDF acts as a compensatory antifibrotic cytokine in pancreatitis.

Topics: Animals; Cells, Cultured; Ceruletide; Collagen Type I; Extracellular Matrix; Eye Proteins; Fibrosis; Mice; Mice, Inbred C57BL; Nerve Growth Factors; Pancreas; Pancreatitis; Platelet-Derived Growth Factor; RNA, Messenger; Serpins; Thrombospondin 1; Transforming Growth Factor beta1

The present study aimed to determine the effects of alpha lipoic acid (ALA) on blood and tissue biochemical parameters, as well as tissue histopathology, in an experimental rat model of cerulein-induced acute pancreatitis (AP).. Three groups consisting of eight rats each were used, as follows: Group 1, controls; Group 2, cerulein-induced pancreatitis group treated with saline; and Group 3, cerulein-induced pancreatitis group treated with ALA. AP was induced by intraperitoneal administration of cerulein (20 µg/kg) 4 times at 1-hour intervals. The animals were decapitated 12 hours after the last dose of cerulein. Blood amylase, lipase, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α levels, pancreas tissue glutathione (GSH) and malondialdehyde (MDA) levels, as well as myeloperoxidase (MPO) and Na+-K+-ATPase activity were measured. Pancreatic tissue samples were also evaluated histopathologically under a light microscope.. While plasma amylase, lipase, IL-1ß, and TNF-α levels, and tissue MDA and MPO levels significantly increased in rats with cerulean-induced AP, tissue GSH and Na+-K+-ATPase activity significantly reduced. These changes were reversed and improved with ALA treatment.. Our findings suggest that ALA may significantly reduce morbidity and mortality by preventing organ dysfunction induced by free radicals in the pancreas.

Topics: Animals; Antioxidants; Ceruletide; Female; Injections, Intraperitoneal; Male; Pancreatitis; Rats; Rats, Sprague-Dawley; Thioctic Acid

Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.

Topics: Animals; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Lineage; Ceruletide; Disease Progression; Gene Targeting; Humans; Integrases; Intermediate Filament Proteins; Mice; Mice, Transgenic; Nerve Tissue Proteins; Nestin; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Precancerous Conditions; Proto-Oncogene Proteins p21(ras); STAT3 Transcription Factor; Stem Cells; Transgenes

Curcuma longa (turmeric) has a long history of use in Ayurvedic medicine as a treatment for inflammatory conditions. The purpose of the present study was to investigate the preventive effects of curcumin against acute pancreatitis (AP) induced by caerulein in mouse and to elucidate possible mechanism of curcumin action.. Curcumin (50 mg/kg/day) was intraperitoneally injected to Kun Ming male mice for 6 days, followed by injection of caerulein to induce AP. GW9662 (0.3 mg/kg), a specific peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, was intravenously injected along with curcumin. Murine macrophage RAW264.7 cells were treated with 100 μmol/l curcumin for 2 h, and then stimulated with 0.1 μ g/ml lipopolysaccharide (LPS). Serum amylase and transaminase levels were measured at 10 h after AP. TNF-α level in mouse serum and cell culture medium were detected by ELISA. Expression of PPARγ and NF-κB were analyzed by RT-PCR and Western blot.. Curcumin significantly decreased the pancreas injury and reversed the elevation of serum amylase, ALT and AST activities and TNF-α level in mice with AP. Curcumin treatment inhibited the elevation of NF-κB-p65 in the nucleus of mouse pancreas AP group and RAW264.7 cells, but significantly increased the expression of PPARγ. GW9662 could abolish the effects of curcumin on serum levels of amylase, ALT, AST, TNF-α, and NF-κB level.. Our results suggest that curcumin could attenuate pancreas tissue and other organ injury by inhibiting the release of inflammatory cytokine TNF-α. These effects may involve upregulation of PPARγ and subsequent downregulation of NF-κB.

Topics: Alanine Transaminase; Amylases; Anilides; Animals; Cell Nucleus; Ceruletide; Curcuma; Curcumin; Disease Models, Animal; Gene Expression Regulation; Inflammation; Lipopolysaccharides; Macrophages; Male; Mice; NF-kappa B; Pancreatitis; Plant Extracts; PPAR gamma; Transaminases; Tumor Necrosis Factor-alpha

To study the potential role of tumor necrosis factor receptor-associated factor 6 (TRAF6) as the key adaptor of the toll-like receptor 4 (TLR4) signaling pathway in acute pancreatitis (AP) in mice.. Acute pancreatitis was induced by 7 intraperitoneal injections of cerulein in TLR4-deficient (TLR4-Def) and TLR4 wild-type (TLR4-WT) mice. Inflammatory severity was scored and evaluated based on pathological study. TRAF6 expression was determined by reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemistry.. Acute pancreatitis was successfully induced in both mice strains, but the inflammatory progression was different. In TLR4-Def mice, pancreatic inflammation was blunt and mild first, then became increasingly intensive and peaked at the later stage, whereas in the TLR4-WT mice, the response was fast initiated and peaked at the early stage of AP, then alleviated gradually. TRAF6 expression in TLR4-Def mice was significantly higher than that in the TLR4-WT mice. Immunohistochemistry located TRAF6 expressed mainly in the pancreatic acinar cells.. The TLR4-TRAF6 signaling pathway is critically involved in AP. Other signaling pathways beyond TLR4 may participate in the pancreatic inflammatory process via TRAF6. As a convergence point of the TLR4-dependent and the TLR4-independent signaling pathways, TRAF6 plays an important role in AP.

Topics: Acute Disease; Animals; Ceruletide; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Pancreatitis; Signal Transduction; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4

The inflammatory response during pancreatitis regulates necrotic and apoptotic rates of parenchymal cells. Neutrophil depletion by use of anti-polymorphonuclear serum (anti-PMN) increases apoptosis in experimental pancreatitis but the mechanism has not been determined. Our study was designed to investigate signaling mechanisms in pancreatic parenchymal cells regulating death responses with neutrophil depletion. Rats were neutrophil depleted with anti-PMN treatment. Then cerulein pancreatitis was induced, followed by measurements of apoptosis signaling pathways. There was greater activation of executioner caspases-3 in the pancreas with anti-PMN treatment compared with control. There were no differences between these groups of animals in mitochondrial cytochrome c release or in activities of initiator caspase-8 and -9. However, there was greater activation of caspase-2 with anti-PMN treatment during cerulein pancreatitis. The upstream regulation of caspases-2 includes p53, which was increased; the p53 negative regulator, Mdm2, was decreased by anti-PMN treatment during cerulein pancreatitis. In vitro experiments using isolated pancreatic acinar cells a pharmacological inhibitor of Mdm2 increased caspase-2/-3 activities, and an inhibitor of p53 decreased these activities during cholecystokinin-8 treatment. Furthermore, experiments using the AR42J cell line Mdm2 small interfering RNA (siRNA) increased caspase-2/-3 activities, and p53 siRNA decreased these activities during cholecystokinin-8 treatment. These results suggest that during acute pancreatitis the inflammatory response inhibits apoptosis. The mechanism of this inhibition involves caspase-2 and its upstream regulation by p53 and Mdm2. Because previous findings indicate that promotion of apoptosis decreases necrosis and severity of pancreatitis, these results suggest that strategies to inhibit Mdm2 or activate p53 will have beneficial effects for treatment of pancreatitis.

Topics: Acute Disease; Animals; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspases; Cells, Cultured; Ceruletide; Cysteine Endopeptidases; Cytochromes c; Disease Models, Animal; Male; Necrosis; Neutrophils; Pancreatitis; Proto-Oncogene Proteins c-mdm2; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Tumor Suppressor Protein p53

The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G-protein-coupled, cell surface bile acid receptor.. Acute pancreatitis was induced in wild-type and Gpbar1(-/-) mice by either retrograde ductal infusion of taurolithocholic acid-3-sulfate (TLCS) or supramaximal secretagogue stimulation with caerulein. In vitro experiments were performed in which acini obtained from wild-type and Gpbar1(-/-) mice were exposed to either submicellar concentrations of TLCS (200-500 microM) or a supramaximally stimulating concentration of caerulein (10 nM).. Gpbar1 is expressed at the apical pole of acinar cells and its genetic deletion is associated with reduced hyperamylasemia, edema, inflammation, and acinar cell injury in TLCS-induced, but not caerulein-induced, pancreatitis. In vitro, genetic deletion of Gpbar1 is associated with markedly reduced generation of pathological calcium transients, intracellular activation of digestive zymogens, and cell injury when these responses are induced by exposure to TLCS, but not when they are induced by exposure to caerulein.. Gpbar1 may play a critical role in the evolution of bile-acid-induced pancreatitis by coupling exposure to bile acids with generation of pathological intracellular calcium transients, intra-acinar cell zymogen activation, and acinar cell injury. Acute biliary pancreatitis may be a "receptor-mediated" disease and interventions that interfere with Gpbar1 function might prove beneficial in the treatment and/or prevention of biliary acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Bile Acids and Salts; Calcium Signaling; Ceruletide; Disease Models, Animal; Enzyme Precursors; GTP-Binding Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Receptors, G-Protein-Coupled; Severity of Illness Index; Taurolithocholic Acid

Acute pancreatitis is characterized by an activation cascade of digestive enzymes in the pancreas. The first of these, trypsinogen, can be converted to active trypsin by the peptidase cathepsin B (CTSB). We investigated whether cathepsin L (CTSL) can also process trypsinogen to active trypsin and has a role in pancreatitis.. In CTSL-deficient (Ctsl(-/-)) mice, pancreatitis was induced by injection of cerulein or infusion of taurocholate into the pancreatic duct. Human tissue, pancreatic juice, mouse pancreatitis specimens, and recombinant enzymes were studied by enzyme assay, immunoblot, N-terminal sequencing, immunocytochemistry, and electron microscopy analyses. Isolated acini from Ctsl(-/-) and Ctsb(-/-) mice were studied.. CTSL was expressed in human and mouse pancreas, colocalized with trypsinogen in secretory vesicles and lysosomes, and secreted into pancreatic juice. Severity of pancreatitis was reduced in Ctsl(-/-) mice, whereas apoptosis and intrapancreatic trypsin activity were increased. CTSL-induced cleavage of trypsinogen occurred 3 amino acids toward the C-terminus from the CTSB activation site and resulted in a truncated, inactive form of trypsin and an elongated propeptide (trypsinogen activation peptide [TAP]). This elongated TAP was not detected by enzyme-linked immunosorbent assay (ELISA) but was effectively converted to an immunoreactive form by CTSB. Levels of TAP thus generated by CTSB were not associated with disease severity, although this is what the TAP-ELISA is used to determine in the clinic.. CTSL inactivates trypsinogen and counteracts the ability of CTSB to form active trypsin. In mouse models of pancreatitis, absence of CTSL induces apoptosis and reduces disease severity.

Topics: Amylases; Animals; Apoptosis; Cathepsin B; Cathepsin L; Ceruletide; Disease Models, Animal; Humans; Hydrogen-Ion Concentration; Lipase; Mice; Mice, Knockout; Pancreatitis; Severity of Illness Index; Taurocholic Acid; Trypsin; Trypsinogen

Our aim was to determine if total parenteral nutrition (TPN)-induced pancreatic atrophy and Hsp70 expression attenuates cerulein-induced pancreatitis in rats.. Rats were randomized to a 7-day course of saline infusion plus a semipurified diet or TPN, with or without an intravenous cerulein injection or vehicle on day 7, and killed 1 or 6 hours after the injection. Based on a pilot study, 1 hour was the primary time point. Pancreatic atrophy was determined by mass, protein, and DNA contents. Pancreatic heat shock protein 70 (Hsp70) expression was measured by Western analysis. Histological examination of the pancreas assessed for edema, inflammation, vacuolization, and apoptosis. Serum amylase activity was measured using the Phadebas assay. Pancreatic trypsinogen activation was measured using a fluorometric substrate assay.. The saline-infused rats fed orally gained significantly more weight than TPN rats. The TPN decreased the pancreatic mass and protein content and the protein-DNA ratio and increased the pancreatic DNA content compared with the saline. The TPN increased the pancreatic Hsp70 expression by 91% compared with the saline. The TPN reduced the cerulein-induced pancreatic histological edema, the vacuolization, and the inflammation compared with the saline. The increase in the serum amylase level after cerulein injection was significantly attenuated, and trypsinogen activation was reduced in TPN animals compared with the saline group.. Lack of luminal nutrients with a 7-day course of TPN provides moderate protection against cerulein-induced pancreatitis in rats.

Topics: Amylases; Animals; Ceruletide; HSP70 Heat-Shock Proteins; Male; Pancreatitis; Parenteral Nutrition, Total; Rats; Rats, Sprague-Dawley; Trypsinogen

Nardostachys jatamansi belonging to the family Valerianaceae has been used as a remedy for stomach and skin ailments in Korea. The effect of N. jatamansi on acute pancreatitis (AP) has not been defined. Therefore, we investigated the effect of N. jatamansi on cerulein-induced AP.. In the pretreatment group, N. jatamansi was administered orally to mice at 10 and 20 mg/kg for 5 days, and the mice were intraperitoneally injected with the stable cholecystokinin analogue cerulein hourly for 6 hours. In the posttreatment group, cerulein was injected hourly for 6 hours, and N. jatamansi was administered at the indicated time (1, 3, and 5 hours after the first cerulein injection) and dose (10 and 20 mg/kg) during the cerulein injection. Blood samples were taken 6 hours later to determine the serum amylase, the lipase, and the cytokine levels. The pancreas and the lung were rapidly removed for morphologic examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction.. Nardostachys jatamansi treatment attenuated the AP, as shown by the histological examination results of the pancreas and the lung, reductions in pancreatic edema, neutrophil infiltration, serum amylase and lipase levels, serum cytokine levels, and messenger RNA expressions of inflammatory mediators.. These results suggest that N. jatamansi attenuates the severity of AP and pancreatitis-associated lung injury.

Topics: Acute Disease; Amylases; Animals; Body Weight; Cell Survival; Cells, Cultured; Ceruletide; Cytokines; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Female; Lipase; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Nardostachys; Organ Size; Pancreas; Pancreatitis; Peroxidase; Phytotherapy; Plant Extracts; Reverse Transcriptase Polymerase Chain Reaction

Both galanin and substance P have been separately implicated in the pathogenesis of acute pancreatitis. We compared the efficacy of the combination of the galanin antagonist galantide and the neurokinin-1 receptor antagonist L703,606 with that of either alone in the treatment of acute pancreatitis. Acute pancreatitis was induced in mice with 7-hourly caerulein injections. Galantide was co-administered with each caerulein injection commencing with the first injection (prophylactic) or 2h after the first injection (therapeutic). L703,606 was administered either 30 min before (prophylactic), or 2h after the first caerulein injection (therapeutic). Combination of the two agents was also administered. Control groups received galantide, L703,606, or saline, without caerulein. Pancreata were harvested for histological examination and estimation of myeloperoxidase activity. Plasma amylase activity was measured. Prophylactic and therapeutic administration of galantide reduced the hyperamylasemia by 37% and 30% respectively whereas only prophylactic L703,606 reduced hyperamylasemia (by 34%). Prophylactic administration of the combined antagonists reduced the hyperamylasemia by 44%. In contrast, therapeutic administration of the combination significantly increased plasma amylase levels by 27%. The plasma amylase activity in the control groups was similar to basal levels. Prophylactic and therapeutic administration of either antagonist or the combination significantly reduced myeloperoxidase activity. Galantide and L703,606 individually, and in combination, significantly reduced the acute pancreatitis-induced necrosis score. The administration of the combined antagonists does not offer any further benefit as compared to galantide alone. An interaction between neurokinin-1 and galanin receptors may occur to modulate amylase secretion.

Topics: Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Ceruletide; Edema; Galanin; Mice; Neurokinin-1 Receptor Antagonists; Pancreas; Pancreas, Exocrine; Pancreatitis; Peroxidase; Quinuclidines; Receptors, Galanin; Receptors, Neurokinin-1; Substance P

Acute pancreatitis (AP) is characterized by pancreatic microcirculatory and secretory disturbances. As galanin can modulate pancreatic vascular perfusion, we sought to determine if galanin plays a role in AP.. Acute pancreatitis was induced in wild-type and galanin gene knockout mice by intraperitoneal injections of cerulein. The severity of AP was evaluated (plasma amylase and lipase, myeloperoxidase activity, and acinar cell necrosis) with and without treatment with galanin or the antagonist galantide. Galanin receptor messenger RNA expression in mouse pancreas was measured by reverse transcription-polymerase chain reaction and Western blot analysis.. Galantide ameliorated AP, reducing all indices by 25% to 40%, whereas galanin was without effect. In galanin knockout mice, all indices of AP were reduced 25% to 50% compared with wild-type littermates. Galanin administration to the knockout mice exacerbated AP such that it was comparable with the AP induced in the wild-type mice. Conversely, administration of galantide to the galanin knockout mice did not affect the AP, whereas AP was ameliorated in the wild-type mice. The 3 galanin receptor subtypes are expressed in mouse pancreas, with receptor subtype 3 expression predominating.. These data implicate a role for galanin in AP and suggest a potential clinical application for galanin antagonists in treatment.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Female; Galanin; Mice; Mice, Inbred BALB C; Mice, Knockout; Pancreas; Pancreatitis; Receptors, Galanin; RNA, Messenger; Severity of Illness Index; Substance P

Severe acute pancreatitis is a life threatening disease with a high rate of mortality, and its treatments are still controversial. The purpose of this study is to investigate the potential effects of proteasome inhibitor PS-341 on severe acute pancreatitis induced by cerulein and lipopolysaccharide in mice.. Severe acute pancreatitis was induced by seven intraperitoneal injections of 50 ug/kg cerulein at hourly intervals and one injection of 10mg/kg lipopolysaccharide in mice. Thirty min before the administration of lipopolysaccharide, mice were treated either with PS-341 or vehicle. The severity of acute pancreatitis was then evaluated by serum and pancreatic biochemical assays as well as histologic examination. Positron emission tomography (PET) was used for the first time to determine the therapeutic effects of interventions in situ.. PS-341 significantly inhibited NF-kappaB activation, while the pancreatic cell apoptosis was significantly enhanced, resulting in the improved parameters such as serum amylase, C-reactive protein, lactate dehydrogenase, interleukin-1beta, interleukin-6, and pancreatic myeloperoxidase activity. Accordingly, pancreatic damage, including inflammatory cell infiltration, hemorrhage, and necrosis, was markedly reduced. (18)F-fluorodeoxyglucose-positron emission tomography demonstrated that PS-341 significantly reduced the uptake of (18)F-fluorodeoxyglucose within the pancreas.. These observations demonstrate that PS-341 was able to significantly reduce the severity of acute pancreatitis induced by cerulein and lipopolysaccharide in mice. The potential effect is associated with the inhibition of NF-kappaB activation and increased pancreatic cell apoptosis within the pancreas. (18)F-fluorodeoxyglucose-positron emission tomography could be a sensitive and promising means in evaluating the therapeutic effect and adjusting medical interventions for pancreatitis.

Topics: Amylases; Animals; Apoptosis; Boronic Acids; Bortezomib; C-Reactive Protein; Ceruletide; Female; Inflammation; Interleukin-1beta; L-Lactate Dehydrogenase; Mice; Mice, Inbred ICR; Pancreatitis; Positron-Emission Tomography; Protease Inhibitors; Pyrazines; Radiography

The neuropeptide substance P (SP) has emerged to be an important proinflammatory mediator in acute pancreatitis (AP). The presence of substance P and its receptor, neurokinin-1 receptor (NK1R) has been shown in the pancreas and the pancreatic acinar cells. In this study, we investigated the unexplored mechanisms that mediate SP and NK1R expression using an in vitro AP model. Pancreatic acinar cells were obtained from pancreas of male Swiss mice. Isolated cells were treated with caerulein to mimic secretagogue pancreatitis. A concentration-dependent study that subjected the cells to 60 min of stimulation by caerulein showed that SP and the transcript from its gene preprotachykinin-A (PPT-A), and NK1R were up-regulated at a supraphysiological concentration of 10(-7) M. A concentration-dependent study on intracellular kinases, extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) and also transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) showed that they were activated when the caerulein concentration was 10(-7) M. Inhibition of JNK reversed the up-regulation of PPT-A, SP, and NK1R. However, inhibition of ERK1/2 reversed the up-regulation of NK1R but not of PPT-A and SP. Furthermore, we found that specific ERK1/2 and JNK inhibitors reduce NF-kappaB and AP-1 activity. Taken together, our results suggest that supraphysiological concentrations of caerulein up-regulate the expression of SP and NK1R in pancreatic acinar cells, and the signaling molecules that are involved in this up-regulation include ERK1/2, JNK, NF-kappaB, and AP-1.

Topics: Acute Disease; Animals; Ceruletide; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Pancreas; Pancreatitis; Phosphorylation; Protein Precursors; Receptor, Cholecystokinin A; Receptors, Neurokinin-1; Signal Transduction; Substance P; Tachykinins; Transcription Factor AP-1

We surveyed changes of the gene expression profile in caerulein-exposed pancreas using Affymetrix GeneChip system (39,000 genes). Up-regulation of genes coding for claudin 4, claudin 7, F11 receptor, cadherin 1, integrin beta 4, syndecan 1, heat shock proteins b1/90aa1, Serpinb6a, Serpinb6b, Serpinb9, Bax, Bak1, calpain 2, calpain 5, microtubule-associated protein 1 light chain 3 alpha, S100 calcium-binding proteins A4/A10 were found in mouse pancreas exposed to caerulein for 12h. In contrast, the anti-apoptotic gene Bcl2 was down-regulated. The functions of these genes concern tight junction formation, cell-cell/cell-matrix adhesions, stress response, protease inhibition, apoptosis, autophagy, and regulation of cytoskeletal dynamics. Caerulein-exposed pancreatic acinar cells were immunohistochemically stained for claudin 4, cadherin 1, integrin beta 4, heat shock protein b1, and Serpinb6a. In conclusion, we have newly identified a set of genes that are likely to be involved in endogenous self-protection mechanisms against acute pancreatitis.

Topics: Acute Disease; Animals; Apoptosis Regulatory Proteins; Autophagy; Ceruletide; Gene Expression; Gene Expression Profiling; Heat-Shock Proteins; Intercellular Junctions; Male; Mice; Mice, Inbred Strains; Pancreatitis; Proteinase Inhibitory Proteins, Secretory; S100 Proteins

Endogenous trypsin inhibitors are synthesized, stored, and secreted by pancreatic acinar cells. It is believed that they play a protective role in the pancreas by inhibiting trypsin within the cell should trypsinogen become prematurely activated. Rodent trypsin inhibitors are highly homologous to human serine protease inhibitor Kazal-type 1 (SPINK1). The mouse has one pancreatic trypsin inhibitor known as SPINK3, and the rat has two trypsin inhibitors commonly known as pancreatic secretory trypsin inhibitors I and II (PSTI-I and -II). Rat PSTI-I is a 61-amino acid protein that shares 65% sequence identity with mouse SPINK3. It was recently demonstrated that mice with genetic deletion of the Spink3 gene (Spink3(-/-)) do not survive beyond 15 days and lack normal pancreata because of pancreatic autophagy. We have shown that targeted transgenic expression of the rat Psti1 gene to acinar cells in mice [TgN(Psti1)] protects mice against caerulein-induced pancreatitis. To determine whether the autophagic phenotype and lethality in Spink3(-/-) mice were due to lack of pancreatic trypsin inhibitor, we conducted breeding studies with Spink3(+/-) heterozygous mice and TgN(Psti1) mice. We observed that, whereas Spink3(+/+), Spink3(+/-), and Spink3(-/-)/TgN(Psti1) mice had similar survival rates, no Spink3(-/-) mice survived longer than 1 wk. The level of expression of SPINK3 protein in acini was reduced in heterozygote mice compared with wild-type mice. Furthermore, endogenous trypsin inhibitor capacity was reduced in the pancreas of heterozygote mice compared with wild-type or knockout mice rescued with the rat Psti1 gene. Surprisingly, the lesser amount of SPINK3 present in the pancreata of heterozygote mice did not predispose animals to increased susceptibility to caerulein-induced acute pancreatitis. We propose that a threshold level of expression is sufficient to protect against pancreatitis.

Topics: Amino Acid Sequence; Amylases; Animals; Body Size; Ceruletide; Female; Glycoproteins; Heterozygote; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Molecular Sequence Data; Organ Size; Pancreas; Pancreas, Exocrine; Pancreatitis; Prostatic Secretory Proteins; Rats; Sequence Homology, Amino Acid; Survival Rate; Transgenes; Trypsin; Trypsin Inhibitor, Kazal Pancreatic

To analyze the expression modulation of pancreatic protein tyrosine phosphatase (PTP)1B during the development of cerulein (Cer)-induced acute pancreatitis (AP) and the effect of inhibition of type 4 phosphodiesterase and c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 on its expression levels.. Acute pancreatitis was induced in rats by subcutaneous injections of 20 microg Cer per kilogram body weight at hourly intervals, and the animals were killed at 2, 4, or 9 hours after the first injection. Neutropenia was induced with vinblastine sulfate. Phosphodiesterase and the mitogen-activated protein kinases were inhibited with rolipram and SP600125, respectively, before the induction of AP.. Protein tyrosine phosphatase 1B increases its expression at the levels of both protein and messenger RNA during the early phase of Cer-induced AP. The increase in protein expression persisted along the development of the disease, and neutrophil infiltration seemed to play a central role. Rolipram and SP600125 pretreatments mostly suppressed the increase in the expression of PTP1B during the early phase of AP.. Cerulein-induced AP is associated with an increase in the expression of PTP1B in its early phase. An increase in cyclic adenosine monophosphate levels in inflammatory cells and the inhibition of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 are able to suppress the increase in PTP1B protein level.

Topics: Animals; Anthracenes; Ceruletide; Cyclic AMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Disease Models, Animal; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neutropenia; Pancreatitis; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Rats; Rats, Wistar; Rolipram; Vinblastine

To analyze susceptibility to acute pancreatitis, five mouse strains including Japanese Fancy Mouse 1 (JF1), C57BL/6J, BALB/c, CBA/J, and C3H/HeJ were treated with either a cholecystokinin analog, cerulein, or a choline-deficient, ethionine-supplemented (CDE) diet. The severity of acute pancreatitis induced by cerulein was highest in C3H/HeJ and CBA/J, moderate in BALB/c, and mildest in C57BL/6J and JF1. Basal protein expression levels of the serine protease inhibitor, Kazal type 3 (Spink3) were higher in JF1 and C57BL/6J mice than those of the other three strains under normal feeding conditions. After treatment with cerulein, expression level of Spink3 increased remarkably in JF1 and mildly in C57BL/6J, BALB/c, CBA/J, and C3H/HeJ strains. Increased proteinase, serine, 1 (Prss1) protein expression accompanied by increased trypsin activity with cerulein treatment was observed in susceptible strains such as CBA/J and C3H/HeJ. Similar results were obtained with a CDE diet. In the 3 kb Spink3 promoter region, 92 or 8 nucleotide changes were found in JF1 or C3H vs C57BL/6J, respectively, whereas in the Prss1 promoter region 39 or 46 nucleotide changes were found in JF1 or C3H vs C57BL/6J, respectively. These results suggest that regulation of Prss1 and Spink3 expression is involved in the susceptibility to experimentally induced pancreatitis. The JF1 strain, which is derived from the Japanese wild mouse, will be useful to examine new mechanisms that may not be found in other laboratory mouse strains.

Topics: Acute Disease; Animals; Base Sequence; Blotting, Northern; Blotting, Western; Ceruletide; Diet; Gene Expression; Genetic Predisposition to Disease; Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred CBA; Molecular Sequence Data; Pancreatitis; Prostatic Secretory Proteins; Sequence Homology, Nucleic Acid; Severity of Illness Index; Species Specificity; Trypsin; Trypsin Inhibitor, Kazal Pancreatic

We have previously shown that galantide ameliorates mild acute pancreatitis (AP), and the salivary tripeptide analogue, feG, ameliorates severe AP in mice. In this study, we compared the efficacy of combining galantide and feG with that of the individual agents in treating mild AP induced in mice with 7-hourly caerulein injections. Galantide was co-administered with each caerulein injection commencing with the first injection. feG was co-administered with the first injection of caerulein as a single intraperitoneal injection. Combination of the agents was also administered. Control animals received galantide, feG, or saline alone. Pancreata were harvested for histological examination and estimation of myeloperoxidase (MPO) activity. Plasma enzyme activities were measured. Galantide significantly reduced AP-induced hyperenzymemia by 41-49%. The combination of galantide and feG significantly reduced AP-induced hyperenzymemia by 39-40%, whereas feG alone was without effect. Plasma enzyme activity in the control groups was comparable with pre-treatment activity. Galantide, feG, and their combination significantly reduced MPO activity by 83, 44 and 74% respectively, and % abnormal acinar cells by 32, 29 and 36% respectively. This study demonstrates for the first time the beneficial effect of feG in mild caerulein-induced AP. Moreover the data indicate that the hyperenzymemia in mild caerulein-induced AP at 12h possibly reflect a larger secretory component as compared to enzyme release due to neutrophil-mediated acinar cell damage. The effects of the treatment with both peptides indicate a possible role for galantide in modulating neutrophil chemotaxis/activation and supports the hypothesis that galantide may influence neurogenic inflammation in AP.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Drug Therapy, Combination; Galanin; Lipase; Mice; Oligopeptides; Pancreas; Pancreatitis; Stereoisomerism; Substance P

Alcoholic chronic pancreatitis (ACP) is characterized by pancreatic necrosis, inflammation, and scarring, the latter of which is due to excessive collagen deposition by activated pancreatic stellate cells (PSC). The aim of this study was to establish a model of ACP in mice, a species that is usually resistant to the toxic effects of alcohol, and to identify the cell type(s) responsible for production of connective tissue growth factor (CTGF), a pro-fibrotic molecule. C57Bl/6 male mice received intraperitoneal ethanol injections for 3 weeks against a background of cerulein-induced acute pancreatitis. Peak blood alcohol levels remained consistently high in ethanol-treated mice as compared with control mice. In mice receiving ethanol plus cerulein, there was increased collagen deposition as compared with other treatment groups as well as increased frequency of alpha-smooth muscle actin and desmin-positive PSC, which also showed significantly enhanced CTGF protein production. Expression of mRNA for collagen alpha1(I), alpha-smooth muscle actin or CTGF were all increased and co-localized exclusively to activated PSC in ACP. Pancreatic expression of mRNA for key profibrotic markers were all increased in ACP. In conclusion, a mouse model of ACP has been developed that mimics key pathophysiological features of the disease in humans and which shows that activated PSC are the principal producers of collagen and CTGF. PSC-derived CTGF is thus a candidate therapeutic target in anti-fibrotic strategies for ACP.

Topics: Alcoholics; Alcoholism; Animals; Ceruletide; Collagen; Connective Tissue Growth Factor; Ethanol; Extracellular Matrix; Fibrosis; Male; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Pancreatitis, Alcoholic; Pancreatitis, Chronic; RNA, Messenger

Topics: Acute Disease; Animals; Ceruletide; Cytoplasm; Dyneins; Golgi Apparatus; Kinesins; Male; Pancreatitis; Rats; Rats, Wistar

To examine the pharmacological effect of etanercept and methylprednisolone (MP) on acute pancreatitis (AP) induced by cerulein in an experimental rat model.. The present study was carried out in the Experimental Research Center, Ondokuz Mayis University, Samsun, Turkey between December 2008 and October 2009. Forty adult Sprague-Dawley rats were divided into 5 groups (n=8): 1--sham, 2--cerulein induced pancreatitis (over 20 hours), 3--etanercept (5 mg/kg, intraperitoneal), 4--MP (10 mg/kg, intramuscular), 5--etanercept plus MP. The rats in groups 3, 4, and 5 were cerulein-induced pancreatitis at 20 hours, as well. After the treatment, the pancreas and blood were taken for histopathological and biochemical analysis.. All cerulein-treated rats developed biochemical and histopathological AP after 20 hours. Histological findings of pancreatitis and serum levels of amylase and lipase were lower in group 5 compared to group 2. Pancreatic inflammation and total pathological score were statistically reduced in the tissues of the pancreas at 20 hours after the treatment of etanercept plus MP in group 5 compared to groups 2, 3, and 4.. In the early stage of cerulein induced AP, the administration of etanercept plus MP attenuated pancreatic inflammation and significant damage in rats.

Topics: Animals; Anti-Inflammatory Agents; Ceruletide; Drug Therapy, Combination; Etanercept; Immunoglobulin G; Methylprednisolone; Pancreatitis; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha

To study the immunologic mechanism in pathogenesis of the acute pancreatitis (AP) and the intervention effects of sandostatin and magnolol.. Ninety BALB/c mice were divided into negative control group, caerulein-induced model group, sandostatin-treatment group, magnolol-treatment group, combined sandostatin- and magnolol-treatment group by means of random number table, with 18 mice in each group. AP model was reproduced by seven intraperitoneal injections of caerulein at an interval of 1 hour. Every 30 minutes before the caerulein challenge, sandostatin was injected sub- cutaneously. Magnolol was injected intravenously immediately after the AP model was reproduced. Then at 9, 12, 24 hours after modelling, blood was drawn from orbital vein and serum was separated. Serum amylase (SA), interleukin-10 (IL-10) and gamma-interferon (IFN-gamma) were determined after the mice were sacrificed, and pancreas and spleen were harvested . The pathological changes of pancreas were observed, and the number and the ratio of myeloid- dendritic cells (MDCs) to lymphoid dendritic cells (LDCs) were measured with flow cytometry.. Compared with control group [SA (1.12 + or - 0.05) kU/L, pancreatic score (PS) 0.09 + or - 0.10], both indexes increased progressively in the model group [SA (26.11 + or - 1.96) kU/L, PS 5.32 + or - 0.19, both P<0.01]. The ratio of MDCs/LDCs showed downward tendency at every time-point especially at 9th hour (0.421 + or - 0.049 vs. 1.712 + or - 0.372, P<0.05), while the ratio of IL-10 to IFN-gamma did not show significant differences. Compared with model group, both SA and PS significantly decreased in all the three drug intervention groups [SA (kU/L): 18.25 + or - 1.09, 17.32 + or - 1.26, 17.62 + or - 0.56, PS: 4.55 + or - 0.15, 4.16 + or - 0.18, 4.10 + or - 0.13, all P<0.01]. There was no significant difference in the two ratios of MDCs/LDCs and IL-10/IFN-gamma between sandostatin-treatment group and model group. However, the ratio of MDCs/LDCs of the magnolol-treatment group was higher than that in sandostatin-treatment group 9, 12, 24 hours after modelling (9 hours: 4.694 + or - 0.527 vs. 0.819 + or - 0.182, 12 hours: 2.566 + or - 0.463 vs. 1.421 + or - 0.163, 24 hours : 2.343 + or - 0.359 vs. 1.421 + or - 0.113, P<0.05 or P<0.01). At every time-point, the ratio of IL-10/IFN-gamma in the magnolol-treatment group was significantly higher compared with the model group, and at the 12-hour point, it was higher than that of sandostatin-treatment group (8.000 + or - 1.738 vs. 3.558 + or - 0.362, P<0.05 ). The combined treatment group showed similar changes as the magnolol-treatment group.. When AP occurs, the differentiation from T helper (Th0) to Th1 is augmented, while differentiation of Th0 to Th2 decreases, thus inducing an imbalance in the relationship of pro- and anti-inflammatory response. Magnolol can induce the differentiation from Th0 to Th2 by modulating the different subtype dendritic cells, thus improving the anti-inflammatory response, resulting in attenuating local and systemic inflammatory response in AP. However, sandostatin did not show such effect.

Topics: Amylases; Animals; Biphenyl Compounds; Ceruletide; Dendritic Cells; Disease Models, Animal; Interferon-gamma; Interleukin-10; Lignans; Mice; Mice, Inbred BALB C; Octreotide; Pancreas; Pancreatitis

Acute pancreatitis is a painful, inflammatory disorder for which adequate treatments are lacking. An early, critical step in its development is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. This Ca(2+) release is modulated by the intracellular Ca(2+) channel the ryanodine receptor (RYR). We have previously shown that RYR inhibition reduces pathological intra-acinar protease activation, an early marker of pancreatitis. In this study, we examined whether pretreatment with the RYR inhibitor dantrolene attenuates the severity of caerulein-induced pancreatitis in mice. Immunofluorescent labeling for RYR from mouse pancreatic sections showed localization to the basolateral region of the acinar cell. After 1 h of caerulein hyperstimulation in vivo, dantrolene 1) reduced pancreatic trypsin activity by 59% (P < 0.05) and 2) mitigated early ultrastructural derangements within the acinar cell. Eight hours after pancreatitis induction, dantrolene reduced pancreatic trypsin activity and serum amylase by 61 and 32%, respectively (P < 0.05). At this later time point, overall histological severity of pancreatitis was reduced by 63% with dantrolene pretreatment (P < 0.05). TUNEL-positive cells were reduced by 58% (P < 0.05). These data suggest that the RYR plays an important role in mediating early acinar cell events during in vivo pancreatitis and contributes to disease severity. Blockade of Ca(2+) signals and particularly RYR-Ca(2+) may be useful as prophylactic treatment for this disease in high-risk settings for pancreatitis.

Topics: Amylases; Animals; Apoptosis; Calcium Channel Blockers; Calcium Signaling; Ceruletide; Cytoprotection; Dantrolene; Disease Models, Animal; Enzyme Activation; Fluorescent Antibody Technique; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron; Pancreas; Pancreatitis; Ryanodine Receptor Calcium Release Channel; Severity of Illness Index; Time Factors; Trypsin

This study was designed to evaluate the protective effect of the cysteinyl leukotriene receptor antagonist montelukast against pancreatic injury during acute pancreatitis.. Acute pancreatitis was induced in rats by 20-μg/kg (intraperitoneal) cerulein given at 1-hour intervals within 4 hours. Montelukast was administered intraperitoneally at a dose of 10 mg/kg 15 minutes before the first cerulein injection. Six hours after the cerulein or saline injections, the animals were killed by decapitation. Blood samples were collected to analyze amylase, lipase, and the proinflammatory cytokines tumor necrosis factor α and interleukin 1β. Pancreas tissues were taken for the determination of tissue glutathione and malondialdehyde levels and Na,K-adenosine triphosphatase and myeloperoxidase activities. The extent of tissue injury was analyzed microscopically.. Acute pancreatitis caused significant decreases in tissue glutathione level and Na,K-adenosine triphosphatase activity, which were accompanied with significant increases in the pancreatic malondialdehyde level, myeloperoxidase activity, and plasma cytokine level. On the other hand, montelukast treatment reversed all these biochemical indices and histopathological alterations that were induced by cerulein.. These results suggest that cysteinyl leukotrienes may be involved in the pathogenesis of acute pancreatitis and that the cysteinyl leukotriene receptor antagonist, montelukast, might be of therapeutic value for treatment of acute pancreatitis.

Topics: Acetates; Acute Disease; Animals; Ceruletide; Cyclopropanes; Cytokines; Female; Glutathione; Leukotriene Antagonists; Leukotriene B4; Lipid Peroxidation; Male; Pancreas; Pancreatitis; Peroxidase; Quinolines; Rats; Rats, Sprague-Dawley; Receptors, Leukotriene; Sodium-Potassium-Exchanging ATPase; Sulfides

Reg proteins are normally expressed in pancreatic acinar cells, and the level of several of these proteins was significantly induced upon damage to the endocrine or exocrine pancreas. It has been established that Reg1 and pancreatic islet neogenesis-associated protein [INGAP, Reg3delta] promote the growth or regeneration of the endocrine islet cells. Recent reports suggest that Reg2 is an autoantigen normally expressed in islet beta-cells. Reg2 overexpression in vitro offered protection to insulinoma cells. Overexpressed Reg3alpha increased cyclin D1 and CDK4 levels and the rate of proliferation in insulinoma cells. Acinar-specific overexpression of INGAP increased beta-cell mass and protected the animals from streptozotocin-induced diabetes. Moreover, Reg2 gene expression was induced during pancreatitis. We hypothesized that Reg2 is a secreted protein that promotes the growth, survival, and/or regeneration of pancreatic endocrine and exocrine cells. To test its effectiveness, we used elastase-1 promoter (Ela-Reg2) to develop an acinar cell-specific overexpression of the Reg2 gene. Western blot analysis, real-time PCR, and immunohistochemistry revealed barely detectable levels of endogenous Reg2 in the pancreas of normal wild-type mice and increased Reg2 levels in the pancreas of Ela-Reg2 mice that were similar to or higher than Reg2 levels induced in experimental diabetes or pancreatitis. Compared with wild-type littermates, growth, blood glucose and insulin levels, and glucose tolerance were normal in Ela-Reg2 mice; pancreatic histology revealed no change in endocrine or exocrine tissues. Acinar-specific overexpression of the Reg2 gene offered no protection against streptozotocin-induced beta-cell damage and diabetes, in hyperglycemia and weight loss, and no advantage in restoring glucose homeostasis and islet function within 3 mo. Furthermore, serum amylase level and pancreatic histochemistry showed that Reg2 overexpression did not protect acinar cells against caerulein-induced acute pancreatitis. In contrast to INGAP or Reg3beta, exocrine overexpression of Reg2 offered no protection to the endocrine or exocrine pancreas, indicating clear subtype specificities of the Reg family of proteins.

Topics: Animals; Ceruletide; Cyclin D1; Diabetes Mellitus, Experimental; Glucose; Homeostasis; Islets of Langerhans; Mice; Mice, Transgenic; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Proteins; Proto-Oncogene Proteins c-akt; Up-Regulation

Previously, we found that the University of North Carolina cystic fibrosis (UNC-CF) mouse had more severe experimental acute pancreatitis (AP) than wild-type (WT) mice characterized by exuberant pancreatic inflammation and impaired acinar apoptosis. Because exon 10 CFTR gene mutations exhibit different phenotypes in tissues such as the mouse lung, we tested the hypothesis that DeltaF508-CF mice also develop severe AP associated with an antiapoptotic acinar phenotype, which requires indirect effects of the extracellular milieu. We used cerulein hyperstimulation models of AP. More severe pancreatitis occurred in cerulein-injected DeltaF508-CF vs. WT mice based on histological severity (P < 0.01) and greater neutrophil sequestration [P < 0.0001; confirmed by myeloperoxidase activity (P < 0.005)]. In dispersed acini cerulein-evoked necrosis was greater in DeltaF508-CF acini compared with WT (P < 0.05) and in WT acini pretreated with CFTR(inh)-172 compared with vehicle (P < 0.05). Cerulein-injected DeltaF508-CF vs. WT mice had less apoptosis based on poly(ADP-ribose) polymerase (PARP) cleavage (P < 0.005), absent DNA laddering, and reduced terminal deoxynucleotidyltransferase biotin-dUTP nick end labeling (TUNEL) staining (P < 0.005). Unexpectedly, caspase-3 activation was greater in DeltaF508-CF vs. WT acini at baseline (P < 0.05) and during AP (P < 0.0001). Downstream, DeltaF508-CF pancreas overexpressed the X-linked inhibitor of apoptosis compared with WT (P < 0.005). In summary, the DeltaF508-CF mutation, similar to the UNC-CF "null" mutation, causes severe AP characterized by an exuberant inflammatory response and impaired acinar apoptosis. Enhanced acinar necrosis in DeltaF508-CF occurs independently of extracellular milieu and correlates with loss of CFTR-Cl conductance. Although both exon 10 models of CF inhibit acinar apoptosis execution, the DeltaF508-CF mouse differs by increasing apoptosis signaling. Impaired transduction of increased apoptosis signaling in DeltaF508-CF acini may be biologically relevant to the pathogenesis of AP associated with CFTR mutations.

Topics: Acute Disease; Animals; Apoptosis; Caspase 3; Ceruletide; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Activation; Genotype; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation; Necrosis; Pancreas; Pancreatitis; Peroxidase; RNA, Messenger; Severity of Illness Index; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

Acute pancreatitis is an inflammatory disorder of the pancreas. Protein kinase C (PKC) δ plays an important role in mediating chemokine production in mouse pancreatic acinar cells. This study aims to investigate the role of PKC δ in the pathogenesis of acute pancreatitis and to explore the mechanisms through which PKC δ mediates pro-inflammatory signaling. Acute pancreatitis was induced in mice by ten hourly intraperitoneal injections of caerulein. PKC δ translocation inhibitor peptide (δV1-1) at a dose of 1.0 mg/kg or Tat (carrier peptide) at a dose of 1.0 mg/kg was administered to mice either 1 h before or 1 h after the first caerulein injection. One hour after the last caerulein injection, the mice were killed and pancreas, lungs, and blood were collected. Prophylactic and therapeutic treatment with δV1-1 attenuated caerulein-induced plasma amylase levels and pancreatic edema. Treatment with δV1-1 decreased myeloperoxidase activity and monocyte chemotactic protein-1 levels in both pancreas and plasma. PKC δ mediated acute pancreatitis by activating pancreatic nuclear factor κB, activator protein-1, and mitogen-activated protein kinases. Moreover, blockade of PKC δ attenuated lung myeloperoxidase activity and edema. Histological examination of pancreatic and lung sections confirmed protection against acute pancreatitis. Treatment with Tat had no protective effect on acute pancreatitis. Blockade of PKC δ represents a promising prophylactic and/or therapeutic tool for the treatment of acute pancreatitis.

Topics: Amylases; Animals; Ceruletide; Chemokines; Disease Models, Animal; Humans; Ice; Inflammation; Lung Injury; Male; Mice; Mitogen-Activated Protein Kinases; Neutrophils; NF-kappa B; Pancreatitis; Peroxidase; Protein Kinase C-delta; Signal Transduction; Transcription Factor AP-1

We used a peptidomic approach for the analysis of the low molecular weight proteome in rat pancreatic tissue extracts. The goal was to develop a method that allows identifying endogenous peptides produced in the pancreas in the course of acute pancreatitis. The workflow combines peptides enrichment by centrifugal ultrafiltration, fractionation by isoelectric focusing, and LC-MS/MS analysis without prior enzymatic digestion. The method was assessed on pancreatic extracts from 3 rats with caerulein-induced pancreatitis and 3 healthy controls. A qualitative analysis of the peptide patterns obtained from the different samples was performed to determine the main biological processes associated to the identified peptides. Comparison of peptidomic and immunoblot data for alpha-tubulin, beta-tubulin and coatomer gamma showed that the correlation between the number of identified peptides and the protein abundance was variable. Nevertheless, peptidomic analysis highlighted inflammatory and stress proteins, which peptide pattern was related to acute pancreatitis pathobiology. For these proteins, the higher number of peptides in pancreatitis samples reflected an increase in protein abundance. Moreover, for murinoglobulin-1 or carboxypeptidase B, peptide pattern could be related to protein function. These data suggest that peptidomic analysis is a complementary approach to proteomics for investigating pathobiological processes involved in acute pancreatitis.

Topics: Acute Disease; Amino Acid Sequence; Animals; Ceruletide; Chromatography, Liquid; Disease Models, Animal; Heat-Shock Proteins; Immunoblotting; Inflammation; Male; Molecular Sequence Data; Molecular Weight; Pancreatitis; Peptides; Proteins; Proteome; Proteomics; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry

Cannabinoids (CBs) evoke their effects by activating the cannabinoid receptor subtypes CB1-r and CB2-r and exert anti-inflammatory effects altering chemokine and cytokine expression. Various cytokines and chemokines are produced and released by rodent pancreatic acini in acute pancreatitis. Although CB1-r and CB2-r expressed in rat exocrine pancreatic acinar cells do not modulate digestive enzyme release, whether they modulate inflammatory mediators remains unclear. We investigated the CB-r system role on exocrine pancreas in unstimulated conditions and during acute pancreatitis.. We evaluated in vitro and in vivo changes induced by WIN55,212 on the inflammatory variables amylasemia, pancreatic edema and morphology, and on acinar release and content of the cytokine interleukin-6 (IL-6) and chemokine monocyte chemo-attractant protein-1 (MCP-1) in untreated rats and rats with caerulein (CK)-induced pancreatitis.. In the in vitro experiments, WIN55,212 (10(-6) mol L(-1)) inhibited IL-6 and MCP-1 release from acinar cells of unstimulated rats and after CK-induced pancreatitis. In vivo, when rats were pretreated with WIN55,212 (2 mg kg(-1), intraperitoneally) before experimentally-induced pancreatitis, serum amylase, pancreatic edema and IL-6 and MCP-1 acinar content diminished and pancreatic morphology improved. Conversely, when rats with experimentally-induced pancreatitis were post-treated with WIN55,212, pancreatitis worsened.. These findings provide new evidence showing that the pancreatic CB1-r/CB2-r system modulates pro-inflammatory factor levels in rat exocrine pancreatic acinar cells. The dual, time-dependent WIN55,212-induced changes in the development and course of acute pancreatitis support the idea that the role of the endogenous CB receptor system differs according to the local inflammatory status.

Topics: Amylases; Animals; Benzoxazines; Body Water; Cannabinoids; Ceruletide; Chemokine CCL2; Edema; Enzyme-Linked Immunosorbent Assay; Gastrointestinal Agents; Interleukin-6; Male; Morpholines; Naphthalenes; Pancreas; Pancreatitis; Quinolines; Rats

We have previously shown that galantide, a non-specific galanin receptor antagonist, ameliorates acute pancreatitis (AP) induced in mice. Octreotide, a somatostatin analogue, has been used in the treatment of AP with inconsistent outcomes. This study set out to compare the efficacy of a combined treatment of galantide and octreotide with the efficacy of each agent individually in experimental AP.. Acute pancreatitis was induced in mice with 7-hourly caerulein injections. Galantide and/or octreotide were co-administered with each caerulein injection commencing with the first injection. Control animals received galantide, octreotide or saline alone. Pancreata were harvested for histological examination and estimation of myeloperoxidase (MPO) activity. Plasma amylase and lipase activities were measured.. Galantide significantly reduced AP-induced hyperenzymaemia by 39-45%. Octreotide alone, or in combination with galantide, did not significantly alter AP-induced hyperenzymaemia. Plasma enzyme activity in the control groups was comparable with pre-treatment activity. Galantide and octreotide administered individually reduced MPO activity by 79% and 50%, respectively; however their combination was without effect. Galantide, octreotide and their combination significantly reduced the percentage of abnormal acinar cells by 28-45%.. Treatment with galantide alone ameliorated most of the indices of AP studied, whereas treatment with octreotide reduced pancreatic MPO activity and acinar cell damage. Combining the two peptides appears to negate their individual benefits, which suggests an interaction in their mechanism of action.

Topics: Acute Disease; Amylases; Animals; Biomarkers; Ceruletide; Disease Models, Animal; Drug Therapy, Combination; Galanin; Lipase; Male; Mice; Octreotide; Pancreas; Pancreatitis; Peroxidase; Substance P; Time Factors

Recent studies have shown that pretreatment with ghrelin exhibits protective effect in the gut. Administration of ghrelin reduces gastric mucosal damage, as well as inhibits the development of experimental pancreatitis. However, this protective effect requires administration of ghrelin before gastric or pancreatic damage and thus has a limited clinical value. The aim of present study was to assess the influence of ghrelin administered after development of acute pancreatitis on the course of this disease. Acute pancreatitis was induced by cerulein. Ghrelin was administered twice a day for 1, 2, 4, 6 or 9 days at the dose of 4, 8 or 16 nmol/kg/dose. The first dose of ghrelin was given 24 hours after last injection of cerulein. The severity of acute pancreatitis was assessed between 0 h and 10 days after cessation of cerulein administration. Administration of caerulein led to the development of acute edematous pancreatitis and maximal severity of this disease was observed 24 hours after induction of pancreatitis. Treatment with ghrelin reduced morphological signs of pancreatic damage such as pancreatic edema, leukocyte infiltration and vacuolization of acinar cells, and led to earlier regeneration of the pancreas. Also biochemical indexes of the severity of acute pancreatitis, serum activity of lipase and amylase were significantly reduced in animals treated with ghrelin. These effects were accompanied by an increase in the pancreatic DNA synthesis and a decrease in serum level of pro-inflammatory interleukin-1b. Administration of ghrelin improved pancreatic blood flow in rats with acute pancreatitis. We conclude that: (1) treatment with ghrelin exhibits therapeutic effect in caerulein-induced experimental acute pancreatitis; (2) this effect is related, at least in part, to the improvement of pancreatic blood flow, reduction in proinflammatory interleukin-1beta and stimulation of pancreatic cell proliferation.

Topics: Acute Disease; Animals; Blood Flow Velocity; Blood Glucose; Cell Proliferation; Ceruletide; Dose-Response Relationship, Drug; Down-Regulation; Ghrelin; Inflammation Mediators; Insulin; Interleukin-1beta; Male; Organ Size; Pancreas; Pancreatitis; Rats; Rats, Wistar; Up-Regulation

Acute pancreatitis is an inflammatory disease of the pancreas, which can result in serious morbidity or death. Acute pancreatitis severity can be reduced in experimental models by preconditioning animals with a short hyperthermia prior to disease induction. Heat shock proteins 27 and 70 are key effectors of this protective effect. In this study, we performed a comparative proteomic analysis using a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and isobaric tagging to investigate changes in pancreatic proteins expression that were associated with thermal stress, both in healthy rats and in a model of caerulein-induced pancreatitis. In agreement with previous studies, we observed modulation of heat shock and inflammatory proteins expression in response to heat stress or pancreatitis induction. We also identified numerous other proteins, whose pancreatic level changed following pancreatitis induction, when acute pancreatitis severity was reduced by prior thermal stress, or in healthy rats in response to hyperthermia. Interestingly, we showed that the expression of various proteins associated with the secretory pathway was modified in the different experimental models, suggesting that modulation of this process is involved in the protective effect against pancreatic tissue damage.

Topics: Acute Disease; Animals; Ceruletide; Fever; Heat-Shock Response; Pancreatitis; Protective Agents; Proteomics; Rats

Our aim in this study was to investigate the changes of inflammatory response by protein inhibitor of activated signal transducer and activator of transcription 1 (PIAS1) gene silencing treatment in cerulein-stimulated AR42J cells, and relate them to changes in cell injury, thus providing evidence for developing clinical therapies. This study examined the effects of cerulein on the activity of P38 mitogen activated protein kinase (P38MAPK), c-jun NH2-terminal kinase/stress-activated protein kinase and the inflammatory mediators released by PIAS1 gene-silenced AR42J cells. Consequently, the markers including DNA ladder, cell apoptotic rat, cell cycles, levels of cell cycle and apoptotic related factors were used to determine the effects of PIAS1 gene silencing on the cerulein-induced cell injury. The results indicated that in the cerulein-stimulated PIASI silencing cells, the activity of P38MAPK was enhanced, while at the same time, the levels of inflammatory mediators such as the tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and matrix metallopeptidase-9, were markedly higher than those of other cerulein-stimulated cells. Thus, the cerulein-stimulated PIASI gene-silenced cells obviously increased cell arrest in the G1/M phase by increasing P21 and P27 expression, and also induced apoptosis by regulating the P53 signaling pathway. This study suggests that the down-regulation of PIAS1 is efficacious at enhancing the expression of inflammatory mediators and inducing cell injury in acute pancreatitis (AP), thus deteriorating the severity of disease. It provides evidence that PIAS1 is a potential therapeutic target for AP.

Topics: Animals; Apoptosis; Cell Line; Ceruletide; Gene Silencing; Inflammation; Interleukin-6; JNK Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Pancreas; Pancreatitis; Protein Inhibitors of Activated STAT; Rats; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor-alpha

Many plant-derived flavonoids including quercetin exhibit antioxidant and antiinflammatory properties. Proinflammatory cytokines and oxidative stress play an important role in acute pancreatitis. This study aimed to evaluate the effect of quercetin on cerulein-induced acute pancreatitis in mice. Animal groups were pretreated with quercetin (25, 50, 100 mg/kg, per os (p.o.)), thalidomide (200 mg/kg, p.o.) or vehicle (2% dimethyl sulfoxide (DMSO)) 1 h before hourly (x5) intraperitoneal injections of cerulein. A saline (0.9%, NaCl)-treated control group was included for comparison. Cerulein significantly enhanced the serum levels of amylase and lipase, and pancreatic myeloperoxidase activities, malondialdehyde and the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6, as well as the pancreatic wet weight/body weight ratio. Cerulein significantly reduced the serum levels of IL-10. Histological assessment of the pancreas showed tissue edema, neutrophil infiltration, acinar vacuolization, and cell necrosis and a marked increase in the immunoreactivity staining for TNF-alpha. Pretreatment with quercetin or thalidomide significantly attenuated the severity of cerulein-induced acute pancreatitis as evidenced by effective reductions in the pancreatic wet weight/body weight ratio, biochemical indices, proinflammatory cytokines, myeloperoxidase activity, malondialdehyde formation, and an increase in antiinflammatory cytokine IL-10. Quercetin treatment also markedly suppressed the histological changes such as pancreatic edema, inflammatory cell infiltration, acinar cell necrosis, and the expression of TNF-alpha. Taken together, these results indicate that quercetin ameliorates the severity of cerulein-induced acute pancreatitis by acting as an antiinflammatory and antioxidant agent.

Topics: Animals; Ceruletide; Dose-Response Relationship, Drug; Flavonoids; Male; Mice; Pancreatitis; Quercetin

Oxidative stress with free radicals plays a crucial role in acute pancreatitis (AP). Pantoprazole (PPZ), widely used as a proton pump inhibitor, possesses reactivity towards hydroxyl radicals. The aim of the study was to examine the effect of PPZ on the course of experimental AP.. Mild AP was induced in rats by caerulein (n=12). Severe AP was induced by infusion of glycodeoxycholic acid (10mM) into the pancreatic duct combined with caerulein (n=12). Both AP models were randomized to PPZ treatment (20mg/kg at baseline and after 12h) or placebo. Control animals received Ringer solution (n=6) without AP induction. After 24h severity of AP was examined by histology, enzyme levels, edema and inflammatory markers (myeloperoxidase, protein profiling). Furthermore, CD62P and CD31 for leukocyte and platelet activation were investigated.. Histology showed that PPZ treatment reduced tissue infiltration of inflammatory cells and acinar cell necrosis in severe AP. After PPZ treatment CD62P expression in mild AP and CD31 expression in severe pancreatitis decreased, indicating an inhibition of platelet activation. In mild and severe AP, PPZ significantly decreased amylase, LDH, edema and myeloperoxidase activity. Protein profile of pancreatic juice and serum revealed different spectra and less pancreatic juice proteins in PPZ treated groups indicating less acinar cell leakage.. PPZ possesses anti-inflammatory in vivo properties and attenuates the course of AP. This is mediated via a reduced expression of inflammatory and adhesive proteins with a consecutive decrease in platelet and leukocyte activation as key steps in the pathogenesis of AP.

Topics: 2-Pyridinylmethylsulfinylbenzimidazoles; Animals; Cell Movement; Ceruletide; H(+)-K(+)-Exchanging ATPase; Inflammation Mediators; Male; Oxidative Stress; Pancreatitis; Pantoprazole; Proton Pump Inhibitors; Rats; Rats, Wistar

The aim of this study was to determine the effects of mycophenolate mofetil (MMF) on acute pancreatitis with evaluation of biochemical and histopathological findings.. Cerulein was administered to induce acute pancreatitis in rats. Three groups of 10 rats each were formed. Animals in group 1 received physiologic saline solution. In group 2 animals received MMF at a dose of 14 mg/kg and group 3 had double doses of MMF. Alanine aminostransferase, aspartate aminotransferase (AST), amylase and bilirubin levels were assessed. Pancreatic tissues were evaluated under light microscopy for the presence of edema, acinar necrosis, hemorrhage, inflammation and perivascular infiltration.. Amylase, serum AST, edema and inflammatory infiltration levels differed between groups (amylase: p = 0.0001, serum AST: p = 0.001, edema: p = 0.0001 and inflammatory infiltration: p = 0.002), group 1 showing the highest amylase, serum AST and edema levels. The lowest levels of edema were found in group 3.. In an experimental pancreatitis model in rats, MMF proved to exert a beneficial effect on biochemical and histopathological parameters.

Topics: Alanine Transaminase; Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartate Aminotransferases; Bilirubin; Ceruletide; Female; Immunosuppressive Agents; Male; Mycophenolic Acid; Pancreatitis; Rats; Rats, Wistar

Proteinase-activated receptor-2 (PAR2) and transient receptor potential vanilloid-1 (TRPV1) are co-localized in the primary afferents, and the trans-activation of TRPV1 by PAR2 activation is involved in processing of somatic pain. Given evidence for contribution of PAR2 to pancreatic pain, the present study aimed at clarifying the involvement of TRPV1 in processing of pancreatic pain by the proteinase/PAR2 pathway in mice.. Acute pancreatitis was created by repeated administration of cerulein in conscious mice, and the referred allodynia/hyperalgesia was assessed using von Frey filaments. Injection of PAR2 agonists into the pancreatic duct was achieved in anesthetized mice, and expression of Fos in the spinal cord was determined by immunohistochemistry.. The established referred allodynia/hyperalgesia following cerulein treatment was abolished by post-treatment with nafamostat mesilate, a proteinase inhibitor, and with capsazepine, a TRPV1 antagonist, in mice. Injection of trypsin, an endogenous PAR2 agonist, or SLIGRL-NH(2), a PAR2-activating peptide, into the pancreatic duct caused expression of Fos protein in the spinal superficial layers at T8-T10 levels in the mice. The spinal Fos expression caused by trypsin and by SLIGRL-NH(2) was partially blocked by capsazepine, the former effect abolished by nafamostat mesilate.. Our data thus suggest that the proteinase/PAR2/TRPV1 cascade might impact pancreatic pain, in addition to somatic pain, and play a role in the maintenance of pancreatitis-related pain in mice.

Topics: Acute Disease; Animals; Benzamidines; Capsaicin; Ceruletide; Disease Models, Animal; Gene Expression Regulation; Guanidines; Hyperalgesia; Male; Mice; Oligopeptides; Pain; Pancreatitis; Proto-Oncogene Proteins c-fos; Receptor, PAR-2; Spinal Cord; TRPV Cation Channels

The endoplasmic reticulum (ER) is abundant in the acinar cells of the exocrine pancreas. To test the role of ER homeostasis in acute pancreatitis, we manipulated GRP78 levels, a major ER chaperone, in mice. Grp78(+/+) and (+/-) littermates were fed either a regular diet (RD) or a high-fat diet. Acinar cells were examined for ER structure by electron microscopy, and ER chaperone levels were assessed by immunoblotting. Pancreatitis was induced by cerulein injection, and multiple pathological parameters were analyzed. Grp78(+/-) mice showed decreased GRP78 expression in acinar cells. Exocrine pancreata of RD-fed Grp78(+/-) mice in an outbred C57BL/6 × 129/sv genetic background exhibited ER lumen dilation, a reduction in chaperones calnexin (CNX) and calreticulin (CRT), and exacerbated pancreatitis associated with high CHOP induction. With the high-fat diet regimen, Grp78 heterozygosity triggered GRP94 up-regulation and restoration of GRP78, CNX, and CRT to wild-type levels, corresponding with mitigated pancreatitis on cerulein insult. Interestingly, after backcrossing into the C57BL/6 background, RD-fed Grp78(+/-) mice exhibited an increase in GRP94 and levels of CNX and CRT equivalent to wild type, associated with decreased experimental pancreatitis severity. Administration of a chemical chaperone, 4-phenolbutyrate, was protective against cerulein-induced death. Thus, in exocrine pancreata, Grp78 heterozygosity regulates ER chaperone balance, in dietary- and genetic background-dependent manners, and improved ER protein folding capacity might be protective against pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Dietary Fats; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gastrointestinal Agents; Heat-Shock Proteins; Heterozygote; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Chaperones; Pancreas, Exocrine; Pancreatitis; Stress, Physiological; Unfolded Protein Response

To investigate the origin and localization of pancreatic stem cells in adult pancreatic tissues and to determine the primary mechanism underlying the participation of these cells in repairing pancreatic injuries.. Sprague-Dawley rats were divided into experimental and control groups. The experimental group was given intraperitoneal injections of cerulein to induce acute pancreatitis. At 6 h, 1, 2, 3, 5 and 7 days, 5 rats from the experimental group and 2 rats from the control group were sacrificed; all sacrificed animals were intraperitoneally injected with 5-bromo-2'-deoxyuracil nucleotides (BrdU) 6 and 3 h prior to sacrifice. The pathological changes of pancreatic tissue were observed. The stem cell marker nestin and the cell proliferation marker BrdU were detected with immunohistochemistry. Pancreatic duodenal homeobox-1 (PDX-1) was determined by real-time PCR.. (1) The pathological changes of acute pancreatitis can be divided into three phases: the edema and apoptosis phase, the hemorrhagic necrosis phase, and the reconstruction phase. (2) Nestin-positive cells mainly appeared in the interlobular vascular lumen after cerulein injection, and they peaked at day 3 when the positive cells spread all over the pancreatic tissues. (3) BrdU-positive cells began to appear in the area surrounding the interlobular region, and the number of positive cells peaked on day 7. (4) The expression of PDX-1 mRNA initially increased, then decreased and gradually got close to a normal level.. Primary pancreatic stem cells may not exist in the adult pancreatic tissues. The so-called pancreatic stem cells may actually originate from bone marrow stem cells. When pancreatic tissue is injured, bone marrow stem cells may participate in the repair.

Topics: Adult Stem Cells; Animals; Bone Marrow Cells; Bromodeoxyuridine; Ceruletide; Homeodomain Proteins; Intermediate Filament Proteins; Nerve Tissue Proteins; Nestin; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Stem Cells; Trans-Activators

The lysosomal protease cathepsin B is thought to play a crucial role in the intracellular activation cascade of digestive proteases and in the initiation of acute pancreatitis. Although cathepsin B has been shown to be physiologically present in the secretory pathway of pancreatic acinar cells it has been suggested that premature activation of zymogens requires an additional redistribution of cathepsin B into the secretory compartment. Here, we studied the role of cathepsin B targeting during caerulein-induced pancreatitis in mouse mutants lacking the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) which normally mediates the trafficking of cathepsin B to lysosomes. Absence of the CI-MPR led to redistribution of cathepsin B to the zymogen granule enriched subcellular fraction and to a substantial formation of large cytoplasmic vacuoles that contained both, trypsinogen and cathepsin B. However, this did not cause premature intracellular trypsin activation in saline-treated control animals lacking the CI-MPR. During caerulein-induced pancreatitis, trypsinogen activation in the pancreas of CI-MPR-deficient animals was about 40% higher than in wild-type animals but serum amylase levels were reduced and lung damage was unchanged. These data suggest that subcellular redistribution of cathepsin B, in itself, induces neither spontaneous trypsinogen activation nor pancreatitis. Furthermore, we clearly show that a marked increase in intracellular trypsinogen activation is not necessarily associated with greater disease severity.

Topics: Amylases; Animals; Cathepsin B; Ceruletide; Disease Progression; Insulin-Like Growth Factor II; Lysosomes; Mice; Mice, Knockout; Pancreas; Pancreatitis; Peptide Hydrolases; Receptor, IGF Type 2; Secretory Vesicles; Trypsin; Trypsinogen; Vacuoles

Ghrelin is a ligand for growth hormone secretagogue receptor and stimulates release of growth hormone (GH). Recent studies have shown that treatment with ghrelin exhibits protective and therapeutic effect in the course of experimental pancreatitis. The aim of present study was to examine the role of GH and insulin-like growth factor-1 (IGF-1) in these effects. Acute pancreatitis was induced by cerulein. Study was performed on pituitary-intact hypophysectomized rats. Ghrelin was administered twice a day at the dose of 8 nmol/kg/dose. IGF-1 was given twice a day at the dose of 20 nmol/kg/dose. The severity of acute pancreatitis was assessed 0 h or 1, 2, 3, 5 and 10 days after the last dose of cerulein. Administration of cerulein led to the development of acute edematous pancreatitis. In pituitary-intact rats, treatment with ghrelin reduced biochemical indexes of the severity of acute pancreatitis and morphological signs of pancreatic damage, leading to faster regeneration of the pancreas reduction in serum concentration of pro-inflammatory interleukin-1β and decrease in serum activity of amylase and lipase. These effects were accompanied with an improvement of pancreatic blood flow and an increase in pancreatic DNA synthesis. Hypophysectomy delayed the healing of the pancreas and abolished the therapeutic effect of ghrelin. In hypophysectomized rats with pancreatitis, treatment with IGF-1 exhibits therapeutic effect similar to that observed in ghrelin-treated rats with the intact pituitary. We conclude that therapeutic effect of ghrelin in cerulein-induced pancreatitis is indirect and depends on the release of GH and IGF-1.

Topics: Amylases; Animals; Ceruletide; Disease Progression; Ghrelin; Growth Hormone; Hypophysectomy; Insulin-Like Growth Factor I; Interleukin-1beta; Lipase; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Receptors, Ghrelin

To investigate chronic stress as a susceptibility factor for developing pancreatitis, as well as tumor necrosis factor-α (TNF-α) as a putative sensitizer.. Rat pancreatic acini were used to analyze the influence of TNF-α on submaximal (50 pmol/L) cholecystokinin (CCK) stimulation. Chronic restraint (4 h every day for 21 d) was used to evaluate the effects of submaximal (0.2 μg/kg per hour) cerulein stimulation on chronically stressed rats.. In vitro exposure of pancreatic acini to TNF-α disorganized the actin cytoskeleton. This was further increased by TNF-α/CCK treatment, which additionally reduced amylase secretion, and increased trypsin and nuclear factor-κB activities in a protein-kinase-C δ and ε-dependent manner. TNF-α/CCK also enhanced caspases' activity and lactate dehydrogenase release, induced ATP loss, and augmented the ADP/ATP ratio. In vivo, rats under chronic restraint exhibited elevated serum and pancreatic TNF-α levels. Serum, pancreatic, and lung inflammatory parameters, as well as caspases'activity in pancreatic and lung tissue, were substantially enhanced in stressed/cerulein-treated rats, which also experienced tissues' ATP loss and greater ADP/ATP ratios. Histological examination revealed that stressed/cerulein-treated animals developed abundant pancreatic and lung edema, hemorrhage and leukocyte infiltrate, and pancreatic necrosis. Pancreatitis severity was greatly decreased by treating animals with an anti-TNF-α-antibody, which diminished all inflammatory parameters, histopathological scores, and apoptotic/necrotic markers in stressed/cerulein-treated rats.. In rats, chronic stress increases susceptibility for developing pancreatitis, which involves TNF-α sensitization of pancreatic acinar cells to undergo injury by physiological cerulein stimulation.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Amylases; Animals; Antibodies; Calcium Signaling; Caspases; Ceruletide; Cholecystokinin; Chronic Disease; Cytoskeleton; Disease Models, Animal; Enzyme Activation; Lung Injury; Male; Necrosis; NF-kappa B; Pancreas, Exocrine; Pancreatitis; Protein Kinase C-delta; Protein Kinase C-epsilon; Protein Transport; Rats; Rats, Wistar; Restraint, Physical; Severity of Illness Index; Stress, Psychological; Tissue Culture Techniques; Trypsin; Tumor Necrosis Factor-alpha

Toll-like receptor 4 (TLR4) plays an important role in the occurrence and development of acute pancreatitis (AP). Apoptosis of pancreatic cells is closely related to the severity of AP. TLR4 is known to induce apoptosis in some cell types and therefore it is of importance to investigate potential associations between TLR4 activity and apoptosis in the setting of AP.. A total of 50 wild-type (C57BL/10J) and TLR4-deficient (C57BL/10ScNJ) mice were divided into three groups: 2-hour, 4-hour, and control groups. Each group was divided into two equal subgroups: TLR4-wild-type mice and TLR4-deficient mice. AP was experimentally induced by 7 intraperitoneal injections of 50 μg/kg cerulein at hourly intervals. Control mice received 7 injections of equal volumes of saline. The severity of pancreatic injury during AP was assessed by serum amylase concentration and histopathology. The level of apoptosis of pancreatic cells in response to AP was evaluated by calculating the apoptotic index (AI) and comparing the expression levels of cytochrome C and Fas-associated protein with death domain (FADD) between TLR4-wild-type and TLR4-deficient mice at 2 time points.. The AI was found to be significantly lower in the pancreas of TLR4-deficient mice with AP compared to TLR4-wild-type mice at two hours after the last treatment injection. Enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction also revealed significantly lower expression of cytochrome C and FADD in the pancreas of TLR4-deficient mice than in TLR4-wild-type animals at the same time point. Serum amylase concentration and morphological severity of AP in pancreatic tissue were found to be similar in the two strains of mice at both time points.. We postulate that TLR4 can mediate apoptosis of pancreatic cells during the early stages of AP, via the activation of both intrinsic and extrinsic apoptotic signaling pathways.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Ceruletide; Fas-Associated Death Domain Protein; Female; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Pancreas; Pancreatitis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Toll-Like Receptor 4

In this study we aimed to investigate the effect of pentoxifylline on caerulein-induced acute pancreatitis (AP) by detecting oxidative stress markers and performing histopathological examination. Twenty-one adult female Sprague-Dawley rats were divided into three groups as follows: control, caerulein, and caerulein + pentoxifylline groups. Pancreatic tissues of rats from all groups were removed for light and electron microscopic examination and determination of oxidative stress markers. Pancreatic oxidative stress markers were evaluated by the measurements of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and total glutathione (GSH). Serum amylase and lipase levels were determined spectrophotometrically. The pancreatic damage score was significantly increased (P < 0.005) in the caerulein group, whereas it was decreased (P < 0.05) in the caerulein+ with pentoxifylline group. MDA levels, CAT, SOD, GPx, and GSH activities were significantly altered (P < 0.05, P < 0.005) in the caerulein group and indicated increased oxidative stress. Oxidative stress markers were normalized with pentoxifylline administration. Caerulein administration resulted in significant increase (P < 0.05) in amylase and lipase levels; pentoxifylline reduced the levels of these enzymes. Pentoxifylline is potentially capable of limiting pancreatic damage produced during AP by restoring the fine structure of acinar cells and tissue antioxidant enzyme activities. We concluded that pentoxifylline may have beneficial effects in the treatment of caerulein-induced AP.

Topics: Animals; Ceruletide; Female; Oxidative Stress; Pancreas; Pancreatitis; Pentoxifylline; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley

Platelet-activating factor (PAF) is an important mediator of inflammation and postulated to be involved in the pathogenesis of acute pancreatitis. In this study, we evaluated the therapeutic effect of PAF antagonist WEB 2086 in acute experimental pancreatitis of graded severity in rats.. According to a block design, 64 animals were randomly allocated to 8 groups. Severe necrotizing pancreatitis was induced by intraductal infusion of taurocholic acid (4%, 0.4 mL), and the combination of glycodeoxycholic acid (10 mmol/L, 1.0 mL/kg, intraductal infusion) and cerulein (5 microg/kg per hour, intravenous) was applied to induce intermediate pancreatitis, or cerulein alone (5 microg/kg per hour, intravenous) to establish edematous pancreatitis. WEB 2086 was given 15 minutes after beginning the induction of pancreatitis. Pancreatic microcirculation was analyzed in vivo with an epiluminescent microscope. Histopathology was evaluated by a validated score. Trypsinogen-activating peptide and serum amylase were analyzed sequentially.. WEB 2086 had no significant influence on the breakdown of microcirculation, leukocyte adherence, histopathological damage, and amylase levels in severe necrotizing pancreatitis, intermediate pancreatitis, and edematous pancreatitis. Only in intermediate pancreatitis was there a significant reduction of trypsinogen-activating peptide levels.. In our study, PAF antagonist WEB 2086 had no beneficial effect on microcirculation in acute experimental pancreatitis.

Topics: Amylases; Animals; Azepines; Capillaries; Cell Adhesion; Ceruletide; Disease Models, Animal; Edema; Female; Glycodeoxycholic Acid; Leukocytes; Microcirculation; Oligopeptides; Pancreas; Pancreatitis; Pancreatitis, Acute Necrotizing; Platelet Activating Factor; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Regional Blood Flow; Severity of Illness Index; Taurocholic Acid; Time Factors; Triazoles

Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia in patients with various gene mutations in lipoprotein lipase (LPL) or apolipoprotein CII. However, the exact pathogenetic mechanism has not yet been defined.. Susceptibility to pancreatitis in LPL-deficient mice was compared with that of wild-type mice after intraperitoneal injections of caerulein by determination of amylase release and pancreatic pathological scores. The effect of chylomicrons and fatty acids on enzyme release, Ca(2+) signalling and cell injury in isolated pancreatic acinar cells from wild-type and LPL-deficient mice was investigated.. Caerulein induced higher levels of serum amylase and more severe inflammation in the pancreas of LPL-deficient mice than in wild-type mice. Addition of free fatty acids or chylomicrons to isolated pancreatic acinar cells led to the release of amylase and caused cell injury at higher concentrations. The effect of chylomicrons was partially blocked by orlistat, an inhibitor of pancreatic lipase. These results suggest that increased concentrations of free fatty acids from chylomicron hydrolysis by pancreatic lipase can induce acinar cell injury. Surprisingly, pancreatic lipase, whether in its active or inactive state could act like an agonist by inducing amylase secretion without cell injury. It caused an increase in cGMP levels and conversion of cell-damaging sustained elevations of [Ca(2+)] to normal Ca(2+) oscillations.. LPL-deficient mice with severe hypertriglyceridaemia display enhanced susceptibility to acute pancreatitis. High levels of chylomicrons and free fatty acids result in pancreatic cell injury. Pancreatic lipase has a dual effect: generating free fatty acids from chylomicrons and preventing Ca(2+) overload in pancreatic acinar cells.

Topics: Animals; Ceruletide; Endothelial Cells; Enzyme Inhibitors; Female; Genetic Predisposition to Disease; Hypertriglyceridemia; Lipoprotein Lipase; Mice; Pancreas; Pancreatitis; Signal Transduction

Systemic inflammatory mediators, including the protein high-mobility group box 1 (HMGB1), play an important role in the development of acute pancreatitis. Anticoagulants such as danaparoid sodium (DA) may be able to inhibit sepsis-induced inflammation, but the mechanism of action is not well understood. We hypothesized that DA would act as an inhibitor of inflammation and prevent cerulein-induced acute pancreatitis. Male Wistar rats were used as subjects in this study. Each received a bolus of 50 U/kg of DA or saline-injected into the tail vein, followed by 4 injections of 50 mg/kg cerulean (i.p.) at 1-h intervals. Cytokine (IL-6), NO, and HMGB1 levels in serum and pancreatic tissue were measured after the cerulein injection. Pancreas histopathology and wet-dry ratio significantly improved in the DA-injected (50 U/kg) animals compared with saline-injected rats. Serum and pancreatic HMGB1 levels decreased over time in DA-treated animals. Danaparoid sodium also decreased cytokine, NO, and HMGB1 levels during cerulein-induced inflammation. As a result, DA ameliorated pancreas pathology in the rat model of cerulein-induced acute pancreatitis. This study demonstrates that DA treatment prevents cerulein-induced acute pancreatitis in a rat model. This effect may be mediated through inhibition of cytokines, NO, and HMGB1.

Topics: Animals; Blotting, Western; Ceruletide; Chondroitin Sulfates; Dermatan Sulfate; Fibrinolytic Agents; Heparitin Sulfate; HMGB1 Protein; Immunohistochemistry; Interleukin-6; Male; NF-kappa B; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Pancreas; Pancreatitis; Peroxidase; Rats

The renin-angiotensin system contributes to pathological processes in a variety of organs. In the pancreas, blocking the angiotensin II (AII) type 1 receptor (AT1) attenuates pancreatic fibrogenesis in animal models of pancreatitis. Because the role of the AII type 2 receptor (AT2) in modulating pancreatic injury is unknown we investigated the role of AT2 in pancreatic injury and fibrosis. Pancreatic fibrosis was induced by repetitive cerulein administration in C57BL/6 wild-type (WT) or AT2-deficient (AT2-/-) mice and assessed by morphology and gene expression at 10 days. There was no difference between WT and AT2-/- mice in the degree of acute pancreatic injury as assessed by amylase release at 9 and 12 h and by histological examination of the pancreas at 12 h. In contrast, parenchymal atrophy and fibrosis were more pronounced in AT2-/- mice compared with WT mice at 10 days. Fibrosis was accompanied by activation of pancreatic stellate cells (PSC) evaluated by Western blot analysis for alpha-smooth muscle actin and by immunocytochemistry; PSC activation was further increased in AT2-/- mice compared with WT mice. The level of pancreatic transforming growth factor-beta1 mRNA and protein after repetitive cerulein treatment was higher in AT2-/- mice than in WT mice. Our results demonstrate that, in contrast to AT1 receptor signaling, AT2 receptor signaling modulates protective antifibrogenic effects in a mouse model of cerulein-induced pancreatic fibrogenesis. We propose that the effects of AII on injury-induced pancreatic fibrosis may be determined by the balance between AT1 and AT2 receptor signaling.

Topics: Actins; Acute Disease; Amylases; Angiotensins; Animals; Ceruletide; Collagen; Disease Models, Animal; Female; Fibrosis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; RNA, Messenger; Severity of Illness Index; Signal Transduction; Time Factors; Transforming Growth Factor beta1

The role of nitric oxide (NO) has been increasingly implicated in the pathophysiology of acute pancreatitis (AP). Studies have shown increased NO production in AP although not all are agreeable on whether NO is beneficial or detrimental in AP. This study aims to profile NO production and NO synthase (NOS) expression in the pancreas and lungs in the progression of AP in mice to gain insights to the role played by different NOS isoforms.. AP was induced in mice by hourly administration of cerulein. NO production was determined by measuring the total nitrite and nitrate (NOx) content while NOS expression was measured by Western blot.. Pancreatic NO production increased sharply and was sustained throughout AP. iNOS expression was greatly increased while eNOS was downregulated at the later stages. In the lungs, there was an unexpected early increase in the constitutive NOS expression; however iNOS was also significantly overexpressed at the later time point along with a significant increase in NO. Acinar cells were found to overproduce NO in response to cerulein hyperstimulation with iNOS again being the major contributor.. These data show that NO production and NOS expression are differentially regulated temporally and in magnitude in the pancreas and lungs in response to cerulein hyperstimulation which suggests differing roles for each NOS isoform. and IAP.

Topics: Acute Disease; Animals; Ceruletide; Lung; Lung Injury; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pancreas; Pancreatitis

Intestinal barrier failure during acute pancreatitis (AP) is associated with translocation of luminal bacteria, resulting in infectious complications. We examined the effects of multispecies probiotics on the intestinal barrier impairment in a murine model of AP.. Mice were injected with cerulein to induce AP and were sacrificed 11 (early AP) or 72 hours (late AP) after start of induction. AP and associated systemic effects were confirmed by histology of pancreas and lung. Animals received daily probiotics starting 2 days prior to AP induction (pretreatment) or at the moment of AP induction (treatment). Mucosal barrier function of the distal ileum was assessed in Ussing chambers by measurement of the epithelial electrical resistance and the permeability to Na-fluorescein.. Histological analysis revealed pancreatic injury in both phases of AP, and lung damage in the early phase. Epithelial resistance of the ileum was reduced and permeability increased in both phases of AP, indicating impairment of the intestinal barrier. Pretreatment had no effect on resistance or permeability in the early phase of AP. In the late phase of AP, pretreatment but not treatment abolished the AP induced resistance decrease and permeability increase. Administration of probiotics as such (ie, without induction of AP) had no effect on intestinal barrier function.. Pretreatment with multispecies probiotics for 2 days abolishes intestinal barrier dysfunction in the late phase of AP, while treatment does not. The effectiveness of probiotics in this model depends on the timing of administration. Clinical trials with probiotics should seek conditions where treatment can be started prior to onset of disease or elective surgical intervention.

Topics: Animals; Bacterial Translocation; Bifidobacterium; Ceruletide; Ileal Diseases; Lacticaseibacillus casei; Lactobacillus acidophilus; Lactococcus lactis; Lung; Male; Mice; Pancreas; Pancreatitis; Probiotics

There is no clinical treatment that reduces acinar injury during pancreatitis. Human immunodeficiency virus (HIV) protease inhibitors (PI), including nelfinavir (NFV) and ritonavir (RTV), may reduce the rate of pancreatitis in HIV-infected patients. Since permeability transition pore (PTPC)-mediated mitochondrial dysfunction occurs during pancreatitis, and we have shown that PI prevents PTPC opening, we studied its effects in a model of pancreatitis. The effect of NFV plus RTV (NFV/RTV) or vehicle on caerulein-induced pancreatitis in mice was compared by measuring changes in mitochondrial membrane potential in vitro and cytochrome c leakage in vivo. Histological and inflammatory makers were also compared. NFV/RTV improved DiOC6 retention in acini exposed to caerulein in vitro. In vivo NFV prevented cytosolic leakage of cytochrome c and reduced pancreatic acinar injury, active caspase-3 staining, TUNEL-positive acinar cells, and serum amylase (P < 0.05). Conversely, trypsin activity, serum cytokine levels, and pancreatic and lung inflammation were unaffected. NFV/RTV reduces pancreatic injury and acinar cell death in experimental mouse caerulein-induced pancreatitis but does not impact inflammation.

Topics: Amylases; Animals; Apoptosis; Caspase 3; Ceruletide; Cytochromes c; Disease Models, Animal; Drug Therapy, Combination; HIV Protease Inhibitors; Inflammation Mediators; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Necrosis; Nelfinavir; Pancreas; Pancreatitis; Ritonavir; Trypsin

Acute lung injury is one of the critical complications of acute pancreatitis (AP). Tumor necrosis factor-associated factor 6 (TRAF6) is a key adaptor that regulates various inflammatory signaling pathways, including those mediated by Toll-like receptors (TLRs). This study was performed to investigate the potential role of TRAF6 in the pathogenesis of AP and pancreatitis-associated acute lung injury using a mouse model of caerulein-induced AP (CAP). CAP was induced by intraperitoneal injection of caerulein hourly for 7 times (50 microg/kg), and control mice were treated with saline of the same volume. Typical pancreatic and lung inflammation was observed in the early stage (1 h) of CAP, as judged by morphological changes. Likewise, in CAP mice, the pancreatic myeloperoxidase activity and serum levels of interleukin-6 and interleukin-10 were significantly increased after 2 h, peaked at 4h, and then decreased by 24 h. The expression of TRAF6 was then studied by real time-PCR, immunohistochemistry, and Western blot analysis. Compared with control group, TRAF6 mRNA level was decreased in CAP group within the first 12 h, and then significantly increased after 24 h, which was in accordance with the protein level detected by Western blot analysis and immunohistochemistry. Moreover, TRAF6 protein was expressed in both pancreatic acinar cells and lung bronchial epithelial cells. In conclusion, the down-regulation of TRAF6 was associated with increased inflammatory severity in the pancreas and lung, suggesting that TRAF6 is involved in the anti-inflammatory process during AP. TRAF6 may be a potential molecular target for treating AP.

Topics: Acute Lung Injury; Animals; Blotting, Western; Ceruletide; Down-Regulation; Immunohistochemistry; Interleukin-10; Interleukin-6; Mice; Pancreatitis; Peroxidase; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; TNF Receptor-Associated Factor 6

In the present study, we have addressed the possible protective role of acetyl-L-carnitine in caerulein-induced acute pancreatitis in male Swiss albino rats. Acute pancreatitis paradigm was developed by challenging animals with a supramaximal dose of caerulein (20 microg/kg, SC) four times at hourly intervals. Caerulein induced acute pancreatitis that was well-characterized morphologically and biochemically. Severe oedema with marked increased relative pancreatic weight, marked atrophy of acini with increased interacinar spaces, vacuolization, and extensive leucocytic infiltration were diagnostic fingerprints of the pancreatitis phenotype. A biochemical test battery that confirmed the model comprised increased plasma amylase and lipase activities, calcium levels as well as increased pancreatic enzymatic myeloperoxidase and glutathione-S-transferase activities, beside increased pancreatic contents of nitric oxide and malondialdehyde and reduced pancreatic glutathione level. Prior administration of acetyl-L-carnitine (200 mg/kg, IP) for seven consecutive days ahead of caerulein challenge alleviated all the histological and biochemical manifestations of acute pancreatitis. These results suggest a possible protective role of the carnitine ester in such a murine acute pancreatitis model probably via regulation of the oxidant/antioxidant balance, beside modulation of the myeloperoxidase and nitric oxide systems, which are involved in the inflammatory cascade that most often associate the disease.

Topics: Acetylcarnitine; Amylases; Animals; Calcium; Ceruletide; Disease Models, Animal; Glutathione; Glutathione Transferase; Injections, Intraperitoneal; Injections, Subcutaneous; Lipase; Lipid Peroxidation; Male; Malondialdehyde; Nitric Oxide; Pancreas; Pancreatitis; Peroxidase; Protective Agents; Rats

Toll-like receptor 4 (TLR4) is potentially associated with acute pancreatitis (AP), but its exact role remains controversial. IL-1 receptor-associated kinase 4 (IRAK-4) is a common mediator of Toll-like receptors pathways, with an essential role in transducing downstream signals. This study investigates the potential role of the TLR4 pathway, in particular IRAK-4, in a murine model of AP.. Acute pancreatitis was induced in wild-type and TLR4-deficient mice by intraperitoneal injections of caerulein (50 microg/kg). Pancreatic pathological scores and myeloperoxidase activity were dynamically measured, along with pancreatic TLR4 and IRAK-4 mRNA and protein.. In wild-type mice, pathological scores and myeloperoxidase activity were rapidly increased at 1, 2 and 4 h, followed by alleviation at 12 and 24 h. In TLR4-deficient mice, they were slightly increased within 2 h, but became more severe at 12 and 24 h. IRAK-4 mRNA and protein were significantly down-regulated at 1, 2 and 4 h in wild-type mice. Unexpectedly, TLR4-deficient mice showed more profound reductions of IRAK-4 mRNA and protein at the same time.. TLR4 deficiency delayed the initiation of pancreatitis, implying a potential role for TLR4 during AP. IRAK-4 might function during AP, but independently of TLR4.

Topics: Animals; Ceruletide; Interleukin-1 Receptor-Associated Kinases; Male; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peroxidase; Random Allocation; Signal Transduction; Toll-Like Receptor 4

Protease activation within the pancreatic acinar cell is a key early event in acute pancreatitis and may require low pH intracellular compartments. Clinical studies suggest that acidosis may affect the risk for developing pancreatitis. We hypothesized that exposure to an acid load might sensitize the acinar cell to secretagogue-induced pancreatitis.. Secretagogues (cerulein, carbachol, and bombesin) can induce protease activation in acinar cells at high (100 nmol/L, 1 mmol/L, and 10 micromol/L, respectively) but not at physiologically relevant concentrations. The effects of decreasing extracellular pH (pHe) in early secretagogue-induced pancreatitis (zymogen activation and injury) were examined in rats (1) in vitro with isolated acini and (2) in vivo with an acid challenge.. In acini, lowering pHe from 7.6 to 6.8 enhanced secretagogue-induced zymogen activation and injury, but did not affect secretion. For cerulein, this sensitization was seen over a range of concentrations (0.01-100.00 nmol/L). However, reduced pHe alone had no effect on zymogen activation, amylase secretion, or cell injury. We have reported that zymogen activation is mediated by the vacuolar ATPase (vATPase), a proton transporter. vATPase inhibition, using concanamycin (100 nmol/L), blocked the low pHe effects on zymogen activation. An acute acid load given in vivo enhanced cerulein-induced (50 microg/kg) trypsinogen activation and pancreatic edema.. These studies suggest that acid challenge sensitizes the pancreatic acinar cell to secretagogue-induced zymogen activation and injury and may increase the risk for the development and severity of acute pancreatitis.

Topics: Adenosine Triphosphatases; Amylases; Animals; Carbachol; Ceruletide; Chymotrypsin; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Hydrogen-Ion Concentration; In Vitro Techniques; Lactic Acid; Macrolides; Male; Pancreas; Pancreatitis; Propionates; Rats; Rats, Sprague-Dawley; Trypsin

Clinical reports link use of the glucagon-like peptide-1 receptor (GLP-1R) agonists exenatide and liraglutide to pancreatitis. However, whether these agents act on the exocrine pancreas is poorly understood.. We assessed whether the antidiabetic agents exendin (Ex)-4, liraglutide, the dipeptidyl peptidase-4 inhibitor sitagliptin, or the biguanide metformin were associated with changes in expression of genes associated with the development of experimental pancreatitis. The effects of Ex-4 when administered before or after the initiation of caerulein-induced experimental pancreatitis were determined. The importance of endogenous GLP-1R signaling for gene expression in the exocrine pancreas and the severity of pancreatitis was assessed in Glp1r(-/-) mice.. Acute administration of Ex-4 increased expression of egr-1 and c-fos in the exocrine pancreas. Administration of Ex-4 or liraglutide for 1 week increased pancreas weight and induced expression of mRNA transcripts encoding the anti-inflammatory proteins pancreatitis-associated protein (PAP) (RegIIIbeta) and RegIIIalpha. Chronic Ex-4 treatment of high-fat-fed mice increased expression of PAP and reduced pancreatic expression of mRNA transcripts encoding for the proinflammatory monocyte chemotactic protein-1, tumor necrosis factor-alpha, and signal transducer and activator of transcription-3. Sitagliptin and metformin did not significantly change pancreatic gene expression profiles. Ex-4 administered before or after caerulein did not modify the severity of experimental pancreatitis, and levels of pancreatic edema and serum amylase were comparable in caerulein-treated Glp1r(-/-) versus Glp1r(+/+) mice.. These findings demonstrate that GLP-1 receptor activation increases pancreatic mass and selectively modulates the expression of genes associated with pancreatitis. However, activation or genetic elimination of GLP-1R signaling does not modify the severity of experimental pancreatitis in mice.

Topics: Animals; Ceruletide; Dietary Fats; Disease Models, Animal; Early Growth Response Protein 1; Exenatide; Gene Expression; Genes, fos; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Hypoglycemic Agents; Liraglutide; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Pancreas, Exocrine; Pancreatitis; Pancreatitis-Associated Proteins; Peptides; Receptors, Glucagon; Severity of Illness Index; Signal Transduction; Venoms

Smac mimetics induce apoptosis synergistically with TNF-alpha by triggering the formation of a caspase-8-activating complex containing receptor interacting protein kinase-1 (RIPK1). Caspase inhibitors block this form of apoptosis in many types of cells. However, in several other cell lines, caspase inhibitors switch the apoptotic response to necrosis. A genome wide siRNA screen revealed another member of the RIP kinase family, RIP3, to be required for necrosis. The expression of RIP3 in different cell lines correlates with their responsiveness to necrosis induction. The kinase activity of RIP3 is essential for necrosis execution. Upon induction of necrosis, RIP3 is recruited to RIPK1 to form a necrosis-inducing complex. Embryonic fibroblasts from RIP3 knockout mice are resistant to necrosis and RIP3 knockout animals are devoid of inflammation inflicted tissue damage in an acute pancreatitis model. These data indicate RIP3 as the determinant for cellular necrosis in response to TNF-alpha family of death-inducing cytokines.

Topics: Animals; Cell Line, Tumor; Ceruletide; Humans; Mice; Mutation; Necrosis; Pancreatitis; Protein Structure, Tertiary; Receptor-Interacting Protein Serine-Threonine Kinases; RNA, Small Interfering; Tumor Necrosis Factor-alpha

Inflammatory cytokines and oxidative stress have a central role in the pathogenesis of acute pancreatitis. Propolis is a resinous hive product collected by honeybees from various plant sources and has anti-inflammatory and anti-oxidant effects. The present work aimed to investigate the therapeutic role of ethanolic extract of propolis on a cerulein-induced acute pancreatitis model in rats.. Seventy male Wistar albino rats were used in the study. Acute edematous pancreatitis was induced by subcutaneous cerulein injection (20 microg/kg) four times at one-hour intervals. Ethanolic extract of propolis 300 mg/kg was given subcutaneously at the beginning of the procedure (ethanolic extract of propolis-1 group) or 12 h after the last cerulein injection (ethanolic extract of propolis-2 group). Serum amylase and lipase levels, white blood cell count and serum tumor necrosis factor-alpha levels were measured and pancreatic tissue was evaluated histologically.. In the acute pancreatitis group, serum amylase and lipase levels were found to be elevated and the histopathological evaluation of the tissue revealed massive edema and inflammation with less fatty necrosis when compared to the sham and control groups. Serum amylase and lipase levels and edema formation were significantly decreased in the ethanolic extract of propolis-treated groups (p<0.001). In the ethanolic extract of propolis-2 group, in particular, tissue edema was improved markedly (p=0.001). Tissue inflammation and fatty necrosis were decreased with ethanolic extract of propolis treatment; however, the improvement was not statistically significant.. Treatment with ethanolic extract of propolis improved the biochemical and histopathological findings in a rat model of experimental pancreatitis. Although our findings suggest that ethanolic extract of propolis might be considered an effective agent for the treatment of acute pancreatitis, this notion should be supported with further experimental and clinical investigations.

Topics: Acute Disease; Amylases; Animals; Anti-Infective Agents; Ceruletide; Disease Models, Animal; Edema; Gastrointestinal Agents; Lipase; Male; Pancreas; Pancreatitis; Propolis; Rats; Rats, Wistar; Treatment Outcome

Systemic inflammatory mediators, including the protein high-mobility group box 1 (HMGB1), play an important role in the development of acute pancreatitis. Anticoagulants, such as antithrombin III (AT III), inhibit inflammation resulting from various causes, but their mechanism of action is not well understood. Because acute pancreatitis is a severe inflammatory disease, we hypothesized that AT III would inhibit inflammation and prevent cerulein-induced acute pancreatitis.. Experimental animals received or were saline injected with a bolus of 250 IU/kg of AT III followed by intraperitoneal injections of 50 mg/kg of cerulein. Levels of cytokines (interleukin 6 and tumor necrosis factor alpha), nitric oxide (NO), and HMGB1 were measured in serum and pancreatic tissue at regular intervals for 12 hours after the cerulein injection.. Pancreas histopathology and wet-dry ratio significantly improved in the AT III-injected (250 IU/kg) animals compared with the saline-injected rats. Serum and pancreas HMGB1 levels decreased over time in AT III-treated animals. Antithrombin III also decreased cytokine, NO, and HMGB1 levels during cerulein-induced inflammation. As a result, AT III ameliorated the pathologic pancreas in the rat model of cerulein-induced acute pancreatitis.. Antithrombin III treatment inhibited the secretion of cytokines, NO, and HMGB1 and prevented cerulein-induced acute pancreatitis in the rat model.

Topics: Acute Disease; Animals; Anticoagulants; Antithrombin III; Blotting, Western; Ceruletide; HMGB1 Protein; Immunohistochemistry; Interleukin-6; Male; Nitric Oxide; Pancreas; Pancreatitis; Random Allocation; Rats; Rats, Wistar

Receptor-stimulated Ca(2+) influx is a critical component of the Ca(2+) signal and mediates all cellular functions regulated by Ca(2+). However, excessive Ca(2+) influx is highly toxic, resulting in cell death, which is the nodal point in all forms of pancreatitis. Ca(2+) influx is mediated by store-operated channels (SOCs). The identity and function of the native SOCs in most cells is unknown.. Here, we determined the role of deletion of Trpc3 in mice on Ca(2+) signaling, exocytosis, intracellular trypsin activation, and pancreatitis.. Deletion of TRPC3 reduced the receptor-stimulated and SOC-mediated Ca(2+) influx by about 50%, indicating that TRPC3 functions as an SOC in vivo. The reduced Ca(2+) influx in TRPC3(-/-) acini resulted in reduced frequency of the physiologic Ca(2+) oscillations and of the pathologic sustained increase in cytosolic Ca(2+) levels caused by supramaximal stimulation and by the toxins bile acids and palmitoleic acid ethyl ester. Consequently, deletion of TRPC3 shifted the dose response for receptor-stimulated exocytosis and prevented the pathologic inhibition of digestive enzyme secretion at supramaximal agonist concentrations. Accordingly, deletion of TRPC3 markedly reduced intracellular trypsin activation and excessive actin depolymerization in vitro and the severity of pancreatitis in vivo.. These findings establish the native TRPC3 as an SOC in vivo and a role for TRPC3-mediated Ca(2+) influx in the pathogenesis of acute pancreatitis and suggest that TRPC3 should be considered a target for prevention of pancreatic damage in acute pancreatitis.

Topics: Actins; Acute Disease; Animals; Calcium Signaling; Carbachol; Ceruletide; Cholinergic Agonists; Disease Models, Animal; Dose-Response Relationship, Drug; eIF-2 Kinase; Enzyme Activation; Enzyme Inhibitors; Exocytosis; Indoles; Membrane Potentials; Mice; Mice, Knockout; Pancreas; Pancreatitis; Phosphorylation; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Severity of Illness Index; Sincalide; Taurocholic Acid; TRPC Cation Channels; Trypsin

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 microM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 microM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins alphaII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, mu- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of alphaII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Topics: Actins; Acute Disease; Animals; Blotting, Western; Cadherins; Calcium-Binding Proteins; Calpain; Ceruletide; Cholecystokinin; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Cytoskeleton; Dipeptides; Enzyme Activation; Female; Gene Expression; Microscopy, Confocal; Microscopy, Electron; Models, Animal; Organ Culture Techniques; Pancreas; Pancreatitis; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrin; Stimulation, Chemical; Time Factors; Vinculin

Fibroblast growth factor 21 (FGF21) acts as a hormonal regulator during fasting and is involved in lipid metabolism. Fgf21 gene expression is regulated by peroxisome proliferator-activated receptor (PPAR)-dependent pathways, which are enhanced during pancreatitis. Therefore, the aim of this study was to investigate FGF21's role in pancreatic injury.. Fgf21 expression was quantified during cerulein-induced pancreatitis (CIP) or following mechanical or thapsigargin-induced stress through Northern blot analysis, in situ hybridization, and quantitative reverse transcription polymerase chain reaction. FGF21 protein was quantified by Western blot analysis. Isolated acinar cells or AR42J acinar cells were treated with recombinant FGF21 protein, and extracellular regulated kinase 1/2 activation was examined. The severity of CIP was compared between wild-type mice and mice overexpressing FGF21 (FGF21Tg) or harboring a targeted deletion of Fgf21 (Fgf21(-/-)).. Acinar cell Fgf21 expression markedly increased during CIP and following injury in vitro. Purified FGF21 activated the extracellular regulated kinase 1/2 pathway in pancreatic acinar cells. The severity of CIP is inversely correlated to FGF21 expression because FGF21Tg mice exhibited decreased serum amylase and decreased pancreatic stellate cell activation, whereas Fgf21(-/-) mice had increased serum amylase and tissue damage. The expression of Fgf21 was also inversely correlated to expression of Early growth response 1, a proinflammatory and profibrotic transcription factor.. These studies suggest a novel function for Fgf21 as an immediate response gene protecting pancreatic acini from overt damage.

Topics: Animals; Cell Culture Techniques; Ceruletide; Early Growth Response Protein 1; Fibroblast Growth Factors; Mice; Mice, Inbred C57BL; Pancreas, Exocrine; Pancreatitis; Rats; Receptors, Fibroblast Growth Factor; RNA, Messenger

Development of human chronic pancreatitis is associated with intrapancreatic accumulation of pituitary adenylate cyclase-activating polypeptide (PACAP) accompanied with an altered inflammatory response (Michalski et al., Am J Physiol Gastrointest Liver Physiol. 2008;294:G50-G57). To investigate the role of pancreatic PACAP in the development of acute pancreatitis, we employed transgenic mice over-expressing PACAP in pancreatic beta-cells (PACAP-Tg). In comparison to wild-type mice, PACAP-Tg mice exhibited more severe pathophysiological signs of the cerulein-induced pancreatitis at 12 h, as evidenced by higher serum amylase and lipase levels accompanied by the exacerbation of pancreatic edema, necrosis, and inflammation. Cerulein treatment increased mRNA expression of several proinflammatory cytokines (TNFalpha, IL-1beta, and IL-6) at 12 h with similar magnitude both in wild-type and PACAP-Tg mice. In addition, the mRNA and protein levels of regenerating gene III beta (RegIIIbeta), a key factor in the pancreatic response to acute pancreatitis, were up-regulated at 24 h in wild-type mice upon cerulein administration, whereas they were attenuated in PACAP-Tg mice. These data indicate that over-expressed PACAP in pancreas enhances the cerulein-induced inflammatory response of both acinar cells, leading to aggravated acute pancreatitis, which was accompanied by a down-regulation of RegIIIbeta, an anti-inflammatory factor.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Cytokines; Disease Models, Animal; Down-Regulation; Insulin-Secreting Cells; Lipase; Mice; Mice, Transgenic; Pancreatitis; Pancreatitis-Associated Proteins; Pituitary Adenylate Cyclase-Activating Polypeptide; Proteins; RNA, Messenger; Up-Regulation

Green tea polyphenols (GTPs) are naturally occurring antioxidants acting through pathways that include reactive oxygen species and nuclear factor kappa B (NF-kappaB). This study investigates the effect of GTPs in a cerulein-induced murine model of acute pancreatitis (AP).. Male CD mice (median weight, 37.7 g) were divided into 4 groups: mice administered with cerulein alone, cerulein and GTP, saline alone (sham), and GTP alone. Acute pancreatitis was induced by serial intraperitoneal administration of cerulein (50 microg/kg, x6). Green tea polyphenol was administered intraperitoneally at 25 mg/kg on the first, third, and sixth hours after pancreatitis induction.We analyzed histologic and biochemical features of AP, NF-kappaB pathway activity, leukocyte-mediated damage, cytokine levels, oxidative stress injury, lipid peroxidation, expression of poly-(adenosine diphosphate-ribose) synthetase, and presence of apoptosis.. Treatment with GTP reduced the histologic and biochemical features of AP. Western blot revealed significant NF-kappaB inactivation. Immunostaining for P selectin and intercellular adhesion molecule 1, tumor necrosis factor alpha, transforming growth factor beta, vascular endothelial growth factor, nitrotirosine, poly-(adenosine diphosphate ribose) synthetase, and malondialdheide levels were significantly reduced. There was a significant down-regulation of apoptotic markers.. Our results demonstrated that GTP significantly ameliorated the effects of cerulein-induced AP in mice. These effects of GTP are mediated by actions at the NF-kappaB/IkB (inhibitor kB) proteins and oxidative stress pathways.

Topics: Acute Disease; Animals; Apoptosis; Blotting, Western; Ceruletide; I-kappa B Proteins; Immunohistochemistry; In Situ Nick-End Labeling; Injections, Intraperitoneal; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Male; Mice; Neutrophil Infiltration; NF-kappa B; P-Selectin; Pancreas; Pancreatitis; Phenol; Poly(ADP-ribose) Polymerases; Tea; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Aquaporin 12 (AQP12) is the most recently identified member of the mammalian AQP family and is specifically expressed in pancreatic acinar cells. In vitro expression studies have revealed that AQP12 is localized at intracellular sites. To determine the physiological roles of AQP12 in the pancreas, we generated knockout mice for this gene (AQP12-KO). No obvious differences were observed under normal conditions between wild-type (WT) and AQP12-KO mice in terms of growth, blood chemistry, pancreatic fluid content, or histology. However, when we induced pancreatitis through the administration of a cholecystokinin-8 (CCK-8) analog, the AQP12-KO mice showed more severe pathological damage to this organ than WT mice. Furthermore, when we analyzed exocytosis in the pancreatic acini using a two-photon excitation imaging method, the results revealed larger exocytotic vesicles (vacuoles) in the acini of AQP12-KO mice at a high CCK-8 dose (100 nM). From these results, we conclude that AQP12 may function in the mechanisms that control the proper secretion of pancreatic fluid following rapid and intense stimulation.

Topics: Acute Disease; Amylases; Animals; Aquaporins; Ceruletide; Cholecystokinin; Diet; Disease Susceptibility; Endoplasmic Reticulum; Exocytosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreas, Exocrine; Pancreatitis; Peptide Fragments; Permeability; Photons; Tissue Distribution; Water

The aim of this study was to evaluate the role of oxidative damage in pancreatitis-induced hepatic injury. Thirty-five rats were divided into five groups (each of 7 rats): control, cerulein (100 microg/kg body weight), cerulein and pentoxifylline (12 mg/kg body weight), cerulein plus L-NAME (10 mg/kg body weight) and cerulein plus L-arginine (160 mg/kg body weight). The degree of hepatic cell degeneration differed significantly between groups. Mean malondialdehyde levels were 7.00 +/- 2.29, 20.89 +/- 10.13, 11.52 +/- 4.60, 18.69 +/- 8.56, and 8.58 +/- 3.68 nmol/mg protein for the control, cerulein, pentoxifylline, L-NAME, and L-arginine groups, respectively. Mean catalase activity was 3.20 +/- 0.83, 1.09 +/- 0.35, 2.05 +/- 0.91, 1.70 +/- 0.60, and 2.85 +/- 0.47 U/mg protein for the control, cerulein, pentoxifylline, L-NAME, and L-arginine groups, respectively, and mean glutathione peroxidase activity was 0.72 +/- 0.25, 0.33 +/- 0.09, 0.37 +/- 0.04, 0.34 +/- 0.07 and 0.42 +/- 0.1 U/mg protein for the control, cerulein, pentoxifylline, L-NAME, and L-arginine groups, respectively. Cerulein-induced liver damage was accompanied by a significant increase in tissue malondialdehyde levels (P < 0.05) and a significant decrease in catalase (P < 0.05) and GPx activities (P < 0.05). L-arginine and pentoxifylline, but not L-NAME, protected against this damage. Oxidative injury plays an important role not only in the pathogenesis of AP but also in pancreatitis-induced hepatic damage.

Topics: Acute Disease; Animals; Arginine; Ceruletide; Female; Free Radical Scavengers; Lipid Peroxidation; Liver Diseases; NG-Nitroarginine Methyl Ester; Pancreatitis; Pentoxifylline; Rats; Rats, Wistar; Reactive Oxygen Species

Topics: Animals; Ceruletide; Fibroblast Growth Factors; Humans; Mice; Pancreatitis; Receptors, Fibroblast Growth Factor

Since respiratory dysfunction is the main cause of death in patients with severe acute pancreatitis (SAP), elucidating the critical period of acute pancreatitis-associated lung injury (APALI) is of important clinical value. This study aimed to define the risk period of APALI by a series of studies including a dynamic analysis of total water content, ultrastructure and number of type II alveolar epithelial cells, and reactive oxygen metabolites (ROMs) of lung tissue in a mouse model of SAP, and a clinical analysis of APALI patients.. ICR mice were selected to establish a SAP model. They were given 7 intraperitoneal injections of cerulein (50 microg/kg body weight) at hourly intervals, followed by an intraperitoneal injection of lipopolysaccharide (15 mg/kg body weight). The total water content, ultrastructure, and number of type II alveolar epithelial cells, and ROMs of lung tissue were assessed before (0 hour) and after the establishment of SAP model (6 hours, 12 hours, 1 day, 4 days, and 7 days). In addition, we analyzed the data from 215 patients with APALI (PaO(2) <60 mmHg) treated at our hospital between January 1998 and December 2006. Statistical analyses were made using the F test. P values less than 0.05 were regarded as statistically significant.. The total water content and ultrastructure of type II alveolar epithelial cells (mitochondria and lamellar bodies) of the lung in the SAP mice were significantly altered at 12 hours after the establishment of SAP model, and reached a maximum at 1 to 4 days. The number of type II alveolar epithelial cells and ROMs increased maximally at 1 day after the establishment of the model. Furthermore, clinical results showed that lung injury occurred at a mean of 3.1435+/-1.0199 days in patients with SAP. These clinical data were almost consistent with the results of the SAP model.. The risk period for APALI is between the first and fourth day during the course of SAP.

Topics: Acute Lung Injury; Animals; Ceruletide; Disease Models, Animal; Disease Progression; Epithelial Cells; Lipopolysaccharides; Malondialdehyde; Mice; Mice, Inbred ICR; Pancreatitis; Pulmonary Alveoli; Reactive Oxygen Species; Risk Factors; Time Factors; Water

Obestatin is a peptide derived from the proghrelin, a common prohormone for ghrelin and obestatin. Obestatin, like the ghrelin has been originally extracted from rat stomach, and the stomach seems to be a major source of circulating obestatin. Previous studies have shown that administration of ghrelin exhibits protective effect in the pancreas, inhibiting the development of acute pancreatitis. Recent study has shown that obestatin promotes survival of beta-cells and pancreatic islets. Aim of the present study was to investigate the influence of obestatin administration on the development of cerulein-induced pancreatitis. Studies were performed on male Wistar rats. Acute pancreatitis was induced by cerulein given intraperitoneally 5 times at a dose of 50 microg/kg/dose with 1-h intervals. Obestatin was injected twice intraperitoneally at the dose of 4, 8 or 16 nmol/kg/dose. In control saline-treated rats, obestatin was without effect on pancreatic morphology, serum activity of pancreatic enzymes, serum level of pro-inflammatory interleukin-1beta or pancreatic cells proliferation. In animals with induction of acute pancreatitis, morphological examination showed that administration of obestatin decreased pancreatic leukocyte infiltration and vacuolization of acinar cells. These effects were accompanied by reduction in the pancreatitis-evoked increase in serum level of pancreatic digestive enzymes, lipase amylase and poly-C ribonuclease. Obestatin administered at the highest dose of 16 nmo/kg/dose reduced serum activity of these enzymes by 33, 42 and 44%, respectively. Also serum concentration of pro-inflammatory interleukin-1beta was decreased by obestatin in rats with acute pancreatitis; whereas the pancreatitis-evoked decrease in pancreatic blood flow and pancreatic DNA synthesis was partially reversed. Administration of obestatin reduces the severity of cerulein-induced acute pancreatitis. This effect is related, at least in part, to the improvement of pancreatic blood flow and reduction in proinflammatory interleukin-1beta release.

Topics: Acute Disease; Amylases; Animals; Cell Proliferation; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Intraperitoneal; Interleukin-1beta; Lipase; Male; Pancreas; Pancreatitis; Peptide Hormones; Protective Agents; Rats; Rats, Wistar; Splanchnic Circulation

To evaluate the therapeutic role of caffeic acid phenethyl ester (CAPE) in a rat model of cerulean-induced acute pancreatitis (AP).. Seventy male Wistar albino rats were divided into seven groups. Acute edematous pancreatitis was induced by subcutaneous cerulein injection (20 microg/kg) four times at 1-h intervals. CAPE (30 mg/kg) was given by subcutaneous injection at the beginning (CAPE 1 group) and 12 h after the last cerulein injection (CAPE 2 group). Serum amylase, lipase, white blood cell count, and tumor necrosis factor (TNF)-alpha levels were measured, and pancreatic histopathology was assessed.. In the AP group, amylase and lipase levels were found to be elevated and the histopathological evaluation showed massive edema and inflammation of the pancreas, with less fatty necrosis when compared with sham and control groups. Amylase and lipase levels and edema formation decreased significantly in the CAPE therapy groups (P < 0001); especially in the CAPE 2 group, edema was improved nearly completely (P = 0001). Inflammation and fatty necrosis were partially recovered by CAPE treatment. The pathological results and amylase level in the placebo groups were similar to those in the AP group. White blood cell count and TNF-alpha concentration was nearly the same in the CAPE and placebo groups.. CAPE may be useful agent in treatment of AP but more experimental and clinical studies are needed to support our observation of beneficial effects of CAPE before clinical usage of this agent.

Topics: Acute Disease; Amylases; Animals; Caffeic Acids; Ceruletide; Cytotoxins; Disease Models, Animal; Edema; Leukocyte Count; Lipase; Male; Pancreas; Pancreatitis; Phenylethyl Alcohol; Rats; Rats, Wistar; Treatment Outcome; Tumor Necrosis Factor-alpha

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Ceruletide; Enzyme Activation; HSP70 Heat-Shock Proteins; Interleukin-6; Lung; Male; Mice; Mice, Inbred Strains; Pancreas; Pancreatic alpha-Amylases; Pancreatitis; Peptide Hydrolases; Peroxidase; Tacrolimus; Trypsin

The objective of this study was to investigate the role of MAPKAP kinase 2 (MK2) and heat shock protein (HSP) HSP60 in the pathogenesis of a new model of severe acute pancreatitis (AP). MK2 plays a significant role in the regulation of cytokines. It has been shown that induction and expression of several HSPs can protect against experimental pancreatitis. Interplay between both systems seems of high interest. Mice with a homozygous deletion of the MK2 gene were used. Severe AP was induced by combined intraperitoneal injections of cerulein with lipopolysaccharide (LPS). Severity of AP was assessed by biochemical markers and histology. The serum IL-6 and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. Expression of HSP25, HSP60, HSP70, and HSP90 was analyzed by Western blotting. Repeated injections of cerulein alone or cerulein plus LPS (Cer+LPS) resulted in local inflammatory responses in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the Cer+LPS group. Compared with the C57Bl wild-type mice, the MK2-/- mice presented with significant milder pancreatitis and attenuated responses of serum amylase and trypsinogen activity. Furthermore, serum IL-6 was decreased as well as lung MPO activity. Injection of LPS alone displayed neither pancreatic inflammatory responses nor alterations of pancreatic enzyme activities but evidently elevated serum IL-6 levels and increased lung MPO activity. In contrast hereto, in the MK2-/- mice, these changes were much milder. Increased expression of HSP25 and HSP60 occurred after induction of AP. Especially, HSP60 was robustly elevated after Cer+LPS treatment, in both MK2-/- and wild-type mice. Thus the homozygous deletion of the MK2 gene ameliorates the severity of acute pancreatitis and accompanying systemic inflammatory reactions in a new model of severe acute pancreatitis. Our data support the hypothesis that MK2 participates in the multifactorial regulation of early inflammatory responses in AP, independently of the regulation of stress proteins like HSP25 and HSP60 and most likely due to its effect on cytokine regulation.

Topics: Animals; Ceruletide; Chaperonin 60; Gene Deletion; Heat-Shock Proteins; Interleukin-6; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Chaperones; Neoplasm Proteins; Pancreas; Pancreatic alpha-Amylases; Pancreatitis; Peroxidase; Protein Serine-Threonine Kinases; Trypsinogen

Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.

Topics: Acute Disease; Animals; Bile Acids and Salts; Ceruletide; Enzyme Activation; Female; Male; Mice; Mice, Inbred C57BL; Models, Biological; Pancreas; Pancreatitis; Peptides; Protein Structure, Tertiary; Receptor, PAR-2

Although both endocrine and the exocrine pancreas display a significant capacity for tissue regeneration and renewal, the existence of progenitor cells in the adult pancreas remains uncertain. Using a model of cerulein-mediated injury and repair, we demonstrate that mature exocrine cells, defined by expression of an Elastase1 promoter, actively contribute to regenerating pancreatic epithelium through formation of metaplastic ductal intermediates. Acinar cell regeneration is associated with activation of Hedgehog (Hh) signaling, as assessed by up-regulated expression of multiple pathway components, as well as activation of a Ptch-lacZ reporter allele. Using both pharmacologic and genetic techniques, we also show that the ability of mature exocrine cells to accomplish pancreatic regeneration is impaired by blockade of Hh signaling. Specifically, attenuated regeneration in the absence of an intact Hh pathway is characterized by persistence of metaplastic epithelium expressing markers of pancreatic progenitor cells, suggesting an inhibition of redifferentiation into mature exocrine cells. Given the known role of Hh signaling in exocrine pancreatic cancer, these findings may provide a mechanistic link between injury-induced activation of pancreatic progenitors and subsequent pancreatic neoplasia.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Epithelial Cells; Genes, Reporter; Hedgehog Proteins; Intermediate Filament Proteins; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Tissue Proteins; Nestin; Pancreas, Exocrine; Pancreatic Ducts; Pancreatic Elastase; Pancreatitis; Receptors, G-Protein-Coupled; Regeneration; Signal Transduction; Smoothened Receptor; Stem Cells; Time Factors; Veratrum Alkaloids

Pancreatitis is a disease with high morbidity and mortality. In vitro experiments on pancreatic acini showed that supramaximal but not submaximal cholecystokinin (CCK) stimulation induces effects in the acinar cell that can be correlated with acinar morphological changes observed in the in vivo experimental model of cerulein-induced pancreatitis. The GTPase Rac1 was previously reported to be involved in CCK-evoked amylase release from pancreatic acinar cells. Here, we demonstrate that pretreatment with the Rac1 inhibitor NSC23766 (100 microM, 2 h) effectively blocked Rac1 translocation and activation in CCK-stimulated pancreatic acini, without affecting activation of its closely related GTPase, RhoA. This specific Rac1 inhibition decreased supramaximal (10 nM) CCK-stimulated acinar amylase release (27.% reduction), which seems to be connected to the reduction observed in serum amylase (46.6% reduction) and lipase levels (46.1% reduction) from cerulein-treated mice receiving NSC23766 (100 nmol h(-1)). The lack of Rac1 activation also reduced formation of reactive oxygen species (ROS; 20.8% reduction) and lactate dehydrogenase release (LDH; 24.3% reduction), but did not alter calcium signaling or trypsinogen activation in 10 nM CCK-stimulated acini. In the in vivo model, the cerulein-treated mice receiving NSC23766 also presented a decrease in both pancreatic and lung histopathological scores (reduction in oedema, 32.4 and 66.4%; haemorrhage, 48.3 and 60.2%; and leukocyte infiltrate, 53.5 and 43.6%, respectively; reduction in pancreatic necrosis, 65.6%) and inflammatory parameters [reduction in myeloperoxidase, 52.2 and 38.9%; nuclear factor kappaB (p65), 61.3 and 48.6%; and nuclear factor kappaB (p50), 46.9 and 44.9%, respectively], together with lower serum levels for inflammatory (TNF-alpha, 40.4% reduction) and cellular damage metabolites (LDH, 52.7% reduction). Collectively, these results suggest that pharmacological Rac1 inhibition ameliorates the severity of pancreatitis and pancreatitis-associated lung injury through the reduction of pancreatic acinar damage induced by pathological digestive enzyme secretion and overproduction of ROS.

Topics: Aminoquinolines; Amylases; Animals; Calcium; Cell Membrane; Ceruletide; Cholagogues and Choleretics; Cholecystokinin; Cytosol; Disease Models, Animal; Dose-Response Relationship, Drug; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Neuropeptides; Pancreatitis; Pyrimidines; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Reactive Oxygen Species; Severity of Illness Index

Obesity is a risk factor for acute pancreatitis (AP), but the molecular mechanism remains unclear. Adiponectin, an adipose tissue-derived secretory factor, has anti-inflammatory properties in addition to various biological functions, and its plasma concentrations are reduced in obese subjects. However, the role of adiponectin in AP has not been investigated.. To determine the effects of adiponectin on AP.. We investigated the effects of adiponectin on experimental AP by using adiponectin-knockout (APN-KO) mice and adenovirus-mediated adiponectin over-expression. AP was induced by 10 hourly intraperitoneal injections of low-dose caerulein (10 microg/kg) after 2 week feeding of normal chow or a high-fat diet (HFD) in wild-type (WT) and APN-KO mice. We evaluated the severity of AP biochemically and morphologically.. Low-dose caerulein treatment did not induce pancreatic damage in either WT or APN-KO mice under normal chow feeding. APN-KO mice, but not WT mice, fed a HFD and then treated with caerulein developed pancreatic damage and inflammation, accompanied by increased macrophage/neutrophil infiltration and upregulation of pro-inflammatory mediators such as tumour necrosis factor alpha in the pancreas. Adenovirus-mediated over-expression of adiponectin attenuated the severity of HFD/caerulein-induced AP in APN-KO mice.. Adiponectin plays a protective role in caerulein-induced AP in HFD-fed mice.

Topics: Acute Disease; Adiponectin; Adipose Tissue; Animals; Ceruletide; Dietary Fats; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Obesity; Pancreatitis; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

This study was undertaken to examine the cause of variation in determined values of myeloperoxidase activity from sequestered neutrophils in pancreas and lungs during pancreatitis and to develop a reproducible method for the extraction and measurement of myeloperoxidase in these tissues.. We measured myeloperoxidase in pancreatic and lung homogenates at different steps and evaluated the extent of inhibitory activity by measuring enzyme activity in the presence of homogenates from normal lungs and pancreata. To remove inhibitory activity from the homogenates, different methods like heat inactivation, inclusion of catalase inhibitor, and membranous pellet washing were evaluated.. Significant myeloperoxidase inhibitory activity was observed in pancreatic and lung homogenates, which could be effectively removed by the newly developed protocol. In extracts, myeloperoxidase activity can be determined by a spectrophotometric method, which is not only reproducible but is also adaptable for use in a plate reader or an autoanalyzer. Using this method, we studied the pattern of neutrophil sequestration over time in both pancreatic and lung tissue during caerulein-induced pancreatitis.. Myeloperoxidase inhibition in the pancreas and lungs contributes to the variation observed in measurement of the enzyme.

Topics: Animals; Ceruletide; Disease Models, Animal; Lung; Male; Mice; Neutrophils; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Wistar; Reproducibility of Results; Spectrophotometry; Time Factors

Obesity is clearly an independent risk factor for increased severity of acute pancreatitis (AP), although the mechanisms underlying this association are unknown. Adipokines (including leptin and adiponectin) are pleiotropic molecules produced by adipocytes that are important regulators of the inflammatory response. We hypothesized that the altered adipokine milieu observed in obesity contributes to the increased severity of pancreatitis. Lean (C57BL/6J), obese leptin-deficient (LepOb), and obese hyperleptinemic (LepDb) mice were subjected to AP by six hourly intraperitoneal injections of cerulein (50 microg/kg). Severity of AP was assessed by histology and by measuring pancreatic concentration of the proinflammatory cytokines IL-1beta and IL-6, the chemokine MCP-1, and the marker of neutrophil activation MPO. Both congenitally obese strains of mice developed significantly more severe AP than wild-type lean animals. Severity of AP was not solely related to adipose tissue volume: LepOb mice were heaviest; however, LepDb mice developed the most severe AP both histologically and biochemically. Circulating adiponectin concentrations inversely mirrored the severity of pancreatitis. These data demonstrate that congenitally obese mice develop more severe AP than lean animals when challenged by cerulein hyperstimulation and suggest that alteration of the adipokine milieu exacerbates the severity of AP in obesity.

Topics: Acute Disease; Adipokines; Adiponectin; Amylases; Animals; Blood Glucose; Body Weight; Ceruletide; Chemokines; Cytokines; Disease Models, Animal; Female; Insulin; Leptin; Lung; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Pancreas; Pancreatitis; Peroxidase; Severity of Illness Index

Autophagy is mostly a nonselective bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and killer of the cell depending on the stage of the disease or the surrounding cellular environment (for review see Cuervo, A.M. 2004. Trends Cell Biol. 14:70-77). We found that cytoplasmic vacuoles induced in pancreatic acinar cells by experimental pancreatitis were autophagic in origin, as demonstrated by microtubule-associated protein 1 light chain 3 expression and electron microscopy experiments. To analyze the role of macroautophagy in acute pancreatitis, we produced conditional knockout mice lacking the autophagy-related 5 gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice. Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.

Topics: Animals; Autophagy; Autophagy-Related Protein 5; Ceruletide; Enzyme Activation; Integrases; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Pancreas, Exocrine; Pancreatitis; Trypsin; Trypsinogen

Some recent studies indicate that cannabis may induce acute pancreatitis in humans and administration of anandamide increases the severity of acute pancreatitis; whereas another study exhibits some therapeutic effects in acute pancreatitis. Aim of the present study was to discover what is the reason for these opposite confusing results and to determine the role of sensory nerves in this effect. Acute pancreatitis was induced in rats by cerulein. Anandamide, an endogenous cannabinoid, was administered i.p. (1.5 micromol/kg) before or 2 h after cerulein administration. Stimulation of sensory nerves was performed by capsaicin (0.5 mg/kg s.c.). In rats treated with combination of anandamide plus capsaicin, capsaicin was given 10 min after each dose of anandamide. After the last injection of cerulein or 4 h later, the study was terminated. In our study we observed that stimulation of sensory nerves by capsaicin, before administration of cerulein, reduced the severity of acute pancreatitis. Anandamide, administered alone before cerulein, increased pancreatic damage in acute pancreatitis. Anandamide administered in combination with capsaicin, before cerulein, abolished the capsaicin-induced protective effect on the pancreas. Opposite effects were observed when capsaicin and anandamide were administered after injection of cerulein. Capsaicin increased the severity of acute pancreatitis, whereas anandamide reduced pancreatic damage and reversed the deleterious effect of capsaicin. We conclude that the effect of anandamide on the severity of acute pancreatitis depends on the phase of this disease. Administration of anandamide, before induction of pancreatitis, aggravates pancreatic damage; whereas anandamide administered after induction of pancreatitis, reduces the severity of acute pancreatitis. Sensory nerves are involved in the mechanism of this biphasic effect of anandamide.

Topics: Acute Disease; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Ceruletide; Disease Models, Animal; Drug Administration Schedule; Endocannabinoids; Male; Neurons, Afferent; Pancreatitis; Polyunsaturated Alkamides; Rats; Rats, Wistar; Severity of Illness Index; Time Factors

Some authors have found beneficial effect of statins in certain inflammatory conditions, but the effect of statins on acute pancreatitis is not yet defined.. The aim of this study was to evaluate the effect of simvastatin on an experimental model of mild and severe acute pancreatitis.. One hundred and one Wistar rats with cerulein or taurocholate-induced acute pancreatitis were used in this study.. The rats were divided into two groups: Group I (n=51) received two previously i.p. injections (18+/-2 and 3+/-1 hours) of simvastatin (200 microg/kg) and Group II (n=50) received two previously i.p. injections of saline. Both groups were subdivided into two subgroups: mild pancreatitis (cerulein-induced; IA, n=10; IIA, n=10) and severe pancreatitis (taurocholate-induced; IB, n=41; IIB, n=40).. The parameters evaluated were: pancreatic vascular permeability, tissue water content, histologic lesion, amylase serum levels in rats with mild pancreatitis (subgroups A); mortality rate, serum levels of IL-6, IL-10, amylase, pulmonary myeloperoxidase activity and ascitic levels of TNF-alpha in rats with severe pancreatitis (subgroups B).. Serum levels of IL-10 were significantly lower in the simvastatin-treated group as well as the myeloperoxidase activity. There was no significant difference in any of other studied parameters.. Simvastatin appears to reduce inflammatory cytokines and pulmonary neutrophilic activation in the severe acute pancreatitis model, but there is no significant effect on survival curve, in spite of a clear trend towards a better survival in the simvastatin group.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-10; Interleukin-6; Lung; Male; Pancreatitis; Peroxidase; Rats; Simvastatin; Survival Rate; Taurocholic Acid; Tumor Necrosis Factor-alpha

To evaluate the systemic effects of moderate hypothermia (MH) and the timing of induction on acute pancreatitis (AP) and endotoxemia in rats.. The effects of MH were compared in 4 groups, that is, sham group (38 degrees C), control group (38 degrees C), early MH group (32 degrees C on administration of lipopolysaccharide [LPS]), and delayed MH group (32 degrees C 1 hour after LPS). AP and endotoxemia were induced by intramuscular injection of caerulein and intraperitoneal injection of LPS.. Serum interleukin 6 (IL-6) in both MH groups was significantly lower than that in the control group at 3 hours. Serum interleukin 10 (IL-10) in the early MH group was significantly higher than those in the other 3 groups at 1 hour. IL-10/IL-6 ratios in both MH groups were significantly higher than that in the control group at 3 hours. Serum soluble intercellular adhesion molecule (sICAM-1) in both MH groups was significantly lower than that in the control group at 3 hours. Serum sICAM-1 in the early MH group was significantly lower than that in the delayed MH group. The tendency of pancreatic ICAM-1 was similar to that of serum sICAM-1.. Early induction of MH might be protective against pancreatic injury and systemic inflammation in AP and endotoxemia.

Topics: Animals; Ceruletide; Cytokines; Endotoxemia; Hypothermia, Induced; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-10; Interleukin-6; Lipopolysaccharides; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar

Topics: Acute Disease; Animals; Antioxidants; Ceruletide; Male; Oxidative Stress; Pancreatitis; Pyruvic Acid; Rats; Rats, Wistar

Acute pancreatitis (AP) is an inflammatory disease of the pancreas, which evolves in approximately 20% of the patients to a severe illness associated with a high mortality rate. In this study, we performed a comparative proteomic analysis of pancreatic tissue extracts from rats with AP and healthy rodent controls in order to identify changes in protein expression related to the pathobiological processes of this disease. Pancreatic extracts from diseased and controls rats were analyzed by 2-DE and MS/MS. A total of 125 proteins were identified from both samples. Comparative analysis allowed the detection of 42 proteins or protein fragments differentially expressed between diseased and control pancreas, some of them being newly described in AP. Interestingly, these changes were representative of the main pathobiological pathways involved in this disease. We observed activation of digestive proteases and increased expression of various inflammatory markers, including several members of the alpha-macroglobulin family. We also detected changes related to oxidative and cell stress responses. Finally, we highlighted modifications of 14-3-3 proteins that could be related to apoptosis regulation. These results showed the interest of proteomic analysis to identify changes characterizing pancreatic tissue damage and, therefore, to highlight new potential biomarkers of AP.

Topics: 14-3-3 Proteins; Acute Disease; Animals; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; Ceruletide; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Lectins, C-Type; Lithostathine; Male; Oxidative Stress; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Proteomics; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry

A noninvasive model of necrohemorrhagic pancreatitis induced by simultaneous intravenous cerulein/enterokinase (EK) infusion has recently been established in rats. The aim of the present study was to establish this new model in mice and to compare it with the rat model.. Male Balb/C mice (20 to 25 g) were used for the experiments. Pancreatitis was induced by simultaneous intravenous infusion of cerulein and EK. Controls were infused with either 0.9% NaCl, cerulein, or EK. Animals were humanely killed 6 hours after start of infusions. Pancreatic and pulmonary injury was assessed by histology, wet-to-dry weight ratio, and myeloperoxidase activity. Systemic cytokine, amylase, and lactate dehydrogenase (LDH) levels in blood were measured to assess pancreatic and systemic inflammatory response. To evaluate the role of protease activity in this model, trypsin, cathepsin B, and elastase activity were measured in pancreatic tissue. Survival experiments were performed to determine survival time and tissue injury in the later course of the disease.. Mice with simultaneous cerulein/EK infusion developed marked local and systemic organ injury compared with those animals who received cerulein or EK alone. Pancreatic and pulmonary injury increased with high concentrations of cerulein/EK infusions. Survival decreased in these animals. Whereas acinar cell apoptosis was an early finding, pancreatic necrosis was observed later in the course of the disease. Serum levels of LDH, interleukin (IL)-1 alpha, and IL-1 beta reflected cell damage and the systemic inflammatory response. Protease activity in pancreatic tissue was greatest in animals with simultaneous cerulein/EK infusion.. Using intravenous cerulein/EK infusions, a model of lethal acute pancreatitis has been established in mice. Major pancreatic edema, acinar cell apoptosis and necrosis, and pulmonary leukocyte sequestration are characteristic findings in this model. Although pancreatic injury was not as strong as in the rat model, this model may prove useful for future studies in transgenic mice.

Topics: Animals; Ceruletide; Disease Models, Animal; Enteropeptidase; Gastrointestinal Agents; Male; Mice; Mice, Inbred BALB C; Pancreatitis; Rats

During acute pancreatitis, protease-activated receptor 2 (PAR2) can be activated by interstitially released trypsin. In the mild form of pancreatitis, PAR2 activation exerts local protection against intrapancreatic damage, whereas, in the severe form of pancreatitis, PAR2 activation mediates some systemic complications. This study aimed to identify the molecular mechanisms of PAR2-mediated protective effects against intrapancreatic damage. A mild form of acute pancreatitis was induced by an intraperitoneal injection of caerulein (40 microg/kg) in rats. Effects of PAR2 activation on intrapancreatic damage and on mitogen-activated protein (MAP) kinase signaling were assessed. Caerulein treatment activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) within 15 min and maintained phosphorylation of ERK and JNK for 2 h in the rat pancreas. Although PAR2 activation by the pretreatment with PAR2-activating peptide (AP) itself increased ERK phosphorylation in rat pancreas, the same treatment remarkably decreased caerulein-induced activation of ERK and JNK principally by accelerating their dephosphorylation. Inhibition of ERK and JNK phosphorylation by the pretreatment with MAP/ERK kinase (MEK) or JNK inhibitors decreased caerulein-induced pancreatic damage that was similar to the effect induced by PAR2-AP. Notably, in caerulein-treated rats, PAR2-AP cotreatment highly increased the expression of a group of MAP kinase phosphatases (MKPs) that deactivate ERK and JNK. The above results imply that downregulation of MAP kinase signaling by MKP induction is a key mechanism involved in the protective effects of PAR2 activation on caerulein-induced intrapancreatic damage.

Topics: Animals; Butadienes; Ceruletide; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinase Phosphatases; Nitriles; Pancreatitis; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Signal Transduction

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. Although lysosomal hydrolases, such as cathepsin B, play a key role in intrapancreatic trypsinogen activation, it remains unclear where and how trypsinogen meets these lysosomal enzymes. Autophagy is an intracellular bulk degradation system in which cytoplasmic components are directed to the lysosome/vacuole by a membrane-mediated process. To analyze the role of autophagy in acute pancreatitis, we produced a conditional knockout mouse that lacks the autophagy-related (Atg) gene Atg5 in the pancreatic acinar cells. The severity of acute pancreatitis induced by cerulein is greatly reduced in these mice. In addition, Atg5-deficient acinar cells show a significantly decreased level of trypsinogen activation. These data suggest that autophagy exerts a detrimental effect in pancreatic acinar cells by activation of trypsinogen to trypsin. We propose a theory in which autophagy accelerates trypsinogen activation by lysosomal hydrolases under acidic conditions, thus triggering acute pancreatitis in its early stage.

Topics: Acute Disease; Animals; Autophagy; Autophagy-Related Protein 5; Ceruletide; Cytoplasm; Enzyme Activation; Mice; Mice, Knockout; Microtubule-Associated Proteins; Pancreatitis; Trypsinogen; Vacuoles

Pancreatitis is a severe debilitating disease with high morbidity and mortality. Treatment is mostly supportive, and until now there are no clinically useful strategies for anti-inflammatory therapy. Although omega-3 polyunsaturated fatty acids (n-3 PUFA) are known to have anti-inflammatory effects, the utility of these fatty acids in the alleviation of pancreatitis remained to be investigated. The aim of this study was to examine the effect of n-3 PUFA on both acute and chronic pancreatitis in a well-controlled experimental system. We used the fat-1 transgenic mouse model, characterized by endogenously increased tissue levels of n-3 PUFA, and their wild-type littermates to examine the effect of n-3 PUFA on both acute and chronic cerulein-induced pancreatitis. Disease activity and inflammatory status were assessed by both histology and molecular methods. In acute pancreatitis, fat-1 mice showed a trend towards decreased necrosis and significantly reduced levels of plasma IL-6 levels as well as reduced neutrophil infiltration in the lung. In chronic pancreatitis there was less pancreatic fibrosis and collagen content accompanied by decreased pancreatic stellate cell activation in the fat-1 animals with increased n-3 PUFA tissue levels as compared to wild-type littermates with high levels of omega-6 (n-6) PUFA in their tissues. Our data provide evidence for a reduction of systemic inflammation in acute pancreatitis and of tissue fibrosis in chronic pancreatitis by increasing the tissue content of omega-3 polyunsaturated fatty acids. These results suggest a beneficial potential for n-3 PUFA supplementation in acute and particularly chronic pancreatitis.

Topics: Animals; Ceruletide; Fatty Acids; Fatty Acids, Omega-3; Female; Inflammation; Interleukin-6; Male; Mice; Mice, Transgenic; Necrosis; Pancreatitis

Factors determining severity of acute pancreatitis (AP) are poorly understood. Oxidative stress causes acinar cell injury and contributes to the severity, whereas prophylactic probiotics ameliorate experimental pancreatitis. Our objective was to study how probiotics affect oxidative stress, inflammation, and acinar cell injury during the early phase of AP. Fifty-three male Sprague-Dawley rats were randomly allocated into groups: 1) control, 2) sham procedure, 3) AP with no treatment, 4) AP with probiotics, and 5) AP with placebo. AP was induced under general anesthesia by intraductal glycodeoxycholate infusion (15 mM) and intravenous cerulein (5 microg.kg(-1).h(-1), for 6 h). Daily probiotics or placebo were administered intragastrically, starting 5 days prior to AP. After cerulein infusion, pancreas samples were collected for analysis including lipid peroxidation, glutathione, glutamate-cysteine-ligase activity, histological grading of pancreatic injury, and NF-kappaB activation. The severity of pancreatic injury correlated to oxidative damage (r = 0.9) and was ameliorated by probiotics (1.5 vs. placebo 5.5; P = 0.014). AP-induced NF-kappaB activation was reduced by probiotics (0.20 vs. placebo 0.53 OD(450nm)/mg nuclear protein; P < 0.001). Probiotics attenuated AP-induced lipid peroxidation (0.25 vs. placebo 0.51 pmol malondialdehyde/mg protein; P < 0.001). Not only was AP-induced glutathione depletion prevented (8.81 vs. placebo 4.1 micromol/mg protein, P < 0.001), probiotic pretreatment even increased glutathione compared with sham rats (8.81 vs. sham 6.18 miccromol/mg protein, P < 0.001). Biosynthesis of glutathione (glutamate-cysteine-ligase activity) was enhanced in probiotic-pretreated animals. Probiotics enhanced the biosynthesis of glutathione, which may have reduced activation of inflammation and acinar cell injury and ameliorated experimental AP, via a reduction in oxidative stress.

Topics: Animals; Apoptosis; Ceruletide; Glutathione; Glycodeoxycholic Acid; Male; Oxidative Stress; Pancreatitis; Probiotics; Rats; Rats, Sprague-Dawley; Specific Pathogen-Free Organisms

The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

Topics: Animals; Carbachol; Ceruletide; Cholecystokinin; Cholinergic Agonists; Mice; NF-kappa B; Pancreas, Exocrine; Pancreatitis; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Protein Kinases; Rats

To investigate the mechanism of Chaiqin Chengqi Decoction (CQCQD), a compound of traditional Chinese herbal medicine, acting on the pancreatic acinar cell calcium overload in rats with acute pancreatitis (AP).. A total of 30 SD rats were randomly divided into normal control group, untreated group and CQCQD group (n=10, respectively). AP was induced in rats by caerulein (5x50 mug/kg) intraperitoneal injection within 4 h. The pancreatic tissue SERCA1 and SERCA2 mRNA expressions were detected by fluorescent quantization polymerase chain reaction method; intracellular calcium fluorescence intensity (FI) of pancreatic acinar cells and the pancreatic pathological score were measured by laser scanning confocal microscopy and light microscopy respectively.. There were no SERCA1 mRNA expressions in pancreatic acinar cells of rats in the normal control group and the untreated group. The expression of pancreatic SERCA2 mRNA in the untreated group was down-regulated compared with that in the normal control group (expression ratio=0.536; P=0.001); the expression of pancreatic SERCA2 mRNA in the CQCQD group was up-regulated compared with that in the untreated group (expression ratio=2.00; P=0.012). The pancreatic pathological score in the CQCQD group was lower than that in the untreated group and the FI of Ca(2+) was also lower.. CQCQD can up-regulate the expression of pancreatic SERCA2 mRNA, release the calcium overload, and hence reduce the pathological changes in pancreatic tissue.

Topics: Acute Disease; Animals; Calcium; Calcium Channel Blockers; Ceruletide; Drugs, Chinese Herbal; Male; Pancreas, Exocrine; Pancreatitis; Random Allocation; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases

Bacterial endotoxin (lipopolysaccharide, LPS), is the component of the cellular wall of Gram negative bacteria. Endotoxemia (sepsis) could produce multiorgan failure and could be particularly danger in the early period of life. The effects of endotoxemia induced in the neonatal period of life on the pancreatic secretory function and on pancreatic defense of adult organism have not been investigated yet. To induce endotoxemia suckling rats (30 g) have been injected intraperitoneally with LPS from E. coli (5, 10 or 15 mg/kg-day) during 5 consecutive days. Three months later in these animals (300 g) the studies on pancreatic secretion and acute pancreatitis were carried out. In the adult rats, which have been subjected in infancy to endotoxemia, basal pancreatic secretion was unaffected, whereas amylase secretions stimulated by caerulein or by diversion of pancreatic-biliary juice to the exterior were significantly, and dose-dependently reduced as compared to the untreated control. In the rats pretreated with LPS in the suckling period of life caerulein-induced amylase release from isolated pancreatic acini was significantly decreased, and dose-dependent reduction of mRNA signal for CCK1 receptor on pancreatic acini have been observed. Caerulein infusion (25 microg/kg) produced caerulein induced pancreatitis (AP) in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS (10 or 15 mg/kg-day x 5 days) all manifestations of AP have been reduced. In these animals acute inflammatory changes of pancreatic tissue have been significantly diminished. Pancreatic weight and plasma lipase activity, have been markedly decreased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in an antioxidative enzyme; SOD concentration was reversed and accompanied by significant reduction of lipid peroxidation products; MDA+ 4 HNE in the pancreatic tissue.. 1/ neonatal endotoxemia reduces gene expression for CCK1 receptor and could produce impairment of the exocrine pancreatic function at adult age; 2/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age and this effect could be related to the increased concentration of antioxidative enzyme SOD in the pancreatic tissue.

Topics: Actins; Acute Disease; Amylases; Animals; Animals, Newborn; Animals, Suckling; Ceruletide; Cytokines; Dose-Response Relationship, Drug; Endotoxemia; Heat-Shock Proteins; Lipase; Lipid Peroxidation; Lipopolysaccharides; Pancreas; Pancreatitis; Rats; Receptor, Cholecystokinin A; Superoxide Dismutase

In patients suffering from acute pancreatitis, the pathogenesis is not completely understood, and several recent studies in vitro suggested that heat shock proteins might play an important role in cell signaling. To investigate the possible role of extracellular heat shock protein 70 (Hsp70) in pancreatitis, toll-like receptor-4 (TLR4)-deficient and wild-type mice were administered with exogenous Hsp70 during the course of cerulein-induced pancreatitis (CIP).. Acute pancreatitis was induced by 5 intraperitoneal injections of cerulein at hourly intervals, and then treated with recombinant Hsp70 through the caudal vein 4 hours after the start of cerulein injections. Subsequently serum amylase and serum cytokines levels were detected. Histologic alteration of the pancreas was evaluated. Tumor necrosis factor alpha (TNF-alpha) concentrations and myeloperoxidase (MPO) activity in both pancreas and lungs were analyzed. The nuclear factor kappa B (NF-kappaB) activation in pancreatic tissue was measured using a sensitive RelA enzyme-linked immunosorbent assay.. Treatment with recombinant Hsp70 to wild-type mice in CIP resulted in significant aggravation of inflammation in pancreas, elevated levels of serum cytokines, up-regulation of pulmonary MPO activity and increase of lung tissues TNF-alpha concentrations. In contrast, treatment with Hsp70 to TLR4-deficient mice had little effect on serum cytokines levels, pancreatic inflammation, pulmonary MPO activity and TNF-alpha concentrations.. The results suggest that extracellular Hsp70 might induce systemic inflammatory response syndrome (SIRS)-like response in vivo and TLR4 might be involved in the Hsp70-mediated activation of inflammatory reaction in the progression of CIP without infection.

Topics: Acute Disease; Animals; Ceruletide; Female; HSP70 Heat-Shock Proteins; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Systemic Inflammatory Response Syndrome; Toll-Like Receptor 4

To investigate the effect of Gardenia jasminoides (GJ) on cerulein-induced acute pancreatitis (AP) in mice.. C57BL/6 mice weighing 18-20 g were divided into three groups. (1) Normal saline-treated group, (2) treatment with GJ at a dose of 0.1 g/kg, (3) treatment with GJ at a dose of 1 g/kg. GJ was administered orally (n = 6 per group) for 1 wk. Three hours later, the mice were given an intraperitoneal injection of cerulein (50 microg/kg), a stable cholecystokinin (CCK) analogue, every hour for a total of 6 h as described previously. The mice were sacrificed at 6 h after completion of cerulein injections. Blood samples were obtained to determine serum amylase, lipase and cytokine levels. The pancreas was rapidly removed for morphologic examination and scoring. A portion of pancreas was stored at -70 degree and prepared for the measurement of tissue myeloperoxidase (MPO) activity, an indicator of neutrophil sequestration, and for reverse-transcriptase PCR (RT-PCR) and real-time PCR measurements.. Treatment with GJ decreased significantly the severity of pancreatitis and pancreatitis-associated lung injury. Treatment with GJ attenuated the severity of AP compared with saline-treated mice, as shown by reduction in pancreatic edema, neutrophil infiltration, serum amylase and lipase levels, serum cytokine levels, and mRNA expression of multiple inflammatory mediators.. These results suggest that GJ attenuated the severity of AP as well as pancreatitis-associated lung injury.

Topics: Acute Disease; Administration, Oral; Amylases; Animals; Anti-Inflammatory Agents; Body Weight; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Gardenia; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Lipase; Lung; Lung Injury; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Organ Size; Pancreas; Pancreatitis; Peroxidase; Plant Extracts; RNA, Messenger; Tumor Necrosis Factor-alpha

The prognosis of acute pancreatitis (AP) depends upon the degree of pancreatic necrosis and the intensity of multisystem organ failure. The liver contributes to the systemic manifestations of AP by releasing some cytokines. This study was undertaken to examine comparative effects of melatonin, antioxidant mixture containing L(+)-ascorbic acid and N-acetyl cysteine, pentoxifylline and L-arginine on hepatic damage induced by caerulein-pancreatitis.. The liver specimens of all groups showed histopathological alterations such as hepatocyte necrosis, intracellular vacuolization, vascular congestion, sinusoidal dilatation and inflammatory infiltration. TEM studies revealed vacuole formation, mitochondrial degeneration, lysosome accumulation and necrosis. The mean histopathological score of the caerulein group was significantly different from that of each treatment group.. L-Arginine and antioxidant administration be important for reducing hepatic damage induced by AP. Improvement of hepatic damage, in turn, might be beneficial for the prognosis of AP.

Topics: Acetylcysteine; Acute Disease; Animals; Antioxidants; Arginine; Ascorbic Acid; Ceruletide; Female; Liver Diseases; Melatonin; Pancreatitis; Pentoxifylline; Rats; Rats, Wistar; Statistics, Nonparametric

Heat shock proteins (HSPs), induced by a variety of stresses, are known to protect against cellular injury. Recent studies have demonstrated that prior beta-adrenergic stimulation as well as thermal or culture stress induces HSP70 expression and protects against cerulein-induced pancreatitis. The goal of our current studies was to determine whether or not a non-thermal, chemical stressor like sodium arsenite also upregulates HSP70 expression in the pancreas and prevents secretagogue-induced trypsinogen and NF-kappaB activation. We examined the effects of sodium arsenite preadministration on the parameters of cerulein-induced pancreatitis in rats and then monitored the effects of preincubating pancreatic acini with sodium arsenite in vitro. Our results showed that sodium arsenite pretreatment induced HSP70 expression both in vitro and in vivo and significantly ameliorated the severity of cerulein-induced pancreatitis, as evidenced by the markedly reduced degree of hyperamylasemia, pancreatic edema, and acinar cell necrosis. Sodium arsenite pretreatment not only inhibited trypsinogen activation and the subcellular redistribution of cathepsin B, but also prevented NF-kappaB translocation to the nucleus by inhibiting the IkappaBalpha degradation both in vivo and in vitro. We also examined the effect of sodium arsenite pretreatment in a more severe model of pancreatitis induced by L-arginine and found a similarly protective effect. Based on our observations we conclude that, like thermal stress, chemical stressors such as sodium arsenite also induce HSP70 expression in the pancreas and protect against acute pancreatitis. Thus, non-thermal pharmacologically induced stress can help prevent or treat pancreatitis.

Topics: Actins; Adenosine Triphosphate; Animals; Arginine; Arsenites; Cell Survival; Ceruletide; Dose-Response Relationship, Drug; Enzyme Activation; HSP70 Heat-Shock Proteins; NF-kappa B; Pancreas; Pancreatitis; Protein Transport; Rats; Rats, Wistar; Sodium Compounds; Time Factors; Trypsin; Trypsinogen; Up-Regulation

The proteins expressed in pancreatic acinar cells during the initiation of acute pancreatitis may determine the severity of the disease. Cerulein pancreatitis is one of the best characterized models for acute pancreatitis. Present study aims to determine the differentially expressed proteins in cerulein-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. Rat pancreatic acinar AR42J cells were treated with 10(-8)M cerulein for 12h. The protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were compared between the cells treated without cerulein and those with cerulein. The changed proteins were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. As a result, 10 proteins (Orp150 protein, protein disulfide isomerase related protein, dnaK-type molecular chaperone hsp72-ps1, mitochondrial glutamate dehydrogenase, similar to chaperonin containing TCP-1 beta subunit, RuvB-like protein 1, heterogeneous nuclear ribonucleoprotein H1, aldehyde reductase 1, triosephosphate isomerase 1, peroxiredoxin 2) were up-regulated while four proteins (vasolin-containing protein, 78 kDa glucose-regulated protein precursor, heat shock protein 8, adenosylhomocysteinase) were down-regulated by cerulein in pancreatic acinar AR42J cells. These proteins are related to chaperone, cell defense mechanism against oxidative stress or DNA damage, anti-apoptosis and energy generation. The differentially expressed proteins by ceruein share their functional roles in pancreatic acinar cells, suggesting the possible involvement of oxidative stress, DNA damage, and anti-apoptosis in pathogenesis of acute pancreatitis. Proteins involved in cellular defense mechanism and energy production may protect pancreatic acinar cells during the development of pancreatitis.

Topics: Acute Disease; Animals; Cell Line, Tumor; Ceruletide; Disease Models, Animal; Down-Regulation; Electrophoresis, Gel, Two-Dimensional; Pancreatitis; Protein Array Analysis; Proteins; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Up-Regulation

C/EBP homologous protein (CHOP) is one of the main mediating factors in the ER stress pathway. To elucidate the role of the ER stress-CHOP pathway in experimental pancreatitis, wild-type (Chop(+/+)) and Chop deficient (Chop(-/-)) mice were administered cerulein, a cholecystokinin analogue, or both cerulein and lipopolysaccharide (LPS). In cerulein-induced acute pancreatitis, ER stress, serum amylase elevation and histological interstitial edema were induced. However, there was no remarkable activation downstream of the CHOP pathway regardless of the presence or absence of CHOP. Whereas, in the cerulein and LPS model, inflammation-associated caspases (caspase-11, caspase-1) and IL-1beta, but not apoptosis-associated caspases, were activated. In Chop(-/-) mice, the expression levels of these mediators returned to basal levels resulting in a milder pancreatitis and decreased serum amylase level. These results indicated that the ER stress-CHOP pathway has a pivotal role in the acceleration of pancreatitis through the induction of inflammation-associated caspases and IL-1beta.

Topics: Amylases; Animals; Apoptosis; Base Sequence; Blotting, Western; Ceruletide; Disease Models, Animal; Inflammation; Interleukin-1beta; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Pancreatitis; Transcription Factor CHOP

The Dmbt1 gene encodes alternatively spliced glycoproteins that are either membrane-associated or secreted epithelial products. Functions proposed for Dmbt1 include it being a tumor suppressor, having roles in innate immune defense and inflammation, and being a Golgi-sorting receptor in the exocrine pancreas. The heavily sulfated membrane glycoprotein mucin-like glycoprotein (Muclin) is a Dmbt1 product that is strongly expressed in organs of the gastrointestinal (GI) system. To explore Muclin's functions in the GI system, the Dmbt1 gene was targeted to produce Muclin-deficient mice. Muclin-deficient mice have normal body weight gain and are fertile. The Muclin-deficient mice did not develop GI tumors, even when crossed with mice lacking the known tumor suppressor p53. When colitis was induced by dextran sulfate sodium, there was no significant difference in disease severity in Muclin-deficient mice. Also, when acute pancreatitis was induced with supraphysiological caerulein, there was no difference in disease severity in the Muclin-deficient mice. Exocrine pancreatic function was impaired, as measured by attenuated neurohormonal-stimulated amylase release from Muclin-deficient acinar cells. Also, by [(35)S]Met/Cys pulse-chase analysis, traffic of newly synthesized protein to the stimulus-releasable pool was significantly retarded in Muclin-deficient cells compared with wild type. Thus Muclin deficiency impairs trafficking of regulated proteins to a stimulus-releasable pool in the exocrine pancreas.

Topics: Amylases; Animals; Blotting, Western; Calcium-Binding Proteins; Ceruletide; Colitis; Dextran Sulfate; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Gastrointestinal Neoplasms; Gastrointestinal Tract; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucins; Pancreas; Pancreatitis; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice.. Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays.. Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain.. Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

Topics: Acute Disease; Amyloid Precursor Protein Secretases; Animals; beta Catenin; Cell Line, Tumor; Ceruletide; Dibenzazepines; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatic Neoplasms; Pancreatitis; Receptor, Notch1; Regeneration; Signal Transduction; Wnt Proteins

Glutathione depletion is a key factor in the development of acute pancreatitis. Our aim was to study the regulation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, in edematous or necrotizing pancreatitis in rats. Glutathione levels were kept low in necrotizing pancreatitis for several hours, with no increase in protein or mRNA levels of glutamate cysteine ligase subunits, despite binding of RNA polymerase II to their promoters and coding regions. The survival signal pathway mediated by ERK and c-MYC was activated, and c-MYC was recruited to the promoters. The failure in gene up-regulation seems to be due to a marked increase in cytosolic ribonuclease activity. In contrast, in edematous pancreatitis glutathione levels were depleted and rapidly restored, and protein and mRNA expression of glutamate cysteine ligase increased markedly due to enhanced transcription mediated by recruitment of c-MYC, NF-kappaB, and SP-1 to the promoters. No increase in cytosolic ribonuclease activity was found in this case. We propose a novel pathophysiological mechanism to differentiate necrotizing from edematous pancreatitis, which is the inefficient up-regulation of glutamate cysteine ligase caused by increased cytosolic ribonuclease activity in the severe form of the disease. This mechanism would abrogate a rapid recovery of glutathione levels.

Topics: Animals; Ceruletide; Edema; Gene Expression Regulation, Enzymologic; Glutamate-Cysteine Ligase; Glutathione; Male; Pancreatitis; Pancreatitis, Acute Necrotizing; Rats; Rats, Wistar; Ribonucleases; RNA Polymerase II; RNA Stability; RNA, Messenger; Taurocholic Acid; Transcription Factors; Up-Regulation

Protein tyrosine phosphatases (PTPs) are important regulators of cell functions but data on different PTP expression and dynamics in acute pancreatitis (AP) are very scarce. Additionally, both c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (ERK1/2), together with intracellular cAMP levels in inflammatory cells, play an essential role in AP. In this study we have detected an increase in PTP SHP-1 and SHP-2 in the pancreas at the level of both protein and mRNA as an early event during the development of Cerulein (Cer)-induced AP in rats. Nevertheless, while SHP-2 protein returned to baseline levels in the intermediate or later phases of AP, SHP-1 protein expression remained increased throughout the development of the disease. The increase in SHP-2 protein expression was associated with changes in its subcellular distribution, with higher percentages located in the fractions enriched in lysosomes+mitochondria or microsomes. Furthermore, while the increase in SHP-2 protein was also observed in sodium-taurocholate duct infusion or bile-pancreatic duct obstruction AP, that of SHP-1 was specific to the Cer-induced model. Neutrophil infiltration did not affect the increase in SHP-1 protein, but favoured the return of SHP-2 protein to control levels, as indicated when rats were rendered neutropenic by the administration of vinblastine sulfate. Inhibition of JNK and ERK1/2 with SP600125 pre-treatment further increased the expression of both SHP-1 and SHP-2 proteins in the early phase of Cer-induced AP, while the inhibition of type IV phosphodiesterase with rolipram only suppressed the increase in SHP-2 protein expression during the same phase. Our results show that AP is associated with increases in the expression of SHP-1 and SHP-2 and changes in the dynamics of SHP-2 subcellular distribution in the early phase of Cer-induced AP. Finally, both JNK and ERK1/2 and intracellular cAMP levels are able to modulate the expression of these PTPs.

Topics: Animals; Anthracenes; Ceruletide; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 3; Neutrophils; Pancreatitis; Phosphodiesterase 4 Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Rats; Rats, Wistar; Rolipram

Trypsinogen activation and inflammation are early events in acute pancreatitis. This experimental study aimed to show the effects of dexamethasone on them.. Cerulein and taurocholate pancreatitis were induced in 2 groups of 12 Wistar rats each. Six animals per group were injected with dexamethasone 1 h prior to the induction of acute pancreatitis. Amylase, phospholipase A2, TNF-alpha, IL-6, IL-10, alpha2-antiplasmin in plasma and trypsinogen activation peptide (TAP) in urine were measured in healthy rats, then 0.5 and 6 h after pancreatitis induction. A severity score based on edema, necrosis and ascites was calculated at 6 h. TNF-alpha, IL-6 and IL-10 were measured 0.5 h after laparotomy in a control sham-operated group of 6 rats.. Inflammatory markers increased early in the course of both mild and severe acute pancreatitis and were significantly lowered by dexamethasone. The severity score was higher in taurocholate than in cerulein pancreatitis. It was significantly decreased by dexamethasone only in rats with mild pancreatitis. TAP remained unchanged in mild pancreatitis compared to healthy animals but increased late in the course of taurocholate pancreatitis. Trypsinogen activation was not affected by dexamethasone at all.. Inflammation occurred earlier than the increase in urinary TAP in severe pancreatitis in rats. Dexamethasone inhibited inflammation but had no influence on TAP levels in experimental mild and severe acute pancreatitis.

Topics: Animals; Anti-Inflammatory Agents; Ceruletide; Dexamethasone; Female; Oligopeptides; Pancreatitis; Rats; Rats, Wistar; Taurocholic Acid; Trypsinogen

Acute pancreatitis (AP) is associated with significant morbidity and mortality; however, there is no specific treatment for this disease. A novel salivary tripeptide analog, feG, reduces inflammation in several different animal models of inflammation. The aims of this study were to determine whether feG reduced the severity of AP and modifies the expression of pancreatic ICAM-1 mRNA during AP in a mouse model. AP was induced in mice by hourly (x12) intraperitoneal injections of caerulein. A single dose of feG (100 microg/kg) was coadministered with caerulein either at time 0 h (prophylactic) or 3 h after AP induction (therapeutic). Plasma amylase and pancreatic MPO activities and pancreatic ICAM-1 mRNA expression (by RT-PCR) were measured. Pancreatic sections were histologically assessed for abnormal acinar cells and interstitial space. AP induction produced a sevenfold increase in plasma amylase, a tenfold increase in pancreatic MPO activity, and a threefold increase in interstitial space, and 90% of the acinar cells were abnormal. Prophylactic treatment with feG reduced the AP-induced plasma amylase activity by 45%, pancreatic MPO by 80%, the proportion of abnormal acinar cells by 30%, and interstitial space by 40%. Therapeutic treatment with feG significantly reduced the AP-induced abnormal acinar cells by 10% and the interstitial space by 20%. Pancreatic ICAM-1 mRNA expression was upregulated in AP and was reduced by 50% with prophylactic and therapeutic treatment with feG. We conclude that feG ameliorates experimental AP acting at least in part by modulating ICAM-1 expression in the pancreas.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Ceruletide; Disease Models, Animal; Injections, Intraperitoneal; Intercellular Adhesion Molecule-1; Male; Mice; Oligopeptides; Pancreas; Pancreatitis; Peroxidase; RNA, Messenger; Severity of Illness Index; Time Factors

Suprastimulation of pancreatic acini is a well-known model for pancreatitis, and it is characterized by actin reorganization and cell blebbing. Currently, however, the mechanisms underlying regulation of these aberrant cytoskeletal and membrane dynamics and how they contribute to cell injury are unclear. We observed that suprastimulation results in a rapid activation of Src and relocalization of the actin-binding protein cortactin from the apical to the basolateral domain at the necks of membrane blebs. Furthermore, Src-mediated cortactin tyrosine phosphorylation was markedly increased after suprastimulation. Pretreatment of acini with Src inhibitors or expression of a cortactin tyrosine phospho-inhibitory mutant reduced actin redistribution and bleb formation induced by suprastimulation in vitro. Importantly, inhibition of Src activity in rat models of suprastimulation-induced pancreatitis substantially reduced disease severity, as indicated by a reduction in serum amylase and pancreatic edema and a striking improvement in tissue histology. These findings indicate a novel, disease-relevant role for Src-mediated cortactin phosphorylation in aberrant reorganization of the actin cytoskeleton, a mechanism that is likely to have implications in other types of cell injury. In addition, they suggest a potential use for Src inhibitors as an approach to reduce cell injury.

Topics: Actins; Animals; Cell Surface Extensions; Ceruletide; Cortactin; Cytoskeleton; Disease Models, Animal; Enzyme Activation; Male; Mutant Proteins; Pancreas, Exocrine; Pancreatitis; Phosphorylation; Phosphotyrosine; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins pp60(c-src); Rats; Rats, Sprague-Dawley

Protease inhibitors showed protective effects on animal models of acute pancreatitis when administered before induction of pancreatitis, and results when administered after induction are uncertain. We assessed the effects of nafamostat mesilate in a mouse model of cerulein-induced pancreatitis comparing results of before and after induction.. Cerulein was injected to mice intraperitoneally to induce pancreatitis, and they received intravenous nafamostat mesilate before and after induction. Serum concentrations of amylase and lipase, histological changes, and tissue expression of myeloperoxidase were measured. In addition, tissue activation of p38 mitogen-activated protein kinase (MAPK) and interleukin-6 was evaluated.. Development of pancreatitis was prevented by pretreatment with nafamostat mesilate. However, such effect was not shown when given after induction, although it partially suppressed myeloperoxidase expression and infiltration of inflammatory cells. Tissue expression of phospho-p38 MAPK was prominent in mice with pancreatitis and suppressed by pretreatment with nafamostat mesilate. Interleukin-6 expression was not influenced by either cerulein or nafamostat mesilate.. The development of pancreatitis was prevented by treating mice with nafamostat mesilate before induction, however, this finding was not observed if administered after injection of cerulein. Pretreatment with nafamostat mesilate suppressed activation of p38 MAPK.

Topics: Amylases; Animals; Benzamidines; Ceruletide; Female; Guanidines; Interleukin-6; Lipase; Mice; Mice, Inbred BALB C; p38 Mitogen-Activated Protein Kinases; Pancreatitis; Peroxidase; Protease Inhibitors

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Pancreas; Pancreatitis; Rats; RNA, Messenger; Time Factors; Toll-Like Receptor 9; Up-Regulation

This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 microg/kg caerulein, L-arginine used for NO induction and N(omega)-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.

Topics: Acute Disease; Amylases; Animals; Arginine; Ceruletide; Female; Gene Expression Regulation; Lipase; Microscopy, Electron, Transmission; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidative Stress; Pancreatitis; Rats; Rats, Sprague-Dawley

Activation of nuclear factor kappaB (NF-kappaB) and caspases may greatly amplify inflammation and cell damage in addition to that directly exerted by free radicals. Since reactive oxygen species (ROS) are involved in acute pancreatitis, we studied whether the administration of chondroitin-4-sulphate (C4S), in addition to its antioxidant activity, was able to modulate NF-kappaB and caspase activation in an experimental model of caerulein-induced acute pancreatitis in mice. Hyperstimulating doses of caerulein (50 microg/ kg), five injections per mouse given at hourly intervals produced the following: high serum lipase and amylase activity; lipid peroxidation, evaluated by 8-isoprostane concentrations; loss of antioxidant defenses such as glutathione reductase (GR) activity; NF-kappaB activation and loss of cytoplasmic IkappaBalpha protein; increases in tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), caspase-3, and caspase-7 gene expression and their related protein; accumulation and activation of neutrophils in the damaged tissue, evaluated by elastase (ELA) determination; and pancreatic injury, evaluated by histologic analysis. Pretreatment of mice with different doses of C4S, given 1 hr before caerulein injections and 1 and 2 hrs after the last caerulein injection, reduced lipid peroxidation, inhibited NF-kappaB translocation and cytoplasmic IkappaBalpha protein loss, decreased TNF-alpha, IL-6, and caspase gene expression and their related protein levels, limited endogenous antioxidant depletion, and reduced tissue neutrophils accumulation and tissue damage. Since molecules with antioxidant activity can block NF-kappaB and apoptosis activation, we suggest that C4S administration is able to block NF-kappaB and caspase activation by reducing the oxidative burst.

Topics: Animals; Apoptosis; Ceruletide; Chondroitin Sulfates; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Glutathione Reductase; Interleukin-6; Lipase; Male; Mice; NF-kappa B; Oxidative Stress; Pancreatitis; Tumor Necrosis Factor-alpha

To investigate the effect of Chaiqinchengqi decoction (CQCQD) on sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) mRNA expression of pancreatic tissues in acute pancreatitis (AP) rats.. Thirty Sprague-Dawley (SD) rats were randomized into control group, AP group and CQCQD group (n = 3 x 10). The rats in the CQCQD group were intragastrically administered with CQCQD (10 mL/kg every 2 h) after induction of AP by intraperitoneal injection of caerulein (50 microg/kg.h x 5) within 4 h. At 6 h after the induction of AP model, pancreatic tissues were collected for the pathological observation, mRNA extraction for determination of SERCA1 and SERCA2 mRNA expression or pancreatic acinar cell isolation for measurement of fluorescence intensity (FI) of intracellular calcium ion concentration [Ca2+]i.. There was no expression of pancreatic SERCA1 mRNA in the control group and the AP group. The expression of pancreatic SERCA2 mRNA in the AP group was down-regulated (expression ratio = 0.536; P = 0.001) compared with the control group, while that in the CQCQD group was up-regulated (expression ratio = 2.00; P = 0.012) compared with AP group. The FI of intracellular [Ca2+] of pancreatic acinar cells in the AP group (138.2 +/- 23.1) was higher than the C group (111.0 +/- 18.4) and the CQCQD group (118.7 +/- 15.2 ) (P < 0.05) and the pancreatic pathological score in the CQCQD group was lower than that in the AP group (5.7 +/- 1.9 vs 9.2 +/- 2.7, P < 0.05).. CQCQD can up-regulate the expression of SERCA2 mRNA of pancreatic tissues, reduce intracellular calcium overload and relieve pancreatic tissue lesions.

Topics: Acute Disease; Animals; Calcium; Ceruletide; Disease Models, Animal; Drugs, Chinese Herbal; Gene Expression Regulation, Enzymologic; Male; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases

Hydrogen sulphide (H(2)S), a novel gasotransmitter, has been recognized to play an important role in inflammation. Cystathionine-gamma-lyase (CSE) is a major H(2)S synthesizing enzyme in the cardiovascular system and DL-propargylglycine (PAG) is an irreversible inhibitor of CSE. Substance P (SP), a product of preprotachykinin-A (PPT-A) gene, is a well-known pro-inflammatory mediator which acts principally through the neurokinin-1 receptor (NK-1R). We have shown an association between H(2)S and SP in pulmonary inflammation as well as a pro-inflammatory role of H(2)S and SP in acute pancreatitis. The present study was aimed to investigate the interplay between pro-inflammatory effects of H(2)S and SP in a murine model of caerulein-induced acute pancreatitis. Acute pancreatitis was induced in mice by 10 hourly intraperitoneal injections of caerulein (50 (g/kg). PAG (100 mg/kg, i.p.) was administered either 1 hr before (prophylactic) or 1 hr after (therapeutic) the first caerulein injection. PAG, given prophylactically as well as therapeutically, significantly reduced plasma H(2)S levels and pancreatic H(2)S synthesizing activities as well as SP concentrations in plasma, pancreas and lung compared with caerulein-induced acute pancreatitis. Furthermore, prophylactic as well as therapeutic administration of PAG significantly reduced PPT-A mRNA expression and NK-1R mRNA expression in both pancreas and lung when compared with caerulein-induced acute pancreatitis. These results suggest that the pro-inflammatory effects of H(2)S may be mediated by SP-NK-1R pathway in acute pancreatitis.

Topics: Acute Disease; Alkynes; Animals; Ceruletide; Cystathionine beta-Synthase; Glycine; Hydrogen Sulfide; Inflammation; Male; Mice; Pancreatitis; Random Allocation; Receptors, Neurokinin-1; RNA, Messenger; Substance P

The present study investigated whether chemokines are involved in hydrogen sulfide (H2S)-associated pathogenesis of acute pancreatitis and associated lung injury.. We have examined the effect of DL-propargylglycine, a cystathionine gamma-lyase inhibitor, on the synthesis of CC chemokine monocyte chemotactic protein 1, Regulated upon Activation, Normal T-cell Expressed, and Secreted, and macrophage inflammatory protein-1alpha (MIP-1alpha), and CXC chemokine MIP-2 in an in vitro and in vivo model of cerulein-induced acute pancreatitis and associated lung injury. In addition, the pancreatic acinar cells were treated with H2S donor drug, sodium hydrosulfide. The expression of these chemokines in the pancreatic acini, pancreas, and lungs was determined by quantitative real-time reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry.. After treatment with DL-propargylglycine, reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay demonstrated down-regulation of cerulein-induced increase in monocyte chemotactic protein 1, MIP-1alpha, and MIP-2 expression but had no apparent effect on Regulated upon Activation, Normal T-cell Expressed, and Secreted expression.. These results suggest that the proinflammatory effect of H2S may be mediated by chemokines.

Topics: Acute Disease; Alkynes; Animals; Ceruletide; Chemokine CCL5; Chemokines; Cystathionine gamma-Lyase; DNA Primers; Glycine; Hydrogen Sulfide; Male; Mice; Pancreatitis; Respiratory Distress Syndrome; Reverse Transcriptase Polymerase Chain Reaction

Transient receptor potential subtype vanilloid 1 (TRPV1) is an ion channel that is primarily expressed by primary sensory neurons where it mediates pain and heat sensation and participates in neurogenic inflammation. In this study, we examined the role of TRPV1 during neurogenic activation of pancreatic inflammation using a secretagogue-induced model in mice.. A supramaximal dose of caerulein (50 microg/kg) was injected hourly for 12 hours. Mice lacking TRPV1 were compared to wild-type animals.. All the parameters: serum amylase, pancreatic myeloperoxidase activity, histological scoring, pancreatic wet weight/body weight ratio, and quantification of neurokinin-1 receptor internalization indicated that null mice were not protected from acute pancreatitis. However, when primary sensory neurons were ablated by injection of the neurotoxin and TRPV1 agonist, resiniferatoxin, pancreatitis was ameliorated in wild-type mice but not in null mice, indicating that nerves bearing TRPV1 are part of the inflammatory pathway in acute pancreatitis because disappearance significantly reduced the inflammatory response.. Nerves expressing TRPV1 participate in the neurogenic inflammation during acute pancreatitis. The lack of protection in TRPV1 null mice suggests that an alternate pathway to TRPV1 coexists in the same neurons.

Topics: Acute Disease; Animals; Ceruletide; Crosses, Genetic; Disease Models, Animal; Endocytosis; Female; Gene Deletion; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Neurons, Afferent; Pancreatitis; TRPV Cation Channels

Leukocyte infiltration is an early and critical event in the development of acute pancreatitis. However, the mechanism of leukocyte transmigration into the pancreas and the function of leukocytes in initiating acute pancreatitis are still poorly understood. Here, we studied the role of S100A9 (MRP14), a calcium binding protein specifically released by polymorph nuclear leukocytes (PMN), in the course of acute experimental pancreatitis. Acute pancreatitis was induced by repeated supramaximal caerulein injections in S100A9 deficient or S100A9 wild-type mice. We then determined S100A9 expression, trypsinogen activation peptide (TAP) levels, serum amylase and lipase activities, and tissue myeloperoxidase (MPO) activity. Cell-cell contact dissociation was analyzed in vitro with biovolume measurements of isolated acini after incubation with purified S100A8/A9 heterodimers, and in vivo as measurement of Evans Blue extravasation after intravenous application of S100A8/A9. Pancreatitis induced increased levels of S100A9 in the pancreas. However, infiltration of leukocytes and MPO activity in the lungs and pancreas during acute pancreatitis was decreased in S100A9-deficient mice and associated with significantly lower serum amylase and lipase activities as well as reduced intrapancreatic TAP-levels. Incubation of isolated pancreatic acini with purified S100A8/A9-heterodimers resulted in a rapid dissociation of acinar cell-cell contacts which was highly calcium-dependent. Consistent with these findings, in vivo application of S100A8/A9 in mice was in itself sufficient to induce pancreatic cell-cell contract dissociation as indicated by Evans Blue extravasation. These data show that the degree of intrapancreatic trypsinogen activation is influenced by the extent of leukocyte infiltration into the pancreas which, in turn, depends on the presence of S100A9 that is secreted from PMN. S100A9 directly affects leukocyte tissue invasion and mediates cell contact dissociation via its calcium binding properties.

Topics: Animals; Biomarkers; Calcium; Calgranulin A; Calgranulin B; Ceruletide; Cholecystokinin; Enzyme Activation; Humans; Intercellular Junctions; Leukocytes; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; S100 Proteins; Trypsinogen

Smad6 is implicated in the inhibition of bone morphogenetic protein signalling. However, the function of Smad6 in the pancreas remains obscure.. To elucidate the unknown function of Smad6, we developed transgenic mice selectively expressing Smad6 in pancreatic acinar cells using a plasmid construct coding rat elastase 1 enhancer/promoter.. Smad6 transgenic mice had no specific distinguishing phenotype such as body weight, pancreatic wet weight and concentrations of pancreatic protein. However, Smad6 transgenic mice reacted to hyperstimulation by caerulein injection or a diet containing 0.5% ethionine. Maximal amylase release stimulated by CCK-8 was significantly decreased in Smad6 transgenic mice acini, and trypsin activities in transgenic mice acini were significantly increased after stimulation of CCK-8. There was no difference in effect of CCK-8 stimulation on the subsequent increase in intracellular free Ca2+ concentration ([Ca2+](i)) between wild-type and transgenic mice acini. These findings suggest that reduced pancreatic enzyme secretion was caused by the disorder of its downstream signal transduction pathways in acinar cells. The amino acid sequence at the N-terminus of Smad6 was similar to that of synaptosome-associated protein (SNAP) 25 interacting protein, which plays an important role in regulating exocytosis of pancreatic enzymes in acinar cells. Pancreatic SNAP25 protein levels in transgenic mice were decreased after caerulein-induced pancreatitis.. These results suggest that elevated expression of Smad6 inhibits normal function of SNAP25-interacting protein and SNAP25, reduces amylase secretion in acinar cells, and increases the susceptibility of acinar cells to the onset of pancreatitis.

Topics: Acute Disease; Amino Acid Sequence; Amylases; Animals; Ceruletide; Disease Models, Animal; Disease Progression; Genetic Predisposition to Disease; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Pancreas, Exocrine; Pancreatitis; Phenotype; RNA, Messenger; Sequence Alignment; Smad6 Protein; Synaptosomal-Associated Protein 25; Transforming Growth Factor beta1; Up-Regulation

Pancreatic and neutrophil elastase can aggravate or induce acute pancreatitis. Although increased elastase levels in the plasma of pancreatitis patients and animal models have been reported, the mechanism by which elastase is involved in the pathogenesis of acute pancreatitis has not yet been elucidated. We aimed to investigate the effects and the possible mechanism of a new human leukocyte elastase inhibitor (recombinant guamerin) in the treatment of cerulein-induced acute pancreatitis in rats.. Fifty Sprague-Dawley rats were divided into three groups: a saline-infused control group (I), a cerulein-induced acute pancreatitis group (II), and a cerulein plus guamerin infusion group (III). Guamerin (1-2 micromol/kg/h) was infused continuously in group III. The severity of pancreatitis was determined biochemically, histologically, and by cytokine changes between groups I, II and III.. Significant differences in serum amylase, lipase, and pancreatic wet weight were observed in each group, respectively (group I; 2346.2 IU/L, 9.9 IU/L, 1.4+/-0.3 g, group II; 13,596.8 IU/L, 7439.4 IU/L, 2.2+/-0.5 g, group III; 9443.2 IU/L, 4516.3 IU/L, 1.7+/-0.6 g). Serum IL-6 and TNF-alpha [AU1]level peaked 1-4 h and 1-2 h. After the induction of pancreatitis, IL-6 and TNF-alpha levels were decreased in group III than group II, (group I; 13.1/4.0 pg/mL, group II; 198.5/63.2 pg/mL, group III; 102.1/13.1 pg/mL), but no significant difference in IL-1beta was observed. Histologic gradings and severity, such as vacuolization, inflammation, lobular disarray, and edema of the pancreas, were significantly lower in the cerulein plus guamerin infusion group III.. Recombinant guamerin, a new human leukocyte elastase inhibitor, may decrease the severity of pancreatitis and diminish pancreatic acinar cell injury by inhibition of neutrophilic infiltration and cytokine activation in the initial stage of cerulein-induced acute pancreatitis in rats.

Topics: Amylases; Animals; Ceruletide; Cytokines; Gabexate; Glycoproteins; Invertebrate Hormones; Leukocyte Elastase; Male; Pancreatitis; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Serine Proteinase Inhibitors

Accumulating evidence suggests the neuropeptide substance P (SP) and its receptor neurokinin-1 receptor (NK-1R) play a pivotal role in the pathogenesis of acute pancreatitis (AP). However, the mechanisms remain unclear. The present study investigated whether chemokines as proinflammatory molecules are involved in SP-NK-1R-related pathogenesis of this condition. We observed temporally and spatially selective chemokine responses in secretagogue caerulein-induced AP in mice. CC chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein-1alpha (MIP-1alpha) and CXC chemokine MIP-2 were elevated after AP induction. Time-dependent, tissue-specific analysis of their mRNA and protein expression suggested that they are early mediators in the condition and mediate local as well as systemic inflammatory responses. In contrast, another CC chemokine regulated on activation, T cells expressed and secreted (RANTES) was only involved in local pancreatic inflammation at a later stage of the disease. Either prophylactic or therapeutic treatment with a potent selective NK-1R antagonist CP-96,345 significantly suppressed caerulein-induced increase in MCP-1, MIP-1alpha, and MIP-2 expression but had no apparent effect on RANTES expression. The suppression effect of CP-96,345 on MCP-1, MIP-1alpha, and MIP-2 expression was concordantly demonstrated by immunohistochemistry, which, additionally, suggested that chemokine immunoreactivity was localized to acinar cells and the infiltrating leukocytes in the pancreas and alveolar macrophages, epithelial cells, and endothelial cells in the lungs. Our data suggest that SP, probably by acting via NK-1R on various chemokine-secreting cells in the pancreas and lungs, stimulates the release of chemokines that aggravate local AP and the development of its systemic sequelae.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents, Non-Steroidal; Biphenyl Compounds; Ceruletide; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokine CXCL2; Chemokines; Disease Models, Animal; Gene Expression Regulation; Lung; Lung Injury; Macrophage Inflammatory Proteins; Macrophages, Alveolar; Male; Mice; Neurokinin-1 Receptor Antagonists; Pancreatitis; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transcription, Genetic

The STAT pathways are integral to the inflammatory response and these proteins provide a direct link between the cytokine receptors and cytokine-induced gene transcription. We examined the roles of STAT4 and STAT6 in lung injury after caerulein-induced severe acute pancreatitis. We hypothesized that a modified organ expression of cytokines and chemokines that occurs in transgenic mice may affect the systemic response to severe acute pancreatitis.. Acute pancreatitis [13-hourly intraperitoneal injections of caerulein (50 microg/kg body weight, 0.2 mL) or the same volume of saline] was induced in wild-type (BALB/c) and transgenic (STAT4 or STAT6) mice of the same background, 7 to 8 weeks old. The pancreatic and lung tissues were collected at 1, 6, 12, and 24 h after the completion of caerulein administration. Tissue leukocyte sequestration was assessed by myeloperoxidase (MPO) activity. Standard histological staining hematoxylin and eosin was performed and blindly scored by a pathologist for evidence of lung injury (pulmonary edema, accumulations of neutrophils and mononuclear cells, thickness of alveolar-capillary membrane, perivascular infiltrate, and hemorrhage).. Caerulein-treated wild-type mice exhibited increased lung injury score at 1 through 12 h, as compared to saline controls. As compared to wild-type, STAT6-deficient mice had increased lung injury from 1 to 6 h, with full recovery by 12 h. An opposite pattern was observed in STAT4-deficient mice with mild injury seen at 1 and 6 h, and maximal injury at 12 h. MPO activity was significantly increased at 6 h in caerulein-treated wild-type mice compared to saline-treated controls. Caerulein-treated STAT6 and STAT4 mice had markedly increased MPO activity as compared with their saline controls during the first 6 h. Both caerulein-treated STAT4- and STAT6-deficient mice had significantly increased MPO activity in comparison with wild-type mice with pancreatitis at 6 h.. We found the maximal lung injury after caerulein-induced pancreatitis occurred at different time-points in STAT4 and STAT6-deficient mice. These temporal differences may suggest alternative roles in the systemic inflammatory response associated with pancreatitis.

Topics: Acute Disease; Animals; CD4-Positive T-Lymphocytes; Ceruletide; Disease Models, Animal; Female; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Transgenic; Pancreas; Pancreatitis; Peroxidase; Respiratory Distress Syndrome; STAT4 Transcription Factor; STAT6 Transcription Factor

Protease-activated receptor-2 (PAR-2) is present in the pancreas, where it has been shown to play a protective role during pancreatitis. However, the mechanism by which it protects against pancreatitis still remains to be elucidated. Acute pancreatitis is associated with premature zymogen activation and a blockage in digestive enzyme secretion.. To examine the effects of PAR-2 activation on the severity of pancreatitis, and to determine whether its protective effects are mediated by affecting either premature activation or secretory blockage, or both.. The results confirmed that PAR-2 -/- mice have more severe pancreatitis than wild-type mice. Interestingly, intrapancreatic trypsin levels in the PAR-2 knockouts remained high after 6 h of pancreatitis, whereas they reverted to normal in the wild types. During pancreatitis, PAR-2 mRNA levels were upregulated in wild-type mice in response to supramaximal caerulein administration. Further, after a single injection of supramaximal caerulein, PAR-2 mRNA levels were also elevated, reaching a peak at 3 h. Stimulating PAR-2 with trypsin or the PAR-2-activating peptide, serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL), induced significantly more secretion from the acini of these caerulein-sensitised mice compared with the controls. PAR-2 activation also reversed the inhibition of secretion observed in both the caerulein and arginine models.. Trypsin released during the early stages of pancreatitis activates PAR-2 receptors on the acinar cells and stimulates secretion from these cells. Thus, PAR-2 activation may decrease pancreatic injury and limit the severity of pancreatitis by allowing extracellular trypsin to act as a secretagogue.

Topics: Acute Disease; Amylases; Animals; Arginine; Ceruletide; Disease Models, Animal; Enzyme Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatitis; Receptor, PAR-2; RNA, Messenger; Tissue Culture Techniques; Transcription Factors; Trypsin; Up-Regulation

Transforming growth factors betas (TGF-betas) are implicated in pancreatic tissue repair but their role in acute pancreatitis is not known. To determine whether endogenous TGF-betas modulate the course of caerulein induced acute pancreatitis, caerulein was administered to wild-type (FVB-/-) and transgenic mice that are heterozygous (FVB+/-) for expression of a dominant negative type II TGF-beta receptor.. After 7 hourly supramaximal injections of caerulein, the pancreas was evaluated histologically and serum was assayed for amylase and lipase levels. Next, the effects of caerulein on amylase secretion were determined in mouse pancreatic acini, and cholecystokinin (CCK) receptor expression was assessed.. The normal mouse pancreas was devoid of inflammatory cells whereas the pancreas from transgenic mice contained lymphocytic infiltrates. Caerulein injection in wild-type mice resulted in 6- and 36-fold increases in serum amylase and lipase levels, respectively, increased serum trypsinogen activation peptide (TAP) levels, gross oedema and a marked inflammatory response in the pancreas that consisted mainly of neutrophils and macrophages. By contrast, FVB+/- mice exhibited minimal alterations in response to caerulein with attenuated neutrophil-macrophage infiltrates. Moreover, acini from FVB+/- mice did not exhibit restricted stimulation at high caerulein concentrations, even though CCK receptor mRNA levels were not decreased.. Our findings indicate that a functional TGF-beta signalling pathway may be required for caerulein to induce acute pancreatitis and for the CCK receptor to induce acinar cell damage at high ligand concentrations. Our results also support the concept that restricted stimulation at high caerulein concentrations contributes to the ability of caerulein to induce acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Blotting, Northern; Ceruletide; Edema; Ligands; Lipase; Mice; Mice, Transgenic; Oligopeptides; Pancreatic Diseases; Pancreatitis; Receptors, Cholecystokinin; Severity of Illness Index; Signal Transduction; Transforming Growth Factor beta

Current markers of exocrine pancreatic toxicity have historically been poor indicators for both early diagnosis of disease and prediction of disease severity. Recently we identified two peptide markers (RA1609 and RT2864) of pancreatic toxicity that are target organ specific. In order to evaluate sensitivity of these markers versus current standard tests for pancreatic damage (i.e., lipase), we measured amylase and lipase, as well as RA1609 and RT2864 marker levels, in serum from rats treated with four doses (50-200 mg/kg) of the model pancreatic toxicant cyanohydroxybutene (CHB). In addition, to determine whether these peptide markers could detect pancreatic injury induced by different toxicants and in different species, we measured RA1609 and RT2864 marker levels in rats treated with the pancreatic toxicant caerulein, and in mice treated with CHB. RA1609 and RT2864 peptide markers proved to be more sensitive than amylase or lipase in detecting pancreatic damage, especially at an early time point (8 h) following CHB administration. The peptide markers also accurately predicted pancreatic injury induced by caerulein in rats. These markers were sensitive in detecting very mild pancreatic damage following CHB administration in mice, which are less susceptible to CHB-induced pancreatic toxicity. In addition, a species comparison of the RA1609 albumin fragment sequence indicated that cleavage of albumin from pancreatic proteases produces a similar fragment marker in several species, including humans. To determine whether the comparable human albumin fragment could be detected in sera from pancreatitis patients, we analyzed sera from normal individuals and from patients with diabetes, vasculitis, pancreatic cancer, and pancreatitis. It was found that markers corresponding to the fragments found in rat serum (RA1609 and RT2864) were present in human serum, and changes in these were indicative of and specific to pancreatitis. In conclusion, the RA1609 and RT2864 peptides are sensitive indicators of exocrine pancreatic damage that may be useful as safety markers for general pancreatic toxicity in multiple species.

Topics: Acute Disease; Alkenes; Amylases; Animals; Biomarkers; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Humans; Lipase; Male; Mice; Nitriles; Pancreas, Exocrine; Pancreatitis; Peptide Fragments; Predictive Value of Tests; Protein Array Analysis; Proteomics; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Severity of Illness Index; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors

Several animal models have been developed to investigate the pathobiology of pancreatitis, but few studies have examined the effects that altered pancreatic gene expression have in these models. In this study, the sensitivity to secretagogue-induced pancreatitis was examined in a mouse line that has an altered acinar cell environment due to the targeted deletion of Mist1. Mist1 is an exocrine specific transcription factor important for the complete differentiation and function of pancreatic acinar cells. Mice lacking the Mist1 gene [Mist1 knockout (KO) mice] exhibit cellular disorganization and functional defects in the exocrine pancreas but no gross morphological defects. Following the induction of pancreatitis with caerulein, a CCK analog, we observed elevated serum amylase levels, necrosis, and tissue damage in Mist1 KO mice, indicating increased pancreatic damage. There was also a delay in the regeneration of acinar tissue in Mist1 KO animals. Molecular profiling revealed an altered activation of stress response genes in Mist1 KO pancreatic tissue compared with wild-type (WT) tissue following the induction of pancreatitis. In particular, Western blot analysis for activating transcription factor 3 and phosphorylated eukaryotic initiation factor 2alpha (eIF2alpha), mediators of endoplasmic reticulum (ER) stress, indicated limited activation of this pathway in Mist1 KO animals compared with WT controls. Conversely, Mist1 KO pancreatic tissue exhibits increased expression of growth arrest and DNA damage inducible 34 protein, an inhibitor of eIF2alpha phosphorylation, before and after the induction of pancreatitis. These finding suggest that activation of the ER stress pathway is a protective event in the progression of pancreatitis and highlight the Mist1 KO mouse line as an important new model for studying the molecular events that contribute to the sensitivity to pancreatic injury.

Topics: Activating Transcription Factor 3; Acute Disease; Amylases; Animals; Antigens, Differentiation; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle Proteins; Cells, Cultured; Ceruletide; Cholecystokinin; Disease Models, Animal; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Eukaryotic Initiation Factor-2; Gene Expression; Immediate-Early Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatitis; Protein Phosphatase 1; Regeneration; RNA, Messenger; Severity of Illness Index; Stress, Physiological; Time Factors

The present study was conducted to examine the contribution of substance P to the pancreatic microcirculatory dysfunction during acute pancreatitis.. Pancreatitis was elicited by up to 6 hourly injections of cerulein (50 microg/kg IP) in male C57Bl/6 mice. At 0, 1, 3, and 6 hours after cerulein treatment, the pancreatic microvasculature in anesthetized mice was studied using established high-resolution in vivo microscopic methods.. Treatment of mice with cerulein for 6 hours caused a 30% decrease in capillary perfusion and the diameter of the capillaries and an increase in microvascular permeability (20%) and interstitial space (30-fold). The administration of the substance P receptor antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) (2 mg/kg IP) minimized the pancreatic microcirculatory dysfunction 3 hours after cerulein treatment. The superfusion of substance P for 0.5 hours decreased the diameter (by 22%) and increased microvascular permeability (by 23%) along with interstitial space (22-fold increase). Blockade of substance P receptor attenuated substance P-induced pancreatic microcirculatory dysfunction.. These results suggest that substance P mediates pancreatic microcirculatory dysfunction during the development of acute pancreatitis.

Topics: Acute Disease; Animals; Capillary Permeability; Ceruletide; Male; Mice; Mice, Inbred C57BL; Microcirculation; Microscopy, Video; Pancreas; Pancreatitis; Substance P

We investigated the effect of a specific neurokinin-1 receptor (NK1R) antagonist, CP-96,345, on the regulation of the expression of adhesion molecules ICAM-1, VCAM-1, E-selectin, and P-selectin as well as leukocyte recruitment during acute pancreatitis (AP). AP was induced in male Balb/C mice by 10 consecutive hourly intraperitoneal injections of caerulein. In the treatment groups, CP-96,345 was administered at 2.5 mg/kg ip either 30 min before or 1 h after the first caerulein injection. Animals were killed, and the lungs and pancreas were isolated for RNA extraction and RT-PCR or for immunohistochemical staining. mRNA expression of the four adhesion molecules was upregulated in the pancreas during AP. Treatment with CP-96,345 effectively reduced the mRNA expression of P-selectin and E-selectin but not ICAM-1 and VCAM-1. In the lung, ICAM-1, E-selectin, and P-selectin mRNA expression increased during AP. Antagonist treatment suppressed this elevation. Similar expression patterns were seen in the immunohistochemical stainings. Intravital microscopy of the pancreatic microcirculation revealed the effect of CP-96,345 on leukocyte recruitment. The present study provides important information on the relationship between NK1R activation and the regulation of adhesion molecules. Also, this study points to the differential regulation of inflammation in the pancreas and lung with AP.

Topics: Acute Disease; Amylases; Animals; Biphenyl Compounds; Cell Adhesion; Ceruletide; Immunohistochemistry; Intercellular Adhesion Molecule-1; Lung; Mice; Mice, Inbred BALB C; Neurokinin-1 Receptor Antagonists; Pancreas; Pancreatitis; Peroxidase; Substance P; Vascular Cell Adhesion Molecule-1

We have hypothesized that the colocalization of digestive zymogens with lysosomal hydrolases, which occurs during the early stages of every experimental pancreatitis model, facilitates activation of those zymogens by lysosomal hydrolases such as cathepsin B and that this activation triggers acute pancreatitis by leading to acinar cell injury. Some, however, have argued that the colocalization phenomenon may be the result, rather than the cause, of zymogen activation during pancreatitis. To resolve this controversy and explore the causal relationships between zymogen activation and other early pancreatitis events, we induced pancreatitis in mice by repeated supramaximal secretagogue stimulation with caerulein. Some animals were pretreated with the cathepsin B inhibitor CA-074 me to inhibit cathepsin B, prevent intrapancreatic activation of digestive zymogens, and reduce the severity of pancreatitis. We show that inhibition of cathepsin B by pretreatment with CA-074 me prevents intrapancreatic zymogen activation and reduces organellar fragility, but it does not alter the caerulein-induced colocalization phenomenon or subcellular F-actin redistribution or prevent caerulein-induced activation of NF-kappaB, ERK1/2, and JNK or upregulated expression of cytochemokines. We conclude 1) that the colocalization phenomenon, F-actin redistribution, activation of proinflammatory transcription factors, and upregulated expression of cytochemokines are not the results of zymogen activation, and 2) that these early events in pancreatitis are not dependent on cathepsin B activity. In contrast, zymogen activation and increased subcellular organellar fragility during caerulein-induced pancreatitis are dependent on cathepsin B activity.

Topics: Actins; Acute Disease; Amylases; Animals; Arylsulfatases; Cathepsin B; Ceruletide; Chemokine CCL2; Dipeptides; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lysosomes; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Pancreas; Pancreatitis; Protein Transport; Secretory Vesicles; Severity of Illness Index; Time Factors; Trypsin; Trypsinogen

Clusterin is overexpressed in pancreas during the acute phase of pancreatitis. We intended to clarify the role of clusterin expression in stressed exocrine pancreas. We performed in vitro experiments in transfected AR4-2J cells with modified expression levels of clusterin and in vivo studies in clusterin-deficient mice. AR4-2J cells were exposed to agents mimicking cell-stress during pancreatitis (cerulein, hydrogen peroxide, staurosporine or lysophosphatidylcholine). Clusterin-overexpressing AR4-2J cells showed higher viability after cell stress and accordingly reduced rates of apoptosis and lessened caspase-3 activation. Blockage of endogenous clusterin expression reduced viability and enhanced apoptosis. Presence of clusterin reduced NF-kappaB activation and expression of the NF-kappaB target genes TNF-alpha and MOB-1 under cell stress. Clusterin-deficient mice showed a more severe course of acute experimental pancreatitis with enhanced rates of apoptosis and inflammatory cell infiltration. We concluded that clusterin was protective during inflammation of exocrine pancreas because of its anti-apoptotic and anti-inflammatory functions.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Ceruletide; Clusterin; Disease Models, Animal; Drug Interactions; Mice; NF-kappa B; Pancreatitis; Rats; Transfection

PAP/HIP was first reported as an additional pancreatic secretory protein expressed during the acute phase of pancreatitis. It was shown in vitro to be anti-apoptotic and anti-inflammatory. This study aims to look at whether PAP/HIP plays the same role in vivo.. A model of caerulein-induced pancreatitis was used to compare the outcome of pancreatitis in PAP/HIP(-/-) and wild-type mice.. PAP/HIP(-/-) mice showed the normal phenotype at birth and normal postnatal development. Caerulein-induced pancreatic necrosis was, however, less severe in PAP/HIP(-/-) mice than in wild-type mice, as judged by lower amylasemia and lipasemia levels and smaller areas of necrosis. On the contrary, pancreas from PAP/HIP(-/-) mice was more sensitive to apoptosis, in agreement with the anti-apoptotic effect of PAP/HIP in vitro. Surprisingly, pancreatic inflammation was more extensive in PAP/HIP(-/-) mice, as judged from histological parameters, increased myeloperoxidase activity and increased pro-inflammatory cytokine expression. This result, in apparent contradiction with the limited necrosis observed in these mice, is, however, in agreement with the anti-inflammatory function previously reported in vitro for PAP/HIP. This is supported by the observation that activation of the STAT3/SOCS3 pathway was strongly decreased in the pancreas of PAP/HIP(-/-) mice and by the reversion of the apoptotic and inflammatory phenotypes upon administration of recombinant PAP/HIP to PAP/HIP(-/-) mice.. The anti-apoptotic and anti-inflammatory functions described in vitro for PAP/HIP have physiological relevance in the pancreas in vivo during caerulein-induced pancreatitis.

Topics: Acute Disease; Animals; Apoptosis; Autoantigens; Ceruletide; Disease Models, Animal; Lithostathine; Mice; Mice, Knockout; Necrosis; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Phenotype; Proteins; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral pre-administration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of caerulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role.

Topics: Abdominal Pain; Animals; Ceruletide; Dose-Response Relationship, Drug; Esters; Gabexate; Guanidines; Male; Pain, Referred; Pancreatitis; Physical Stimulation; Posterior Horn Cells; Protease Inhibitors; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Trypsin

TNF-alpha plays a pivotal role in the pathogenesis of acute pancreatitis. Recent studies have shown that TNF-alpha inhibition significantly ameliorates the course of experimental acute pancreatitis, but in this context, the effects of Etanercept, a novel anti-TNF-alpha agent, have not been investigated so far. The aims of the present study are (i) to assess the effects of pharmacological inhibition of TNF-alpha by means of Etanercept on the inflammatory response and apoptosis in a murine model of necrotizing acute pancreatitis and (ii) to compare the results to those observed in TNF-alpha receptor 1 knockout (TNFR1-KO) mice. Necrotizing acute pancreatitis was induced in TNF-alpha wild type for TNFR1 (WT) and TNFR1-KO mice by intraperitoneal injection of cerulein (hourly x5, 50 microg/kg). In another group of WT mice, Etanercept was administered (5 or 10 mg/kg, s.c.) at 1 h after first cerulein injection. Control groups received saline treatment. After 24 h, biochemical, histological, and immunohistochemical evidences of acute pancreatitis developed in all cerulein-treated mice; apoptosis was also present in the pancreas. Contrarily, pancreatitis histological features, amylase and lipase levels, pancreas water content, and myeloperoxidase activity were reduced in a similar degree in Etanercept-treated and TNFR1-KO mice. Likewise, in these two groups, immunohistochemical stainings and terminal deoxynucleotidyltransferase-mediated UTP nick-end labeling assay were found negative. TNF-alpha receptor 1 gene deletion and Etanercept administration ameliorate the course of experimental acute pancreatitis in a similar degree. Future studies on clinical applications of Etanercept in pancreatitis seem promising.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Ceruletide; Enzyme-Linked Immunosorbent Assay; Etanercept; Fas Ligand Protein; Gene Deletion; Immunoglobulin G; Immunohistochemistry; Inflammation; Intercellular Adhesion Molecule-1; Lymphotoxin-alpha; Mice; Mice, Knockout; P-Selectin; Pancreatitis; Peroxidase; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Bacterial endotoxin (lipopolysaccharide, LPS), at high concentration is responsible for sepsis, and neonatal mortality, however low concentration of LPS protected the pancreas against acute damage. The aim of this study was to investigate the effect of exposition of suckling rats to LPS on the course of acute pancreatitis at adult age. Suckling rat (30-40g) received intraperitoneal (i.p.) injection of saline (control) or LPS from Escherichia coli or Salmonella typhi (5, 10 or 15 mg/kg-day) during 5 consecutive days. Two months later these rats have been subjected to i.p. cearulein infusion (25 microg/kg) to produce caerulein-induced pancreatitis (CIP). The following parameters were tested: pancreatic weight and morphology, plasma amylase and lipase activities, interleukin 1beta (IL-1 beta), interleukin 6 (IL-6), and interleukin 10 (IL-10) plasma concentrations. Pancreatic concentration of superoxide dismutase (SOD) and lipid peroxidation products; malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) have been also measured. Caerulein infusion produced CIP in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS at doses 10 or 15 mg/kg-day x 5 days, all manifestations of CIP have been reduced. In these animals acute inflammatory infiltration of pancreatic tissue and pancreatic cell vacuolization have been significantly diminished. Also pancreatic weight, plasma lipase and alpha-amylase activities, as well as plasma concentrations of IL-1beta and IL-6 have been markedly decreased, whereas plasma anti-inflammatory IL-10 concentration was significantly increased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in pancreatic SOD concentration was reversed and accompanied by significant reduction of MDA + 4 HNE in the pancreatic tissue. The effects of LPS derived from E. coli or S. typhi were similar. Pretreatment of suckling rats with LPS at dose of 10 mg/kg-day x 5 days resulted in the most prominent attenuation of acute pancreatitis at adult age, whereas LPS at dose of 5 mg/kg-day x 5 days given to the neonatal rats failed to affect significantly acute pancreatitis induced in these animals 2 months later. We conclude that: 1/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age. 2/ This effect could be related to

Topics: Acute Disease; Aldehydes; alpha-Amylases; Animals; Animals, Newborn; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Endotoxemia; Interleukins; Lipase; Lipid Peroxidation; Lipopolysaccharides; Male; Malondialdehyde; Organ Size; Pancreas; Pancreatitis; Rats; Rats, Wistar; Severity of Illness Index; Superoxide Dismutase; Time Factors

Topics: Acute Disease; Animals; Ascitic Fluid; Ceruletide; Deoxycholic Acid; Disease Models, Animal; High Mobility Group Proteins; HMGB1 Protein; Intestine, Small; Kidney; Liver; Lung; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Repressor Proteins; Severity of Illness Index; Time Factors

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists, such as the thiazolidinediones (TZDs), decrease acute inflammation in both pancreatic cell lines and mouse models of acute pancreatitis. Since PPAR-gamma agonists have been shown to exert some of their actions independent of PPAR-gamma, the role of PPAR-gamma in pancreatic inflammation has not been directly tested. Furthermore, the differential role of PPAR-gamma in endodermal derivatives (acini, ductal cells, and islets) as opposed to the endothelial or inflammatory cells is unknown. To determine whether the effects of a TZD, rosiglitazone, on caerulein-induced acute pancreatitis are dependent on PPAR-gamma in the endodermal derivatives, we created a cell-type specific knock out of PPAR-gamma in pancreatic acini, ducts, and islets. PPAR-gamma knockout animals show a greater response in some inflammatory genes after caerulein challenge. The anti-inflammatory effect of rosiglitazone on edema, macrophage infiltration, and expression of the proinflammatory cytokines is significantly decreased in pancreata of the knockout animals compared with control animals. However, rosiglitazone retains its effect in the lungs of the pancreatic-specific PPAR-gamma knockout animals, likely due to direct anti-inflammatory effect on lung parenchyma. These data show that the PPAR-gamma in the pancreatic epithelia and islets is important in suppressing inflammation and is required for the anti-inflammatory effects of TZDs in acute pancreatitis.

Topics: Animals; Ceruletide; Chemokine CCL2; Intercellular Adhesion Molecule-1; Mice; Mice, Knockout; Pancreatitis; PPAR gamma; Proto-Oncogene Proteins c-jun; Rosiglitazone; Thiazolidinediones

The functional involvement of the endocannabinoid system in modulation of pancreatic inflammation, such as acute pancreatitis, has not been studied to date. Moreover, the therapeutic potential of cannabinoids in pancreatitis has not been addressed.. We quantified endocannabinoid levels and expression of cannabinoid receptors 1 and 2 (CB1 and CB2) in pancreas biopsies from patients and mice with acute pancreatitis. Functional studies were performed in mice using pharmacological interventions. Histological examination, serological, and molecular analyses (lipase, myeloperoxidase, cytokines, and chemokines) were performed to assess disease pathology and inflammation. Pain resulting from pancreatitis was studied as abdominal hypersensitivity to punctate von Frey stimuli. Behavioral analyses in the open-field, light-dark, and catalepsy tests were performed to judge cannabinoid-induced central side effects.. Patients with acute pancreatitis showed an up-regulation of cannabinoid receptors and elevated levels of endocannabinoids in the pancreas. HU210, a synthetic agonist at CB1 and CB2, abolished abdominal pain associated with pancreatitis and also reduced inflammation and decreased tissue pathology in mice without producing central, adverse effects. Antagonists at CB1- and CB2-receptors were effective in reversing HU210-induced antinociception, whereas a combination of CB1- and CB2-antagonists was required to block the anti-inflammatory effects of HU210 in pancreatitis.. In humans, acute pancreatitis is associated with up-regulation of ligands as well as receptors of the endocannabinoid system in the pancreas. Furthermore, our results suggest a therapeutic potential for cannabinoids in abolishing pain associated with acute pancreatitis and in partially reducing inflammation and disease pathology in the absence of adverse side effects.

Topics: Acute Disease; Animals; Biopsy; Cannabinoid Receptor Modulators; Cannabinoids; Ceruletide; Dronabinol; Female; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Pain; Pancreas; Pancreatitis; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

We explored the pathophysiological roles of IFN-gamma in cerulein-induced acute pancreatitis. In wild-type (WT) mice, cerulein injection caused acute pancreatitis as evidenced by increased serum amylase levels and pathological changes such as interstitial edema, vacuolization, acinar cell necrosis, and neutrophil infiltration in pancreas. Concomitantly, cerulein treatment augmented intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, cyclooxygenase-2 (COX-2), and IFN-gamma in WT mice. In situ hybridization combined with immunofluorescence analyses demonstrated that infiltrating neutrophils expressed IFN-gamma mRNA. Unexpectedly, IFN-gamma(-/-) mice exhibited exacerbated cerulein-induced pancreatic injury, with enhanced neutrophil recruitment. Moreover, intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2 were significantly exaggerated in IFN-gamma(-/-) mice, compared with WT mice. Cerulein activated NF-kappaB, an indispensable transcription factor for gene transcription of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2, in pancreas of cerulein-treated WT mice as evidenced by the increases in nuclear amount and DNA-binding activity of NF-kappaB p65. In comparison with WT mice, IFN-gamma(-/-) mice exhibited exaggerated and prolonged NF-kappaB activation, probably due to reduced acetylation of Stat1, a main signal transducer of IFN-gamma, because acetylated Stat1 can inhibit NF-kappaB activation. Indeed, IFN-gamma acetylated Stat1 and reciprocally reduced NF-kappaB activation and COX-2 expression in neutrophils. Finally, even when administered 4 h after the first cerulein injection, IFN-gamma remarkably attenuated acute pancreatitis in both WT and IFN-gamma(-/-) mice, with reduced NF-kappaB activation and COX-2 expression. Thus, IFN-gamma can have anti-inflammatory effects on acute pancreatitis by depressing the proinflammatory consequences of NF-kappaB activation.

Topics: Acute Disease; Adjuvants, Immunologic; Animals; Cell Movement; Ceruletide; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Injections, Intraperitoneal; Interferon-gamma; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Neutrophils; NF-kappa B; Pancreas; Pancreatitis; Repressor Proteins; RNA, Messenger; Up-Regulation

Activation of the inhibitor of NF-kappaB kinase/NF-kappaB (IKK/NF-kappaB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were generated to mediate acinar cell-specific expression of IKK2 alleles. Expression of dominant-negative IKK2 ameliorated cerulein-induced pancreatitis but did not affect activation of trypsin, an initial event in experimental pancreatitis. Notably, expression of constitutively active IKK2 was sufficient to induce acute pancreatitis. This acinar cell-specific phenotype included edema, cellular infiltrates, necrosis, and elevation of serum lipase levels as well as pancreatic fibrosis. IKK2 activation caused increased expression of known NF-kappaB target genes, including mediators of the inflammatory response such as TNF-alpha and ICAM-1. Indeed, inhibition of TNF-alpha activity identified this cytokine as an important effector of IKK2-induced pancreatitis. Our data identify the IKK/NF-kappaB pathway in acinar cells as being key to the development of experimental pancreatitis and the major factor in the inflammatory response typical of this disease.

Topics: Animals; Ceruletide; Disease Models, Animal; Enzyme Activation; Humans; I-kappa B Kinase; Mice; Mice, Transgenic; Pancreas; Pancreatitis; Rats

Activation of the transcription factor NF-kappaB/Rel has been shown to be involved in inflammatory disease. Here we studied the role of RelA/p65, the main transactivating subunit, during acute pancreatitis using a Cre-loxP strategy. Selective truncation of the rela gene in pancreatic exocrine cells led to both severe injury of the acinar cells and systemic complications including lung and liver damage. Our data demonstrated that expression and induction of the protective pancreas-specific acute phase protein pancreatitis-associated protein 1 (PAP1) depended on RelA/p65. Lentiviral gene transfer of PAP1 cDNA reduced the extent of necrosis and infiltration in the pancreata of mice with selective truncation of RelA/p65. These results provide in vivo evidence for RelA/p65 protection of acinar cell death via upregulation of PAP1. Moreover, our data underscore the pancreas-specific role of NF-kappaB/Rel and suggest multidimensional roles of NF-kappaB/Rel in different cells and contexts during inflammation.

Topics: Animals; Base Sequence; Cell Death; Ceruletide; Disease Models, Animal; Female; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Pancreatitis; Pancreatitis-Associated Proteins; Proteins; RNA, Messenger; RNA, Small Interfering; Sequence Deletion; Transcription Factor RelA

To determine the effect of exogenous leptin on acute lung injury (ALI) in cerulein-induced acute pancreatitis (AP).. Forty-eight rats were randomly divided into 3 groups. AP was induced by intraperitoneal (i.p.) injection of cerulein (50 microg/kg) four times, at 1 h intervals. The rats received a single i.p. injection of 10 mug/kg leptin (leptin group) or 2 mL saline (AP group) after cerulein injections. In the sham group, animals were given a single i.p. injection of 2 mL saline. Experimental samples were collected for biochemical and histological evaluations at 24 h and 48 h after the induction of AP or saline administration. Blood samples were obtained for the determination of amylase, lipase, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, macrophage inflammatory peptide (MIP)-2 and soluble intercellular adhesion molecule (sICAM)-1 levels, while pancreatic and lung tissues were removed for myeloperoxidase (MPO) activity, nitric oxide (NOx) level, CD40 expression and histological evaluation.. Cerulein injection caused severe AP, confirmed by an increase in serum amylase and lipase levels, histopathological findings of severe AP, and pancreatic MPO activity, compared to the values obtained in the sham group. In the leptin group, serum levels of MIP-2, sICMA-1, TNF-alpha, and IL-1beta, pancreatic MPO activity, CD40 expression in pancreas and lung tissues, and NOx level in the lung tissue were lower compared to those in the AP group. Histologically, pancreatic and lung damage was less severe following leptin administration.. Exogenous leptin attenuates inflamma-tory changes, and reduces pro-inflammatory cytokines, nitric oxide levels, and CD40 expression in cerulein-induced AP and may be protective in AP associated ALI.

Topics: Acute Disease; Animals; CD40 Antigens; Ceruletide; Chemokine CXCL2; Chemokines, CXC; Female; Interleukin-1beta; Leptin; Lung; Nitric Oxide; Pancreas; Pancreatitis; Peroxidase; Random Allocation; Rats; Rats, Wistar; Respiratory Distress Syndrome; Tumor Necrosis Factor-alpha

Previous studies have shown that ischemic preconditioning protects several organs, including the pancreas, from ischemia/reperfusion-induced injury. The aim of the investigation was to determine whether ischemic preconditioning affects the course edematous pancreatitis.. In rats, ischemic preconditioning was performed by short-term clamping the celiac artery. Acute pancreatitis was induced by caerulein. The severity of acute pancreatitis was evaluated between the first and tenth day of inflammation.. Ischemic preconditioning applied alone caused a mild pancreatic damage. Combination of ischemic preconditioning with caerulein attenuated the severity of pancreatitis in histological examination and reduced the pancreatitis-evoked increase in plasma lipase and pro-inflammatory interleukin-1beta. This effect was associated with an increase in plasma level of anti-inflammatory interleukin-10 and partial reversion of the pancreatitis-evoked drop in pancreatic DNA synthesis and pancreatic blood flow. In secretory studies, ischemic preconditioning in combination with induction of acute pancreatitis attenuated the pancreatitis-evoked decrease in secretory reactivity of isolated pancreatic acini to stimulation by caerulein. In the initial period of acute pancreatitis, ischemic preconditioning alone and in combination with caerulein-induced acute pancreatitis prolonged the activated partial thromboplastin time (APTT), increased plasma level of D-dimer and shortened the euglobulin clot lysis time. The protective effect of ischemic preconditioning was observed during entire time of experiment and led to acceleration of pancreatic regeneration.. Ischemic preconditioning reduces the severity of caerulein-induced pancreatitis and accelerates pancreatic repair; and this effect is related to the activation of fibrinolysis and reduction of inflammatory process.

Topics: Acute Disease; Amylases; Animals; Blood Coagulation; Ceruletide; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Interleukin-10; Ischemic Preconditioning; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar

Protein kinase C-zeta (PKC-zeta) regulates cell death via NF-kappaB; therefore, we tested the hypothesis that PKC-zeta plays a critical role in pancreatitis-induced Kupffer cell apoptosis. Acute pancreatitis was induced in rats by cerulein injection 24 h later, livers were assayed for PKC-zeta, IKKalpha, IKKbeta, IKKgamma, NF-kappaB, Fas/FasL, and apoptosis was assessed with Caspase-3 and DNA fragmentation. Kupffer cells from unoperated rats were infected with a PKC-zeta domain-negative adenovirus (AdPKCzeta-DN) to inhibit PKC-zeta, or transfected with pCMVPKC-zeta to overexpress PKC-zeta, and then stimulated with pancreatic elastase; cellular extracts were assayed for PKC-zeta, IKKalpha, IKKbeta, IKKgamma, NF-kappaB, Fas/FasL, Caspase-3, and DNA fragmentation. Cerulein-induced pancreatitis upregulated PKC-zeta protein and activity, IKKbeta, IKKgamma, NF-kappaB, Fas/FasL, Caspase-3 and increased DNA fragmentation in rat livers (all p < 0.001 vs control). AdPKCzeta-DN abolished elastase-induced upregulation of PKC-zeta activity, IKKbeta, IKKgamma, NF-kappaB, Fas/FasL, Caspase-3 and DNA fragmentation (all p < 0.001 vs infection control), whereas overexpression of PKC-zeta augmented elastase-induced upregulation of IKKbeta, IKKgamma, Fas/FasL, Caspase-3 and DNA fragmentation (p < 0.001 vs control). PKC-zeta plays a critical role in pancreatitis-induced Kupffer cell apoptosis via NF-kappaB and Fas/FasL. The ability of Kupffer cells to autoregulate their stress response by upregulating their death receptor/ligand and key proapoptotic cell signaling systems warrants further investigation.

Topics: Acute Disease; Animals; Apoptosis; Caspase 3; Ceruletide; DNA Fragmentation; I-kappa B Kinase; In Vitro Techniques; Isoenzymes; Kupffer Cells; Male; NF-kappa B; Pancreatitis; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Up-Regulation

Unlike edematous pancreatitis, induction of severe necrotizing pancreatitis in rats generally requires an invasive laparotomy with infusion and/or ligation of the pancreatic duct or duodenal or arterial occlusion. The aim of this study was to establish and characterize a noninvasive model of severe acute pancreatitis in rats.. Wistar rats were infused intravenously with cerulein or a combination of cerulein and enterokinase. Saline (154-mmol/L NaCl) or enterokinase only was infused in controls. In a first set of experiments, intrapancreatic protease activation and the release of cytokines were correlated with the severity of organ injury. Pancreatic and pulmonary injuries were determined at 6 h. In a second set of experiments, we assessed 24-h survival, serum parameters possibly reflecting the course of the disease, and morphologic changes later in the course of the disease.. The severity of pancreatic injury and survival were correlated strongly with the amount of enterokinase infused simultaneously with cerulein. Trypsin as well as elastase and cathepsin B activity in pancreatic tissue samples were increased markedly in these animals. Marked pancreatic hemorrhage, necrosis, and leukocyte infiltration were present in animals with the greatest amounts of enterokinase infused. IL-6 and LDH, but not IL-1beta, CRP, and amylase, in serum correlated with the severity of pancreatitis.. This noninvasive rat model of acute pancreatitis is characterized by major pancreatic necrosis, hemorrhage, and fatality. The simple and noninvasive induction technique may have advantages for future studies on inflammatory changes and sepsis in necrotizing pancreatitis compared with other currently available invasive models.

Topics: Animals; Cathepsin B; Ceruletide; Disease Models, Animal; Drug Combinations; Enteropeptidase; Interleukin-6; L-Lactate Dehydrogenase; Male; Necrosis; Pancreas; Pancreatic Elastase; Pancreatitis; Rats; Rats, Wistar; Severity of Illness Index

Hypothermia is a frequent event in severe acute pancreatitis (AP) and its real effects on the normal pancreas have not been well demonstrated. Moreover, neither have its effects on the outcome of acute pancreatitis been fully investigated. One hypothesis is that oxidative stress may be implicated in lesions caused or treated by hypothermia.. To investigate the effect of hypothermia in cerulein-induced acute pancreatitis (CIAP) in rats and the role played by oxidative stress in this process.. Male Wistar rats were divided into hypothermic and normothermic groups. Hypothermia was induced with a cold mattress and rectal temperature was kept at 30 masculineC for one hour. Acute pancreatitis was induced with 2 doses of cerulein (20 ìg/kg) administered at a one-hour interval. Serum amylase, pancreas vascular permeability by Evan's blue method, pancreas wet-to-dry weight ratio and histopathology were analyzed in each group.. When compared with normothermic rats, hypothermic animals, with cerulein-induced acute pancreatitis, showed higher levels of pancreatic vascular permeability (p < 0.05), pancreas wet-to-dry weight ratio (p = 0.03), and histologically verified edema (p < 0.05), but similar serum amylase levels. The hypothermic group showed a higher oxidized-reduced glutathione ratio than the normothermic group.. Moderate hypothermia produced a greater inflammatory response in established acute pancreatitis induced by cerulein in rats. Moreover, this study suggests that oxidative stress may be one of the mechanisms responsible for the worse outcome in hypothermic rats with cerulein-induced acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Capillary Permeability; Ceruletide; Glutathione; Hypothermia; Male; Oxidative Stress; Pancreatitis; Rats; Rats, Wistar

Ghrelin and its receptor are expressed abundantly in the stomach and pituitary. Recently, a ghrelin system, consisting of both ligand and receptor, has also been found to exist in the endocrine cells of pancreatic islets. This ghrelin system may play a role in regulating insulin secretion and glucose homeostasis. The aim of the present study was to investigate whether a functional ghrelin system also exists in the exocrine pancreas.. Precise localization and expression of ghrelin and its receptor in rat pancreatic acinar cells were examined by immunocytochemistry and Western blot, whereas messenger RNA levels were examined by semiquantitative reverse transcription-polymerase chain reaction. The roles of physiological and pathophysiological conditions, such as gastric acid inhibition, starvation, and acute pancreatitis, in regulation of ghrelin and its receptor were also examined.. Both ghrelin and its receptor were detected, at both protein and messenger RNA levels, in the acinar cells of the exocrine pancreas. Ghrelin receptor expression was up-regulated by gastric acid inhibition and down-regulated by acute pancreatitis, whereas levels remained unchanged after food deprivation. In contrast, ghrelin expression did not exhibit significant changes in any condition.. Our data indicate that a ghrelin system exists in the acinar cells of the exocrine pancreas. This system is subject to regulation by physiological and pathophysiological stimuli and may thus regulate exocrine functions by paracrine and/or autocrine mechanisms.

Topics: Achlorhydria; Acute Disease; Animals; Autocrine Communication; Ceruletide; Food Deprivation; Gastric Acid; Gene Expression Regulation; Ghrelin; Male; Omeprazole; Pancreas, Exocrine; Pancreatitis; Paracrine Communication; Peptide Hormones; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Ghrelin; RNA, Messenger

We investigated the effect of Adrenomedullin (AM) on cerulein-induced acute pancreatitis in rats. AM treatment (100 ng/kg per rat, subcutaneous) after one hour of cerulein injection reduced the plasma amylase levels, pancreatic weight, pancreatic malondialdehyde (MDA) levels, and the severity of the lesions microscopically. These data suggest that AM has a protective effect on cerulein-induced acute pancreatitis. These could be due to anti-inflammatory properties of AM, inhibition of proinflammatory cytokine secretion, reducing the endothelial permeability increased by reactive oxygen species, endotoxins or cytokines.

Topics: Acute Disease; Adrenomedullin; Amylases; Animals; Ceruletide; Female; Injections, Subcutaneous; Lipid Peroxidation; Male; Malondialdehyde; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley

To observe the apoptosis and oncosis of pancreatic acinar cells and secondary inflammatory reaction in pancreatic tissue from rats with acute pancreatitis (AP), and the influences of artemisinin on them.. AP was induced by 4 intraperitoneal injections of caerulein at 1 h intervals. To induce apoptosis, solution of artemisinin (50 mg/kg) was given intraperitoneally 1, 12, 24 and 36 h after the last caerulein injection. Histological examination of impairment of pancreatic tissue and detection of serum amylase were performed to evaluate the severity of acute pancreatitis. Apoptosis and oncosis were detected with acridine orange (AO) and ethylene dibromide (EB) staining. Caspase-3 and myeloperoxidase (MPO) activity were measured by colorimetric assay. Nuclear factor-kappa B (NF-kappaB) activation was detected by flow cytometry. Macrophage inflammatory protein-1alpha (MIP-1alpha) protein was measured by Western blot. Interleukin-1beta (IL-1beta) mRNA was detected by RT-PCR.. Addition of artemisinin increased the number of apoptotic cells (11.7% +/- 1.4% vs 6.3% +/- 0.7%, P < 0.05), while reduced the number of oncotic cells (13.0% +/- 2.4% vs 17.5% +/- 2.2%, P < 0.05). The activity of caspase-3 speeded up (1.52 +/- 0.21 vs 1.03 +/- 0.08, P < 0.05), the pancreas pathological impairment was relieved (3.0 +/- 0.5 vs 4.0 +/- 0.5, P < 0.05) and the level of serum amylase decreased (5642 +/- 721 U/dL vs 7821 +/- 653 U/dL, P < 0.05). The activation of NF-kB (29% +/- 4.1% vs 42% +/- 5.8%), MIP-1alpha protein (3.7 +/- 0.5 vs 5.8 +/- 0.7), MPO (0.52 +/- 0.06 U/g vs 0.68 +/- 0.09 U/g), IL-1beta mRNA (1.7 +/- 0.3 vs 2.4 +/- 0.4) in the apoptosis inducing group was obviously decreased (P < 0.05).. Inducing apoptosis can relieve pathological impairment and inflammatory reaction in AP rats.

Topics: Acute Disease; Animals; Apoptosis; Artemisinins; Caspase 3; Ceruletide; Male; NF-kappa B; Pancreatitis; Peroxidase; Rats; Rats, Wistar

The aim of the present study was to elucidate the pathogenic role of endogenous 5-HT in pancreatitis. Injections of cerulein at hourly intervals caused edematous pancreatitis in mice characterized by hyperenzymemia and histological alterations. While the cerulein-induced hyperenzymemia was attenuated in mice pretreated with p-CPA, a 5-HT depletor, it was exaggerated by the preferential 5-HT2A agonist (DOI), but not by the preferential 5-HT2B agonist (BW723C86) or the preferential 5-HT2C agonist (mCPP). Selective 5-HT2A antagonists (risperidone, spiperone, ketanserin, AMI-193, and MDL 11,939) dose-dependently attenuated the hyperenzymemia; and their potency order, excepting that of ketanserin which has considerable affinity at the 5-HT2C receptor as well, paralleled their reported pKi values at the 5-HT2A receptor. Selective 5-HT2B (SB204741) and 5-HT2C (SB242084) antagonists hardly affected the hyperenzymemia. Although the non-selective 5-HT2A/2B/2C antagonists (metergoline, ritanserin, and methysergide) dose-dependently attenuated the hyperenzymemia, they were relatively less potent compared to their high pKi values at the 5-HT2A receptor. In another set of experiments, risperidone, but not SB204741 and SB242084, dose-dependently reversed the cerulein-induced histological alteration of the pancreas (inflammatory cell infiltration). These results suggest that endogenously released 5-HT activates 5-HT2A receptors to aggravate cerulein-induced pancreatitis. We propose that selective 5-HT2A antagonists may provide a new therapy for acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Female; Mice; Mice, Inbred ICR; Pancreas; Pancreatitis; Receptor, Serotonin, 5-HT2A; Receptors, Cholecystokinin; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Spiro Compounds

Glycogen synthase kinase (GSK)-3 is a ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and signal transduction pathways. It also plays an important role in the pathophysiology of a number of diseases characterized by an enhanced or unregulated inflammatory response. Here we investigate the effects of GSK-3beta inhibition on the development of experimental acute pancreatitis induced by cerulein in mice.. Prospective, randomized study.. University-based research laboratory.. One-hundred and sixty anesthetized male CD mice.. Pancreatitis was induced by intraperitoneal injection of cerulein (hourly x5, 50 microg/kg). In the treatment group, the potent and selective GSK-3beta inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) was administered 1 hr and 6 hrs after the first injection of cerulein (10 mg/kg, intraperitoneally). Sham groups were treated with vehicle (0.1 mL of 0.9% NaCl, intraperitoneally) and TDZD-8. In another set of experiments, mice were monitored for 24 days to determine their mortality rate.. The injection of cerulein resulted in acute necrotizing pancreatitis. TDZD-8 significantly reduced the degree of pancreas injury, amylase, and lipase serum levels (p < .01); nuclear factor-kappaB activation (p < .01); the production of tumor necrosis factor-alpha and interleukin-1beta (p < .01); the expression of adhesion molecules and neutrophil accumulation (p < .01); the formation of oxygen and nitrogen-derived radicals (p < .01); the degree of lipid peroxidation (p < .01); the expression of transforming growth factor-beta and vascular endothelial growth factor (p < .01); and-ultimately-the mortality rate (p < .01).. Inhibition of GSK-3beta reduces the degree of cerulein-induced acute pancreatitis and the associated mortality rate in mice. Blocking protein kinase activity may be a novel approach to treatment of this inflammatory condition.

Topics: Acute Disease; Animals; Cell Adhesion Molecules; Ceruletide; Cytokines; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Male; Mice; Mice, Inbred Strains; Neutrophil Activation; NF-kappa B; Oxidative Stress; Pancreatitis; Prospective Studies; Random Allocation; Survival Analysis; Thiadiazoles

To test the hypothesis that disruption of acinar cell membranes is the earliest event that takes place after the onset of acute pancreatitis.. Cerulein and taurocholate pancreatitis were induced in rats. Furthermore, stimulation with different doses of bombesin, pilocarpine, and cerulein was performed. Five to 180 minutes after initiation of treatment, animals were killed. Disruption of cell membranes was detected by the penetration of the experimental animal's own albumin or immunoglobulin G (IgG) into acinar cells by immunocytological localization. Tissue was further analyzed by electron microscopy and electron microscopic immunostaining.. Animals with pancreatitis displayed significantly greater antialbumin and anti-IgG immunostaining in the cytoplasm of acinar cells and in vacuoles in comparison with controls, confirming membrane disruption. This was not detectable after stimulation with bombesin, pilocarpine, and nonsupramaximal doses of cerulein. The first changes were seen after 5 minutes of induction of pancreatitis. Results were verified by electron microscopy and electron microscopic immunohistochemistry.. The penetration of albumin and IgG into acinar cells indicates that wounding of their plasma membrane occurs at the onset of acute pancreatitis. Disruption of the membranes could be expected to allow the influx of calcium ions, causing massive intracellular alterations, and exit of molecules, such as enzymes from acinar cells.

Topics: Acute Disease; Animals; Capillary Permeability; Cell Membrane; Ceruletide; Cytoplasm; Disease Models, Animal; Edema; Immunoglobulin G; Male; Microscopy, Electron, Transmission; Microscopy, Immunoelectron; Pancreas, Exocrine; Pancreatitis; Rats; Rats, Sprague-Dawley; Serum Albumin; Taurocholic Acid; Time Factors; Vacuoles

To study if the course of cerulein-induced pancreatitis in rats changes in a state of triglyceride-rich lipoprotein metabolism alteration.. Two groups of rats received control diet during a 90-day period (A) and sucrose-rich diet to induce endogenous hypertriglyceridemia (B). Subgroups A2 and B2 received i.p. 45 microg cerulein/kg body weight (to induce acute pancreatitis). Histological examination of pancreas tissue, serum pancreatic lipase, lipoprotein profile and VLDL chemical composition were assessed. Then, pancreatic lipase hydrolytic activity on VLDL-triglycerides was evaluated in vitro.. Cellular vacuolization was observed in all of the cerulein-injected rats, but only in subgroup B2 fat necrosis was present. Serum triglycerides were higher in subgroup B1 than in subgroup A1 (mean +/- SEM, mg/dl 123,77 +/- 25.7 vs. 65.8 +/- 7, p < 0.01). Triglycerides from rats fed with sucrose-rich diet, decreased after cerulein-induced pancreatitis (80.38 +/- 11.3 vs. 123,77 +/- 25.7, p < 0.02). Moreover, the endogenous hypertriglyceridemic rats showed an increment of VLDL triglyceride content, which decreased when rats were injected with cerulein. A negative correlation was found between VLDL-triglyceride content and serum pancreatic lipase activity (r = 0.58, p < 0.02). The in vitro assay showed a decrease in VLDL-triglyceride content post incubation with pancreatic lipase enriched serum (mean +/- SD: 59.2 +/- 27.7%, p < 0.01).. The endogenous hypertriglyceridemia intensifies the course of cerulein-induced pancreatitis and it could be related to the decrease in VLDL-triglycerides as a consequence of pancreatic lipase hydrolytic activity.

Topics: Acute Disease; Animals; Ceruletide; Cholesterol, VLDL; Hypertriglyceridemia; Lipase; Lipoproteins, VLDL; Male; Pancreatitis; Random Allocation; Rats; Rats, Wistar; Triglycerides

Melatonin has been used to treat experimental pancreatitis, although not all the drug's therapeutic mechanisms of melatonin have been defined. Prostaglandins (PGs) are proinflammatory mediators that exert their effects mainly locally during inflammatory diseases. The present study was undertaken to examine whether treatment with melatonin influences local PG production. An acute pancreatitis model in male Sprague-Dawley rats (225-275 g) was established by continuously infusing caerulein (15 mg/kg/hr). Mean arterial pressure and pancreatic perfusion were monitored continuously. Melatonin was delivered via the intraperitoneal route at doses of either 2 or 10 mg/kg, 30 min after caerulein injection. Malondialdehyde and glutathione levels of the pancreas and liver and the trypsinogen activation peptide levels in the serum were measured at the end of the experiment (8 hr after infusion of caerulein). Intraperitoneal injection of melatonin (2 and 10 mg/kg) reduced the reduction in systemic arterial pressure and decreased pancreatic perfusion in the rat model of caerulein pancreatitis. Moreover, melatonin treatment changed local PG production toward control level. Higher dose of melatonin was somewhat more effective in preventing the caerulein-induced alterations than was the lower dose.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Blood Pressure; Ceruletide; Dinoprostone; Glutathione; Leukotriene B4; Male; Malondialdehyde; Melatonin; Microdialysis; Oligopeptides; Pancreas; Pancreatitis; Prostaglandins; Rats; Rats, Sprague-Dawley; Regional Blood Flow

The role of oxidative stress has been evaluated in experimental models of acute pancreatitis (AP). The aim of this study is to investigate the effect of melatonin on the ultrastructural changes in cerulein-induced AP in rats. Acute pancreatitis was induced by two i.p. injections of cerulein at 2-hr intervals (50 microg/kg BW). One group received additionally melatonin (20 mg/kg BW) i.p. before each injection of cerulein. The rats were sacrificed 12 hr after the last injection. Pancreatic oxidative stress markers were evaluated by changes in the amount of lipid peroxides and changes in the antioxidant enzyme levels, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) levels. Ultrastructural examination was performed using a transmission electron microscope. Formation of numerous, large autophagosomes, mitochondrial damage, dilatation of rough endoplasmic reticulum (RER) and Golgi apparatus, margination and clumping of nuclear chromatin were the major ultrastructural alterations observed in the AP group. Melatonin administration prevented mitochondrial and nuclear changes and dilatation of RER and Golgi apparatus. Rare, small autophagosomes were present within the cytoplasm of some of the acinar cells. Pancreatic damage was accompanied by a significant increase in tissue MDA levels (P < 0.05) and a significant decrease in CAT, SOD, GPx activities and GSH levels (P < 0.005). Melatonin administration significantly reduced MDA levels but increased CAT, SOD, GPx activities and GSH levels (P < 0.005). Melatonin also reduced serum amylase and lipase activities, which were significantly elevated in AP (P < 0.05 and P < 0.005 respectively). These results suggest that oxidative injury is important in the pathogenesis of AP. Melatonin is potentially capable of limiting pancreatic damage produced during AP by protecting the fine structure of acinar cells and tissue antioxidant enzyme activities.

Topics: Acute Disease; Animals; Apoptosis; Autophagy; Catalase; Ceruletide; Female; Glutathione; Glutathione Peroxidase; Lipid Peroxidation; Melatonin; Microscopy, Electron; Oxidative Stress; Pancreas; Pancreatitis; Phagosomes; Rats; Rats, Sprague-Dawley; Superoxide Dismutase

We have investigated the involvement of cholecystokinin (CCK) receptor subtypes in haemodynamic changes in the pancreas of anaesthetized rats during submaximal and supramaximal stimulation with the CCK analogue, caerulein.. For submaximal stimulation, caerulein (0.4 nmol/kg/h) was infused intravenously, while acute pancreatitis was induced by intraperitoneal injections of high doses of caerulein (3 x 25 nmol/kg). Pancreatic blood flow was measured by hydrogen clearance.. Low caerulein doses increased pancreatic blood flow by 26 +/- 8% and vascular conductance by 24 +/- 4%. This effect was mimicked by the CCK2 agonist gastrin-17. All effects were abolished by a CCK2 antagonist while a CCK1 antagonist remained inactive. Conversely, amylase output by caerulein was abolished by CCK1 receptor blockade, but not by inhibition of CCK2 receptors. During caerulein-induced pancreatitis, vascular conductance increased by 109 +/- 26% and remained elevated throughout the experiment; vascular flow initially increased by 62 +/- 27% and then returned to baseline. The vascular effects were prevented by a CCK2 receptor antagonist, while the induction of pancreatitis was due to CCK1 receptor stimulation.. Caerulein increases pancreatic vascular flow via activation of CCK2 receptors. This effect occurs both at submaximal and at supramaximal levels of exocrine stimulation.

Topics: Animals; Bradykinin B2 Receptor Antagonists; Ceruletide; Female; Gastrins; Indoles; Pancreas, Exocrine; Pancreatitis; Rats; Rats, Sprague-Dawley; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Regional Blood Flow; Stimulation, Chemical

It remains unclear whether interleukin (IL) 6 plays a role in initiating either the inflammatory or antiapoptotic responses in severe acute pancreatitis. This study examined the effect of neutralizing antibody against IL-6 on the induction of pancreatic acinar cell apoptosis and attenuation of the severity of severe acute pancreatitis.. Experiments were conducted on laboratory mice with severe acute pancreatitis induced by lipopolysaccharide injection following six injections of caerulein at intervals of 6 h. Neutralizing monoclonal anti-IL-6 antibody was administered either 5 min or 2 h after the first caerulein injection. Apoptosis in pancreatic sections was determined by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labelling method.. Administration of caerulein and LPS induced an increase in serum amylase and IL-6 levels, severe acute pancreatitis, pancreatitis-associated lung injury, and phosphorylation of signal transducer and activator of transcription (STAT) 3 in the pancreas. A neutralizing antibody against IL-6 effectively suppressed these responses. Application of IL-6 neutralizing antibody caused the induction of apoptosis in the pancreatic acinar cells of mice with acute pancreatitis.. Blocking IL-6 suppresses STAT-3 activation in the pancreas and consequently attenuates the severity of severe acute pancreatitis by promotion of pancreatic acinar cell apoptosis.

Topics: Amylases; Animals; Apoptosis; Blotting, Western; Ceruletide; Interleukin-6; Lipopolysaccharides; Male; Mice; Mice, Inbred ICR; Pancreatitis; STAT3 Transcription Factor

Acute pancreatitis (AP) is an inflammatory disease characterized by tissue edema, necrosis and hemorrhage. The mortality rate associated with this disease is particularly high when the inflammation has become systemic. Recently, activation of the pancreatic renin-angiotensin system (RAS) was shown to play a role in AP. The present study investigated whether administering an AT1 receptor antagonist decreases the severity of AP and pancreatitis-induced systemic inflammation, particularly pulmonary injury. Rats with AP-associated lung injury were induced by multiple doses of caerulein, which was demonstrated in the previous studies. Three injections of losartan (200 microg/ kg/h) were given 30 min prior to the first injection of caerulein. The results demonstrated that caerulein injections resulted in significant increases in pancreatic and pulmonary myeloperoxidase (MPO) activities, and losartan treatment attenuates these effects. Lung microvascular permeability was also significantly improved by losartan treatment. Losartan prevented caerulein-induced pancreatic and pulmonary morphological alterations, but not elevations in serum alpha-amylase or pancreas/body weight ratio. These data indicate that losartan treatment can attenuate pancreatic and lung injury. Thus, the implication is that a blockade of AT1 receptors may have a clinical application for the treatment of AP and, perhaps more importantly, subsequent pulmonary complications.

Topics: alpha-Amylases; Angiotensin II Type 1 Receptor Blockers; Animals; Capillary Permeability; Ceruletide; Losartan; Lung; Lung Diseases; Male; Organ Size; Pancreas; Pancreatitis; Peroxidase; Rats; Receptor, Angiotensin, Type 1

To investigate the role of oxidative injury in pancreatitis-induced hepatic damage and the effect of antioxidant agents such as melatonin, ascorbic acid and N-acetyl cysteine on caerulein-induced pancreatitis and associated liver injury in rats.. Thirty-eight female Wistar rats were used. Acute pancreatitis (AP) was induced by two i.p. injections of caerulein at 2-h intervals (at a total dose of 100 microg/kg b.wt). The other two groups received additional melatonin (20 mg/kg b.wt) or an antioxidant mixture containing L(+)-ascorbic acid (14.3 mg/kb.wt.) and N-acetyl cysteine (181 mg/kg b.wt.) i.p. shortly before each injection of caerulein. The rats were sacrificed by decapitation 12 h after the last injection of caerulein. Pancreatic and hepatic oxidative stress markers were evaluated by changes in the amount of lipid peroxides measured as malondialdehyde (MDA) and changes in tissue antioxidant enzyme levels, catalase (CAT) and glutathione peroxidase (GPx). Histopathological examination was performed using scoring systems.. The degree of hepatic cell degeneration, intracellular vacuolization, vascular congestion, sinusoidal dilatation and inflammatory infiltration showed a significant difference between caerulein and caerulein + melatonin (P = 0.001), and careulein and caerulein + L(+)-ascorbic acid + N-acetyl cysteine groups (P = 0.002). The degree of aciner cell degeneration, pancreatic edema, intracellular vacuolization and inflammatory infiltration showed a significant difference between caerulein and caerulein + melatonin (P = 0.004), and careulein and caerulein + L(+)-ascorbic acid + N-acetyl cysteine groups (P = 0.002). Caerulein-induced pancreatic and liver damage was accompanied with a significant increase in tissue MDA levels (P = 0.01, P = 0.003, respectively) whereas a significant decrease in CAT (P = 0.002, P = 0.003, respectively) and GPx activities (P = 0.002, P = 0.03, respectively). Melatonin and L(+)-ascorbic acid+N-acetyl cysteine administration significantly decreased MDA levels in pancreas (P=0.03, P=0.002, respectively) and liver (P = 0.007, P = 0.01, respectively). Administration of these agents increased pancreatic and hepatic CAT and GPx activities. Melatonin significantly increased pancreatic and hepatic CAT (P = 0.002, P = 0.001, respectively) and GPx activities (P = 0.002, P = 0.001). Additionally, L(+)-ascorbic acid + N-acetyl cysteine significantly increased pancreatic GPx (P = 0.002) and hepatic CAT and GPx activities (P = 0.001, P = 0.007, respectively).. Oxidative injury plays an important role not only in the pathogenesis of AP but also in pancreatitis-induced hepatic damage. Antioxidant agents such as melatonin and ascorbic acid + N-acetyl cysteine, are capable of limiting pancreatic and hepatic damage produced during AP via restoring tissue antioxidant enzyme activities.

Topics: Acetylcysteine; Acute Disease; Animals; Antioxidants; Ascorbic Acid; Ceruletide; Female; Lipid Peroxidation; Liver; Melatonin; Pancreatitis; Rats; Rats, Wistar; Reactive Oxygen Species

1 Proteinase-activated receptor-2 (PAR2), a receptor activated by trypsin and tryptase, is abundantly expressed in the gastrointestinal tract including the C-fiber terminal, and might play a role in processing of visceral pain. In the present study, we examined and characterized the roles of PAR2 in pancreatitis-related abdominal hyperalgesia/allodynia in mice. 2 Caerulein, administered i.p. once, caused a small increase in abdominal sensitivity to stimulation with von Frey hairs, without causing pancreatitis, in PAR2-knockout (KO) mice, but not wild-type (WT) mice. 3 Caerulein, given hourly six times in total, caused more profound abdominal hyperalgesia/allodynia in PAR2-KO mice, as compared with WT mice, although no significant differences were detected in the severity of pancreatitis between the KO and WT animals. 4 The PAR2-activating peptide, 2-furoyl-LIGRL-NH(2), coadministered repeatedly with caerulein six times in total, abolished the caerulein-evoked abdominal hyperalgesia/allodynia in WT, but not PAR2-KO, mice. Repeated doses of 2-furoyl-LIGRL-NH(2) moderately attenuated the severity of caerulein-induced pancreatitis in WT animals. 5 Our data from experiments using PAR2-KO mice provide evidence that PAR2 functions to attenuate pancreatitis-related abdominal hyperalgesia/allodynia without affecting pancreatitis itself, although the PAR2AP applied exogenously is not only antinociceptive but also anti-inflammatory.

Topics: Abdominal Pain; Analgesics; Animals; Ceruletide; Female; Hyperalgesia; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligopeptides; Pain Measurement; Pain Threshold; Pancreatitis; Receptor, PAR-2; Touch

A considerable body of recent evidence suggests that oxidative stress and exaggerated production of reactive oxygen species play a major role in several aspects of inflammation and shock. Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Because polyphenolic compounds have high antioxidant potential, in this study we evaluated the effect of Hypericum perforatum extract on acute pancreatitis induced by cerulein administration in male CD mice. Intraperitoneal injection of cerulein in mice resulted in a severe, acute pancreatitis, which was characterized by edema, neutrophil infiltration, tissue hemorrhage, and cell necrosis as well as increases in the serum levels of amylase and/or lipase in comparison to sham-treated mice. The infiltration of the pancreatic tissue of these animals with neutrophils (measured as increase in myeloperoxidase activity) was associated with expression of the adhesion molecule ICAM-1. Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for nitrotyrosine and poly(ADP-ribose) (PAR) in the pancreas of cerulein-treated mice in comparison to sham-treated mice. In contrast, the degree of (a) pancreatic inflammation and tissue injury (histological score), (b) expression of ICAM-1, (c) the staining for nitrotyrosine and PAR, and (d) myeloperoxidase activity was markedly reduced in pancreatic tissue sections obtained from cerulein-treated mice administered Hypericum perforatum extract (30 mg/kg, suspended in 0.2 mL of saline solution, o.s.). Moreover, the treatment with Hypericum perforatum extract significantly reduced the mortality rate at 5 days after cerulein administration. Taken together, our results indicate that Hypericum perforatum extract reduces the development of acute pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Flavonoids; Hypericum; Inflammation; Male; Mice; Pancreatitis; Phenols; Phytotherapy; Plant Extracts; Polyphenols

To determine whether neutrophil depletion and Kupffer cell inhibition might combine their protective effects to decrease the severity of acute pancreatitis.. Mice had cerulein administration to induce acute pancreatitis and were pretreated with either anti-mouse neutrophil serum or gadolinium chloride (GdCl3) to prevent Kupffer cell activation, or both treatments. Injury was assessed in pancreas and lungs. Myeloperoxidases (MPO) assessed neutrophil infiltration. Interleukin-6 (IL-6) and IL-10 were measured in serum, pancreas, lungs and liver.. In mice with acute pancreatitis, neutrophil depletion reduced the severity of pancreatitis and pancreatitis-associated lung injury. Kupffer cell inactivation by GdCl3 had less protective effect, although IL-6 and IL-10 concentrations were significantly decreased. The protective treatment brought by neutrophil depletion was not enhanced by Kupffer cell inactivation and both treatments did not combine their protective effects.. Our results confirm the role of activated neutrophils in aggravating organ injury in acute pancreatitis while the role of Kupffer cell activation is less obvious.

Topics: Acute Disease; Amylases; Animals; Cell Count; Ceruletide; Gadolinium; Interleukins; Kupffer Cells; Liver; Lung; Macrophage Activation; Male; Mice; Mice, Inbred C57BL; Neutrophil Activation; Neutrophils; Pancreas; Pancreatitis; Peroxidase; Random Allocation; Severity of Illness Index

Acute pancreatitis accompanied by a subsequent infectious attack can often lead to multisystem organ dysfunction, including acute lung injury (ALI), but the molecular mechanisms are poorly defined. In this study, we explored the role of the priming insult by induction of cerulein pancreatitis, which was followed by the second attack due to endotoxemia, in the development of ALI in mice. Experiments revealed that LPS injection in mice with acute pancreatitis caused the development of ALI, as indicated by blood-gas derangements, pulmonary vascular hyperpermeability, increased inflammatory cell counts in bronchoalveolar lavage, and histologic lung damage. This was associated with the pancreatitis-induced increase in expression of macrophage migration inhibitory factor (MIF) in the lungs, together with elevated expression of Toll-like receptor (TLR)-4, both of which were inhibited by administration of anti-protease-activated receptor (PAR)-2 antibody. Furthermore, anti-MIF antibody treatment suppressed the pancreatitis-induced elevation of TLR-4 pulmonary expression. Genetic removal of MIF from mice resulted in less development of ALI in the setting of acute pancreatitis complicated by endotoxemia. These findings demonstrate that activation of protease-activated receptor-2 with trypsin, which can be released after pancreatitis induction, positively regulates the transcript level of MIF, and increased MIF results in exaggerated pulmonary expression of TLR-4, leading to the development of ALI with a subsequent infectious attack. We thus suggest that interventions designed to modulate MIF may have therapeutic advantages in treating ALI in patients with acute pancreatitis complicated by bacterial infection.

Topics: Acute Disease; Animals; Bronchoalveolar Lavage Fluid; Ceruletide; Endotoxemia; Lipopolysaccharides; Lung; Lung Injury; Macrophage Migration-Inhibitory Factors; Male; Mice; Mice, Inbred BALB C; Pancreatitis; Receptors, Proteinase-Activated; Toll-Like Receptor 4

Bacterial infection of the pancreas aggravates the course of acute pancreatitis. Since bacterial translocation from the gut is likely to be an early event, in an animal model of pancreatitis, we investigated the effect of early bacterial supra-infection of the pancreas on the course of the disease.. Six hours after the induction of acute pancreatitis in male Wistar rats (n = 180) by supramaximal stimulation with cerulein (or placebo in a control group), the animals were operated and a suspension of Helicobacter pylori, Escherichia coli or saline were introduced either in the pancreatic duct or interstitium (12 groups of 15 rats each); after 24 h, animals were killed and the following parameters analysed: macroscopic and histologic appearance of the pancreas (score), wet-to-dry weight ratio, pancreas trypsinogen activation peptide level, serum amylase, interleukin-6 and phospholipase A2 activity.. All parameters were increased in rats with cerulein-induced pancreatitis in comparison to placebo. Interstitial and intraductal application of bacteria increased the pancreatic damage. This effect was more evident with the application of E. coli in both cerulein and placebo groups. Application of E. coli but not of H. pylori determined pancreatic activation of trypsinogen, increased mortality and induced the production of interleukin-6.. Bacterial invasion of the pancreas worsens the histologic and clinical picture of disease and induces a systemic inflammatory response.

Topics: Acute Disease; Amylases; Animals; Bacterial Infections; Ceruletide; Disease Models, Animal; Interleukin-6; Male; Organ Size; Pancreas; Pancreatitis; Pancreatitis, Acute Necrotizing; Phospholipases A; Phospholipases A2; Rats; Rats, Wistar; Systemic Inflammatory Response Syndrome

The nervous system, through the vagus nerve, controls inflammation by decreasing the release of tumor necrosis factor-alpha from endotoxin stimulated macrophages. This anti-inflammatory effect is mediated by an interaction of acetylcholine, the principal neurotransmitter of the vagus nerve, with macrophage cholinergic nicotinic receptors expressing the alpha7 subunit.. To determine the role of this "nicotinic anti-inflammatory pathway" in experimental pancreatitis, we induced pancreatitis in mice by 12 hourly intraperitoneal injections of cerulein. Pancreatitis was preceded by unilateral left cervical vagotomy or pretreatment with the nicotinic receptor antagonist mecamylamine or by pretreatment with the selective alpha7 nicotinic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21).. Vagotomy or pretreatment with mecamylamine resulted in an enhanced severity of pancreatitis, as reflected by histology, edema, plasma hydrolases, and interleukin-6 levels. Furthermore, the number of neutrophils migrated to the pancreas was increased in these mice, as shown by myeloperoxidase content and intrapancreatic staining of neutrophils. Conversely, GTS-21 pretreatment strongly decreased the severity of pancreatitis. Pancreatitis-associated pulmonary inflammation was independent of the integrity of the vagus nerve and nicotinic receptors.. This study provides the first evidence for a therapeutic potential of the vagus nerve and the "nicotinic anti-inflammatory pathway" in attenuating inflammation and injury during experimental pancreatitis.

Topics: Animals; Biopsy, Needle; Ceruletide; Disease Models, Animal; Female; Immunohistochemistry; Mice; Mice, Inbred C57BL; Nicotinic Antagonists; Pancreatitis; Probability; Receptors, Nicotinic; Risk Factors; Sensitivity and Specificity; Severity of Illness Index; Vagotomy; Vagus Nerve

The cystic fibrosis (CF) mouse pancreas has constitutively elevated expression of the Reg/PAP cell stress genes (60-fold greater Reg3alpha, and 10-fold greater PAP/Reg3beta and Reg3gamma). These genes are suggested to be involved in protection or recovery from pancreatic injury.. To test this idea the supramaximal caerulein model was used to induce acute pancreatitis in wild type and CF mice. Serum amylase, pancreatic water content (as a measure of edema), pancreatic myeloperoxidase activity, and Reg/PAP expression were quantified.. In both wild type and CF mice caerulein induced similar elevations in serum amylase (maximal at 12 h), pancreatic edema (maximal at 7 h), and pancreatic myeloperoxidase activity (MPO, a marker of neutrophil infiltration; maximal at 7 h). By immunohistochemistry, Reg3alpha was strongly expressed in the untreated CF pancreas but not in wild type. During pancreatitis, Reg3alpha was intensely expressed in foci of inflamed tissue in both wild type and CF.. These data demonstrate that the severity of caerulein-induced pancreatitis is not ameliorated in the CF mouse even though the Reg/PAP stress genes are already highly upregulated. While Reg/PAP may be protective they may also have a negative effect during pancreatitis due to their anti-apoptotic activity, which has been shown to increase the severity of pancreatitis.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Ceruletide; Disease Models, Animal; Female; Gene Expression Regulation; Lectins, C-Type; Male; Mice; Mice, Inbred CFTR; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Peroxidase; Proteins; Regeneration; RNA

To investigate the effects of hyperlipidemia on acute pancreatitis (AP) and the possible mechanisms.. Rat models of hyperlipidemia and AP were established by Triton WR1339 and cerulein respectively. Human albumin was used to treat AP complicated by hyperlipidemia. In each group, we compared the histological score, volume of ascites, ratio of pancreatic wet/dry weight, serum amylase (AMY) and pancreatic acinar cell apoptosis. The level of protein kinase C (PKC) membrane translocation in pancreatic tissue was detected by Western blot.. In the hyperlipidemia model established by Triton WR1339, triglyceride (TG) increased remarkably and reached its peak 6 h after injection, and most rats developed mild acute pancreatitis. Histological score, volume of ascites, ratio of wet/dry weight and serum AMY in AP animals with hyperlipidemia were obviously higher than those in AP animals (P < 0.05) and decreased after albumin therapy but not significantly (P > 0.05). Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) increased in AP animals with hyperlipidemia and did not change distinctly after albumin therapy. PKC membrane translocation level increased in AP animals with hyperlipidemia and decreased remarkably after albumin therapy (P < 0.05).. Hyperlipidemia may induce AP or intensify pancreatic injury. Albumin therapy can not alleviate pancreatic lesion effectively. PKC activation may be one mechanism by which AP is intensified by hyperlipidemia.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Ascites; Ceruletide; Enzyme Activation; Hyperlipidemias; In Situ Nick-End Labeling; Lipids; Male; Organ Size; Pancreas; Pancreatitis; Polyethylene Glycols; Protein Kinase C; Rats; Rats, Sprague-Dawley

Pancreatic duct obstruction induces edematous but not hemorrhagic pancreatitis even when combined with maximal secretory stimulation. The aim of the present study was to test the hypothesis that pancreatic and bile duct obstruction exacerbates edematous pancreatitis induced by supramaximal secretory stimulation by caerulein.. In in vivo studies using rats, biliopancreatic duct ligation was combined with supramaximal stimulation of caerulein, and pancreatic histology, serum amylase level, pancreatic edema, and intrapancreatic trypsin activation were evaluated. In in vitro studies, the pancreatic acini were isolated from the rats with biliopancreatic duct ligation, and amylase secretion, intracellular trypsin activation, and acinar cell fragility were evaluated.. Biliopancreatic duct ligation exacerbated caerulein-induced pancreatitis from edematous to hemorrhagic only when the obstruction preceded caerulein administration. The amylase secretion from the acini was inhibited, and intracellular trypsin activation and the acinar cell fragility on the supramaximal stimulation with cholecystokinin in vitro were enhanced by the preceding in vivo biliopancreatic duct obstruction.. Preceding biliopancreatic duct obstruction exacerbates caerulein-induced pancreatitis. Enhancement of intracellular trypsin activation is possibly involved in this mechanism.

Topics: Amylases; Animals; Ceruletide; Cholecystokinin; Cholestasis; Constriction, Pathologic; Disease Models, Animal; Disease Progression; Hemorrhage; In Vitro Techniques; Male; Pancreas; Pancreatic Ducts; Pancreatitis; Rats; Rats, Wistar; Trypsin

Type IV phosphodiesterase is a key enzyme to metabolize intracellular adenosine 3',5'-cyclic monophosphate (cAMP) expressed in inflammatory cells. The specific type IV phosphodiesterase inhibitor that increases intracellular cAMP is known to be potent suppressor of proinflammatory cytokines. However, the effect of phosphodiesterase inhibitors on the development of pancreatitis has not been well understood. In the present study, we examined the effect of a specific type IV phosphodiesterase inhibitor on experimentally induced pancreatitis.. Severity of cerulein-induced pancreatitis and pancreatic proinflammatory cytokine levels were studied with or without pretreatment with a specific type IV phosphodiesterase inhibitor (rolipram) in Sprague-Dawley rats.. Administration of rolipram clearly ameliorated severity of pancreatitis evaluated by edema, serum amylase (P<0.05), and lipase levels (P<0.05) in rats. Also, the level of pancreatic proinflammatory cytokine (interleukin-1beta (IL-1beta)) was significantly reduced when rats were treated with rolipram prior cerulein injection (P<0.05).. Our results demonstrated that intracellular cAMP and pancreatic proinflammatory cytokine level, which are regulated by type IV phosphodiesterase, might play an important role in the pathogenesis of acute pancreatitis.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Ceruletide; Cyclic Nucleotide Phosphodiesterases, Type 4; Interleukin-1; Male; Pancreas; Pancreatitis; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Rolipram; Tumor Necrosis Factor-alpha

The peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The aim of the present study was to examine the effects of endogenous PPAR-alpha ligand on the development of acute pancreatitis caused by cerulein in mice. Intraperitoneal injection of cerulein into PPAR-alpha wild-type (WT) mice resulted in severe, acute pancreatitis characterized by oedema, neutrophil infiltration and necrosis and by elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as an increase in myeloperoxidase activity) was associated with enhanced expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF) in the pancreas of cerulein-treated PPAR-alpha wild-type (WT) mice in comparison to sham-treated mice. Acute pancreatitis in PPAR-alphaWT mice was also associated with a significant mortality (20% survival at 5 days after cerulein administration). In contrast, the degree of pancreatic inflammation and tissue injury (histological score), up-regulation/formation of ICAM-1 and P-selectin, infiltration of neutrophils, and the expression of TGF-beta and VEGF was markedly enhanced in pancreatic tissue obtained from cerulein-treated PPAR-alpha knockout (KO) mice. Thus, endogenous PPAR-alpha ligands reduce the degree of pancreas injury caused by acute pancreatitis induced by cerulein administration.

Topics: Acute Disease; Amylases; Animals; Biomarkers; Blotting, Western; Ceruletide; Intercellular Adhesion Molecule-1; Islets of Langerhans; Lipase; Mice; Mice, Knockout; Neutrophil Infiltration; P-Selectin; Pancreatitis; Peroxidase; PPAR alpha; Random Allocation; Survival Rate; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

The recruitment of inflammatory cells contributes significantly to tissue injury in acute pancreatitis. This process implies several molecular interactions between circulating and endothelial cells. The adhesion molecule junctional adhesion molecule C (JAM-C) is involved in leukocyte transendothelial migration and it can form homophilic (JAM-C/JAM-C) and heterophilic interactions with the leukocyte integrin alpha(M)beta(2). In this study, the effect of early administration of monoclonal antibodies directed against JAM-C in cerulein-induced acute pancreatitis was assessed. This reagent significantly blocked influx of leukocytes, release of serum amylase, secretion of inflammatory cytokines, and acinar cell necrosis. These effects were rapid and protected against tissue injury throughout the duration of the model. Conversely, cerulein-induced acute pancreatitis was more severe in transgenic mice overexpressing JAM-C on endothelial cells under the control of the Tie2 promoter. It is proposed that JAM-C expressed by endothelial cells contributes to the pathophysiology of acute pancreatitis and could be considered a target for clinical applications.

Topics: Acute Disease; Amylases; Animals; Antibodies, Monoclonal; Blotting, Western; Cell Adhesion Molecules; Ceruletide; Chemotaxis, Leukocyte; Edema; Endothelial Cells; Immunoglobulins; Immunohistochemistry; Interleukin-6; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Animal; Necrosis; Pancreas; Pancreatitis

Primary sensory neurons of the C and Adelta subtypes express the vanilloid capsaicin receptor TRPV1 and contain proinflammatory peptides such as substance P (SP) that mediate neurogenic inflammation. Pancreatic injury stimulates these neurons causing the release of SP in the pancreas resulting in pancreatic edema and neutrophil infiltration that contributes to pancreatitis. Axons of primary sensory neurons innervating the pancreas course through the celiac ganglion. We hypothesized that disruption of the celiac ganglion by surgical excision or inhibition of C and Adelta fibers through blockade of TRPV1 would reduce the severity of experimental pancreatitis by inhibiting neurogenic inflammation. Resiniferatoxin (RTX) is a specific TRPV1 agonist that, in high doses, selectively destroys C and Adelta fibers. Sprague-Dawley rats underwent surgical ganglionectomy or application of 10 microg RTX (vs. vehicle alone) to the celiac ganglion. One week later, pancreatitis was induced by six hourly intraperitoneal injections of caerulein (50 microg/kg). The severity of pancreatitis was assessed by serum amylase, pancreatic edema, and pancreatic myeloperoxidase (MPO) activity. SP receptor (neurokinin-1 receptor, NK-1R) internalization in acinar cells, used as an index of endogenous SP release, was assessed by immunocytochemical quantification of NK-1R endocytosis. Caerulein administration caused significant increases in pancreatic edema, serum amylase, MPO activity, and NK-1R internalization. RTX treatment and ganglionectomy significantly reduced pancreatic edema by 46% (P < 0.001) and NK-1R internalization by 80% and 51% (P < 0.001 and P < 0.05, respectively). RTX administration also significantly reduced MPO activity by 47% (P < 0.05). Neither treatment affected serum amylase, consistent with a direct effect of caerulein. These results demonstrate that disruption of or local application of RTX to the celiac ganglion inhibits SP release in the pancreas and reduces the severity of acute secretagogue-induced pancreatitis. It is possible that selectively disrupting TRPV1-bearing neurons could be used to reduce pancreatitis severity.

Topics: Acute Disease; Animals; Ceruletide; Denervation; Ganglia, Sympathetic; Male; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Substance P

To study the histological and pancreatitis-associated protein mRNA accumulation changes of pancreas from acute phase of caerulin-induced pancreatitis to recuperation in rats.. Acute pancreatitis was induced by caerulein in male Wistar rats and followed up for 90 d by histological and mRNA analyses of pancreas. Pancreases were dissected at 0, 9, 24 h and 3, 5, 15, 30, 60, 90 d post-induction. Edema (E), polymorphonuclear neutrophil (PMN) infiltration, cytoplasmic vacuolization (V), zymogen granule depletion (ZD) and acinar disorganization (AD) were microscopically evaluated. Accumulation of pancreatitis-associated protein (PAP) and L13A mRNAs were quantified by real-time PCR.. The main histological changes appeared at 9 h post-induction for PMN infiltration and cytoplasmic V, while at 24 h and 3 d for E and ZD, respectively. All the parameters were recovered after 5 d, except for ZD which delayed more than 30 d. The main AD was observed after 15 d and values returned to normal after 30 d. Similarly to histological changes, accumulation of the PAP mRNA was increased at 9 h with the highest accumulation at 24 h and differences disappeared after 5 d.. From the acute phase to recuperation of pancreatitis, regeneration and re-differentiation of pancreas occur and PAP expression is exclusively an acute response of pancreatitis.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Ceruletide; Gene Expression; Lectins, C-Type; Male; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Polymerase Chain Reaction; Rats; Rats, Wistar; Recovery of Function; RNA, Messenger

Acute pancreatitis is a disease involving pro-inflammatory mediators. Two complex and multifactorial pathogenetic ways lead to edematous or necrotizing pancreatitis. The course of the disease is thought to be the consequence of an acute inflammatory response.. The authors examined the impact of Escherichia coli LPS on the acute cerulein pancreatitis in rats.. The study was performed on rats using the ceruleine pancreatitis model. The activation status of polymorphonuclear cells, blood IL-6 concentration, oxidative stress parameters, pancreatic enzymes concentration and microscopic alterations were determined at 5th and 9th h observations.. In acute pancreatitis and acute pancreatitis with LPS groups, the peripheral polymorphonuclear cells activity was lower than in control one. Authors noticed the same neutrophil activation in acute pancreatitis after lipopolysaccharide administration although the peripheral blood polymorphonuclear cells count was significantly higher at the 9th h observation. LPS neither changed the oxidative stress within pancreatic gland, nor amylase or serum lipase activity. LPS given to acute pancreatitis animals resulted in significant increase of serum IL-6 concentration at 5th observation turning normal after 9th h.. Collected data supports thesis of early polymorphonuclear cells involvement in acute pancreatitis and oxidative stress evidence in pancreatic parenchyma. However, results did not reveal that administration of LPS amplified inflammatory response during the course of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Disease Models, Animal; Interleukin-6; Leukocyte Count; Lipase; Lipopolysaccharides; Male; Neutrophil Activation; Neutrophils; Oxidative Stress; Pancreas; Pancreatitis; Rats; Rats, Wistar; Shock, Septic

Oxygen free radicals (OFR) have been implicated in the induction of acute pancreatitis (AP).. The aim of this study was to determine the effect of ascorbic acid and N-acetylcysteine (NAC), potent antioxidants, against oxidative stress in AP.. AP was induced by two i.p. injections of caerulein at 2-hour intervals (50 microg/kg BW). One group received additionally an antioxidant mixture composed of L(+)-ascorbic acid (14.3 mg/kg BW) and NAC (181 mg/kg BW) i.p. The rats were sacrificed 12 h after the last injection. Oxidative stress markers were evaluated. Light-microscopic and ultrastructural examination was performed.. Formation of vacuoles, mitochondrial damage, and dilatation of rough endoplasmic reticulum, margination and clumping of chromatin were major ultrastructural alterations in AP group. Ascorbic acid + NAC prevented these changes. Small vacuoles were present within the cytoplasm of some of the acinar cells. Pancreas damage was accompanied by an increase in tissue malondialdehyde (MDA) levels (p < 0.05), whereas a decrease was seen in catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) activities and total glutathione (GSH) levels (p < 0.005). Ascorbic acid + NAC decreased MDA levels but increased CAT, SOD, GPx activities and GSH levels (p < 0.005).. These results suggest that ascorbic acid + NAC is potentially capable of limiting pancreatic damage produced during AP via protecting fine structure of acinar cells and tissue antioxidant enzyme activities.

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Ceruletide; Female; Gastrointestinal Agents; Oxidative Stress; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley

Acute pancreatitis (AP) is an inflammatory disease involving the production of different cytokines and chemokines and is characterized by leukocyte infiltration. Because the chemokine receptor CCR5 and its ligands [the CC chemokines CCL3/MIP-1alpha, CCL4/MIP-1beta, and CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES)] regulate leukocyte chemotaxis and activation, we investigated the expression of CCR5 ligands and the role of CCR5 and its ligands in experimental AP in mice. AP was induced by hourly intraperitoneal injections of cerulein in CCR5-deficient (CCR5(-/-)) or wild-type (WT) mice. Induction of AP by cerulein resulted in an early increase of pancreatic CCL2, CCL3, and CCL4 mRNA expression, whereas CCL5 mRNA expression occurred later. CCR5(-/-) mice developed a more severe pancreatic injury than WT mice during cerulein-induced AP, as assessed by a more pronounced increase in serum amylase and lipase levels and by more severe pancreatic edema, inflammatory infiltrates (mainly neutrophils), and necrosis. CCR5(-/-) mice also exhibited increased production of CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-1beta during the course of cerulein-induced AP. In vivo simultaneous neutralization of CC chemokines with monoclonal antibodies in CCR5(-/-) mice reduced the severity of cerulein-induced AP, indicating a role of CC chemokines in exacerbating the course of AP in the absence of CCR5. Moreover, simultaneous neutralization of CCR5 ligands in WT mice also reduced the severity of cerulein-induced AP. In conclusion, lack of the chemokine receptor CCR5 exacerbates experimental cerulein-induced AP and leads to increased levels of CC chemokines and a more pronounced pancreatic inflammatory infiltrate, suggesting that CCR5 expression can modulate severity of AP.

Topics: Acute Disease; Animals; Ceruletide; Female; Mice; Mice, Knockout; Pancreatitis; Receptors, CCR5

Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the digestive enzyme production and cytoplasmic vacuolization, the death of acinar cells, edema formation, and an infiltration of inflammatory cells into the pancreas. Cytokines are up-regulated in pancreatic acinar cells stimulated with cerulein. In various cells and tissues, Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway mediates inflammatory process. In the present study, we investigated whether the activation of Jak/Stat signaling mediates IL-1beta expression in pancreatic acinar AR42J cells stimulated with cerulein in vitro as well as the rats with cerulein pancreatitis in vivo using AG490, the Jak2 inhibitor. Activation of Jak2 and Stat3 were monitored by Western blot analysis for phosphorylated Jak2 and phosphorylated Stat3. mRNA expression and protein level of IL-1beta were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). Histological examination of pancreatic tissues were performed and serum IL-1beta levels of the rats were determined by ELISA. As a result, cerulein induced the activation of Jak2 and Stat3 as well as IL-1beta expression, which was inhibited by the treatment of AG490 in AR42J cells. In cerulein pancreatitis of the rats, edematous and inflammatory changes of the pancreas and increased serum levels of IL-1beta were suppressed by AG490 treatment. In conclusion, Jak2/Stat3 pathway may be the underlying mechanism in the pathogenesis of pancreatitis by inducing cytokines such as IL-1beta.

Topics: Animals; Cells, Cultured; Ceruletide; Disease Models, Animal; Drug Therapy, Combination; Enzyme Inhibitors; Gene Expression; Interleukin-1beta; Janus Kinase 2; Male; Pancreas; Pancreatitis; Phosphorylation; Rats; Rats, Sprague-Dawley; RNA, Messenger; STAT3 Transcription Factor; Tyrphostins

The plasminogen (plg) system participates in tissue repair in several organs, but its role in pancreas repair remains poorly characterized. To understand better the role of plg in pancreas recovery following injury, we examined the course of cerulein-induced pancreatitis in plg-deficient and -sufficient mice.. Pancreatitis was induced by cerulein administration (50 microg/kg, 7 intraperitoneal injections). Mice were killed either at the acute phase (7 hours after the first cerulein injection) or during recovery (at 2, 4, and 7 days). In pancreatic sections, we examined pancreatic morphology, trypsin activation, inflammatory cell infiltration, acinar cell death, cell proliferation, extracellular matrix deposition, activation of stellate cells (PSCs), and components of the plg and metalloproteinase systems.. In plg-sufficient mice, pancreatic plg levels and plasmin activity increased during the acute phase and remained elevated during recovery. Pancreatitis resolved in plg-sufficient mice within 7 days. Pancreas recovery involved reorganization of the parenchyma structure, removal of necrotic debris, cell proliferation, transient activation of PSCs, and moderate deposition of extracellular matrix proteins. Acute pancreatitis (7 hours) was indistinguishable between plg-deficient and -sufficient mice. In contrast, pancreas recovery was impaired in plg-deficient mice. Plg deficiency led to disorganized parenchyma, extensive acinar cell loss, poor removal of necrotic debris, reduced cell proliferation, and fibrosis. Fibrosis was characterized by deposition of collagens and fibronectin, persistent activation of PSCs, and up-regulation of pancreatic transforming growth factor beta1.. Plg/plasmin deficiency leads to features similar to those found in chronic pancreatitis such as parenchymal atrophy and fibrosis.

Topics: Animals; Apoptosis; Cell Proliferation; Ceruletide; Disease Models, Animal; Fibrinolysin; Follow-Up Studies; Immunoblotting; Immunohistochemistry; Mice; Pancreatitis; Plasminogen; Recovery of Function; Severity of Illness Index; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin

Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the colon injury associated with experimental colitis. The aim of the present study was to examine the effects of 3-aminobenzamide (3-AB), an inhibitor of PARP activity, in the development of acute pancreatitis caused by cerulein in mice. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by oedema, neutrophil infiltration and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced expression of the intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF) in the pancreas of cerulein-treated mice in comparison to sham-treated mice. Acute pancreatitis in vehicle-treated mice was also associated with a significant mortality (40% survival at 5 days after cerulein administration). In contrast, (1) the degree of pancreatic inflammation and tissue injury (histological score), (2) upregulation/formation of ICAM-1 and P-selectin, (4) neutrophils infiltration and (5) the expression of TGF-beta and VEGF was markedly reduced in pancreatic tissue obtained from cerulein-treated mice which have been treated with 3-AB. These findings provide the evidence that PARP inhibition reduce the degree of pancreas injury caused by acute pancreatitis induced by cerulein administration.

Topics: Acute Disease; Animals; Benzamides; Ceruletide; Disease Models, Animal; Enzyme Inhibitors; Immunohistochemistry; Intercellular Adhesion Molecule-1; Male; Mice; Neutrophil Infiltration; P-Selectin; Pancreas; Pancreatitis; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Survival Rate; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

The R122H mutation of the cationic trypsinogen was found in patients with hereditary pancreatitis. A transgenic animal carrying this mutation could be useful as a genetic model system of pancreatitis.. Mice transgenic for the human R122H cationic trypsinogen were generated using the -205 fragment of the rat elastase promoter. The presence of the transgene was assayed in the DNA, in pancreatic mRNA and in zymogen granule lysates. Serum levels of amylase, lipase and cytokines (MCP-1, IL-6) were monitored and the histological appearance of the tissue was investigated. Pancreatitis was induced by 7 hourly injections of 50 mug/kg cerulein. The procedure was repeated twice weekly for 10 consecutive weeks. The animals were sacrificed 24 (n = 8) and 48 hours (n = 8) after the first injection and at the end of the whole treatment (n = 7).. The transgene was detected at the genomic level and in pancreatic mRNA. The corresponding protein was found in low amounts in zymogen granule lysates. R122H mice showed elevated pancreatic lipase, but there was no spontaneous development of pancreatitis within 18 months. After induction of pancreatitis, levels of lipase (after 24 hours) and amylase (after 48 hours) were higher in R122H mice compared to controls. Repeated treatment with cerulein resulted in a slightly more severe pancreatitis in R122H animals. Amylase, lipase, and the cytokine levels were similar to controls.. The R122H transgenic mouse failed to develop a spontaneous pancreatitis but a repeatedly provoked cerulein-induced pancreatitis led to a slightly more severe pancreatitis. The rather small difference in comparison to controls could be due to the low expression of the transgene in the mouse pancreas.

Topics: Amylases; Animals; Arginine; Ceruletide; Cytokines; Disease Models, Animal; DNA; Histidine; Humans; Lipase; Mice; Mice, Transgenic; Mutation; Pancreas; Pancreatitis; Phenotype; RNA, Messenger; Secretory Vesicles; Severity of Illness Index; Time Factors; Transgenes; Trypsin; Trypsinogen

Recent studies have shown that stimulation of cannabinoid 1 (CB1) receptor reduces the area of ischemic myocardial necrosis and affects activity of the digestive tract. The aim of the present study was to check whether the administration of CB1 receptor agonist or antagonist affects the stress-induced gastric ulceration and development of edematous pancreatitis.. Experiments were performed on rats. Gastric lesions were induced by water immersion and restrain stress (WRS). Acute pancreatitis was induced by cerulein. Prior to WRS or before and during cerulein administration, a natural endogenous ligand for CB1 receptor, anandamide was administered intraperitoneally at the dose of 0.8, 1.5 or 3.0 micromol/kg. A synthetic CB1 receptor antagonist, AM 251 (ALEXIS(R) Biochemicals) was administrated at the dose of 4 micromol/kg i.p. alone or in combination with anandamide at the dose of 1.5 micromol/kg.. Administration of anandamide reduced gastric lesions and this effect was associated with am increase in gastric mucosal blood flow and mucosal DNA synthesis; whereas serum level of pro-inflammatory interleukin-1 beta was reduced. Treatment with AM 251 aggravated gastric damage and reversed protective effect of anandamide administration. Opposite effect was observed in the pancreas. Administration of anandamide increased dose-dependently the severity of pancreatitis. In histological examination, we observed an increase in pancreatic edema and inflammatory infiltration. Also, treatment with anandamide augmented the pancreatitis-induced increase in serum level of lipase, amylase, poly-C ribonuclease, and pro-inflammatory interleukin-1 beta; whereas pancreatic DNA synthesis was reduced. Treatment with AM 251 reduced histological and biochemical signs of pancreatic damage and reversed deleterious effect of anandamide in cerulein-induced acute pancreatitis.. Activation of CB1 receptors evokes opposite effects in the stomach and pancreas: in the stomach, exhibits protective effect against stress-induced gastric mucosal lesions; whereas in the pancreas, increases the severity of cerulein-induced pancreatitis.

Topics: Acute Disease; Animals; Arachidonic Acids; Cannabinoids; Ceruletide; DNA; Endocannabinoids; Gastric Mucosa; Interleukin-1beta; Male; Pancreas; Pancreatitis; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Restraint, Physical; Stomach Ulcer; Stress, Physiological

Forty Wistar rats were divided into 5 groups, including the control group, the acute pancreatitis group (AP group, induced by intraperitoneal injections of caerulein), and the AP group treated with baicalin, the AP group treated with LPS, and the AP group treated with LPS and baicalin. Pathological damage of pancreatic tissue was scored with hematoxylin and eosin (HE) staining. The mRNA expression of TNF-alpha was measured with semiquantitative RT-PCR, and activation of NF-kappaB was detected with flow cytometry assay. It was shown in the results that the expression of TNF-alpha mRNA, activation of NF-kappaB, and pathological score of AP group were all obviously higher than those of control group (P < .01). In AP group treated with LPS, further rise of these values were observed (P < .01). In the AP group treated with baicalin, activation of NF-kappaB decreased (P < .05), and expression of TNF-alpha mRNA also obviously decreased (P < .01), while pancreatic pathological damage was alleviated at the same time (P < .01); similar results were observed in AP group treated with LPS and baicalin (P < .01), which indicated that baicalin might be applied to inhibit NF-kappaB activating and TNF-alpha expressing so as to treat AP.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Base Sequence; Ceruletide; DNA Primers; Flavonoids; Inflammation Mediators; Male; NF-kappa B; Pancreatitis; Rats; Rats, Wistar; RNA, Messenger; Tumor Necrosis Factor-alpha

Protease-activated receptor-2 (PAR-2) is a widely expressed tethered ligand receptor that can be activated by trypsin and other trypsin-like serine proteases. In the exocrine pancreas, PAR-2 activation modulates acinar cell secretion of digestive enzymes and duct cell ion channel function. During acute pancreatitis, digestive enzyme zymogens, including trypsinogen, are activated within the pancreas. We hypothesized that trypsin, acting via PAR-2, might regulate the severity of that disease, and to test this hypothesis, we examined the effect of either genetically deleting or pharmacologically activating PAR-2 on the severity of secretagogue-induced experimental pancreatitis. We found that experimental acute pancreatitis is more severe in PAR-2(-/-) than in wild-type mice and that in vivo activation of PAR-2, achieved by parenteral administration of the PAR-2-activating peptide SLIGRL-NH2, reduces the severity of pancreatitis. In the pancreas during the early stages of pancreatitis, the MAPK ERK1/2 is activated and translocated to the nucleus, but nuclear translocation is reduced by activation of PAR-2. Our findings indicate that PAR-2 exerts a protective effect on pancreatitis and that activation of PAR-2 ameliorates pancreatitis, possibly by inhibiting ERK1/2 translocation to the nucleus. Our observations suggest that PAR-2 activation may be of therapeutic value in the treatment and/or prevention of severe clinical pancreatitis, and they lead us to speculate that, from a teleological standpoint, PAR-2 may have evolved in the pancreas as a protective mechanism designed to dampen the injurious effects of intrapancreatic trypsinogen activation.

Topics: Animals; Ceruletide; Enzyme Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Oligopeptides; Pancreatitis; Peptides; Receptor, PAR-2

Central neuropeptides play a role in many physiological functions through the autonomic nervous system. We have recently demonstrated that central injection of a thyrotropin-releasing hormone (TRH) analog increases pancreatic blood flow through vagal and nitric oxide-dependent pathways. In this study, the central effect of a TRH analog on experimental acute pancreatitis was investigated in rats. Acute pancreatitis was induced by two intraperitoneal injections of cerulein (40 microg/kg) at 1-h interval. Either stable TRH analog, RX 77368 (5-100 ng), or saline was injected intracisternally 15 min before the first cerulein injection under ether anesthesia. Serum amylase level was measured before and 5 h after the first cerulein injection. Pancreatic wet/dry weight ratio and histological changes were also evaluated. Intracisternal TRH analog inhibited cerulean-induced elevation of serum amylase level, increase in pancreatic wet/dry weight ratio and pancreatic histological changes, such as interstitial edema, inflammation and vacuolization. The pancreatic cytoprotection induced by central TRH analog was abolished by subdiaphragmatic vagotomy and N(G)-nitro-L-arginine-methyl ester (L-NAME), but not by 6-hydroxydopamine (6-OHDA). Intravenous administration of the TRH analog did not influence cerulein-induced acute pancreatitis. These results indicate that the TRH analog acts in the central nervous system to protect against acute pancreatitis through vagal and nitric oxide-dependent pathways.

Topics: Adrenergic Agents; Amylases; Animals; Central Nervous System; Ceruletide; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Male; Neuropeptides; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidopamine; Pancreas; Pancreatitis; Peptides; Pyrrolidonecarboxylic Acid; Rats; Rats, Wistar; Thyrotropin-Releasing Hormone; Time Factors

Microcirculatory disturbances are important early pathophysiological events in various organs during acute pancreatitis (AP). The aim of the study was to investigate an influence of L-arginine (nitric oxide substrate) and N(G)-nitro-L-arginine (L-NNA, nitric oxide synthase inhibitor) on organ microcirculation in experimental acute pancreatitis induced by four consecutive intraperitoneal cerulein injections (15 microg/kg/h). The microcirculation of pancreas, liver, kidney, stomach, colon and skeletal muscle was measured by laser Doppler flowmeter. Serum interleukin 6 and hematocrit levels were analyzed. AP resulted in a significant drop of microperfusion in all examined organ. L-arginine administration (2 x 100 mg/kg) improved the microcirculation in the pancreas, liver, kidney, colon and skeletal muscle, and lowered hematocrit levels. L-NNA treatment (2 x 25 mg/kg) caused aggravation of edematous AP to the necrotizing situation, and increased IL-6 and hematocrit levels. A further reduction of blood perfusion was noted in the stomach only. It is concluded that L-arginine administration has a positive influence on organ microcirculatory disturbances accompanying experimental cerulein-induced AP. NO inhibition aggravates the course of pancreatitis.

Topics: Acute Disease; Animals; Arginine; Ceruletide; Enzyme Inhibitors; Interleukin-6; Male; Microcirculation; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pancreas; Pancreatitis; Rats; Rats, Wistar; Regional Blood Flow

Earlier studies have shown that mice deficient in NK1 receptors or its ligand, substance P, are protected against acute pancreatitis and associated lung injury. In the current study, the protective effect of NK1 receptor blockage against acute pancreatitis and associated lung injury was investigated, using a specific receptor antagonist, CP-96345. Acute pancreatitis was induced in mice by intraperitoneal (i.p.) injections of caerulein. Substance P levels in plasma, pancreas, and lungs were found to be elevated in a caerulein dose-dependent manner. Mice treated with CP-96345, either prophylactically, or therapeutically, were protected against acute pancreatitis and associated lung injury as evident by attenuation in plasma amylase, pancreatic and pulmonary myeloperoxidase activities, and histological evidence of pancreatic and pulmonary injuries. Pulmonary microvascular permeability was also reduced as a result of CP-96345 treatment. These results point to a key role of NK1 receptors in acute pancreatitis and associated lung injury.

Topics: Amylases; Animals; Biphenyl Compounds; Capillary Permeability; Ceruletide; Lung; Lung Diseases; Mice; Mice, Inbred BALB C; Neurokinin-1 Receptor Antagonists; Pancreatitis; Peroxidase; Receptors, Neurokinin-1; Substance P; Time Factors

Alcohol consumption is a risk factor for chronic pancreatitis (CP), but the mechanism in humans remains obscure because prolonged alcohol consumption in most humans and animal models fails to produce alcoholic chronic pancreatitis (ACP). We hypothesize that the process leading to ACP is triggered by a sentinel acute pancreatitis (AP) event; this event causes recruitment of inflammatory cells, which initiates fibrosis driven by the anti-inflammatory response to recurrent AP and/or chronic oxidative stress. The aim was to determine whether chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in the rat. Wistar male rats were pair-fed control (C) or 5% ethanol (E) Lieber-DeCarli liquid diets. Animals were studied without pancreatitis (P0), with cerulein pancreatitis induced once (P1), or with cerulein-induced pancreatitis weekly for 3 weeks (P3). AP markers, inflammation, and fibrosis were measured histologically, by gene expression profiling and protein expression. Macrophage infiltration was reduced in EP0 versus CP0 rats, but the pattern was reversed after AP. Microabscess, severe necrosis, and early calcification were only induced in the EP3 rats. Fibrosis was significantly induced in the EP3 rats versus EP1, CP1, and CP3 by histology, hydroxyproline content, and mRNA expression for collagen alpha1(1) and procollagen alpha2(1). Proinflammatory cytokine mRNAs were up-regulated shortly after induction of AP, while the anti-inflammatory cytokines (interleukin-10 and transforming growth factor-beta) were strongly up-regulated later and in parallel with fibrogenesis, especially in the EP3 rats. Pancreatic fibrosis develops after repeated episodes of AP and is potentiated by alcohol. Expression of fibrosis-associated genes was associated with expression of anti-inflammatory cytokines in alcohol-fed rats.

Topics: Alcohol Drinking; Alcoholism; Animals; Ceruletide; Chronic Disease; Disease Models, Animal; DNA Primers; Fibrosis; Gene Expression Regulation; Male; Pancreatitis; Polymerase Chain Reaction; Rats; Rats, Wistar; RNA, Messenger

Decreased levels of expression of opsonin receptors (CD11b and CD32/16) on peritoneal exudate neutrophils may lead to susceptibility to infection. Granulocyte colony-stimulating factor (G-CSF) increases the expression levels of CD11b on neutrophils and prolongs neutrophil survival. The effects of G-CSF on neutrophils and opsonin receptor expressions of neutrophils were investigated in cerulein-induced acute pancreatitis.. Forty-two mice were randomly assigned to each group (n = 6). Mice received subcutaneous G-CSF (120 microg/kg body weight) before the induction of acute pancreatitis with cerulein. Saline was used for instead of G-CSF or cerulein solution in control groups. CD11b and CD32/16 expression levels on circulatory and peritoneal exudate neutrophils were investigated 6 and 24 hours after the induction of acute pancreatitis.. Treatment with G-CSF did not aggravate the inflammation of pancreatic tissue evaluated by plasma amylase, acinar necrosis. However, it significantly increased the number of peritoneal exudate neutrophils (P < 0.05) and the CD11b- (P < 0.05) and CD32/16-positive (P < 0.05) peritoneal exudate neutrophils in mice with cerulein-induced acute pancreatitis. The means of fluorescence intensity for CD11b and CD32/16 expressions on circulatory and peritoneal exudate neutrophils were also elevated in the G-CSF groups.. G-CSF administration increases the numbers of neutrophils and improves expression levels of opsonin receptors on neutrophils in mice with cerulein-induced acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; CD11b Antigen; Ceruletide; Female; Granulocyte Colony-Stimulating Factor; Leukocyte Count; Mice; Mice, Inbred BALB C; Neutrophils; Pancreatitis; Receptors, IgG; Receptors, Immunologic

VIP receptor has been clarified to exist on immune cells, indicating its possible involvement in immunity and inflammatory response. Therefore, we investigated the effects of VIP and selective agonists for 2 subtypes of VIP receptor (VPAC1-R and VPAC2-R agonist) on acute pancreatitis.. Acute pancreatitis was induced in mice by 4 intraperitoneal injections of cerulein and an injection of LPS. VIP, VPAC1-R agonist, VPAC2-R agonist, or secretin (5 nmol/body) was administered 30 minutes before and after the administration of LPS. Serum amylase and cytokine levels were determined, and histologic changes were evaluated. In vitro, IL-6 and TNF-alpha production by monocytes from the spleen was determined under the stimulation of LPS with VIP, VPAC1-R agonist, or VPAC2-R agonist, and the expression of VPAC1-R and VPAC2-R mRNA in monocytes was examined.. VPAC1-R agonist significantly decreased serum amylase, IL-6, and TNF-alpha, whereas VPAC2-R agonist markedly increased serum amylase. Histologically, VIP and VPAC1-R agonist attenuated the severity of pancreatitis, although VPAC2-R agonist or secretin showed no significant effect. In vitro, VPAC1-R and VPAC2-R mRNA were obviously expressed in monocytes. Under the stimulation with LPS, VIP presented a biphasic pattern that once decreased IL-6 production from monocytes and then enhanced at high concentration. VPAC1-R agonist reduced IL-6 levels, whereas VPAC2-R agonist increased IL-6 dose-dependently. VPAC1-R agonist reduced TNF-alpha levels in a dose-dependent manner.. VIP attenuated the experimental acute pancreatitis enzymatically and morphologically by inhibiting proinflammatory cytokine production from monocytes mainly through the VPAC1-R.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Cytokines; Interleukin-10; Interleukin-6; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Monocytes; Pancreas; Pancreatitis; Receptors, Vasoactive Intestinal Peptide, Type II; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Severity of Illness Index; Spleen; Tumor Necrosis Factor-alpha; Vasoactive Intestinal Peptide

Pituitary adenylate cyclase activating-peptide (PACAP) is a late member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family of brain-gut peptides. It is unknown whether PACAP takes part in the development of acute pancreatitis and whether PACAP or its antagonists can be used to suppress the progression of acute pancreatitis. We investigated the actions of PACAP and its receptor antagonists in acute pancreatitis on rats.. Acute pancreatitis was induced in rats with caerulein or 3.5% sodium taurocholate. The rats were continuously infused with 5-30 microg/kg PACAP via jugular vein within the first 90 min, while 10-100 microg/kg PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP (PACAP receptor antagonists) were intravenously infused for 1 h. Biochemical and histopathological assessments were made at 4 h after infusion. Pancreatic and duodenal PACAP concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Chinese ink-perfused pancreas was fixed, sectioned and cleared for counting the functional capillary density.. PACAP augmented caerulein-induced pancreatitis and failed to ameliorate sodium taurocholate-induced pancreatitis. ELISA revealed that relative concentrations of PACAP in pancreas and duodenum were significantly increased in both sodium taurocholate- and caerulein-induced pancreatitis compared with those in normal controls. Unexpectedly, PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP could induce mild acute pancreatitis and aggravate caerulein-induced pancreatitis with characteristic manifestations of acute hemorrhagic/necrotizing pancreatitis. Functional capillary density of pancreas was interpreted in the context of pancreatic edema, and calibrated functional capillary density (calibrated FCD), which combined measurement of functional capillary density with dry weight/wet weight ratio, was introduced. Hyperemia or congestion, rather than ischemia, characterized pancreatic microcirculatory changes in acute pancreatitis.. PACAP may take part in the pathogenesis of acute pancreatitis in rats. The two PACAP receptor antagonsits might act as partial agonists. Calibrated functional capillary density can reflect pancreatic microcirculatory changes in acute pancreatitis.

Topics: Acute Disease; Animals; Capillaries; Ceruletide; Cholagogues and Choleretics; Disease Models, Animal; Duodenum; Hormone Antagonists; Male; Nerve Growth Factors; Neuropeptides; Neurotransmitter Agents; Pancreas, Exocrine; Pancreatitis; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Taurocholic Acid; Vasoactive Intestinal Peptide

To investigate the changes of platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on polymorphonuclear leukocytes (PMNs) in peripheral circulation and pancreatic microcirculation in cerulein-induced acute edematous pancreatitis (AEP).. Fifty Wistar rats were randomly divided into control group (n = 10) and AEP group (n = 40). A model of AEP was established by subcutaneous injection of cerulein 5.5 and 7.5 mug/kg at 0 and 1 h after the beginning of experiment respectively. PECAM-1 expression on PMNs from splenic vein and inferior vena cava was determined by RT-PCR at mRNA level and determined by flow cytometry at protein level.. In experimental rats, an increased PECAM-1 mRNA expression was seen from 4 to 8 h of AEP in peripheral circulation (0.77+/-0.25%, 0.76+/-0.28%, 0.89+/-0.30%, 1.00+/-0.21%), while in pancreatic microcirculation, expression decreased from 2 h and reached the lowest level at 6 h of AEP (0.78+/-0.29%, 0.75+/-0.26%, 0.62+/-0.28%, 0.66+/-0.20%). There were significant differences at 8-h time point of AEP between peripheral circulation and pancreatic microcirculation (1.00+/-0.21% vs 0.66+/-0.20%, P<0.05). Meanwhile, the difference at protein level was also found.. A reverse expression of PECAM-1 on PMNs was found between peripheral circulation and pancreatic microcirculation, suggesting that inhibition of PECAM-1 expression may improve the pathological change of AEP.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Edema; Flow Cytometry; Gene Expression; Male; Microcirculation; Neutrophils; Organ Size; Pancreas; Pancreatitis; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction

Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that pancreatic acinar cells produce the chemokine monocyte chemotactic protein (MCP)-1 in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of inflammatory diseases, such as acute pancreatitis. 2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid (bindarit) has been shown to preferentially inhibit MCP-1 production in vitro in monocytes and in vivo without affecting the production of the cytokines IL-1, IL-6, or the chemokines IL-8, protein macrophage inflammatory-1alpha, and RANTES. The present study aimed to define the role of MCP-1 in acute pancreatitis with the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit significantly reduced MCP-1 levels in the pancreas. Also, this treatment significantly protected mice against acute pancreatitis as evident by attenuated hyperamylasemia neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections.

Topics: Acute Disease; Animals; Ceruletide; Chemokine CCL2; Disease Models, Animal; Indazoles; Inflammation; Mice; Necrosis; Pancreas; Pancreatitis; Propionates

Peroxisome proliferator-activated receptor (PPAR)-gamma controls growth, differentiation, and inflammation. PPAR-gamma agonists exert anti-inflammatory effects in vitro and inhibit the activation of pancreas stellate cells, implicated in the formation and progression of fibrosis. We determined the influence of troglitazone, a ligand for PPAR-gamma, on pancreatic damage and fibrosis in experimental chronic pancreatitis. Mice received six hourly intraperitoneal injections with 50 microg/kg of cerulein or saline, three times a week for 6 weeks. One week after the last injection all mice were sacrificed. Untreated mice were compared with mice treated with troglitazone either during weeks 1 to 6 or weeks 4 to 6. All mice that received cerulein injections displayed histopathological signs of chronic pancreatitis at week 7. Troglitazone treatment improved all markers for severity of pancreatitis. Moreover, early and postponed troglitazone treatments were equally effective in diminishing intrapancreatic fibrosis as quantified by Sirius red staining, hydroxyproline content, and laminin staining as well as the increased number of pancreatic stellate cells and pancreas levels of transforming growth factor-beta. Thus, troglitazone attenuated pancreatic damage and inflammation in experimental chronic pancreatitis and remained beneficial in a therapeutic setting when given after initial damage had been established.

Topics: Actins; Animals; Cell Differentiation; Ceruletide; Chromans; Chronic Disease; Collagen; Disease Models, Animal; Fibrosis; Inflammation; Interleukin-6; Ligands; Mice; Mice, Inbred C57BL; Muscle, Smooth; Pancreatitis; Platelet Aggregation Inhibitors; PPAR gamma; Receptors, Tumor Necrosis Factor, Type I; Sodium Chloride; Thiazolidinediones; Transforming Growth Factor beta; Transforming Growth Factor beta1; Troglitazone

To assess the effect of non-selective ET(A/B) (LU 302872) and selective ET(A) (LU 302146) antagonist on pancreatic histology and ultrastructure of acinar cells in connection with trypsinogen activation in early caerulein-induced AP.. Male Wistar rats with caerulein-induced AP, lasting 4 h, were treated i.p. with 10 and 20 mg/kg b.w. of each antagonist. Edema, inflammatory infiltration, necrosis and vacuolization of acinar cells in the pancreas were scored at 0-3 scale. Free active trypsin (FAT), total potential trypsin (TPT) after activation with enterokinase, and index of trypsinogen activation (%FAT/TPT) were assayed in pancreatic homogenates.. In untreated AP, the edema, inflammatory infiltration, necrosis and vacuolization increased as compared to control healthy rats (P<0.01). None of the treatment exerted any meaningful effect on the edema and inflammatory infiltration. The selective antagonist increased slightly the necrosis score to 0.82+/-0.06 at higher dose (P<0.05) vs 0.58+/-0.06 in untreated AP. The non-selective antagonist increased slightly the vacuolization score to 2.41+/-0.07 at higher dose (P<0.01) vs 1.88+/-0.08 in untreated AP. The decrease in the number of zymogen granules, disorganization of endoplasmic reticulum, autophagosomes and cytoplasmic vacuoles were more prominent in treated AP than in untreated AP groups. %FAT/TPT in untreated AP increased about four times (18.4+/-3.8 vs 4.8+/-1.3 in control group without AP, P<0.001). Treatment of AP with both antagonists did not affect significantly augmented trypsinogen activation.. The treatment with endothelin-1 receptors (non-selective ET(A/B) and selective ET(A)) antagonists has essential effect neither on the edema and inflammatory infiltration nor on trypsinogen activation observed in the early course of caerulein-induced AP. Nevertheless a slight increase of the necrosis and vacuolization score and some of the ultrastructural data could suggest the possibility of their undesired effects in caerulein-induced AP at investigated doses.

Topics: Acute Disease; Animals; Benzhydryl Compounds; Ceruletide; Endothelin A Receptor Antagonists; Male; Microscopy, Electron; Pancreas; Pancreatitis; Propionates; Pyrimidines; Rats; Rats, Wistar; Trypsinogen

Endogenous trypsin inhibitors are believed to inhibit protease activity if trypsin becomes inadvertently activated within the acinar cell. However, this action remains unproven, and the role of endogenous pancreatic trypsin inhibitors in acute pancreatitis is unknown. In this study, we tested whether increased levels of pancreatic secretory trypsin inhibitor-I (PSTI-I) in mice could prevent secretagogue-induced pancreatitis.. Rat PSTI-I expression was targeted to pancreatic acinar cells in transgenic mice by creating a minigene driven by the rat elastase I enhancer/promoter. Secretagogue-induced pancreatitis was achieved by 12 hourly intraperitoneal injections of caerulein. The severity of pancreatitis was assessed by measurements of serum amylase, histologic grading, and pancreas wet weight-to-body weight ratio. Trypsinogen activation and trypsin activity were measured in pancreatic extracts.. Targeted expression of PSTI-I to the pancreas increased endogenous trypsin inhibitor capacity by 190% (P <.01) in transgenic vs. nontransgenic mice. Caerulein administration to nontransgenic mice produced histologic evidence of acute pancreatitis, and significantly elevated serum amylase and pancreas weight ratio. In caerulein-treated transgenic mice, the histologic severity of pancreatitis was significantly reduced. There was no difference in trypsinogen activation peptide levels between caerulein-treated transgenic and nontransgenic mice. However, trypsin activity was significantly lower in transgenic mice receiving caerulein compared with nontransgenic mice.. These data demonstrate that the severity of secretagogue-induced pancreatitis is significantly ameliorated in mice with higher pancreatic levels of trypsin inhibitor. We propose that PSTI-I prevents pancreatitis by inhibiting the activity of trypsin, rather than by reducing trypsinogen activation.

Topics: Animals; Ceruletide; Gene Expression; Immunologic Techniques; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred Strains; Mice, Transgenic; Microscopy, Electron; Oligopeptides; Pancreas; Pancreatitis; Rats; Staining and Labeling; Transgenes; Trypsin; Trypsin Inhibitor, Kazal Pancreatic

The mammalian pancreas has a strong regenerative potential, but the origin of organ restoration is not clear, and it is not known to what degree such a process reflects pancreatic development. To define cell differentiation changes associated with pancreatic regeneration in adult mice, we compared regeneration following caerulein-induced pancreatitis to that of normal pancreatic development.. By performing comparative histology for adult and embryonic pancreatic markers in caerulein-treated and control pancreas, we addressed cellular proliferation and differentiation (amylase, DBA-agglutinin, insulin, glucagon, beta-catenin, E-cadherin, Pdx1, Nkx6.1, Notch1, Notch2, Jagged1, Jagged2, Hes1), hereby describing the kinetics of tissue restoration.. We demonstrate that surviving pancreatic exocrine cells repress the terminal exocrine gene program and induce genes normally associated with undifferentiated pancreatic progenitor cells such as Pdx1, E-cadherin, beta-catenin, and Notch components, including Notch1 , Notch2 , and Jagged2 . Expression of the Notch target gene Hes1 provides evidence that Notch signaling is reactivated in dedifferentiated pancreatic cells. Although previous studies have suggested a process of acino-to-ductal transdifferentiation in pancreatic regeneration, we find no evidence to suggest that dedifferentiated cells acquire a ductal fate during this process.. Pancreatic regeneration following chemically induced pancreatitis in the mouse occurs predominantly through acinar cell dedifferentiation, whereby a genetic program resembling embryonic pancreatic precursors is reinstated.

Topics: Animals; Ceruletide; Embryo, Mammalian; Female; Gene Expression Regulation; Homeodomain Proteins; Male; Membrane Proteins; Mice; Mice, Inbred Strains; Mitosis; Pancreas; Pancreas, Exocrine; Pancreatitis; Receptors, Notch; Regeneration; Signal Transduction; Trans-Activators

The transient receptor potential vanilloid 1 (TRPV-1) is an ion channel found on primary sensory afferent neurons. Activation of TRPV-1 leads to the release of the proinflammatory neuropeptide substance P (SP). SP then binds to the neurokinin-1 receptor (NK1-R) on endothelial cells and promotes extravasation of plasma and proteins into the interstitial tissue and neutrophil infiltration, a process called neurogenic inflammation. We tested 2 hypotheses: (1) activation of TRPV-1 in the pancreas leads to interstitial edema and neutrophil infiltration and (2) TRPV-1-induced plasma extravasation is mediated by the release of SP and activation of the NK1-R in the rat.. We measured extravasation of the intravascular tracer Evans blue as an index of plasma extravasation and quantified pancreas tissue myeloperoxidase activity (MPO) as a marker of neutrophil infiltration. The severity of inflammation following intravenous infusion of the secretagogue cerulein (10 microg/kg/h x 4 hours) was assessed using a histologic scoring system.. Intravenous injection of the TRPV-1 agonist capsaicin induced a dose-dependent increase in Evans blue accumulation in the rat pancreas (P < 0.05 vs. vehicle control). This effect was blocked by pretreatment with the TRPV-1 antagonist capsazepine (1.8 mg/kg), or the NK1-R antagonist CP 96,345 (1 mg/kg). Capsazepine also reduced cerulein-induced Evans blue, MPO, and histologic severity of inflammation in the pancreas but had no effect on serum amylase.. Activation of TRPV-1 induces SP-mediated plasma extravasation in the rat pancreas via activation of the NK1-R. TRPV-1 mediates neurogenic inflammation in cerulein-induced pancreatitis in the rat.

Topics: Animals; Capsaicin; Ceruletide; Coloring Agents; Evans Blue; Male; Neuritis; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; TRPV Cation Channels

Uncoupling-protein 2 (UCP2) is a mitochondrial protein that appears to be involved in cellular oxidant defense and in the regulation of oncotic cell death, both of which are important features of acute pancreatitis. However, UCP2 expression in acute pancreatitis has not been previously reported. In the current experiments, pancreatic gene expression was studied by real-time reverse-transcription/polymerase chain reaction and Northern blots. Two models of acute experimental pancreatitis were investigated: cerulein-induced pancreatitis in mice at two different time points and taurocholate-induced pancreatitis in rats at two degrees of severity. After cerulein administration, acinar injury and leukocyte infiltration was significantly higher at 24 h compared with 12 h after the first injection of cerulein (P<0.05, P<0.005, respectively). UCP2 mRNA was unchanged at 12 h but was nearly 12-fold greater than control levels after 24 h (P<0.001). UCP2 gene expression correlated with acinar injury (r=0.69; P<0.001). By 72 h after taurocholate administration, the severe group had more necrosis than the mild group (P<0.005). Pancreatic UCP2 mRNA was increased fourfold in the severe group compared with controls (P<0.01). UCP2 expression correlated with parenchymal necrosis (r=0.61; P<0.01). Thus, pancreatic UCP2 mRNA increased in two models of acute pancreatitis. The increase in UCP2 gene expression was correlated with the severity of the disease. Up-regulation of UCP2 in the pancreas may be a protective response to oxidative stress, but this increase may also have a negative influence on cellular energy metabolism. Therefore, acinar UCP2 may be an important modifier of the severity of acute pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Female; Gene Expression Regulation; Ion Channels; Male; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mitochondrial Proteins; Necrosis; Pancreatitis; Rats; Rats, Zucker; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Taurocholic Acid; Time Factors; Uncoupling Protein 2; Up-Regulation

To detect Toll-like receptor 4 (TLR4) expression and distribution in rat pancreas and the change of TLR4 expression in cerulein-induced pancreatitis (CIP).. Acute pancreatitis was induced by subcutaneous injections of cerulein at a total dose of 20 microg/kg. Immunohistochemistry (IHC) was used to detect and localize TLR4 in rat pancreas, and real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitatively determine the expression of TLR4 mRNA in CIP.. IHC showed the presence of TLR4 in rat pancreas, and its distribution was specifically localized to pancreatic ductal epithelium, vascular endothelium, and islet. No TLR4 staining was detected in exocrine acinar cells. Real-time RT-PCR results revealed low-level TLR4 mRNA expression in the rat pancreas, and the change of TLR4 in CIP only developed within the first 4 hours, which is a rapid up-regulation process that peaks at the first hour. TLR4 mRNA was sustained at baseline level from 4 to 24 hours.. TLR4 protein was expressed in pancreas and localized to epithelial (pancreatic duct) or endothelial (vessels) tissues; TLR4 responded favorably to the inflammatory process, and the change of expression was characterized as a rapid up-regulation in the early stage of CIP.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Cytokines; Endothelium; Female; Gene Expression; Immunohistochemistry; Lipase; Male; Pancreas, Exocrine; Pancreatic Ducts; Pancreatitis; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Toll-Like Receptor 4; Up-Regulation

Oxygen free radicals (OFRs) mediate an important step in the initiation of experimental acute pancreatitis and several clinical findings suggested the possible contribution of OFRs to the pathogenesis of pancreatic fibrosis. So far, there are no studies which reporting potential role of OFRs in development of chronic pancreatitis with the prevention with antioxidants. This study was aimed to establish the mice model of chronic fibrosing pancreatitis and to prove the involvement of OFRs in chronic pancreatitis with fibrosis.. Repeated intraperitoneal cerulein injection was performed to induce chronic pancreatitis in mice. Histological changes in the pancreas were examined, and markers for oxidative stress were measured in the pancreatic tissue and serum of the mice. DA-9601, a phytochemical possessing anti-inflammatory and antioxidative action, was given together with cerulein to the mice.. Repeated intraperitoneal injection of cerulein provoked significant severity of chronic fibrosing pancreatitis after 5 weeks. After treatment of DA-9601, the extents of pancreatic fibrosis were statistically significantly decreased in accordance with lessened pancreatic inflammations. The NF-kappaB binding activities were increased in chronic pancreatitis, which were significantly attenuated after DA-9601 treatment. The levels of myeloperoxidase and iNOS activities were also significantly decreased in DA-9601-treated group compared to the pancreatitis only group. Cytoprotective proteins such as heat shock protein-70 (HSP) and metallothionein were significantly increased in the DA-9601-treated group. DA-9601 decreased the expressions of alpha-SMA and type I collagen in cultured pancreatic stellate cells.. Oxidative stress was principally involved in the pathogenesis of chronic pancreatitis with fibrosis.

Topics: Animals; Antioxidants; Artemisia; Cells, Cultured; Ceruletide; Chronic Disease; Disease Models, Animal; Fibrosis; Free Radicals; Male; Mice; Mice, Inbred ICR; Oxidative Stress; Pancreatitis; Plant Extracts

Acute pancreatitis is an auto-digestive disease resulting in inflammation. At the cellular level, acute pancreatitis disrupts posttranslational protein processing and traffic in the secretory pathway, and zymogens become activated in the acinar cell. To better understand the disruption of the secretory pathway in pancreatitis, pulse-chase [(35)S]met/cys analysis was used to study the effects of supramaximal cerulein stimulation on posttranslational modification in the secretory pathway of the major sulfated glycoprotein of the mouse pancreas, pro-Muclin, and the lysosomal membrane protein LAMP1. Maximal cerulein or high concentration bombesin stimulation had little effect on glycoprotein processing. By contrast, supramaximal cerulein stimulation strongly inhibited pro-Muclin processing as measured by the failure of Muclin to attain its normal mature size of 300 kDa and to become highly sulfated and decreased proteolytic cleavage of pro-Muclin to produce apactin. Digestion of immunoprecipitated [35S]met/cys-labeled Muclin and LAMP1 with endoglycosidase H demonstrated that the supramaximal cerulein-induced block in processing occurred before the medial Golgi compartment. With supramaximal cerulein stimulation, vacuoles formed which contained Muclin, amylase, and LAMP1. Earlier autoradiographic studies showed that newly synthesized proteins end up in pancreatitis-associated vacuoles, so it is likely that glycoproteins with incomplete posttranslational processing are also present in vacuoles. Because glycoproteins are believed to protect the membranes of lysosomes and zymogen granules, when they are not correctly processed, their defensive mechanisms may be impaired, and this could contribute to vacuole fragility in pancreatitis.

Topics: Acute Disease; Animals; Bombesin; Ceruletide; Disease Models, Animal; Glycoproteins; Golgi Apparatus; Lysosomal Membrane Proteins; Membrane Glycoproteins; Mice; Mucins; Pancreas; Pancreatitis; Protein Processing, Post-Translational; Time Factors

The majority of patients with chronic pancreatitis are alcoholics. Our goal was to develop a mouse model of alcohol dependent chronic pancreatitis.. Mice were fed either the non-alcohol containing Lieber-DeCarli diet or the Lieber-DeCarli diet containing 24% of calories as ethanol. After eight weeks and while on their respective diets, mice were subjected to repeated episodes of acute pancreatitis elicited by administration of caerulein. They were sacrificed 1, 3, and 5 weeks after the last dose of caerulein. Pancreatic morphology and collagen deposition were evaluated in samples stained with haematoxylin-eosin and Sirius red. Collagen content was quantitated by measuring OH-proline. Gene expression was determined by quantitative polymerase chain reaction.. Both groups of mice gained weight at the same rate. Those receiving the alcohol containing diet had serum alcohol levels of approximately 100 mM. No histological or gene expression differences were found in mice that were not subjected to acute pancreatitis, regardless of their diet. Necrosis, Sirius red staining, OH-proline content, and expression of alpha-1 collagen I, alpha-smooth muscle actin, transforming growth factor beta1, and tissue inhibitor of metalloproteinase 1 were all increased in mice fed the alcohol containing diet and given caerulein compared with those fed the control diet and given caerulein. Matrix metalloproteinase 9 expression was transiently decreased in mice fed ethanol and given caerulein compared with the group given caerulein but not fed ethanol.. We have developed a mouse model of alcohol dependent chronic pancreatic fibrosis. This mouse model may be useful in studies examining the effects of genetic manipulation on chronic pancreatitis.

Topics: Animals; Ceruletide; Chronic Disease; Collagen; Diet; Disease Models, Animal; Ethanol; Fibrosis; Gene Expression Regulation; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C3H; Pancreas; Pancreatitis; Weight Gain

We have previously shown that cell contacts between pancreatic acinar cells dissociate early in pancreatitis and that this is a prerequisite for the development of pancreatic oedema. Here we studied the underlying mechanism.. Employing experimental caerulein induced pancreatitis in vivo and isolated pancreatic acini ex vivo, in conjunction with protein chemistry, morphology, and electron microscopy, we determined whether cell contact regulation in the pancreas requires or involves: (1) changes in cadherin-catenin protein expression, (2) tyrosine phosphorylation of adhesion proteins, or (3) alterations in the actin cytoskeleton.. During initial cell-cell contact dissociation at adherens junctions, expression of adhesion proteins remained stable. At time points of dissociated adherens junctions, the cadherin-catenin complex was found to be tyrosine phosphorylated and internalised. The receptor type protein tyrosine phosphatase (PTP)kappa was constitutively associated with the cadherin-catenin complex at intact cell contacts whereas following the dissociation of adherens junctions, the internalised components of the cadherin-catenin complex were tyrosine phosphorylated and associated with the cytosolic PTP SHP-1. In isolated acini, inhibition of endogenous protein tyrosine phosphatases alone was sufficient to induce dissociation of adherens junctions analogous to that found with supramaximal caerulein stimulation. Dissociation of actin microfilaments had no effect on adherens junction integrity.. These data identify tyrosine phosphorylation as the key regulator for cell contacts at adherens junctions and suggest a definitive role for the protein tyrosine phosphatases PTPkappa and SHP-1 in the regulation, maintenance, and restitution of cell adhesions in a complex epithelial organ such as the pancreas.

Topics: Actins; Adherens Junctions; Animals; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Ceruletide; Cytoskeletal Proteins; Cytoskeleton; Intracellular Signaling Peptides and Proteins; Male; Pancreas; Pancreas, Exocrine; Pancreatitis; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Receptors, Cell Surface; Tyrosine

Oxidative stress plays a role in the development of pancreatic fibrosis.. In the present study, we hypothesized that the administration of an antioxidant complex could ameliorate cerulein and cyclosporin A pancreatic fibrosis, assessed by changes in oxidative stress and a histopathological study in an experimental rat model.. Four groups of ten rats each. In Group A, the rats served as controls and were treated with intraperitoneal saline solution. In Group B, six courses of cerulein pancreatitis were induced at weekly intervals. In Group C, the rats received cyclosporin A the day before and the day on which pancreatitis was induced in Group B. In Group D, the rats were treated as in Group C but also received antioxidants. All rats were sacrificed at the seventh week.. The presence of fibrosis was evaluated according to a scoring system. Glutathione peroxidase was utilized as an indicator of oxidative stress and total antioxidant status as an indicator of total antioxidant tissue capacity.. The rats in Groups B and C showed more pancreatic fibrosis than those in Groups A and D (90%, 70%, 0%, and 20%, respectively). The glutathione peroxidase increased in Group B (455+/-196 mU/g protein) and Group C (243+/-206 mU/g protein) with respect to those in Group A (137+/-80 mU/g protein) and Group D (135+/-105 mU/g protein). Total antioxidant status was significantly higher in Groups B (1.41+/-0.96 mmol/g protein) and D (1.28+/-0.09 mmol/g protein) with respect to Groups A (0.10+/-0.06 mmol/g protein) and C (0.15+/-0.09 mmoL/g protein).. The administration of cerulein and cyclosporin A caused fibrosis, whereas antioxidant administration showed preventive effects regarding cerulein and cyclosporin A-induced pancreatic fibrosis.

Topics: Animals; Antioxidants; Ceruletide; Chronic Disease; Cyclosporine; Fibrosis; Glutathione Peroxidase; Male; Oxidative Stress; Pancreas; Pancreatitis; Rats; Rats, Wistar

Serine protease inhibitor Kazal type 1 (SPINK1), which is structurally similar to epidermal growth factor, is thought to inhibit trypsin activity and to prevent pancreatitis. Point mutations in the SPINK1 gene seem to predispose humans to pancreatitis; however, the clinical significance of SPINK1 mutations remains controversial. This study aimed to elucidate the role of SPINK1.. We generated Spink3-deficient (Spink3(-/-)) mice by gene targeting in mouse embryonic stem cells. Embryonic and neonatal pancreases were analyzed morphologically and molecularly. Specific probes were used to show the typical autophagy that occurs during acinar cell death.. In Spink3(-/-) mice, the pancreas developed normally up to 15.5 days after coitus. However, autophagic degeneration of acinar cells, but not ductal or islet cells, started from day 16.5 after coitus. Rapid onset of cell death occurred in the pancreas and duodenum within a few days after birth and resulted in death by 14.5 days after birth. There was limited inflammatory cell infiltration and no sign of apoptosis. At 7.5 days after birth, residual ductlike cells in the tubular complexes strongly expressed pancreatic duodenal homeodomain-containing protein 1, a marker of pancreatic stem cells, without any sign of acinar cell regeneration.. The progressive disappearance of acinar cells in Spink3(-/-) mice was due to autophagic cell death and impaired regeneration. Thus, Spink3 has essential roles in the maintenance of integrity and regeneration of acinar cells.

Topics: Animals; Biopsy, Needle; Blotting, Western; Cell Death; Cells, Cultured; Ceruletide; Disease Models, Animal; DNA; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pancreas; Pancreatic Function Tests; Pancreatitis; Phagocytosis; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sensitivity and Specificity; Serine Proteinase Inhibitors; Trypsin Inhibitor, Kazal Pancreatic

To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance.. A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison.. After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF.. Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression; Immediate-Early Proteins; Male; Pancreatitis; Rats; Rats, Wistar; Thromboplastin; Transcription Factors

The severity of acute pancreatitis results from the transmigration and activation of leukocytes within the pancreas and the local synthesis and release of proinflammatory-soluble mediators that transform a local injury into a systemic inflammatory response. Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Therefore, we set out to investigate whether the genetic deletion of PARP-1 or PARP-2 (a new member of the PARP family) genes, or pharmacological inhibition of PARP activity might affect the development and severity of acute pancreatitis and pancreatitis-associated lung injury. Secretagogue-induced acute pancreatitis was achieved by 12 hourly intraperitoneal injections of cerulein in mice deficient in PARP-1 or PARP-2 genes, and wild-type (WT) littermate mice untreated or treated with PARP activity inhibitors. The severity of pancreatitis was assessed by measurements of serum amylase, lipase, interleukin-1beta and IL-6, pancreatic water content, histologic grading and pancreas myeloperoxidase (MPO) activity. Lung injury was evaluated by quantifying MPO activity and morphological changes. We found that the severity of acute pancreatitis and pancreatitis-associated lung injury was significantly attenuated in mice lacking PARP-1, but not PARP-2, compared with WT mice. Interestingly, administration of PARP inhibitors, 3-aminobenzamide or PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethyacetamide HCl), in WT mice markedly decreased acute pancreatitis severity and pulmonary-associated injury in a larger extension than genetic deletion of PARP-1. Our results support the potential therapeutic application of PARP inhibitors in the development and severity of acute pancreatitis and associated lung injury.

Topics: Acute Disease; Amylases; Animals; Benzamides; Ceruletide; Interleukin-1; Interleukin-6; Lipase; Lung Diseases; Mice; Mice, Knockout; Neutrophils; Pancreatitis; Peroxidase; Phenanthrenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

Topics: Cadherins; Cell Communication; Ceruletide; Humans; Pancreas, Exocrine; Pancreatitis; Phosphorylation; Protein Tyrosine Phosphatases

The effects of R-102444 ((2R, 4R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride) and its active metabolite R-96544 ((2R, 4R)-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-4-hydroxy-1-methylpyrrolidine hydrochloride), potent and selective 5-hydroxytryptamine 2A (5-HT2A) receptor antagonists, on development of pancreatitis were investigated in experimental models of acute and chronic pancreatitis. Rat acute pancreatitis was induced by caerulein (20 microg/kg) intraperitoneal injection and by pancreatic duct ligation. In both the models, serum amylase and lipase activities were markedly increased. R-102444 dose-dependently reduced these enzyme activities at a dose range of 10 to 100 mg/kg (p.o.) for the caerulein model and 0.3 to 10 mg/kg (p.o.) for the ligation model. In a mouse model of acute pancreatitis induced by a choline-deficient, ethionine (0.5%)-supplemented diet, subcutaneous administration of R-96544 (10-100 mg/kg, bid) reduced serum amylase activity. Histological analysis showed that R-96544 dose-dependently attenuated pancreatic necrosis, inflammation and vacuolization. The effect of R-102444 was further examined in male Wistar Bonn/Kobori rats (4-9 months of age) which spontaneously show pancreatic fibrosis and parenchymal destruction compatible with human chronic pancreatitis. In Wistar Bonn/Kobori rats (from 3 to 9 months of age) fed a diet containing 0.017% and 0.17% of R-102444, pancreatic weight, pancreatic protein and amylase content were higher compared to those in non-treated pancreatitis control rats. Histological analysis showed that R-102444 suppressed parenchymal destruction and replacement with adipose tissue, indicating inhibition of pancreatic atrophy. These results clearly indicate that R-102444 and R-96544 inhibit the progression of acute and chronic pancreatitis and support the contention of possible involvement of 5-HT2A receptors in the progression of experimental pancreatitis.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Choline; Chronic Disease; Dietary Supplements; Ethionine; Injections, Intraperitoneal; Ligation; Lipase; Male; Organ Size; Pancreas; Pancreatic Ducts; Pancreatitis; Pyrrolidines; Rats; Rats, Inbred Strains; Rats, Sprague-Dawley; Rats, Wistar; Receptor, Serotonin, 5-HT2A; Serotonin 5-HT2 Receptor Antagonists; Serotonin Antagonists; Time Factors

Acute pancreatitis is characterized by the pathologic activation of zymogens within pancreatic acinar cells. The process requires a rise in cytosolic Ca(2+) from undefined intracellular stores. We hypothesized that zymogen activation is mediated by ryanodine receptor (RYR)-regulated Ca(2+) release, because early zymogen activation takes place in a supranuclear compartment that overlaps in distribution with the RYR. Ca(2+) signals in the basolateral, but not apical, region of acinar cells observed during supraphysiologic agonist stimulation were dependent on RYR Ca(2+) release. Inhibition of RYR or depletion of RYR-sensitive Ca(2+) pools each reduced pathologic zymogen activation in isolated acinar cells, but neither treatment affected amylase secretion. Inhibition of RYR also inhibited zymogen activation in vivo. We propose that Ca(2+) release from the RYR mediates zymogen activation but not enzyme secretion. The findings imply a role for the RYR in acute pancreatitis.

Topics: Animals; Calcium; Ceruletide; Dantrolene; Enzyme Precursors; Male; Microscopy, Confocal; Models, Biological; Pancreas, Exocrine; Pancreatitis; Rats; Rats, Sprague-Dawley; Ryanodine Receptor Calcium Release Channel; Secretory Vesicles; Trypsinogen

The aim of this study was to test the effect of gut manipulation by either novel synbiotics or by metronidazole on either endotoxemia or the severity of liver damage in the course of acute pancreatitis from alcohol ingestion.. Sprague-Dawley rats were fed for 1 week through an intragastric tube a liquid diet with either: (i) 1 mL t.i.d. of a mixture of synbiotics (Lactobacillus acidophilus, Lactobacillus helveticus and Bifidobacterium in an enriched medium); (ii) 20 mg/kg t.i.d. metronidazole; or (iii) standard diet. Then, acute pancreatitis was induced by caerulein and when the disease was full-blown, rats were fed an alcohol-rich diet. Synbiotic and metronidazole treatment was given for a further 2 weeks. Transaminase and endotoxemia levels were measured before treatment, after 6 h, after 24 h and 2 weeks later, at the time the rats were killed. Liver samples were obtained for histological analysis.. Synbiotics but not metronidazole improved the acute pancreatitis-induced increase in endotoxemia and transaminase levels. The addition of alcohol worsened these variables to a limited extent in the synbiotic-treated group, while metronidazole had a negative effect on liver damage.. Gut flora pretreatment with synbiotics was able to effectively protect against endotoxin/bacterial translocation, as well as liver damage in the course of acute pancreatitis and concomitant heavy alcohol consumption. The beneficial effect of synbiotics on liver histology seems to be correlated with endotoxemia. Metronidazole did not produce such a beneficial effect; in fact, it further worsened liver damage when alcohol was added to the background of ongoing acute pancreatic inflammation.

Topics: Acute Disease; Animals; Bacterial Translocation; Bifidobacterium; Ceruletide; Disease Models, Animal; Endotoxemia; Endotoxins; Ethanol; Gastrointestinal Tract; Lactobacillus acidophilus; Lactobacillus helveticus; Liver Diseases, Alcoholic; Metronidazole; Pancreatitis; Probiotics; Protective Agents; Rats; Rats, Sprague-Dawley; Transaminases

Effects of dexamethasone and N(G)-nitro-L-arginine methyl ester (L-NAME), the nitric oxide (NO) synthase inhibitor, on caerulein-induced acute pancreatitis were examined in rats. Acute pancreatitis was induced by caerulein (20 mug/kg, s.c.) given repeatedly 2 or 4 times every hour, and serum amylase levels, pancreas weight and myeloperoxidase (MPO) activity were measured 6 h after the first injection of caerulein. Dexamethasone (3 mg/kg) and L-NAME (30 mg/kg) were administered p.o. 30 min before the first injection of caerulein. Caerulein caused moderate or severe pancreatitis, depending on the times of injections, resulting in different degrees of increase in serum amylase levels and pancreas weight, and the marked elevation of MPO activity was observed only after injections of caerulein given 4 times per hour. Both dexamethasone and L-NAME suppressed the severity of pancreatits, yet the effect of L-NAME as compared with dexamethasone was more potent against mild pancreatitis but less potent against severe pancreatitis. These results suggest that caerulein-induced acute pancreatitis shows different responsiveness to L-NAME and dexamethasone, depending on the severity; the former is more effective against pancreatitis with less inflammation, while the latter is more effective against pancreatitis with severe inflammation. It is assumed that endogenous NO may be involved in oedema formation as the early event in the development of acute pancreatitis.

Topics: Acute Disease; Administration, Oral; Amylases; Animals; Anti-Inflammatory Agents; Ceruletide; Dexamethasone; Disease Models, Animal; Enzyme Inhibitors; Injections, Subcutaneous; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organ Size; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Wistar; Severity of Illness Index

To determine whether ischemic preconditioning (IP) affects the development of edematous cerulein-induced pancreatitis and to assess the role of cyclooxygenase-1 (COX-1), COX-2, and heat shock protein 70 (HSP 70) in this process.. In male Wistar rats, IP was performed by clamping of celiac artery (twice for 5 min at 5-min intervals). Thirty minutes after IP or sham operation, acute pancreatitis was induced by cerulein. Activity of COX-1 or COX-2 was inhibited by resveratrol or rofecoxib, respectively (10 mg/kg).. IP significantly reduced pancreatic damage in cerulein-induced pancreatitis as demonstrated by the improvement of pancreas histology, reduction in serum lipase and poly-C ribonuclease activity, and serum concentration of pro-inflammatory interleukin (IL)-1beta. Also, IP attenuated the pancreatitis-evoked fall in pancreatic blood flow and pancreatic DNA synthesis. Serum level of anti-inflammatory IL-10 was not affected by IP. Cerulein-induced pancreatitis and IP increased the content of HSP 70 in the pancreas. Maximal increase in HSP 70 was observed when IP was combined with cerulein-induced pancreatitis. Inhibition of COXs, especially COX-2, reduced the protective effect of IP in edematous pancreatitis.. Our results indicate that IP reduces pancreatic damage in cerulein-induced pancreatitis and this effect, at least in part, depends on the activity of COXs and pancreatic production of HSP 70.

Topics: Animals; Ceruletide; Cyclooxygenase Inhibitors; Edema; HSP70 Heat-Shock Proteins; Interleukin-1; Interleukin-10; Ischemic Preconditioning; Lactones; Male; Pancreatitis; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Resveratrol; Stilbenes; Sulfones

To assess effects of NF-kappaB activation inhibitor (pyrrolidine dithiocarbamate--PDTC) alone or with endothelins (ET-1, ET-2, ET-3) in early course of cerulein-induced acute pancreatitis (AP) in rats.. After 4 h of AP in Wistar rats, treated with PDTC 10 or 40 mg/kg or with PDTC 10 mg/kg and ET-1, ET-2 or ET-3, 0.5 or 1.0 nmol/kg twice i.p. in 1 h interval, free active trypsin (FAT), total potential trypsin (TPT) and lipase in 12000 x g supernatants of pancreatic homogenates, plasma alpha-amylase and histological changes were assayed. %FAT/TPT was an index of trypsinogen activation.. %FAT/TPT significantly increased to 12.42 +/- 2.14%, lipase to 5.51 +/- 0.84 U/mg protein and alpha-amylase to 28.5 +/- 5.61 U/mL in AP vs 1.96 +/- 0.31%, 1.29 +/- 0.11 U/mg and 5.80 +/- 1.38 U/ml in healthy control. Higher dose PDTC attenuated trypsinogen activation to 3.01 +/- 0.53% and alpha-amylase to 15.3 +/- 1.38. PDTC and ET-1 attenuated %FAT/TPT to 2.55 +/- 0.18% with lower and 2.34 +/- 0.44% with higher dose. ET-3 was less effective than ET-1: 6.76 +/- 0.46% with lower dose. Lower doses of ET-1 and ET-2 with PDTC, diminished lipase activity to 2.60 +/- 0.36 and 2.94 +/- 0.33.. Cumulative attenuation of trypsinogen activation after lower dose of PDTC and ET-1 approximated the effect of higher dose of PDTC. Additional effect of ET-3 was weaker than ET-1, and ET-2 was ineffective in this respect. The combination of this NF-kappaB activation inhibitor and ET-1 could be beneficial in early course of edematous AP by attenuating of trypsinogen activation. However, it should be treated with caution because of some unfavorable effects on histological scores of pancreatic injury.

Topics: Acute Disease; alpha-Amylases; Animals; Antioxidants; Ceruletide; Endothelin-1; Endothelin-2; Endothelin-3; Enzyme Activation; Lipase; Male; NF-kappa B; Pancreatitis; Pyrrolidines; Rats; Rats, Wistar; Thiocarbamates; Trypsinogen

To determine the effect of pioglitazone, a specific peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, on the development of acute pancreatitis (AP) and on the expression of heat shock protein 70 (HSP70) in the pancreas.. AP was induced in rats by subcutaneous infusion of cerulein for 5 h. Pancreatic blood flow was measured by laser Doppler flowmetry. Plasma lipase activity, interleukin-1beta (IL-1beta) and IL-10 were determined. Pancreatic weight and histology were evaluated and pancreatic DNA synthesis and blood flow as well as pancreatic mRNA for IL-1beta and HSP70 were assessed in rats treated with pioglitazone alone or in combination with cerulein.. Pioglitazone administered (10-100 mg/kg i.g.) 30 min before cerulein, attenuated dose-dependently the pancreatic tissue damage in cerulein-induced pancreatitis (CIP) as demonstrated by the improvement of pancreatic histology, reduction in plasma lipase activity, plasma concentration of pro-inflammatory IL-1beta and its gene expression in the pancreas and attenuation of the pancreatitis-evoked fall in pancreatic blood flow. CIP increased pancreatic HSP70 mRNA and protein expression in the pancreas and this effect was enhanced by pioglitazone treatment.. Pioglitazone attenuates CIP and the beneficial effect of this pioglitazone is multifactorial probably due to its anti-inflammatory activities, to the suppression of IL-1beta and to the overexpression of HSP70. PPARgamma ligands could represent a new therapeutic option in the treatment of AP.

Topics: Animals; Ceruletide; DNA; Dose-Response Relationship, Drug; HSP70 Heat-Shock Proteins; Interleukin-1; Interleukin-10; Lipase; Male; Pancreas; Pancreatitis; Pioglitazone; PPAR gamma; Protective Agents; Rats; Rats, Wistar; Regional Blood Flow; Thiazolidinediones

Previous studies showed that a local pancreatic renin-angiotensin system (RAS) was upregulated in experimental acute pancreatitis. RAS inhibition could attenuate pancreatic inflammation and fibrosis, which casts a new light on the role of the pancreatic RAS in pancreatitis. The present study explores the prophylactic and therapeutic potentials, and possible molecular mechanism for the antagonism of angiotensin II receptors on the changes in the severity of pancreatic injury induced by acute pancreatitis. Experimental pancreatitis was induced by an intraperitoneal injection of supra-maximal dose of cerulein. The differential effects of angiotensin II receptors inhibitors losartan and PD123319 on the pancreatic injury were assessed by virtue of using the pancreatic water content, biochemical and histological analyses. Blockade of the AT(1) receptor by losartan at a dose of 200microg/kg could markedly ameliorate the pancreatic injury induced by cerulein, as evidenced by biochemical and histopathological studies. However, blockade of the AT(2) receptor by PD123319 appeared not to provide any beneficial role in cerulein-induced pancreatic injury. Both prophylactic and therapeutic treatments with losartan were effective against cerulein-induced pancreatic injury. The protective action of losartan was linked to an inhibition of NAD(P)H oxidase activity, thus consequential oxidative modification of pancreatic proteins in the pancreas. Inhibition of the AT(1) receptor, but not AT(2) receptor, may play a beneficial role in ameliorating the severity of acute pancreatitis. The differential effects of AT(1) and AT(2) inhibitors on cerulein-induced pancreatic injury might be due to the distinctive mechanism of the AT(1) and AT(2) receptors on the activation of NAD(P)H oxidase. Thus the protective role of AT(1) receptor antagonist, losartan, could be mediated by the inhibition of NAD(P)H oxidase-dependent generation of reactive oxygen species (ROS).

Topics: alpha-Amylases; Angiotensin II Type 1 Receptor Blockers; Angiotensin II Type 2 Receptor Blockers; Animals; Ceruletide; Imidazoles; Inflammation; Losartan; NADPH Oxidases; Organ Size; Oxygen; Pancreas; Pancreatitis; Pyridines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

p8 is a transcription cofactor whose expression is strongly and rapidly activated in pancreatic acinar cells during the acute phase of pancreatitis. A p8-deficient mouse strain was generated as a tool to investigate its function. Upon induction of acute pancreatitis, myeloperoxidase activity in pancreas and serum concentrations of amylase and lipase were much higher and pancreatic lesions more severe in p8-deficient mice than in wild-type, indicating that p8 expression decreased pancreatic sensitivity to pancreatitis induction. The protective mechanism might involve the pancreatitis-associated protein (PAP I), whose strong induction during pancreatitis is p8-dependent, because administration of anti-PAP I antibodies to rats increased pancreatic inflammation during pancreatitis. In addition, 100 ng/ml PAP I in the culture medium of macrophages prevented their activation by tumor necrosis factor alpha, strongly suggesting that PAP I was an anti-inflammatory factor. Finally, PAP I was able to inhibit NFkappaB activation by tumor necrosis factor alpha, in macrophages and in the AR42J pancreatic acinar cell line. In conclusion, p8 improves pancreatic resistance to inducers of acute pancreatitis by a mechanism implicating the expression of the anti-inflammatory protein PAP I.

Topics: Alleles; Amylases; Animals; Antigens, Neoplasm; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Blotting, Western; Ceruletide; DNA-Binding Proteins; Female; Inflammation; Lectins, C-Type; Lipase; Macrophages; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Neoplasm Proteins; NF-kappa B; Pancreas; Pancreatic Elastase; Pancreatitis; Pancreatitis-Associated Proteins; Peroxidase; Promoter Regions, Genetic; Protein Transport; Proteins; Rats; Rats, Wistar; Time Factors; Transfection; Trypsin; Tumor Necrosis Factor-alpha

Acute pancreatitis (AP) is a complex disease that may be linked to acinar cell apoptosis and inadequate acinar cell replacement. Differentiation of acinar cells is regulated by p48, a DNA binding subunit of the transcription factor PTF1, and the Notch signaling pathway. Acinar cell apoptosis is triggered by oxygen deprivation, ie, hypoxia, by activation of hypoxia inducible factor-1alpha (HIF-1alpha). The aim of this study was to characterize by Northern blot analyses expression of HIF-1alpha, HIF-1alpha-inducible genes (GLUT-1, VEGF, p53), p48, and genes involved in the Notch signaling pathway (Notch-1, Dll1, RBP-Jk, HES-1) during cerulein-induced AP in mice. Maximal expression of HIF-1alpha, HIF-1alpha-inducible genes, p48, and Notch signaling genes occurred 8-12 hours after induction of AP. Maximal expression of p53 occurred 12-48 hours after induction of AP. These findings demonstrate that multiple pancreatic genes are activated acutely during AP that support pancreatic cell replenishment, regulation of expression of acinar cell-specific genes, and apoptosis.

Topics: Acute Disease; Animals; Basic Helix-Loop-Helix Transcription Factors; Blotting, Northern; Ceruletide; DNA-Binding Proteins; Female; Gene Expression Regulation; Homeodomain Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Membrane Proteins; Mice; Nuclear Proteins; Pancreatitis; Proteins; Receptor, Notch1; Receptors, Cell Surface; Receptors, Notch; RNA, Messenger; Signal Transduction; Time Factors; Transcription Factor HES-1; Transcription Factors

Tumor necrosis factor alpha (TNF-alpha) has a central role in the pathogenesis of acute pancreatitis and related systemic complications. The aim of this study is to investigate the therapeutic effectiveness of monoclonal TNF antibody (infliximab) in acute edematous and severe necrotizing pancreatitis models in rats.. One hundred rats were randomly divided into 10 groups. Acute edematous pancreatitis (AEP) was induced by injection of cerulein 20 microg/kg 4 times subcutaneously at hourly intervals. Severe necrotizing pancreatitis (SNP) was induced by retrograde injection of 3% taurocholate into the common biliopancreatic duct. Infliximab 8 mg/kg was given via intravenous infusion. Serum amylase activity, pancreatic histopathology, myeloperoxidase enzyme activity (MPO), and pulmonary changes were assessed.. Infliximab treatment significantly decreased serum amylase activity (11939 +/- 1914 U/L versus 3458 +/- 915 U/L, P < 0.001) and histopathologic score (4.1 +/- 0.5 versus 1.5 +/- 0.3, P < 0.001) in AEP. It also suppressed neutrophil infiltration and MPO activity of the pancreatic tissue. In SNP, infliximab treatment was found to decrease pathologic score (9.4 +/- 1.2 versus 3.6 +/- 0.8, P < 0.001) and serum amylase activity (20442 +/- 2375 versus 8990 +/- 1730, P < 0.01). It ameliorated both parenchymal and fatty tissue necrosis of the pancreas. Infliximab also alleviated alveolar edema and acute respiratory distress syndrome like pulmonary complications, but the difference was not significant.. Chimeric TNF antibody, infliximab, should be evaluated for treatment of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Antibodies, Monoclonal; Ceruletide; Edema; Infliximab; Male; Necrosis; Pancreas; Pancreatitis; Pancreatitis, Acute Necrotizing; Peroxidase; Pulmonary Edema; Rats; Rats, Wistar; Severity of Illness Index; Taurocholic Acid; Treatment Outcome; Tumor Necrosis Factor-alpha

Oxidative stress involvement in damage to the pancreas in acute pancreatitis (AP) is well documented. However, little is known about oxidative damage occurring in the different subcellular fractions of pancreatic cells. The aim of this study was to ascertain the main targets of oxidative damage inside cells after AP and the role of endogenous nitric oxide (NO) in it.. A model of cerulein-induced AP in rats was used and N-nitro-l-arginine methyl ester (l-NAME) was administered as an NO production inhibitor. After pancreatitis induction, indicative parameters of lipid peroxidation and protein oxidation together with some enzymatic and nonenzymatic endogenous free radical scavengers were assessed in serum and pancreatic subcellular fractions.. In pancreatitic rats, malondialdehyde and protein carbonyl group concentrations were significantly increased (P < 0.05) in serum and some fractions. The increases were higher in l-NAME-treated rats (P < 0.05). Superoxide dismutase and catalase activities were also increased (P < 0.05) but were decreased (P < 0.05) with l-NAME. The alpha-tocopherol concentration diminished (P < 0.05) in serum and all the studied subcellular fractions and the decrease was stronger in l-NAME-treated rats. Our data suggest that microsomes followed by lysosomal + mitochondrial are the fractions most susceptible to oxidative damage in AP. Endogenous NO plays a protective role against oxidative damage to subcellular fractions.

Topics: Acute Disease; alpha-Tocopherol; Animals; Catalase; Ceruletide; Enzyme Inhibitors; Lipid Peroxides; Male; Malondialdehyde; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Pancreas; Pancreatitis; Proteins; Random Allocation; Rats; Rats, Wistar; Subcellular Fractions; Superoxide Dismutase

Supramaximal dosage of the cholecystokinin analog caerulein leads to edematous pancreatitis with subsequent acinar cell destruction predominantly by apoptosis. We have used immunohistochemistry to reveal the expression of the anti-apoptotic protein galectin-3 in pancreatic acinar cells. Galectin-3, which occurs only in duct cells under physiological conditions, is expressed in a subset of acinar cells after the end of a 12-h caerulein infusion, giving rise to a "patchy" staining pattern. During the subsequent period of inflammation and regeneration, galectin-3 expression increases in those acinar cells that undergo apoptosis. By 48 h after the end of caerulein infusion, morphologically normal cells do not contain galectin-3 and participate in regeneration by proliferation. Tubular complexes, being transient structures from degenerative acini, accumulate galectin-3 in the remnants of the epithelium cells. Stimulation with supramaximal dosages of caerulein of the cell line AR4-2J, which is derived from rat pancreatic acinar cells, also results in a marked increase of galectin-3, confirming the in vivo results. We postulate that the high expression of the anti-apoptotic protein galectin-3 regulates the time course of the apoptotic process in pancreatic acinar cells.

Topics: Acute Disease; Amino Acid Sequence; Animals; Apoptosis; Cell Line, Tumor; Ceruletide; Disease Models, Animal; Edema; Fluorescent Antibody Technique, Indirect; Galectin 3; Humans; Male; Molecular Sequence Data; Pancreas; Pancreatic Ducts; Pancreatitis; Rats; Rats, Wistar; Regeneration

It is well established that damage to the outer membrane of cells is a common phenomenon allowing abnormal transmission of substances into the cytosol. Penetration of albumin into acinar cells has been detected in experimental acute pancreatitis, raising the possibility that membrane damage is a very early event, potentially representing the first changes leading to pancreatitis. To determine if direct damage to the cell membrane is a key factor during induction of acute pancreatitis, thus altering the balance of extra- and intracellular substances, fluorescein-dextran was administered with supramaximal doses of caerulein via the jugular vein or by injection directly into the pancreas. This tracer rapidly penetrates into cells. Two patterns of tracer penetration are observed: cytosolic and vesicular/vacuolar. Fluorescein-dextran administered intravenously with caerulein penetrates into the cytosol of acinar cells within 10 min. Strong cytoplasmic fluorescence occurs within 5 min after direct injection. It may be concluded that supramaximal caerulein, administered in vivo, damages the cell membrane of acinar cells, allowing large molecules to enter the cytosol. Thus Ca(2+) and other substances may enter the cells in abnormally high concentrations, initiating the cellular changes characteristic of pancreatitis. The results raise the question whether membrane wounding may play a role in the initiation of human pancreatitis.

Topics: Acute Disease; Animals; Cell Membrane; Cell Membrane Permeability; Ceruletide; Dextrans; Ferritins; Fluoresceins; Fluorescent Dyes; Male; Molecular Weight; Pancreatitis; Rats; Rats, Sprague-Dawley

Recent studies have shown that inosine, a purine nucleoside produced during the breakdown of adenosine, has immunomodulatory and anti-inflammatory properties. The aim of this study was to examine the effects of inosine on the course of acute pancreatitis.. Edematous pancreatitis was induced by the intraperitoneal injection of caerulein (50 micro g/kg), seven times, at 1-h intervals, in male Wistar rats (caerulein pancreatitis). Inosine (100 mg/kg) was administered 30 min before or 1 h after the first injection of caerulein. The effects of inosine on the severity of pancreatitis were assessed by serum amylase, pancreatic edema (wet/dry ratio), myeloperoxidase activity, cytokine-induced neutrophil chemoattractant-1 concentrations, and histological changes.. Prophylactic administration of inosine significantly decreased the elevation of serum amylase, myeloperoxidase activity, and cytokine-induced neutrophil chemoattractant-1 concentrations in the pancreas and the lung. Inosine did not significantly affect edema formation. Histologically, vacuole formation in pancreatic acinar cells, infiltration of inflammatory cells in the pancreas and the lung, and alveolar wall thickening in the lung were reduced. Inosine improved the histological findings and reduced myeloperoxidase activity even if it was administered 1 h after the first injection of caerulein.. Inosine reduced the severity of acute pancreatitis, suggesting a possible application of this compound in the treatment of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Chemokines, CXC; Enzyme-Linked Immunosorbent Assay; Inosine; Intercellular Signaling Peptides and Proteins; Lung; Lung Diseases; Male; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Wistar

The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.

Topics: Acute Disease; Animals; Ceruletide; Cytoprotection; Enzyme Inhibitors; Mice; Mice, Inbred C57BL; Mice, Knockout; Microcirculation; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Pancreas; Pancreatitis; Phosphorylation; Regional Blood Flow; Trypsin

In the present study, we investigated the effects of rosiglitazone (10 mg/kg, i.p.), a PPAR-gamma agonist, on the development of acute pancreatitis.. Intraperitoneal injection of cerulein in mice induced an acute pancreatitis characterized by edema, neutrophil infiltration elevated serum levels of amylase and lipase. This experimental model was performed to test the anti-inflammatory activity of rosiglitazone. SETTING. University research laboratory.. Male CD mice (20-22 g) were allocated into four groups (n=10 for each group): (a) Cerulein+vehicle group. Mice were treated hourly (x 5) with cerulein (50 microg/kg, in saline solution, i.p.); (b) Rosiglitazone group (same as the Cerulein+vehicle group but were administered rosiglitazone, 10 mg/kg bolus, 30 min prior to cerulein); (c) Sham+saline group. Mice were treated with saline instead of cerulein; (d) Sham+Rosiglitazone. Identical to Rosiglitazone group except that the saline was administered instead of cerulein. Mice were killed at 6 h after the induction of pancreatitis. Blood samples, pancreas, and lungs were collected.. Infiltration of pancreatic and lung tissue with neutrophils was associated with enhanced lipid peroxidation. Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine and for ICAM-1 in the pancreas of cerulein-treated mice. In contrast, the degree of (a) pancreatic inflammation and tissue injury, (b) upregulation/formation of ICAM-1 and nitrotyrosine, and (c) neutrophils infiltration was markedly reduced in pancreatic tissue obtained from rosiglitazone-treated mice.. These findings support the view that rosiglitazone and other potent PPAR-gamma agonists may be useful in the therapy of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Cell Adhesion Molecules; Ceruletide; Lipase; Lipid Peroxidation; Male; Mice; Pancreatitis; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazolidinediones; Transcription Factors

The secretin-caerulein test (SCT) is generally considered the gold standard for evaluation of the exocrine pancreatic function. Problems related to enzyme inactivation in the aspirated duodenal juice limit the clinical applicability of the test. Pancreatic zinc, which is mainly secreted as constituent of metalloenzymes, is stable in duodenal juice and easy to quantify. The aim of this study was to analyze the accuracy of the secretin-caerulein-stimulated pancreatic zinc output as pancreatic function test in comparison with the standard SCT.. Forty consecutive patients with suspected chronic pancreatitis and 28 healthy subjects were studied. A SCT was performed after overnight fast by infusing intravenously secretin (1 U/kg/h) and caerulein (100 ng/kg/h) over 90 min. The duodenal content was continuously aspirated and separated at 15-min intervals and immediately analyzed for pH, bicarbonate, amylase, lipase, elastase, carboxypeptidase A, and zinc. Correlation and concordance between standard SCT and quantification of zinc output and the accuracy of the latter for diagnosing and grading the exocrine pancreatic dysfunction were calculated.. The pancreatic zinc output correlated significantly with enzyme and bicarbonate output (r ranging from 0.670 to 0.855; p < 0.001). A highly significant concordance was found between the degree of exocrine pancreatic dysfunction based on the standard SCT (bicarbonate and enzymes output) and that based only on zinc output (k = 0.831; p < 0.001). Quantification of the stimulated pancreatic zinc output has a sensitivity of 97% and a specificity of 91% in the diagnosis of exocrine pancreatic dysfunction.. The determination of pancreatic zinc output during secretin and caerulein stimulation is a simple and accurate method for evaluation of the exocrine pancreatic function. Zinc is stable in duodenal juice, and its determination as a single parameter simplifies the clinical applicability of the SCT.

Topics: Adolescent; Adult; Aged; Amylases; Bicarbonates; Carboxypeptidases A; Ceruletide; Chronic Disease; Duodenum; Female; Humans; Hydrogen-Ion Concentration; Linear Models; Lipase; Male; Middle Aged; Pancreas; Pancreatic Elastase; Pancreatic Function Tests; Pancreatitis; Secretin; Sensitivity and Specificity; Suction; Zinc

The objective was to investigate the effects of vitamin E on collagen deposition induced by Cyclosporin A (CsA) administration in rats with caerulein (Cr) pancreatitis. CsA transforms the fully regenerative, self-limited form of Cr pancreatitis into a chroniclike disease in conjunction with increased transforming growth factor (TGF)-beta and myofibroblast proliferation. Vitamin E inhibits TGF-beta release in mesangial cells and reduces CsA cytotoxicity. Wistar rats received CsA daily (20 mg/kg), and CR pancreatitis was induced on days 1 and 8 (Cr + CsA group). In a separate group, vitamin E (600 mg.kg(-1).day(-1)) was administered starting 4 days before CsA. Three other groups received either vehicle, CsA, or Cr alone. Thiobarbituric acid-reactive substance (TBARS), 8-isoprostanes, and hyaluronic acid were measured in plasma obtained on the day the animals were killed (day 15). Pancreases were weighed and processed for light microscopy to assess connective tissue and myofibroblast number. Pancreatic homogenates were also assayed for collagen (hydroxyproline) and TBARS content. TBARS, 8-isoprostane, and TGF-beta were elevated in CsA and Cr + CsA rats. Vitamin E treatment greatly decreased these parameters. Vitamin E also decreased the fall in pancreatic weight observed in Cr + CsA pancreas. Pancreatic hydroxyproline and plasma hyaluronic acid were increased in Cr + CsA rats but were effectively reduced by vitamin E. Morphology showed improvement in fibrosis score and a decreased number of myofibroblasts in vitamin E-treated rats. Vitamin E reduces oxidative stress and collagen deposition during the development of experimental chronic pancreatitis. Adjuvant antioxidants may be of value in the treatment of chronic pancreatitis.

Topics: Animals; Ceruletide; Chronic Disease; Collagen; Cyclosporine; Fibroblasts; Fibrosis; Lipid Peroxidation; Lipoproteins; Male; Myocytes, Smooth Muscle; Pancreas; Pancreatitis; Rats; Rats, Wistar; Vitamin E

We investigated the effect of alpha-melanocyte stimulating hormone (alpha-MSH) on cerulein induced acute pancreatitis in rats. alpha-MSH treatment (50 microg per rat, intraperitoneally) prior to cerulein reduced the plasma amylase level, pancreatic weight, pancreatic myeloperoxidase activity and the severity of the lesions microscopically. These data suggest that alpha-MSH has a protective effect on cerulein-induced acute pancreatitis and this effect could be attributed, at least in part, to decreased tissue leukocyte infiltration and thus, to decreased pro-inflammatory cytokine production and/or oxygen- and nitrogen-derived reactive metabolite release.

Topics: alpha-MSH; Animals; Ceruletide; Female; Gastrointestinal Agents; Injections, Intraperitoneal; Injections, Subcutaneous; Male; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley

Caerulein-induced pancreatitis is a widely used experimental model for studies on acute pancreatitis, however, the molecular mechanisms underlying pancreatitis in response to caerulein hyperstimulation are incompletely understood. We therefore studied early effects of caerulein on tight junctional integrity. Mice were injected with the cholecystokinin analogue caerulein (50microg/kg BW/h) to induce pancreatitis. In pancreatic tissue occludin, claudin 1, zonula occludens protein 1 (ZO-1) were stained immunohistochemically and F-actin was visualized with phalloidin-TRITC. Stained sections and isolated acini were studied by confocal laser scanning microscopy. Under control conditions occludin, claudin1, ZO-1, and F-actin showed a linear staining pattern delineating the apical membranes of intralobular duct cells and of acinar cells. While in vitro caerulein hyperstimulation induced within 10 minutes disassembly of both occludin and ZO-1, in vivo caerulein hyperstimulation induced disassembly of occludin and claudin1 but not of ZO-1 from the tight junctions. Subsequent progressive disruption of ZO-1 was detected in a time dependent manner. Disruption of the transmembrane tight junction proteins occludin and claudin1 is an early event of caerulein hyperstimulation and may allow evasion of noxious luminal content into the interstitium, which may augment edema formation in acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Blotting, Western; Ceruletide; Claudin-1; Immunohistochemistry; Lipase; Male; Membrane Proteins; Mice; Occludin; Pancreas, Exocrine; Pancreatitis; Phosphoproteins; Tight Junctions; Zonula Occludens-1 Protein

Mechanisms of pain transduction in acute pancreatitis are poorly understood. Increased Fos expression in the spinal cord is a marker of activation of nociceptive neurons. We hypothesized that cerulein pancreatitis leads to increased Fos expression at T9 and T10, which receive sensory input from the pancreas. Rats were injected with cerulein (100 microg/kg, s.c.) or saline carrier (NS). Endpoints at 4, 6, and 10 h were serum amylase, myeloperoxidase activity (MPO), and spinal cord Fos expression (number of immunoreactive nuclei/section dorsal gray matter). Fos-like immunoreactivity (FLI) at T9-T10 was compared to internal controls (T6, T12). An average of 20 spinal cord histologic sections were evaluated per rat. Some animals were injected with the mu-opioid receptor agonist, buprenorphine (90 microg/kg, s.c.), 3 h after cerulein, and their endpoints were measured at 6 h. Analysis of variance and t tests were used for statistical analysis. Results are means +/- SEM. As expected, cerulein induced edematous pancreatitis, with a 4-fold increase in serum amylase at 6 h [cer (n = 8): 14,000 +/- 1,300 U/ml versus NS (n = 10): 3,700 +/- 300, P < 0.005)] and a 2-fold increase in MPO activity (0.25 +/- 0.05) activity units/dry wt versus 0.13 +/- 0.02, P < 0.05). Cerulein induced nearly a 2-fold increase in FLI at T9 and T10 [n = 10 (cer) and n = 13 (NS): T9, 14 +/- 1.5 versus 7.8 +/- 0.88; T10, 15 +/- 1.7 versus 8.3 +/- 0.70; P < 0.05]. Peak effects of cerulein on FLI occurred at 6 h and were greatest at T9/T10 with relative sparing of T6/T12. T6/T12 expression was similar in experimental and control groups. Buprenorphine significantly reduced both serum amylase and FLI and T9/T10. Cerulein-induced acute pancreatitis in rat increases visceral nociceptive signaling at spinal cord levels T9 and T10, with a peak at 6 h. Blockade of this effect by the mu-opioid receptor agonist buprenorphine could occur either by direct activation of central opioid receptors and/or an anti-inflammatory mechanism. FLI is a useful tool for studying the pathophysiology of pain in experimental acute pancreatitis.

Topics: Animals; Buprenorphine; Ceruletide; Male; Nociceptors; Pancreatitis; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Spinal Cord; Thoracic Vertebrae; Time Factors

To investigate the effects of rhubarb on severe acute pancreatitis (SAP) in rats.. Severe acute pancreatitis was induced by two intraperitoneal injections of cerulein (40 microg/kg body weight) plus 5-h restraint water-immersion stress. Rhubarb (75-150 mg/kg) was orally fed before the first cerulein injection. The degree of pancreatic edema, serum amylase level, local pancreatic blood flow (PBF), and histological alterations were investigated. The effects of rhubarb on pancreatic exocrine secretion in this model were evaluated by comparing with those of somatostatin.. In the Cerulein+Stress group, severe edema and diffuse hemorrhage in the pancreas were observed, the pancreatic wet weight (11.60+/-0.61 g/Kg) and serum amylase (458 490+/-43 100 U/L) were markedly increased (P<0.01 vs control). In the rhubarb (150 mg/kg) treated rats, necrosis and polymorphonuclear neutrophil (PMN) infiltration in the pancreas were significantly reduced (P<0.01), and a marked decrease (50%) in serum amylase levels was also observed (P<0.01). PBF dropped to 38% (93+/-5 mL/min per 100 g) of the control in the Cerulein+Stress group and partly recovered in the Cerulein+Stress+Rhubarb 150 mg group (135+/-12 mL/min per 100 g) (P<0.01). The pancreatic exocrine function was impaired in the SAP rats. The amylase levels of pancreatic juice were reduced in the rats treated with rhubarb or somatostatin, comparing with that of untreated SAP group. The bicarbonate concentration of pancreatic juice was markedly elevated only in the rhubarb-treated group (P<0.01).. Rhubarb can exert protective effects on SAP, probably by inhibiting the inflammation of pancreas, improving pancreatic microcirculation, and altering exocrine secretion.

Topics: Acute Disease; Animals; Ceruletide; Cytoprotection; Male; Pancreatitis; Phytotherapy; Rats; Rats, Sprague-Dawley; Rheum; Severity of Illness Index

Keratin polypeptides 8 and 18 (K8/K18) are the major intermediate filament proteins of pancreatic acinar cells and hepatocytes. Pancreatic keratin function is unknown, whereas hepatocyte keratins protect from mechanical and non-mechanical forms of stress. We characterized steady-state pancreatic keratin expression in Balb/c mice after caerulein and choline-deficient ethionine-supplemented diet (CDD), or on exposure to the generalized stresses of heat and water immersion. Keratins were studied at the protein, RNA and organizational levels. Isolated acini were used to study the role of nuclear factor (NF)-kappaB using selective inhibitors. Keratins were found to be abundant proteins making up 0.2%, 0.3% and 0.5% of the total cellular protein of pancreas, liver and small intestine, respectively. Caerulein and CDD caused a threefold transcription-mediated overall increase in K8/K18/K19/K20 proteins. Keratin overexpression begins on tissue recovery, peaks 2 days after caerulein injection, or 1 day after CDD discontinuation, and returns to basal levels after 10 days. K19/K20-containing cytoplasmic filaments are nearly absent pre-injury but form post-injury then return to their original membrane-proximal distribution after 10 days. By contrast, generalized stresses of heat or water-immersion stress do not alter keratin expression levels. Caerulein-induced keratin overexpression is associated with NF-kappaB activation when tested using ex vivo acinar cell cultures. In conclusion, keratins are abundant proteins that can behave as stress proteins in response to tissue-specific but not generalized forms of injury. Pancreatic keratin overexpression is associated with NF-kappaB activation and may serve unique functions in acinar or ductal cell response to injury.

Topics: Animals; Ceruletide; Choline Deficiency; Diet; Ethionine; Fever; Gene Expression Regulation; Hot Temperature; Intestine, Small; Keratins; Liver; Mice; Mice, Inbred BALB C; NF-kappa B; Organ Specificity; Pancreas; Pancreatitis; RNA, Messenger; Transcriptional Activation; Water

Melatonin, produced from L-tryptophan, protects the pancreas against acute damage by improving the antioxidative status of tissue. Melatonin receptors have been detected in the brain, but the contribution of these receptors to the pancreatic protection is unknown. The aim of our study was to compare the effects of melatonin precursor; L-tryptophan given intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) on the course of acute pancreatitis. Acute pancreatitis was induced by subcutaneous infusion of caerulein (5 microg/kg-h x 5 h). L-tryptophan was given i.p. (2.5, 25 or 250 mg/kg) or administered into right cerebral ventricle (0.02, 0.2 or 2.0 mg/rat) 30 min prior to the start of caerulein infusion. Plasma amylase, lipase and TNF alpha activities were measured to determine the severity of caerulein-induced pancreatitis (CIP). The lipid peroxidation products: malonylodialdehyde and 4-hydroksynonenal (MDA + 4-HNE) and activity of superoxide dismutase (SOD) were measured in the pancreas of intact or CIP rats with or without L-tryptophan pretreatment. Melatonin blood level was measured by RIA. CIP was confirmed by histological examination and manifested as an edema and rises of plasma levels of amylase, lipase and TNF alpha (by 550%, 1000% and 600%). MDA + 4-HNE was increased by 600%, whereas SOD activity was reduced by 75% in the pancreas of CIP rats. All manifestations of CIP were significantly reduced by pretreatment of the rats with L-tryptophan given i.c.v. at doses of 0.2 or 2.0 mg/rat, or by peripheral administration of this amino acid used at dose of 250 mg/kg i.p. In control rats plasma level of melatonin averaged about 40 +/- 2 pg/ml and was not significantly affected by CIP, by central application of L-tryptophan (0.02, 0.2 or 2.0 mg/rat) or by peripheral administration of this melatonin precursor used at doses of 2.5 or 25 mg/kg i.p. Plasma melatonin level was markedly increased by pretreatment of the rats with L-tryptophan given i.p. at dose of 250 mg/kg. We conclude that central administration of melatonin precursor; L-tryptophan, as well as peripheral application of high dose of this melatonin precursor prevented the pancreatic damage produced by CIP. The favorable effect of peripherally administered L-tryptophan could be related to the rise of melatonin plasma level and to pancreatoprotective action of this indoleamine. The beneficial effect of centrally administered L-tryptophan could be mediated through activation of central recep

Topics: Acute Disease; Aldehydes; Amylases; Animals; Ceruletide; Dose-Response Relationship, Drug; Drug Administration Schedule; Infusions, Parenteral; Injections, Intraperitoneal; Injections, Intraventricular; Lipase; Male; Malondialdehyde; Melatonin; Organ Size; Pancreas; Pancreatitis; Rats; Rats, Wistar; Reactive Oxygen Species; Superoxide Dismutase; Tryptophan; Tumor Necrosis Factor-alpha

There have been various studies of exocrine pancreatic function after acute pancreatitis, but few have examined the relationship between this function and the etiology of the pancreatitis. The aim of this work was to study pancreatic function in patients who had had acute alcoholic or acute biliary pancreatitis.. Seventy-five patients who had had a single attack of acute pancreatitis were studied. The etiology was alcohol in 36 and cholelithiasis in 39. Pancreatic function was studied between 4 and 18 months after pancreatitis by duodenal intubation in 18 patients (8 alcohol, 10 lithiasis) and by the amino acid consumption test (AACT) in the remaining 57 (28 alcohol, 29 lithiasis). For those who underwent AACT, the test was repeated 1 year after the first examination.. Among the 36 patients with alcoholic pancreatitis, most had impaired pancreatic function at both duodenal intubation (8/8, 100%) and at AACT (22/28, 78.6%); at the second test, the AACT remained pathological (18/23, 82.1%). Of the 39 patients with biliary pancreatitis, only 4 of the 10 (40%) who underwent duodenal intubation and only 5 of the 29 (17.2%) who performed AACT had pancreatic insufficiency; at the second test, only 4 of the 26 (15.4%) who repeated the AACT were pathological. The differences in the frequency and degree of pancreatic insufficiency between patients with alcoholic and those with biliary pancreatitis were statistically significant.. The results show that after alcoholic acute pancreatitis, the pancreatic insufficiency was significantly more frequent and more severe than after biliary pancreatitis. These findings together with the fact that the insufficiency was also more persistent suggest that acute alcoholic pancreatitis may occur in a pancreas that already has chronic lesions.

Topics: Acute Disease; Adolescent; Adult; Aged; Amino Acids; Bicarbonates; Ceruletide; Cholelithiasis; Chymotrypsin; Exocrine Pancreatic Insufficiency; Female; Humans; Lipase; Male; Middle Aged; Pancreas; Pancreatitis; Pancreatitis, Alcoholic

A mouse model using repetitive acinar cell injury caused by supraphysiologic doses of cerulein to induce the characteristic fibrosis and loss of acinar cell mass found in human chronic pancreatitis was employed to identify early changes in gene expression. A gene array was used to detect changes in 18,000 expressed sequence tags; changes in specific transcripts were confirmed by RNase protection assays. These methods identified SPINK3, the mouse homologue of human and rat protease inhibitor genes, as being highly expressed in the pancreas and induced after pancreatic injury. Because SPINK3 may be an important serine protease inhibitor, its up-regulation may reflect an important endogenous cytoprotective mechanism in preventing further injury. The up-regulation of SPINK3 was specific; the mouse homologue of the zymogen-processing protein ZG-16p was also highly expressed in the pancreas but sharply down-regulated early in the course of injury. These findings suggest that the pancreatic acinar cell may respond to injury with a program of self-preservation and loss of normal function.

Topics: Amino Acid Sequence; Animals; Ceruletide; Chronic Disease; Female; Gene Expression Profiling; Gene Expression Regulation; Glycoproteins; Lectins; Membrane Proteins; Mice; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Pancreas; Pancreatitis; Prostatic Secretory Proteins; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Trypsin Inhibitor, Kazal Pancreatic; Trypsin Inhibitors

The pancreas contains a local renin-angiotensin system (RAS), which is subject to activation by experimental pancreatitis. In the exocrine pancreas, angiotensin II receptor subtypes AT1 and AT2 have been localized in the pancreatic ducts, blood vessels and acinar cells. We hypothesize that local RAS activities may have a potential role in regulating pancreatic acinar digestive enzyme secretion. The present study was designed to elucidate firstly the existence of RAS components in pancreatic acinar cells and their regulation by acute pancreatitis. Secondly, the differential roles of AT1 and AT2 receptors in controlling digestive enzyme secretion from dispersed functional pancreatic acini were also investigated. The mRNA levels of RAS components were assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Acinar secretions were assayed by the measurement of alpha-amylase and lipase activities. Induction of acute pancreatitis was achieved by hyperstimulation of two intraperitoneal (i.p.) injections of cerulein (50 microg/kg/h). Results from RT-PCR showed that the mRNA levels of the major RAS components (angiotensinogen, AT1 and AT2 receptors) were expressed in isolated rat pancreatic acinar cells, and they were upregulated during pancreatitis. Exogenous addition of angiotensin II could stimulate a dose-dependent release of digestive enzymes from the acinar cells. Administration of the selective AT1 receptor antagonist losartan significantly inhibited the acinar digestion enzyme secretion in both normal and pancreatitis-induced acini. However, a specific AT2 receptor blocker PD123319 did not exhibit such a suppressive effect. These data indicate the existence of an acinar RAS in the pancreas of potential importance in the physiological regulation of digestive enzyme secretion. The differential actions of AT1 and AT2 receptors and their upregulation may have clinical relevance to the pathogenesis and management of acute pancreatitis.

Topics: Acute Disease; Angiotensin II Type 1 Receptor Blockers; Animals; Ceruletide; Enzymes; Gene Expression Regulation, Enzymologic; Imidazoles; Losartan; Pancreas; Pancreatitis; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Renin-Angiotensin System; Vasoconstrictor Agents

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.

Topics: Acute Disease; Animals; Calcium-Binding Proteins; Calpain; Ceruletide; Dipeptides; Enzyme Activation; Female; Pancreas; Pancreatitis; Rats; Rats, Inbred Lew; Severity of Illness Index

Traditional Chinese medicine is a potent agent in the management of clinical and experimental acute pancreatitis (AP), but the molecular mechanism of its therapeutic action is unclear. Numerous experimental and clinical studies have shown that platelet endothelial cell adhesion molecule-1 (PECAM-1) is pivotal to leukocyte recruitment, which results in microcirculatory injury during inflammation, but its role in acute pancreatitis is poorly understood. We investigated the effects of a compound of traditional Chinese medicine pancreatitis-1 (TCMP-1) on the changes of platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on polymorphonuclear leukocytes (PMNs) in acute edematous pancreatitis (AEP).. The model of acute pancreatitis was established by subcutaneous injection of caerulein, and TCMP-1 treated groups were given TCMP-1 by catheterization from mouth to stomach (20 ml/kg) immediately after first time subcutaneous injection of caerulein. The changes of expression of PECAM-1 on leukocytes from the blood of the splenic vein and inferior vena cava were determined by flow cytometry.. In the AEP group, expression of PECAM-1 on PMNs was not significantly different between pancreatic microcirculation and systemic circulation at AEP2h and AEP4h time point. Then from AEP4h time point to AEP8h time point, expression of PECAM-1 was up-regulated in systemic circulation while it was down-regulated in pancreatic microcirculation and was significantly different between pancreatic microcirculation and systemic circulation at AEP8h time point (P<0.05). In the TCMP-1 treated group, compared with the AEP group, expression of PECAM-1 on PMNs decreased in different levels between pancreatic microcirculation and systemic circulation and was of significant difference at AEP8h time point (P<0.05).. Inhibition of PECAM-1 expression on PMNs may prevent PMNs from transmigration through the endothelium and may be one of the treatment mechanisms of TCMP-1 decoction on AEP.

Topics: Acute Disease; Animals; Ceruletide; Drugs, Chinese Herbal; Female; Gastrointestinal Agents; Leukocytes; Male; Models, Animal; Pancreatitis; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Wistar

Stress is reportedly known to affect the severity of acute pancreatitis, yet the effect has not been without controversy. We investigated the influence of restraint stress on cerulein-induced pancreatitis, especially in relation to endogenous glucocorticoids. In the present study, restraint stress significantly reduced the increase in serum amylase levels but not pancreas weight induced by cerulein, the effect being totally antagonized by pretreatment with mifepristone, a glucocorticoid receptor antagonist. The changes induced by cerulein were prevented by dexamethasone in a dose-dependent manner. Histologically, restraint stress suppressed the intralobular edema, similar to a low dose of dexamethasone, while the latter at a high dose prevented not only the intralobular but also the interlobular edema. These results suggest that restraint stress exerts a beneficial influence on the cerulein-induced pancreatitis, mainly mediated by endogenous glucocorticoids, and it is assumed that short-term steroid therapy has a potential of clinical application for treatment of pancreatitis.

Topics: Amylases; Animals; Ceruletide; Chaperonin 60; Dexamethasone; Dose-Response Relationship, Drug; Glucocorticoids; HSP70 Heat-Shock Proteins; Male; Organ Size; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Wistar; Restraint, Physical

To investigate the role of inducible cyclooxygenase (COX-2) mRNA expression in local microvessels in rats with acute interstitial pancreatitis (AIP) induced by caerulein injection.. The reverse transcription polymerase chain reaction (RT-PCR) was used to detect COX-2 gene expression in pancreatic tissue. Parameters of acute pancreatitis, such as serum amylase (AMS) and plasma myeloperoxidase (MPO) activities, were assayed using spectrophotometry. Intravital fluorescence microscopy with fluorescein isothiocyanate-labelled erythrocytes was used to study the pancreatic microvessels of rats with AIP and normal control rats.. Highly significant increases in COX-2 expression and AMS and MPO activity were seen in rats with AIP compared with controls. After caerulein injection, pancreatic capillary blood flow was decreased (4 hours, p > 0.05; 8 hours, p < 0.001), functional capillary density was reduced (4 hours, p > 0.05; 8 hours, p < 0.001), and there was irregular and intermittent capillary perfusion at 8 hours. There was also a positive correlation between the level of COX-2 expression and MPO activity (plasma, r = 0.5449, p < 0.05; tissue, r = 0.5698, p < 0.05).. The correlations between increased COX-2 expression and decreased capillary perfusion and blood flow and increased oedema following AIP may show that COX-2 expression can induce neutrophil sequestration to the pancreas, which may be one of the cascading inflammatory factors in the development of AIP.

Topics: Acute Disease; Animals; Ceruletide; Cyclooxygenase 2; Gastrointestinal Agents; Gene Expression; Isoenzymes; Male; Microcirculation; Models, Animal; Pancreas; Pancreatitis; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar

Nuclear factor-kappaB (NF-kappaB) is a transcription factor which plays a pivotal role in the induction of genes involved in the response to injury and inflammation. Calpain I inhibitor is a potent antioxidant which is an effective inhibitor of NF-kappaB. This study examined whether the postulate that calpain I inhibitor attenuates experimental acute pancreatitis.. In a murine model we measured NF-kappaB activation, expression of intercellular adhesion molecule (ICAM) 1, nitrotyrosine, inducible nitric oxide synthase (iNOS), nuclear enzyme poly(ADP-ribose) synthetase (PARS), myeloperoxidase, malondialdehyde, amylase and lipase and determined histological evidence of lung and pancreas injury in four groups: control (saline only), cerulein, calpain I inhibitor plus cerulein and calpain I inhibitor plus saline.. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterised by oedema, neutrophil infiltration, tissue haemorrhage and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS and PARS in the pancreas and lung of cerulein-treated mice. In contrast, pre-treatment with calpain I inhibitor markedly reduced: the degree of pancreas and lung injury; upregulation/expression of ICAM-1; staining for iNOS, nitrotyrosine and PARS; and lipid peroxidation. Additionally, calpain I inhibitor treatment significantly prevented the activation of NF-kappaB as suggested by the inhibition of IkappaB-alpha; degradation in the pancreas tissues after cerulein administration.. Taken together, our results clearly demonstrate that prevention of the activation of NF-kappaB by calpain I inhibitor ameliorates experimental murine acute pancreatitis.

Topics: Acute Disease; Analysis of Variance; Animals; Blotting, Western; Calpain; Ceruletide; Disease Models, Animal; Immunohistochemistry; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Male; Mice; NF-kappa B; Nitric Oxide Synthase; Pancreatitis; Poly(ADP-ribose) Polymerases; Random Allocation; Respiratory Distress Syndrome; Tyrosine

We describe the cloning and expression of the mouse gene interferon-inducible-protein 15 (IP15), whose activation is related to the acute phase of experimental pancreatitis. Analysis of its structure indicates that it encodes a putative transmembrane protein of 137 amino acids. This gene contains a predicted IFN-stimulable-response element. In vivo studies showed that IP15 is strongly activated in pancreas early during caerulein-induced pancreatitis. In situ hybridization of IP15 mRNA showed that its expression is restricted to acinar cells. IP15 was also induced in pancreas under systemic-lipopolysaccharide treatment and in intestine under Salmonella infection. In vitro studies using NIH3T3 fibroblasts showed that IP15 is induced by IFN-alpha. Growth rate was significantly lower in cells transfected with pcDNA4/IP15 plasmid. In addition, cells expressing IP15 showed less capacity to develop colonies after antibiotic selection. In conclusion, we identified a new interferon-inducible gene that is activated early in pancreas with pancreatitis and whose expression inhibits cell growth.

Topics: Amino Acid Sequence; Animals; Cell Division; Cell Line; Ceruletide; Cloning, Molecular; Female; Fetus; Humans; In Situ Hybridization; Interferons; Membrane Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Pancreas; Pancreatitis; Pregnancy; Response Elements; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid

Protease-activated receptor 2 can be stimulated by interstitially released trypsin during acute inflammation of the pancreas. In this study, we investigated the roles of pancreatic and circulatory protease-activated receptor 2 in the pathogenesis of acute pancreatitis by using in vitro and in vivo model systems.. Physiological and pathologic effects of protease-activated receptor 2 activation were measured in isolated pancreatic cells and in rats with experimental pancreatitis. Consequences of protease-activated receptor 2 activation on the systemic and inflammatory responses were measured after treatments with trypsin or protease-activated receptor 2-activating peptide.. Stimulation of protease-activated receptor 2 in rat pancreatic acinar cells activated short-lasting (Ca(2+) signaling) and long-lasting (extracellular signal-related kinase) signaling pathways and protected the cells against bile-induced cell damage. More importantly, protease-activated receptor 2 activation ameliorated the pathologic effects observed in the in vivo model of cerulein-induced pancreatitis. Trypsin in the circulation of rats with taurocholate-induced severe acute pancreatitis reached levels sufficient to activate endothelial and immune cells to stimulate nitric oxide and interleukin-8 production, respectively. Most notably, activation of systemic protease-activated receptor 2 by circulating protease-activated receptor 2 agonists induced a hemodynamic response pattern similar to that observed in rats with severe acute pancreatitis. The effects of protease-activated receptor 2 agonists and acute pancreatitis were not additive.. These findings suggest that protease-activated receptor 2 may have a dual role in acute pancreatitis: protecting acinar and duct cells against pancreatitis-induced cell damage while mediating or aggravating the systemic complications of acute pancreatitis, which are the major cause of mortality in the early phase of necrotizing pancreatitis.

Topics: Acute Disease; Animals; Blood Pressure; Cell Survival; Cells, Cultured; Ceruletide; Endopeptidases; Gene Expression; In Vitro Techniques; Male; Monocytes; Pancreas; Pancreatic Ducts; Pancreatitis; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Trypsin

Heat shock protein (Hsp) 27 regulates actin cytoskeletal dynamics, and overexpression of Hsp27 in fibroblasts protects against stress in a phosphorylation-dependent manner. Induction of Hsps occurs in acute pancreatitis, but Hsp27 has not been ascribed a specific role. To examine whether Hsp27 would ameliorate acute pancreatitis, we generated transgenic mice overexpressing human Hsp27 (huHsp27) or Hsp27 with the phosphorylatable residues Ser(15,78,82) mutated to aspartic acid (huHsp27-3D) to mimic phosphorylation or to alanine (huHsp27-3A), which is nonphosphorylatable.. huHsp27 was expressed at high levels in the exocrine pancreas by use of a cytomegalovirus promoter. Protein expression was analyzed by Western blotting and immunofluorescence. Acute pancreatitis was induced with 6 or 12 hourly cerulein injections (50 microg/kg intraperitoneally) and its severity assessed by measuring serum amylase and lipase levels, pancreatic trypsin activity, edema, and morphologic changes by quantitative scoring of multiple histologic sections and visualization of filamentous actin. Systemic inflammatory effects were monitored by measuring lung myeloperoxidase activity (a marker of neutrophil infiltration).. huHsp27 protein was overexpressed in the pancreas and localized to pancreatic acini. Acute pancreatitis was ameliorated by overexpression of huHsp27 and the huHsp27-3D mutant, which were associated with suppression of pancreatic trypsin activity and acinar cell injury and preservation of the actin cytoskeleton. In contrast, these changes were unaffected by overexpression of the nonphosphorylatable huHsp27-3A mutant.. Pancreatic overexpression of huHsp27 protects against cerulein-induced acute pancreatitis in a specific phosphorylation-dependent manner and is associated with preservation of the actin cytoskeleton.

Topics: Actins; Acute Disease; Animals; Ceruletide; Cytoskeleton; Female; Gastrointestinal Agents; Heat-Shock Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Animal; Molecular Chaperones; Neoplasm Proteins; Pancreatitis; Phosphorylation; Severity of Illness Index

To elucidate the role of COX-2 in the development of capillary leakage in rats with acute interstitial pancreatitis.. Rats with acute interstitial pancreatitis were induced by caerulein subcutaneous injection. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of COX-2 in pancreatic tissues, spectrophotometry was used to assay the parameters of acute pancreatitis such as the serum amylase and plasma myeloperoxidase, and determination of capillary permeability in the pancreas by quantifying the permeability index (PI) assisted response of pancreatic microvascular via intravital fluorescence microscope video image analysis system.. A significant increase of COX-2 expression, elevation of serum amylase, and plasma myeloperoxidase were detected in rats with acute edematous pancreatitis compared with control rats. The changes of pancreatic microvascular after caerulein injection were as following: (a) the decrease of pancreatic capillary blood flow (4th h, 0.56+/-0.09 nL/min, P<0.05; 8 th h, 0.34+/-0.10 nL/min, P<0.001); (b) reduction of functional capillary density (4 th h, 381+/-9 cm(-1), P>0.05; 8th h, 277+/-13 cm(-1), P<0.001); (c) irregular and intermittent capillary perfusion was observed at the 8th h and these vessels were also prone to permeation.. COX-2 plays an important role in mediating capillary permeability in pancreatitis, thereby contributing to capillary leakage.

Topics: Animals; Blood Circulation; Capillaries; Capillary Permeability; Ceruletide; Cyclooxygenase 2; Isoenzymes; Male; Microcirculation; Pancreatitis; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Regional Blood Flow

Infection of pancreatic necrosis is a severe complication of acute pancreatitis. Because Toll-like receptor 4 (TLR4) has been identified as a receptor necessary to transduct the signal of bacteria-derived lipopolysaccharide into cells, we investigated the role of TLR4 on pancreatic and pulmonary injury in acute pancreatitis and acute pancreatitis associated with endotoxemia in wild-type and TLR4-deficient mice.. Laboratory investigation.. University laboratory.. Heterozygous TLR4 mice.. Mice were injected intraperitoneally with a supramaximal dose of cerulein each hour for 10 hrs. To mimic infection, additional groups of mice were injected with lipopolysaccharide in the presence or absence of cerulein injections.. The severity of acute pancreatitis was assessed by serum amylase activity, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity. Lung injury was quantitated by lung microvascular permeability and lung myeloperoxidase activity. Injections of cerulein induced an edematous pancreatitis that was of similar severity in wild-type and TLR4-deficient mice. Lipopolysaccharide alone had no toxic effect on pancreas and lungs and did not worsen the pancreatic injury induced by cerulein in wild-type and TRL4-deficient mice. In contrast, lipopolysaccharide worsened pancreatitis-associated lung injury, and the deficiency in TLR4 fully prevented this aggravation.. TLR4 may not play a role in the pancreatitis-associated lung injury but participates in the pulmonary injury mediated by endotoxemia.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Disease Models, Animal; Lipopolysaccharides; Lung Diseases; Membrane Glycoproteins; Mice; Pancreas; Pancreatitis; Receptors, Cell Surface; Toll-Like Receptor 4; Toll-Like Receptors

Microcirculatory disturbances are important early pathophysiological events in various organs during acute pancreatitis. The aim of the study was to evaluate changes in microperfusion of the pancreas, liver, kidney, stomach, colon, skeletal muscle, and to investigate the influence of heparin on the organ microcirculation in caerulein-induced experimental acute pancreatitis.. Acute pancreatitis was induced by 4 intraperitoneal injections of caerulein (Cn) (15 microg/kg). The organ microcirculation was measured by laser Doppler flowmetry. Serum interleukin 6 and hematocrit levels were analysed.. Acute pancreatitis resulted in a significant drop of microperfusion in all examined organs. Heparin administration (2 x 2.5 mg/kg) improved the microcirculation in pancreas (36.9 +/- 4% vs 75.9 +/- 10%), liver (56.6 +/- 6% vs 75.2 +/- 16%), kidney (45.1 +/- 6% vs 79.3 +/- 5%), stomach (65.2 +/- 8% vs 78.1 +/- 19%), colon (69.8 +/- 6% vs 102.5 +/- 19%), and skeletal muscle (59.2 +/- 6% vs 77.9 +/- 13%). Heparin treatment lowered IL-6 (359.0 +/- 66 U/mL vs 288.5 +/- 58 U/mL) and hematocrit level (53 +/- 4% vs 46 +/- 3%).. Heparin administration has a positive influence on organ microcirculatory disturbances accompanying experimental Cn-induced acute pancreatitis.

Topics: Acute Disease; Animals; Anticoagulants; Ceruletide; Hematocrit; Heparin; Male; Microcirculation; Pancreas; Pancreatitis; Rats; Rats, Wistar; Splanchnic Circulation

Oxidative stress plays an important role in the early stage of acute pancreatitis, as well as in the associated multiple organ injury. This study tests the hypothesis that M40401, a new superoxide dismutase mimetic, attenuates experimental acute pancreatitis. Intraperitoneal injection of cerulein in mice resulted in a severe, acute pancreatitis that was characterized by edema, neutrophil infiltration, tissue hemorrhage, and cell necrosis, as well as increases in the serum levels of amylase and/or lipase. The infiltration of the pancreatic tissue of these animals with neutrophils (measured as an increase in myeloperoxidase activity) was associated with expression of intercellular adhesion molecule-1, as well as signs of enhanced lipid peroxidation (e.g., increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for nitrotyrosine and poly (ADP-ribose) polymerase in the pancreas of cerulein-treated mice. In contrast, the degree of pancreatic inflammation and tissue injury (histological score), the expression of intercellular adhesion molecule-1, the staining for nitrotyrosine and poly (ADP-ribose) polymerase, and lipid peroxidation were markedly reduced in pancreatic tissue sections obtained from cerulein-treated mice administered with M40401. These results confirm our hypothesis that superoxide anions play an important role in cerulein-mediated acute pancreatitis and support the possible clinical use of low-molecular-weight synthetic superoxide dismutase mimetics in those conditions that are associated with overproduction of superoxide.

Topics: Acute Disease; Animals; Ceruletide; Male; Manganese; Mice; Mice, Inbred Strains; Organometallic Compounds; Pancreatitis; Peroxidase; Poly(ADP-ribose) Polymerases; Superoxide Dismutase

Among the three major cytofilament proteins, keratin (K8/K18/K19) expression increases nearly threefold upon pancreas or liver injury, while actin and tubulin expressions are considered relatively stable. K8/K18 serves essential hepatocyte cytoprotective functions yet appears dispensable in K8-null mouse pancreata, which led us to hypothesize that actin or tubulin expressions may increase after pancreatic injury. Balb/c and FVB/n mice manifested different susceptibility to injury in two pancreatitis models, with significant induction of actin protein (threefold) and RNA after moderate or severe but not mild injury. Alterations in tubulin expression were less prominent. Basally, K8-null and wild-type pancreata expressed similar actin and tubulin levels, while the injury-induced actin protein but not RNA was more pronounced in K8-null mice. K7/K18/K19/K20 were also induced in K8-null mice after injury. Ex vivo, caerulein-triggered pancreatitis caused protein degradation (actin approximately or = tubulin > keratins) and mRNA up-regulation that was blocked by actinomycin-D (act-D) (actin approximately or = tubulin approximately or = keratin) or by NF-kappaB inhibition (keratins > actin approximately or = tubulin). Hence, actin is not as static as previously held and is overexpressed after moderate to severe pancreatic injury while keratins are induced after minimal injury. Keratin and actin induction may serve protective roles in pancreatic injury.

Topics: Actins; Animals; Ceruletide; Cytoskeleton; Dactinomycin; Disease Susceptibility; Female; Immunoblotting; Keratin-8; Keratins; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; NF-kappa B; Pancreas; Pancreatic Diseases; Pancreatitis; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tubulin; Up-Regulation

Melatonin, an antioxidant, protects the pancreas against acute inflammation but, although this indole is released mainly at night, no study has been undertaken to determine circadian changes of plasma melatonin levels and the severity of acute pancreatitis. The aims of this study were: (a) to compare the severity of caerulein-induced pancreatitis (CIP) produced in the rat during the day and at the night, and (b) to assess the changes of plasma melatonin level and the activity of an antioxidative enzyme; superoxide dismutase (SOD), in the pancreas subjected to CIP during the day time and at night without or with administration of exogenous melatonin or its precursor; l-tryptophan. Rats were kept in 12 hr light/dark cycle. CIP was induced by subcutaneous infusion of caerulein (5 microg/kg/hr for 5 hr). Melatonin (5 or 25 mg/kg) or l-tryptophan (50 or 250 mg/kg) was given intraperitoneally 30 min prior to the start of CIP. CIP induced during the day time was confirmed by histological examination and manifested by pancreatic edema, and rises of amylase and lipase plasma activities (by 400 and 500%, respectively), whereas pancreatic SOD, pancreatic blood flow (PBF) and oxygen consumption by pancreatic tissue (VO(2)) were decreased by 70, 40 and 45%, respectively, as compared with the appropriate controls. All morphological and biochemical parameters of CIP induced at night were significantly less severe, compared with those recorded during the light phase. Plasma melatonin immunoreactivity was significantly higher during the night, than during the day, especially following administration of melatonin or its precursor, which reversed all manifestations of CIP. In conclusion, a circadian rhythm modulates the severity of CIP with a decrease of pancreatitis severity during the night compared with that at the day time and this may be due to the increased plasma level of melatonin and higher activity of SOD in the pancreas.

Topics: Amylases; Animals; Ceruletide; Circadian Rhythm; Darkness; Dose-Response Relationship, Drug; Lipase; Male; Melatonin; Organ Size; Oxygen; Pancreas; Pancreatitis; Rats; Rats, Wistar; Regional Blood Flow; Superoxide Dismutase; Tryptophan

Extracellular regulated kinases (ERK1/2) and c-Jun N-terminal Kinases (JNK), are generally considered to play a key role in signal transduction pathways activated by a wide range of stimuli. We studied the effects of SP600125, a novel inhibitor of both JNK and ERK1/2, in male C57/BL6 mice given with an hyper-stimulating dose of cerulein (50 microg/kg for each of four injections at hourly intervals) to elicit secretagogue-induced pancreatitis. A control group received four intra-peritoneal injections of 0.9% saline at hourly intervals. Animals were randomized to receive either SP600125 (15 mg/kg i.p. administered 2 h before and 30 min after the first injection of cerulein) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). A group of animals was killed 30 minutes after the last cerulein injection to evaluate pancreatic JNK and ERK1/2 activation by Western Blot analysis. Another group was sacrificed 2 hours after the last cerulein injection to evaluate serum lipase and amylase levels, pancreas oedema, pancreatic content of Tumor Necrosis Factor-alpha (TNF-alpha) and Intercellular adhesion molecule-1 (ICAM-1) and the histological alterations. SP600125 inhibited almost totally JNK activation (90%) and partially ERK1/2 activation (45%), reduced the serum lipase and amylase levels and the degree of oedema, blunted the increased pancreatic content of TNF-alpha and ICAM-1 and protected against the histological damage. Our data confirm that both JNK and ERK1/2 activation plays a key role in acute pancreatitis and that SP600125 may represent a potential therapeutic approach to the treatment of patients at high risk of developing this life-threatening condition.

Topics: Amylases; Analysis of Variance; Animals; Anthracenes; Blotting, Western; Ceruletide; Disease Models, Animal; Edema; Enzyme Activation; Histological Techniques; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; Lipase; Male; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Oligonucleotides; Pancreas; Pancreatitis; Peroxidase; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction; Tumor Necrosis Factor-alpha

To investigate the fluid shear stress induced changes of (Ca(2+))i in neutrophils in pancreatic microcirculation of experimental acute pancreatitis (AP).. Wistar rats (n = 36) were randomized into three groups. A model of AP was established by subcutaneous injection of caerulein. Low-shear 30 viscometer was used to provide steady fluid shear stress on separated neutrophils. The mean fluorescent intensity tested by flow cytometry was used as the indication of [Ca(2+)]i quantity.. Under steady shear, cytosolic [Ca(2+)]i showed biphasic changes. The shear rate changed from low to high, [(Ca(2+)]i in different groups decreased slightly and then increased gradually to a high level (P<0.05). A close correlation was observed between the cytosolic [Ca(2+)]i level and the alteration of fluid shear stress in regional microcirculation of AP.. The increase of [Ca(2+)]i is highly related to the activation of neutrophils, which contributes to neutrophil adhesion to endothelium in the early phase of AP. The effect of fluid shear stress on [Ca(2+)]i may play a crucial role in pancreatic microcirculatory failure of AP.

Topics: Acute Disease; Animals; Calcium; Ceruletide; Cytosol; Male; Microcirculation; Neutrophils; Pancreas; Pancreatitis; Rats; Rats, Wistar; Stress, Mechanical

Nosocomial pneumonia is a feared complication in the critically ill patient. Serious acute pancreatitis is frequently complicated by infections. The objectives of this study were to determine the influence of acute pancreatitis on host defense against Pseudomonas pneumonia and to determine the influence of Pseudomonas pneumonia on the severity of concurrent pancreatitis.. A controlled, in vivo laboratory study.. Research laboratory of a health sciences university.. Female C57Bl/6 mice.. Pancreatitis was induced by 12 hourly intraperitoneal injections of cerulein (pancreatitis) or saline (sham) immediately followed by intranasal administration of Pseudomonas aeruginosa (to induce pneumonia) or saline (controls). Mice were killed 24 hrs later. Hence, four groups were studied: sham/control, pancreatitis/control, sham/pneumonia, and pancreatitis/pneumonia mice.. When compared with sham/pneumonia mice, pancreatitis/pneumonia mice demonstrated exaggerated lung inflammation, higher bacterial counts in lungs and pancreas, and enhanced dissemination of the infection. Concurrently, pneumonia prolonged the course of pancreatitis, as reflected by histopathology and higher plasma amylase and relative pancreas weights (all p < .05 for the difference between pancreatitis/pneumonia and pancreatitis/control mice), which was associated with the localization of Pseudomonas in the pancreas.. Acute pancreatitis impairs host defense against Pseudomonas pneumonia, whereas pneumonia prolongs the course of pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Female; Gastrointestinal Agents; Mice; Mice, Inbred C57BL; Models, Animal; Pancreatitis; Pneumonia, Bacterial; Pseudomonas Infections; Severity of Illness Index

Matrix metalloproteinases, in particular gelatinase B/MMP-9, are key mediators in autoimmune diseases like multiple sclerosis and rheumatoid arthritis, but their pathogenic roles in diabetes are not well established. Gelatinase B has previously been shown to be upregulated in pancreas tissue from patients with acute and chronic pancreatitis and was suggested to exacerbate diabetes by cleaving insulin. In this study, the role of gelatinase B in diabetes was investigated using two streptozotocin-induced animal models of type I diabetes. In both a hyperacute and a subacute model, gelatinase B upregulation was found to be associated with disease activity. However, gelatinase B deficiency did not significantly protect against diabetes development, and wild-type and gelatinase B-deficient animals behaved similarly in terms of beta-cell apoptosis or necrosis. The fact that gelatinase B was found almost exclusively as the inactive pro-enzyme in most of the streptozotocin-induced diabetic animals may explain the lack of a gelatinase B effect. On the contrary, gelatinase B was completely activated in a minority (15%) of wild-type animals. This coincided with exocrine pancreatic inflammation, as revealed by the presence of active trypsin. The discovery of in vivo activation of progelatinase B by trypsin in acute pancreatitis is extended in a model of caerulein-induced pancreatitis. In the latter model, trypsinogen activation is systematically achieved and gelatinase B is found in its active form. In conclusion, gelatinase B itself is not a causative factor but, when activated by endogenous trypsin, is a permissive factor for insulin degradation and diabetes.

Topics: Acute Disease; Animals; Apoptosis; Blood Glucose; Ceruletide; Diabetes Mellitus, Experimental; Enzyme Activation; Islets of Langerhans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Pancreas, Exocrine; Pancreatitis; Trypsin; Up-Regulation

To observe the expressions of early growth response 1 (Egr-1) and tissue factor (TF) in rat tissues of acute pancreatitis induced by caerulein and to explore their significance.. Pancreatitis was induced in rats by high-dose intraabdominal caerulein injection. The changes of Egr-1 mRNA and protein in pancreas were measured by quantitative PCR and Western blotting, and the localization of Egr-1 protein in acinar cells was visualized by immunohistochemistry. TF mRNA levels were also measured by quantitative PCR. High-dose bombesin-stimulated rats served as the negative control.. Egr-1 mRNA was rapidly increased in the pancreas of rats stimulated by high-dose cearulein, and reached the peak level 30 min after the stimulation, whereas band for peak Egr-1 protein level was visualized by Western blotting till 2 h after stimulation. Immunohistochemistry showed that almost every acinar cell in the pancreas was Egr-1-positive, especially in the nucleus. In line with Egr-1 activation, TF mRNA was detected 1 h after the stimulation and increased steadily within the initial 4 h. Only a small quantity of Egr-1 mRNA expression was observed in bombesin-stimulated rats, in which no Egr-1 protein or TF mRNA were detected.. Egr-1 mRNA and protein were up-regulated in the early stage of pancreatitis. Egr-1, as a pro-inflammatory transcriptional factor, probably plays an important role in the initiation of acute pancreatitis, and its action might be partially mediated through the up-regulation of TF expression.

Topics: Animals; Ceruletide; Early Growth Response Protein 1; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; RNA, Messenger; Thromboplastin

In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-gamma (PI3K gamma) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3K gamma, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3K gamma significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3K gamma-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3K gamma, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3K gamma. Our results thus suggest that inhibition of PI3K gamma may be of therapeutic value in acute pancreatitis.

Topics: Acute Disease; Animals; Apoptosis; Caspase 3; Caspases; Ceruletide; Choline Deficiency; Class Ib Phosphatidylinositol 3-Kinase; Diet; Dietary Supplements; Ethionine; In Situ Nick-End Labeling; Isoenzymes; Mice; Mice, Knockout; Necrosis; Neutrophils; Pancreatitis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Survival Rate

To assess the effect of endothelins: ET-1, ET-2 and ET-3 on trypsinogen activation, lipase activity and histological changes in the pancreas in early (4 hrs) cerulein acute pancreatitis (AP) in rats.. In 45 Wistar rats with cerulein induced AP (2 x 40 microg/kg i.p. at 1 hour interval, the effect of endothelins at the dose 2 x 0.5 or 2 x 1.0 nmol/kg i.p. was assessed vs untreated AP; 6 healthy rats were control (C). Free active trypsin (FAT), total potential trypsin after activation with enterokinase (TPT), lipase in 12000 xg supernatants of pancreatic homogenates and the plasma alpha-amylase were assayed. The %FAT/TPT was an index of trypsinogen activation.. %FAT/TPT increased from 3.0 +/- 0.6 in C to 16.2 +/- 3.1 in AP (p < 0.01). ET-1 decreased this index to 4.8 +/- 1.1 after higher dose (p < 0.01); the effect of lower dose was insignificant. Attenuating effect of ET-2 was significant: 7.3 +/- 1.7 after higher dose (p < 0.05) and 6.1 +/- 0.9 after lower dose (p < 0.01). ET-3 diminished this index to 4.5 +/- 1.5 (p < 0.01) and to 6.3 +/- 2.2 (p < 0.05) respectively. Lipase activity in supernatant increased from 4.1 +/- 0.6 in C to 6.3 +/- 0.7 U/mg protein in untreated AP (p < 0.05) and plasma alpha-amylase from 7.0 +/- 0.6 in C to 25.9 +/- 4.3 U/ml in AP (p < 0.001), without essential changes in treated groups vs untreated AP. Higher doses of endothelins decreased inflammatory cell infiltration score in AP.. The exogenous endothelins, especially ET-2 and ET-3 and to lesser extent ET-1 exerted some protective effect in early, edematous acute pancreatitis by the attenuation of trypsinogen activation and inflammatory cell infiltration in the pancreas.

Topics: Acute Disease; alpha-Amylases; Animals; Ceruletide; Endothelin-1; Endothelin-2; Endothelin-3; Endothelins; Enzyme Activation; Lipase; Male; Pancreatitis; Rats; Rats, Wistar; Time Factors; Trypsinogen

Severe necrotizing pancreatitis occurs in young female mice fed a choline-deficient and ethionine-supplemented (CDE) diet. Although the mechanism of the pancreatitis is unknown, one consequence of this diet is depletion of hepatic S-adenosylmethionine (SAM). SAM formation is catalyzed by methionine adenosyltransferases (MATs), which are encoded by liver-specific (MAT1A) and non-liver-specific (MAT2A) genes. In this work, we examined changes in pancreatic SAM homeostasis in mice receiving the CDE diet and the effect of SAM treatment. We found that both MAT forms are expressed in normal pancreas and pancreatic acini. After 48 h of the CDE diet, SAM levels decreased 50% and MAT1A-encoded protein disappeared via post-translational mechanisms, whereas MAT2A-encoded protein increased via pretranslational mechanisms. CDE-fed mice exhibited extensive necrosis, edema, and acute pancreatic inflammatory infiltration, which were prevented by SAM treatment. However, old female mice consuming the CDE diet that do not develop pancreatitis showed a similar fall in pancreatic SAM level. SAM was also protective in cerulein-induced pancreatitis in the rat, but the protection was limited. Although the pancreatic SAM level fell by more than 80% in the MAT1A knockout mice, no pancreatitis developed. This study thus provides several novel findings. First, the so-called liver-specific MAT1A is highly expressed in the normal pancreas and pancreatic acini. Second, the CDE diet causes dramatic changes in the expression of MAT isozymes by different mechanisms. Third, in contrast to the situation in the liver, where absence of MAT1A and decreased hepatic SAM level can lead to spontaneous tissue injury, in the pancreas the roles of SAM and MAT1A appear more complex and remain to be defined.

Topics: Administration, Oral; Animals; Ceruletide; Choline Deficiency; Ethionine; Female; Methionine Adenosyltransferase; Mice; Models, Biological; Pancreas; Pancreatitis; S-Adenosylmethionine

Melatonin, a pineal secretory product, synthesized from l-tryptophan, has received increased attention because of its antioxidative and immunomodulatory properties. It has been detected in the gut and shown to protect the gastric mucosa, and liver from acute damage, but the role of melatonin in the protection of the pancreas against acute inflammation is not clear. The aim of this study was to investigate the effects of melatonin and its precursor, l-tryptophan, on caerulein-induced pancreatitis (CIP) and on ischemia/reperfusion (I/R)-provoked pancreatitis in rats. CIP was induced by subcutaneous infusion of caerulein to the rats (25 microg/kg). I/R was induced by clamping of the inferior splenic artery for 30 min followed by 2 hr of reperfusion. Melatonin (10, 25 or 50 mg/hr) or l-tryptophan (50, 100 or 250 mg/kg) was given as a bolus intraperitoneal (i.p.) injection 30 min prior to the onset of pancreatitis. CIP and I/R were confirmed by histologic examination and manifested by typical pancreatic edema, by an increase of plasma levels of amylase (by 500% in CIP and by 40% in I/R) and the pro-inflammatory tumor necrosis factor alpha (TNFalpha) (by 500%). Lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), were increased several fold in the pancreas CIP and I/R, whereas pancreatic blood flow (PBF) was significantly reduced in these animals. Pretreatment of rats subjected to CIP or to I/R with melatonin (25 or 50 mg/kg i.p.) or l-tryptophan (100 or 250 mg/kg i.p.) significantly reduced pancreatic edema, plasma levels of amylase and TNFalpha and diminished pancreatic MDA + 4-HNE contents, while enhancing PBF, pancreatic integrity and plasma levels of the anti-inflammatory interleukin 10 (IL-10). This was accompanied by a marked and dose-dependent rise of plasma melatonin immunoreactivity. Gene expression of N-acetyl transferase, an enzyme involved in melatonin biosynthesis, was detected in the pancreas of normal rats and was significantly enhanced in the rats with CIP. We conclude that exogenous melatonin, and that produced from l-tryptophan, attenuates pancreatic damage induced by CIP or by I/R and this effect may be attributable to the reduction in lipid peroxidation and TNFalpha release combined with an increase of plasma anti-inflammatory IL-10 in rats with acute pancreatitis.

Topics: Adjuvants, Immunologic; Animals; Ceruletide; Interleukin-10; Ischemia; Male; Melatonin; Pancreas; Pancreatitis; Rats; Rats, Wistar; Reperfusion Injury; Tryptophan; Tumor Necrosis Factor-alpha

We examined the effects of a weak base, chloroquine, on the trypsinogen processing in cerulein-induced pancreatitis.. Immunofluorescence studies were performed using newly generated affinity-purified antibodies to the trypsinogen activation peptide (TAP).. The present study showed that chloroquine pretreatment blocked intracellular TAP generation in cerulein-induced pancreatitis.. These results indicate that intracellular trypsinogen activation, which plays an important role in acute pancreatitis, requires a low-pH compartment, as well as serine protease activity.

Topics: Animals; Ceruletide; Chloroquine; Enzyme Inhibitors; Fluorescent Antibody Technique; Hydrogen-Ion Concentration; Male; Oligopeptides; Pancreatitis; Rats; Rats, Wistar; Trypsinogen

Proinflammatory cytokines act as mediators of the local and systemic manifestations of acute pancreatitis (AP).. To investigate whether moderate hypothermia (MH) (32 degrees C) can reduce the severity of AP by inhibiting cytokine production in a rat model of caerulein-induced pancreatitis.. Rats were divided into three groups: control rats (Group I), AP rats treated with normothermia (38 degrees C) (Group II), and AP rats treated with MH (Group III). AP was induced by intramuscular injection of caerulein and intraperitoneal infusion of lipopolysaccharide. MH was induced 4 hours after the first caerulein injection. Serum interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, amylase, and lipase levels were determined 8 hours after the first injection. The pancreas and lung were examined histologically.. MH in comparison with normothermia significantly reduced serum levels of IL-1beta, TNF-alpha, IL-6, amylase, and lipase. Histologically, the MH group showed less vacuolization of the acinar cells and cellular infiltration into the interacinar areas of the pancreas than were shown in the normothermia group, but these effects were not evident in the lung.. Our results suggest that MH may be clinically applicable for reducing the severity of AP.

Topics: Acute Disease; Amylases; Animals; Blood Gas Analysis; Blood Pressure; Ceruletide; Cytokines; Hypothermia, Induced; Interleukin-1; Interleukin-6; Lipase; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

The early events leading to acinar cell injury during acute pancreatitis are poorly characterized. Signaling through gap junction channels contributes to the homeostasis of the exocrine pancreas by coordinating acinar cell activity within an acinus. To explore the role of gap junctional communication in acinar cell response to injury, we analyzed the course of acute pancreatitis induced by injection of cerulein in mice deficient for Cx32, the major gap junction protein expressed in the exocrine pancreas.. The severity of pancreatitis was evidenced by measuring serum amylase activity, pancreatic edema, acinar cell necrosis, pancreatic tumor necrosis factor alpha concentration, and myeloperoxidase activity. Acinar cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), caspase-3 activity, and Bax/Bcl-2 expression. Expression and function of connexin were evaluated by immunofluorescence and dye coupling.. Cx32-deficient mice exhibited a deleterious course of acute pancreatitis with increased necrosis, edema, and inflammation of the exocrine pancreas. In addition, the exocrine pancreas of Cx32-deficient mice showed a decreased number of TUNEL-positive acinar cells and decreased caspase-3 activity but no change in Bax or Bcl-2 pancreatic expression. Interestingly, chemicals known to induce apoptosis in vivo had no effect on Cx32-deficient pancreatic acinar cells.. Deficiency of a pancreatic connexin converts a mild reversible form of acute pancreatitis into a severe disease and decreases the sensitivity of acinar cells to apoptotic stimuli. The results show that acinar cell-to-cell communication plays a key role in the modulation of severity of acute pancreatitis.

Topics: Acute Disease; Animals; Apoptosis; bcl-2-Associated X Protein; Ceruletide; Connexins; Gap Junction beta-1 Protein; Glutathione; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Severity of Illness Index

Topics: Acute Disease; Animals; Bacterial Proteins; Catecholamines; Ceruletide; Chaperonin 60; Chaperonins; Pancreatitis

Acute pancreatitis is an inflammatory disease characterized by pancreatic tissue edema, acinar cell necrosis, hemorrhage and inflammation of the damaged gland. It is believed that acinar cell injury is initiated by the activation of digestive zymogens inside the acinar cells, leading finally to the autodigestion of the pancreas. Previous study in our laboratory demonstrated that cerulein-induced acute pancreatitis was associated with an up-regulation of local renin-angiotensin system (RAS) in rat pancreas. Therefore, the utilization of RAS inhibitors may provide a novel and alternative treatment for acute pancreatitis. By means of a rat model of cerulein-induced acute pancreatitis, results from the present study showed that an intravenous injection of saralasin, an antagonist for angiotensin II receptors, at a dose of 40 microg/kg 30 min before the induction of acute pancreatitis significantly attenuated pancreatic edema. Results from the biochemical measurements showed that pretreatment with saralasin at a dose of 20 microg/kg markedly reduced pancreatic injury, as evidenced by the decreased activities of alpha-amylase and lipase in plasma. However, the same recipe of ramiprilat, a specific inhibitor for angiotensin-converting enzyme, at a dose of 20 microg/kg did not provide any protective effect against acute pancreatitis. On the contrary, pretreatment with ramiprilat at a dose 40 microg/kg enhanced cerulein-induced pancreatic injury. Results from histopathological analysis of these RAS inhibitors further confirmed with those results as obtained from biochemical analysis. These data indicate that administration of saralasin but not ramiprilat could be protective against acute pancreatitis and that activation of pancreatic RAS in acute pancreatitis may play a role in pancreatic tissue injury.

Topics: Acute Disease; alpha-Amylases; Angiotensin Receptor Antagonists; Animals; Ceruletide; Disease Models, Animal; Edema; Injections, Intravenous; Lipase; Necrosis; Pancreatitis; Ramipril; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; Saralasin

To establish a non-traumatic, easy to induce and reproducible mouse model of severe acute pancreatitis (SAP) induced with caerulein and lipopolyasccharide (LPS).. Thirty-two healthy mature NIH female mice were selected and divided at random into four groups (each of 8 mice), i.e., the control group (NS group), the caerulein group (Cn group), the lipopolysaccharide group (LPS group), and the caerulein+LPS group (Cn+LPS group). Mice were injected intraperitoneally with caerulein only, or LPS only, and caerulein and LPS in combination. All the animals were then killed by neck dislocation three hours after the last intraperitoneal injection. The pancreas and exo-pancreatic organs were then carefully removed for microscopic examination. And the pancreatic acinus was further observed under transmission electron microscope (TEM). Pancreatic weight, serum amylase, serum nitric oxide (NO) concentration, superoxide dismutase (SOD) and malondialdehyde (MDA) concentration of the pancreas were assayed respectively.. (1) NS animals displayed normal pancreatic structure both in the exocrine and endocrine. In the LPS group, the pancreas was slightly edematous, with the infiltration of a few inflammatory cells and the necrosis of the adjacent fat tissues. All the animals of the Cn group showed distinct signs of a mild edematous pancreatitis characterized by interstitial edema, infiltration of neutrophil and mononuclear cells, but without obvious parenchyma necrosis and hemorrhage. In contrast, the Cn+LPS group showed more diffuse focal areas of nonviable pancreatic and hemorrhage as well as systemic organ dysfunction. According to Schmidt's criteria, the pancreatic histologic score showed that there existed significant difference in the Cn+LPS group in the interstitial edema, inflammatory infiltration, parenchyma necrosis and parenchyma homorrhage in comparison with those of the Cn group, LPS group and NS group (P<0.01 or P<0.05). (2) The ultrasturcture of acinar cells was seriously damaged in the Cn+LPS group. Chromatin margination of nuclei was present, the number and volume of vacuoles greatly increased. Zymogen granules (ZGs) were greatly decreased in number and endoplasmic reticulum exhibited whorls. The swollen mitochondria appeared, the crista of which was decreased in number or disappeared. (3) Pancreatic weight and serum amylase levels in the Cn +LPS was significantly higher than those of the NS group and the LPS group respectively (P<0.01 or P<0.05). However, the pancreatic wet weight and serum amylase concentration showed no significant difference between the Cn+LPS group and the Cn group. (4) NO concentration in the Cn+LPS group was significantly higher than that of NS group, LPS group and Cn group(P<0.05 or P<0.01). (5) The SOD and MDA concentration of the pancreas in the Cn+LPS group were significantly higher than those of NS, LPS and Cn groups (P<0.05 or P<0.01).. The mouse model of severe acute pancreatitis could be induced with caerulein and LPS, which could be non-traumatic and easy to induce, reproducible with the same pathological characteristics as those of SAP in human, and could be used in the research on the mechanism of human SAP.

Topics: Acute Disease; Animals; Ceruletide; Female; Lipopolysaccharides; Mice; Mice, Inbred Strains; Pancreatitis; Severity of Illness Index

Free radical-mediated pancreatic injury is believed to play a key role in the pathogenesis of acute pancreatitis. Most of these studies have focused on the effects of antioxidant enzymes and free radical scavengers on improving the pancreatic injury. Recent findings showed that cerulein-induced acute pancreatitis was associated with an upregulation of a local pancreatic renin-angiotensin system in the pancreas. In the current study we hypothesized that inhibition of this renin-angiotensin system by saralasin, a nonspecific antagonist for angiotensin II receptor, could attenuate the severity of cerulein-induced pancreatitis.. The effects of saralasin on oxidative stress and tissue injury in cerulein-induced pancreatitis were assessed by histopathologic analysis and on the basis of biochemical changes of plasma alpha-amylase level, pancreatic glutathione status, oxidative modification of protein, and lipid peroxidation.. Data from the biochemical analysis showed that intravenous injections of saralasin at doses of 10 microg/kg to 50 microg/kg 30 minutes before the induction of acute pancreatitis significantly reduced pancreatic injury, as indicated by a decrease in plasma alpha-amylase activity in comparison with the cerulein-treated control. The effect of saralasin was further manifested by significant suppressions of glutathione depletion, oxidative modification of proteins, and lipid peroxidation in cerulein-treated rat pancreas. Histopathologic examination findings were in agreement with the biochemical data.. These data suggest that prophylactic administration of saralasin can ameliorate the oxidative stress and tissue injury in cerulein-induced pancreatitis. Such a protective effect may provide new insight into the potential value of angiotensin II receptor antagonists in the clinical therapy for acute pancreatitis.

Topics: Acute Disease; alpha-Amylases; Angiotensin Receptor Antagonists; Animals; Ceruletide; Glutathione; Lipid Peroxidation; Oxidative Stress; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Saralasin

Impaired lung function in severe acute pancreatitis is the primary cause of morbidity and mortality in this condition. Preprotachykinin-A (PPT-A) gene products substance P and neurokinin (NK)-A have been shown to play important roles in neurogenic inflammation. Substance P acts primarily (but not exclusively) via the NK1 receptor. NKA acts primarily via the NK2 receptor. Earlier work has shown that knockout mice deficient in NK1 receptors are protected against acute pancreatitis and associated lung injury. NK1 receptors, however, bind other peptides in addition to substance P, not all of which are derived from the PPT-A gene. To examine the role of PPT-A gene products in acute pancreatitis, the effect of PPT-A gene deletion on the severity of acute pancreatitis and the associated lung injury was investigated. Deletion of PPT-A almost completely protected against acute pancreatitis-associated lung injury, with a partial protection against local pancreatic damage. These results show that PPT-A gene products are critical proinflammatory mediators in acute pancreatitis and the associated lung injury.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Dose-Response Relationship, Drug; Gene Deletion; Lung; Lung Diseases; Mice; Mice, Knockout; Pancreas; Pancreatitis; Protein Precursors; Tachykinins

Acute pancreatitis is an inflammatory process of variable severity, and leukocytes are thought to play a key role in the development of pancreatitis and pancreatitis-associated lung injury. The effects of mediators released by these inflammatory cells may induce tissue damage. The aim of our study was to evaluate the role of the chemokine, macrophage inflammatory protein-2 (MIP-2), in the pathogenesis of cerulein-induced pancreatitis and pancreatitis-associated lung injury. The severity of pancreatitis was measured by serum amylase, pancreatic edema, acinar cell necrosis, and myeloperoxidase activity. Lung injury was quantitated by evaluating lung microvascular permeability and lung myeloperoxidase activity. To determine the role of MIP-2 in the pathophysiology of the disease, anti-MIP-2 antibody was administered either 1 hour before or 2 hours after the start of cerulein administration. MIP-2 concentrations increased in serum, pancreas, and lung tissues in mice treated with cerulein. Anti-MIP-2 antibody administrated either before or after cerulein partially protected against pancreas and lung injury. These results show that MIP-2 plays a key role in the pathophysiology of acute pancreatitis and that MIP-2 blockade may improve the outcome of the disease.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Chemokine CXCL2; Disease Models, Animal; Edema; Fluorescent Antibody Technique, Indirect; Gastrointestinal Agents; Injections, Intraperitoneal; Lung Diseases; Male; Mice; Mice, Inbred Strains; Monokines; Necrosis; Pancreas; Pancreatitis; Peroxidase

We hypothesized that genes expressed in pancreatic acinar cells during the initiation of acute pancreatitis determine the severity of the disease. Therefore, we utilized microarrays to identify those genes commonly induced in rat pancreatic acinar cells within 1-4 h in two in vivo models, caerulein and taurocholate administration. This strategy yielded 51 known genes representing a complex array of molecules, including those that are likely to either reduce or increase the severity of the disease. Novel genes identified in the current study included ATF3, BRF1, C/EBPbeta, CGRP, EGR-1, ephrinA1, villin2, ferredoxin, latexin, lipocalin, MKP-1, NGFI-B, RhoA, tissue factor (TF), and syndecan. To validate these microarray results, the role of EGR-1 was further investigated using quantitative RT-PCR, Western blotting, and immunocytochemistry. EGR-1 expression occurred within acinar cells and correlated with the development of caerulein-induced acute pancreatitis in rats. Furthermore, the levels of the inflammation-related genes MCP-1, PAI, TF, IL-6, and ICAM-1 and the extent of lung inflammation were reduced during the initiation of caerulein-induced acute pancreatitis in EGR-1-deficient mice. Thus this study identified EGR-1 and several other novel genes likely to be important in the development and severity of acute pancreatitis.

Topics: Acute Disease; Animals; Cells, Cultured; Ceruletide; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression Profiling; Gene Expression Regulation; Immediate-Early Proteins; Inflammation; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Taurocholic Acid; Transcription Factors

In isolated rat pancreatic acini, protein expression of RhoA and Rho-associated kinase, ROCK-II, and the formation of immunocomplex of RhoA with ROCK-II were enhanced by CCK-8, carbachol, and the phorbol ester TPA. The ROCK-specific inhibitor, Y-27632, did not alter basal amylase secretion, whereas it potentiated CCK-stimulated pancreatic enzyme secretion in vitro. During caerulein-induced pancreatitis occurring in mice in vivo, Y-27632 enhanced serum amylase levels and the formation of interstitial edema and vacuolization at 12-18h after the first injection of caerulein. Y-27632 in turn inhibited the recovery of protein expression of ROCK-II at 18h after the first caerulein injection. These results suggest that RhoA and ROCK-II assemble normal CCK-stimulated pancreatic enzyme secretion and prevent caerulein-induced acute pancreatitis.

Topics: Acute Disease; Amides; Amylases; Animals; Cell Polarity; Cells, Cultured; Ceruletide; Enzyme Inhibitors; Intracellular Signaling Peptides and Proteins; Male; Mice; Pancreas; Pancreatitis; Protein Serine-Threonine Kinases; Pyridines; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; rhoA GTP-Binding Protein

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF-kappaB) activation and augmented tumor necrosis factor-alpha (TNF-alpha) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF-kappaB activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250g body weight received administration of cerulein (80 microg/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER + DMSO = 3.075 +/- 0.54 micromol/g; CER + raxofelast = 0.693 +/- 0.18 micromol/g; p < 0.001), decreased myeloperoxidase (MPO) activity (CER + DMSO = 22.2 +/- 3.54 mU/g; CER + raxofelast = 9.07 +/- 2.05 mU/g, p < 0.01), increased glutathione levels (GSH) (CER + DMSO = 5.21 +/- 1.79 micromol/g; CER + raxofelast = 15.71 +/- 2.14 micronol/g; p < 0.001), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase (CER + DMSO = 4063 +/- 707.9 U/l; CER + raxofelast = 1198 +/- 214.4 U/l; p < 0.001), and lipase (CER + DMSO = 1654 +/- 330 U/l; CER + raxofelast = 386 +/- 118.2 U/l; p < 0.001), Furthermore, raxofelast reduced pancreatic NF-kappaB activation and the TNF-alpha mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.

Topics: Amylases; Animals; Benzofurans; Blotting, Western; Cell Nucleus; Ceruletide; Cytoplasm; Dimerization; Glutathione; I-kappa B Proteins; Lipase; Lipid Peroxidation; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha; Vitamin E

Severity of systemic lesions and mortality of experimental acute pancreatitis (AP) are reduced after pancreatic enzyme content reduction induced by cerulein administration. Octreotide has been used both prophylatically and therapeutically in AP. The possible effects of octreotide on pancreatic enzyme content and its influence on pulmonary lesions of experimental AP were assessed in this study.. Wistar male rats were divided in two branches: BRANCH I - Animals divided into three groups: Group Sa (n = 10) intravenous saline infusion; Group Ce (n = 10) intravenous cerulein infusion, (0.133 micro g/kg(-1).h(-1)) and Group Oc (n = 10) SC octreotide (10 micro g/kg(-1)). Trypsin, elastase and amylase pancreatic contents as well as serum amylase were determined thereafter in all three groups; BRANCH II - Rats treated as in branch I, were submitted to sodium taurocholate AP (Groups Sa+AP, Ce+AP and Oc+AP). Two hours thereafter amylase and TAP assays were performed in serum, ascites and pancreatic tissue in eight animals of each group. Pulmonary histology was studied by morphometry 24 h after AP in the remaining animals.. Increased serum amylase and pancreatic enzyme contents were observed in octreotide-treated animals when compared to animals receiving saline or cerulein. After AP increases of serum and ascitic fluid amylase and of pancreatic TAP were observed in octreotide pre-treated animals when compared to saline and cerulein groups. Pulmonary interstitial and alveolar edema after AP was significantly increased in rats receiving octreotide as compared to the cerulein group.. Octreotide administration acutely increases the enzymatic content of the pancreas and thus may have a potential deleterious influence in the evolution of AP.

Topics: Acute Disease; Amylases; Animals; Ascitic Fluid; Ceruletide; Disease Models, Animal; Drug Therapy, Combination; Gastrointestinal Agents; Injections, Intravenous; Male; Octreotide; Pancreas; Pancreatic Elastase; Pancreatitis; Pulmonary Edema; Rats; Rats, Wistar; Taurocholic Acid; Trypsin

1 Kinin B(2) receptor antagonists or tissue kallikrein (t-KK) inhibitors prevent oedema formation and associated sequelae in caerulein-induced pancreatitis in the rat. We have now further investigated the mechanism of kinin generation in the pancreas. 2 Kinins were elevated in the pancreatic tissue already before oedema formation became manifest. Peak values (421+/-59 pmol g(-1) dry wt) were reached at 45 min and remained elevated for at least 2 h; a second increase was observed at 24 h. Pretreatment with the B(2) receptor antagonist icatibant abolished kinin formation, while post-treatment was ineffective. 3 Total kininogen levels were very low in the pancreas of controls, but increased 75-fold during acute pancreatitis. This increase was absent in rats that were pretreated with icatibant. 4 During pancreatitis, t-KK-like and plasma kallikrein (p-KK)-like activity in the pancreas, as well as trypsinogen activation peptide (TAP) increased significantly. Icatibant pretreatment further augmented t-KK about 100-fold, while p-KK was significantly attenuated; TAP levels remained unaffected. 5 Endogenous protease inhibitors (alpha(1)-antitrypsin, alpha(2)-macroglobulin) were low in normal tissues, but increased 45- and four-fold, respectively, during pancreatitis. This increase was abolished when oedema formation was prevented by icatibant. 6 In summary, oedema formation is initiated by t-KK; the ensuing plasma protein extravasation supplies further kininogen and active p-KK to the tissue. Concomitantly, endogenous protease inhibitors in the oedema fluid inhibit up to 99% of active t-KK. Our data thus suggest a complex interaction between kinin action and kinin generation involving positive and negative feedback actions of the inflammatory oedema.

Topics: Acute Disease; alpha 1-Antitrypsin; alpha-Macroglobulins; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bradykinin; Ceruletide; Edema; Enzyme Activation; Female; Kininogens; Kinins; Pancreas; Pancreatitis; Plasma Kallikrein; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Serine Proteinase Inhibitors; Tissue Kallikreins; Trypsinogen

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.

Topics: Acute Disease; Animals; Ceruletide; Eukaryotic Initiation Factor-2B; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4F; Mice; Pancreatitis; Peptide Elongation Factor 2; Phosphorylation; Protein Biosynthesis; Protein Kinases; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

Severe acute pancreatitis leads to a systemic inflammatory response characterized by widespread leucocyte activation and, as a consequence, distant lung injury. In CC chemokines the first two cysteine residues are adjacent to each other. The aim of this study was to evaluate the effect of Met-RANTES, a CC chemokine receptor antagonist, on pancreatic inflammation and lung injury in caerulein-induced acute pancreatitis in mice.. Acute pancreatitis was induced in mice by hourly intraperitoneal injection of caerulein. Met-RANTES was administered either 30 min before or 1 h after starting caerulein injections, and pancreatic inflammation and lung injury were assessed. There were five groups of eight mice each including controls.. Treatment with Met-RANTES had little effect on caerulein-induced pancreatic damage. Met-RANTES, however, reduced lung injury when given either before administration of caerulein (mean(s.e.m.) lung myeloperoxidase (MPO) 1.47(0.19) versus 3.70(0.86)-fold increase over control, P = 0.024; mean(s.e.m.) microvascular permeability 1.15(0.05) versus 3.57(0.63) lavage to plasma fluorescein isothiocyanate-labelled albumin fluorescence ratio (L/P) per cent, P = 0.002) or after caerulein administration (lung MPO 1.96(0.27) versus 3.65(0.63)-fold increase over control, P = 0.029; microvascular permeability 0.94(0.04) versus 2.85(0.34) L/P per cent, P < 0.001).. Treatment with Met-RANTES reduces lung damage associated with caerulein-induced pancreatitis in mice. Chemokine receptor antagonists may be of use for the treatment of the systemic complications of acute pancreatitis.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Ceruletide; Chemokine CCL5; Drug Evaluation, Preclinical; Lung Diseases; Mice; Microcirculation; Pancreatitis; Receptors, Chemokine; Systemic Inflammatory Response Syndrome

The prostaglandin D2 metabolite, 15d-PGJ2, a potent natural ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), exerts antiinflammatory effects by inhibiting the induction of inflammatory response genes and NF-kappaB-dependent transcription. AIM To determine whether 15d-PGJ2 decreases the severity of secretagogue-induced acute pancreatitis (AP) and to assess cellular mechanisms contributing to these effects. METHODOLOGY Swiss Webster mice were injected with either saline or cerulein (50 microg/kg) hourly for 8 hours and received either 15d-PGJ2 (2 mg/kg) or vehicle 1 hour before and 4 hours after induction of AP. RESULTS Treatment with 15d-PGJ2 significantly attenuated AP, as determined by histologic assessment of edema, vacuolization, inflammation, and necrosis. This attenuation was associated with decreased cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression and decreased serum and pancreatic IL-6 levels. Treatment with 15d-PGJ2 markedly inhibited NF-kappaB DNA-binding activity, and, moreover, this decreased activity was associated with a concomitant inhibition of IkappaB protein degradation. CONCLUSION Our findings demonstrate that 15d-PGJ2 attenuates the severity of AP most likely through the inhibition of COX-2 expression, IL-6 production, and NF-kappaB activation. Ligands specific for PPARgamma may represent novel and effective means of clinical therapy for AP.

Topics: Acute Disease; Animals; Blotting, Western; Cell Nucleus; Ceruletide; Cyclooxygenase 2; Electrophoretic Mobility Shift Assay; Female; Gene Expression Regulation; I-kappa B Proteins; Intercellular Adhesion Molecule-1; Interleukin-6; Isoenzymes; Ligands; Mice; NF-kappa B; Pancreatitis; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Protein Transport; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Time Factors; Transcription Factors

To set up a nontraumatic and convenient mouse model of severe acute pancreatitis (SAP). Caerulein(Cn) was injected the mice intraperitonealy with lipopolysaccharide(LPS). Serum amylase and pancreas weight were measured in experiment. The pathological changes of pancreas and other organs were observed under light microscope. The ultrastructure of acini were observed under transmission electron microscope (TEM). Serum NO concentration were measured and the SOD and MDA in pancreas were examined. The results in Cn + LPS group were showed that serum amylase, NO concentration and pancreas weight were increased, SOD deduced and MDA increased. Severe edema, inflammation infiltration, necrosis and different extent of hemorrhage were showed. The acini were damaged severely. And the lesion of other organs were also happened. In Cn group, there were only pancreatic interstitial edema but no parenchmal necrosis or hemorrhage, and the other organs were normal. In LPS group, pancreas were almost normal and the organs besides pancreas were only showed light inflammation infiltration. The SAP mouse model induced by caerulein plus LPS has the same pathological characteristics of human SAP, which can be used in human SAP research. The unbalance of oxygen free radical release-elimination and oxidation-antioxidation mechanisms might be involved in the pathogenesis of mouse model of severe acute pancreatitis induced by intraperitoneal injection of caerulein plus LPS.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Female; Lipopolysaccharides; Mice; Mice, Inbred Strains; Pancreas; Pancreatitis

We demonstrated dynamic aspects of granulocyte activation in rat severe acute pancreatitis, which was induced by cerulein and aggravated following lipopolysaccharide (LPS) injection. Pancreatitis induced by cerulein increased intracellular elastase activity of granulocytes in the blood. However, significant systemic cytokinemia was not provoked under such conditions. After induction of severe pancreatitis by LPS, intracellular elastase activity of circulating granulocytes decreased markedly and immediately. This decrease occurred simultaneous to induction of systemic hypercytokinemia and granulocyte migration into the lung. Overall results imply that: (1) circulating granulocytes are activated by induction of mild pancreatitis; (2) activation of granulocytes is mediated by factors other than systemic cytokinemia, such as locally produced cytokines; (3) those priming granulocytes immediately and significantly migrate from the circulation into the extravascular space by induction of endotoxemia; and (4) migration of granulocytes, in turn, may be mediated by systemic cytokinemia.

Topics: Acute Disease; Animals; Ceruletide; Cytokines; Granulocytes; Interleukin-1; Leukocyte Count; Lipopolysaccharides; Lung; Male; Pancreatic Elastase; Pancreatitis; Peroxidase; Rats; Rats, Wistar; Time Factors; Tumor Necrosis Factor-alpha

The role of nitric oxide (NO) in bacterial translocation (BT) associated with acute pancreatitis is controversial. We investigated the effects of the NO synthase substrate, L-arginine, and the NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), on BT in caerulein-induced acute pancreatitis in rats.. Acute pancreatitis was induced by subcutaneous injections of caerulein (12 microg/kg) at 6-hour intervals for 2 days. Subcutaneous injections of L-arginine (100 mg/kg) or L-NAME (10 mg/kg) were administeredonce daily for 2 days. At 48 h, pancreatic injury and BT to the mesenteric lymph nodes (MLN), liver, and peritoneum were assessed.. Compared with controls, rats that received caerulein injections alone had increased BT to the MLN and pancreatic inflammatory changes. L-Arginine significantly reduced the inflammation and BT caused by caerulein. L-NAME did not significantly alter pancreatic inflammation. Although caerulein + L-NAME-treated rats had increased BT to the peritoneum, MLN, and liver compared with controls, rates of BT did not significantly differ between caerulein alone- and caerulein + L-NAME-treated rats.. In acute edematous pancreatitis, BT is increased and is regulated by NO. NO substrates limit BT and pancreatic inflammation associated with acute pancreatitis, probably by their bactericidal actions and ability to improve pancreatic blood flow.

Topics: Acute Disease; Animals; Arginine; Bacterial Translocation; Ceruletide; Edema; Injections, Subcutaneous; Liver; Lymph Nodes; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Pancreas; Pancreatitis; Peritoneum; Rats; Rats, Wistar

An enhanced formation of nitric oxide (NO), due to the induction of inducible nitric oxide synthase (iNOS), has been implicated in the pathogenesis of shock and inflammation, but its role in acute pancreatitis still remains controversial. To clarify the role of NO in acute pancreatitis, the present experiment investigated the expression of iNOS and the effect of NOS inhibition on cerulein-induced pancreatitis in rats. Group I received intraperitoneal (ip) injection of normal saline. Group II received two ip injections of cerulein (20 microgram/kg). Group III received injections of N(G)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg) with cerulein. Group IV received L-arginine (250 mg/kg) with cerulein and L-NAME. The expression of iNOS in the pancreas was examined by western blot analysis. The plasma concentration of NO metabolites was measured. The severity of pancreatitis was assessed by measuring serum amylase, pancreas water content and histopathological examination. Compared with controls, the cerulein group displayed significantly increased expression of iNOS and raised plasma NO metabolites. Treatment with L-NAME significantly decreased hyperamylasemia, plasma NO level, and the extent of pancreatic injury. Treatment with L-arginine reversed the effects of L-NAME. These findings suggest that an enhanced formation of NO by iNOS plays an important role in the development of acute pancreatitis, and inhibition of NO production has the beneficial effects in reducing pancreas injury.

Topics: Amylases; Animals; Arginine; Blotting, Western; Ceruletide; Enzyme Inhibitors; Inflammation; Male; Necrosis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pancreatitis; Rats; Rats, Sprague-Dawley

Here we compare the degree of pancreatitis caused by cerulein in mice lacking 5-lipoxygenase (5-LO) and in the corresponding wild-type mice. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by oedema, neutrophil infiltration and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for intracellular adhesion molecule-1 (ICAM-1), P-selectin and E-selectin in the pancreas and lung of cerulein-treated mice. In contrast, the degree of (1) pancreatic inflammation and tissue injury (histological score), (2) up-regulation/expression of P-selectin, E-selectin and ICAM-1, and (3) neutrophil infiltration was markedly reduced in pancreatic and lung tissue obtained from cerulein-treated 5-LO-deficient mice. These findings support the view that 5-LO plays an important, pro-inflammatory role in the acute pancreatitis caused by cerulein in mice.

Topics: Acute Disease; Animals; Arachidonate 5-Lipoxygenase; Ceruletide; E-Selectin; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Liver Diseases; Mice; Mice, Knockout; Neutrophil Infiltration; P-Selectin; Pancreatitis; Respiratory Distress Syndrome

Microcirculatory mechanisms have been suggested to be involved in the development of acute pancreatitis. Islet blood flow has not previously been studied in this disease. The present study aimed to investigate the effects of caerulein-induced pancreatitis on pancreatic blood perfusion, especially islet blood flow.. Continuous 4 h caerulein-infusion was used to induce mild, edemateous pancreatitis in anesthetized Sprague-Dawley rats. Some animals were then given an additional 2 h infusion of saline. Thus, at 4 or 6 h after initiating caerulein infusion the blood flow to the pancreas, pancreatic islets, and intestines was measured with a microsphere technique.. All infused animals demonstrated an edemateous pancreatitis, without hemorrhages. Both total pancreatic and islet blood flow was increased after the 4-h infusion. However, the increase was less pronounced in the islets. After an additional 2 h with only saline infused, the blood flow values in rats initially infused with caerulein were lower than at 4 h, but total pancreatic blood was still higher than in control rats. No effects on intestinal blood flow values were seen.. Pancreatic islet blood flow in rats with mild edematous pancreatitis is increased, but not to the same extent as that in the whole pancreas.

Topics: Amylases; Animals; Blood Glucose; Ceruletide; Gastrointestinal Agents; Insulin; Islets of Langerhans; Male; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Splanchnic Circulation

Peptide YY (PYY), a gastrointestinal regulatory peptide, improves survival and histologic parameters in animal models of acute pancreatitis. Its effects on pancreatic cell growth and acute pancreatitis in pancreatic acinar and ductal cells are unknown. We hypothesized that PYY would affect cell growth and attenuate acute pancreatitis in pancreatic acinar and ductal cells in vitro.. Rat pancreatic acinar and ductal cells were cultured in the presence of 1) cerulein, a synthetic cholecystokinin analog that induces pancreatitis, 2) PYY, or 3) a combination group pretreated with PYY prior to addition of cerulein. Cell survival was measured at 48 h using MTT assay. Amylase secretion, as marker for pancreatitis, was measured at 48 h using an amylase activity assay. Statistical significance was calculated using analysis of variance and the Student's t test.. Peptide yy significantly increased cell growth and decreased amylase secretion compared with control and cerulein groups. Pretreatment with PYY significantly protected against the pancreatitis effects of cerulein.. We have shown for the first time that PYY has a mitogenic effect on pancreatic acinar and ductal cells in vitro. In addition, it directly protects against cerulein-induced pancreatitis. Its potential therapeutic benefit in acute pancreatitis would therefore be twofold: amelioration of the inflammatory process, and augmenting growth of normal pancreas to replace necrotic or apoptotic cell loss.

Topics: Acute Disease; Amylases; Animals; Cell Culture Techniques; Cell Division; Cell Survival; Ceruletide; Gastrointestinal Agents; Mitogens; Pancreas; Pancreatitis; Peptide YY; Rats

Hepatic injury is considered one of the critical complications associated with acute pancreatitis. It was proposed that initial insults to the liver in the early phase of the attack have an important priming effect, and the subsequent infectious attack (e.g. infectious pancreatic necrosis, bacterial translocation episode) constitutes a second attack on the liver.. To evaluate the role of priming by induction of cerulein pancreatitis and a following second attack by endotoxemia.. Plasma clearance and biliary excretion of indocyanine green (a hepatophillic hydrophobic organic anion).. A model of acute pancreatitis in rats.. Four groups of rats: untreated control, cerulein pancreatitis, endotoxemia and endotoxemia following the induction of cerulein pancreatitis (pancreatitis + endotoxemia).. Biliary indocyanine green excretion was significantly disturbed only in the pancreatitis + endotoxemia group. Plasma clearance (a reflection of hepatic uptake) of indocyanine green from the blood was only slightly affected in endotoxemia group.. Biliary secretion is quite sensitive to this hepatic injury model. Both the preceding priming insult and the following second attack are important in the development of hepatic injury.

Topics: Acute Disease; Alanine Transaminase; Animals; Bile; Bile Ducts, Intrahepatic; Ceruletide; Disease Models, Animal; Endotoxemia; Indocyanine Green; Lipopolysaccharides; Liver Function Tests; Male; Pancreatitis; Rats; Rats, Wistar

Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiologic concentrations of cholecystokinin (CCK) cause intrapancreatic zymogen activation and pancreatitis. Supraphysiologic concentrations of CCK also cause zymogen activation in isolated pancreatic acini. This activation first occurs in a nonzymogen granule compartment that contains lysosomal markers. A low pH environment may also be needed for activation. To examine the ability of alcohols to sensitize the acinar cell to CCK, the conversion of zymogens to active enzymes in isolated acini was assayed. Alcohols, including 35 mmol/L ethanol, sensitized acini to CCK induced activation. The sensitization increased with chain length and was less in branched compared with unbranched alcohols. The relationship of alcohol's structure to sensitization may be related to the mechanism of sensitization.

Topics: Acute Disease; Alcohols; Animals; Ceruletide; Cholecystokinin; Chymotrypsin; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Precursors; Ethanol; Hydrogen-Ion Concentration; Oligopeptides; Organelles; Pancreas; Pancreatitis; Time Factors; Trypsin

In this study we examine the effect of endotoxin (lipopolysaccharide) on lung injury in the setting of acute pancreatitis (AP).. Twelve hourly injections of cerulein (50 microg/kg/h) were used to induce pancreatitis in mice. Intraperitoneal lipopolysaccharide (LPS [6 mg/kg]) was administered 24 hours after the initial cerulein injection. Twenty-four hours after LPS injection, myeloperoxidase (MPO) activity, nuclear factor (NF)-kappaB activation, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and chemokines MIP-2 and KC levels were measured in pancreas, liver, and lung tissues. Four groups of mice were studied: cerulein-LPS, cerulein-saline, saline-LPS, and saline-saline treated mice.. Elevated serum lipase confirmed pancreatitis in cerulein treated mice. Lung MPO activity was significantly increased in the cerulein-LPS group. NF-kappaB was activated in the liver but not in pancreas and lung tissue. Chemokines MIP-2 and KC were elevated in pancreatic tissue only.. These findings suggest that gram-negative infections may be an important predisposition for the development of adult respiratory distress syndrome in the setting of AP and that hepatic NF-kappaB may mediate multisystem injury.

Topics: Animals; Ceruletide; Cytokines; Endotoxins; Liver; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreatitis; Peroxidase; Respiratory Distress Syndrome

The nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a pivotal role in the induction of genes involved in the response to injury and inflammation. Dithiocarbamates are antioxidants that are potent inhibitors of NF-kappaB. This study tested the hypothesis that pyrrolidine dithiocarbamate (PDTC) attenuates experimental acute pancreatitis. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by edema, neutrophil infiltration, tissue hemorrhage and necrosis, and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine and intracellular adhesion molecule-1 in the pancreas and lung of cerulein-treated mice. In contrast, the degree of 1) pancreas and lung injury, 2) upregulation/expression of intracellular adhesion molecule-1, 3) staining for nitrotyrosine, and 4) lipid peroxidation was markedly reduced by pretreatment with PDTC. This study demonstrates that prevention of the activation of NF-kappaB by PDTC ameliorates the tissue injury associated with experimental murine acute pancreatitis and provides an important insight into the molecular biology of acute pancreatitis.

Topics: Amylases; Animals; Antioxidants; Blotting, Western; Ceruletide; Edema; I-kappa B Proteins; Immunohistochemistry; Inflammation; Intercellular Adhesion Molecule-1; Lipase; Lipid Peroxidation; Male; Mice; Necrosis; Neutrophils; NF-kappa B; NF-KappaB Inhibitor alpha; Pancreatitis; Peroxidase; Pyrrolidines; Rats; Thiocarbamates; Tyrosine; Up-Regulation

Cerulein pancreatitis was shown to be one of the best characterized models for acute pancreatitis. High doses of cerulein induce a dysregulation of the digestive enzyme production and cytoplasmic vacuolization and the death of acinar cells, edema formation, and an infiltration of inflammatory cells into the pancreas, which are similar symptoms shown in human acute pancreatitis. The present study aims to determine the differentially expressed proteins in cerulein-treated pancreatic acinar cells as an in vitro model for acute pancreatitis. Pancreatic acinar AR42J cells were treated with 10(-8) M cerulein for 24 h. The changed protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis of the peptide digests. Five differentially expressed proteins (heat shock protein 90, mitochondrial ATP synthase beta chain precursor, tubulin beta chain, 3-mercaptopyruvate sulfurtransferase, mitochondrial ATP synthase subunit D) were identified in cerulein-treated AR42J cells. These proteins are related to cellular stress such as reactive oxygen species, cytoskeletal function, and cell signaling. In conclusion, the differentially expressed proteins will provide valuable information to understand the pathophysiologic mechanism of acute pancreatitis and may be useful for prognostic indices of acute pancreatitis.

Topics: Amino Acid Sequence; Animals; Cells, Cultured; Ceruletide; Electrophoresis, Gel, Two-Dimensional; HSP90 Heat-Shock Proteins; Humans; Mitochondria; Mitochondrial Proton-Translocating ATPases; Molecular Sequence Data; Pancreas; Pancreatitis; Rats; Reactive Oxygen Species; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfurtransferases; Up-Regulation

Mitochondrion is a vulnerable intracellular target to reactive oxygen species (ROS). ROS have been considered to be important regulators of the pathogenesis of pancreatitis. This study aims to determine whether ROS induces mitochondrial damage by monitoring the expression level of mitochondrial ATP synthase as the key molecular component in mitochondria associated with cellular damage. Pancreatic acinar AR42J cells were treated with cerulein which induces symptoms similar to that associated with human acute pancreatitis. Proteins were separated by two-dimensional electrophoresis using pH gradients of 5-8 and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MS), quadrupole time-of-flight MS and MS/MS with nano-electrospray. Following cerulein treatment, mitochondrial ATP synthase beta chain was highly expressed compared to nontreated cell. The protein was identified by its pI of 5.2 and molecular weight (56 354 Da) with 27 matched peptides. Among the MS spectrum, precursor ions m/z 488.28, 544.81, 631.82, 693.34, 718.38, 729.41, 801.40, 809.39, 825.94, and 994.52 were further identified using MS/MS and confirmed the isolated protein to be mitochondrial ATP synthase beta chain. In conclusion, cerulein-induced oxidative injury may result in the induction of mitochondrial ATP synthase, which may act as an adaptive pathophysiological process in the pancreas.

Topics: Amino Acid Sequence; Animals; Cells, Cultured; Ceruletide; Electrophoresis, Gel, Two-Dimensional; Humans; Mitochondria; Mitochondrial Proton-Translocating ATPases; Molecular Sequence Data; Pancreas; Pancreatitis; Rats; Reactive Oxygen Species; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Up-Regulation

Nuclear factor (NF)-kappaB plays a central role in acute pancreatitis. We studied cerulein (CER)-induced pancreatitis in NF-kappaB knockout (KO) mice. NF-kappaB KO mice and normal control littermate wild-type (WT) mice were given four hyperstimulating doses of cerulein every hour to elicit secreatagogue-induced pancreatitis. Malonildialdehyde activity, glutathione levels, myeloperoxidase activity, TNF-alpha, and NF-kappaB binding activity and its inhibitory protein IkappaBalpha were studied in the pancreas. Furthermore, we measured plasma lipase and amylase and the histological damage. KO mice had reduced malonildialdehyde levels (WT + CER = 4.083 +/- 0.95 micromol/g; KO + CER = 1.513 +/- 0.63 microol/g), decreased myeloperoxidase activity (WT + CER = 19.3 +/- 2.39 mU/g; KO + CER = 10.21 +/- 2.05 mU/g), increased glutathione levels (WT + CER 6.22 +/- 2.46 micromol/g; KO + CER = 15. 516 +/- 2.92 micromol/g), and reduced serum levels of amylase (WT + CER = 2519 +/- 656.9 U/L; KO + CER = 916 +/- 280.4 U/L) and lipase (WT + CER = 1420 +/- 170 U/L; KO + CER = 861 +/- 172. 3 U/L). KO mice showed reduced pancreatic NF-kappaB activation, decreased TNF-alpha tissue content, and reduced histologic alterations. Our data suggest that KO mice have an attenuated cerulein-induced pancreatitis and help to define the possible interaction between NF-kappaB activation and oxidative stress in this deleterious event.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Disease Models, Animal; Glutathione; Lipase; Lymphotoxin-alpha; Malondialdehyde; Mice; Mice, Knockout; NF-kappa B; Pancreas; Pancreatitis; Peroxidase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha; Up-Regulation

Diabetes occurs in 20-30% of patients above forty years old with chronic pancreatitis (CP). The aim of the present study was to determine the relationship between development of diabetes mellitus in CP patients and insufficiency of pancreatic exocrine secretion as well as changes in composition of pancreatic juice. Ninety CP patients with diagnosis confirmed by endoscopic retrograde pancreatography (ERP) were studied. They were divided into 3 groups in dependency on ERP changes (according to Cambridge classification) and oral glucose tolerance test (OGTT): group A--equivocal or mild changes in ERP and normal OGTT (control group); group B--moderate or marked changes in ERP and normal OGTT: group C--moderate or marked changes in ERP and diabetes mellitus. The exocrine pancreatic function was determined by the secretin-caerulein test; volume of duodenal content and bicarbonate, protein, alpha-amylase activity outputs were measured.. All exocrine pancreatic function parameters were diminished in B and C groups compared with A group--differences were statistically significant. However in group C values of volume and bicarbonate, protein and amylase activity were especially low.. Exocrine pancreatic insufficiency is strongly associated with anatomical changes of the pancreas but also depends on endocrine function.

Topics: Adult; Aged; Ceruletide; Cholangiopancreatography, Endoscopic Retrograde; Chronic Disease; Diabetes Mellitus; Female; Glucose Tolerance Test; Humans; Male; Middle Aged; Pancreas; Pancreatitis; Secretin

Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis.. Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis.. Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow.. (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems Background: Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis. Methods: Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis. Results: Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta conc; concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow. Conclusions: (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems to be related the inhibition in inflammatory process and the reduction in liberation o

Topics: Acute Disease; Animals; Ceruletide; DNA; Dose-Response Relationship, Drug; Drug Administration Schedule; Ghrelin; Humans; Injections, Intraperitoneal; Interleukin-1; Interleukin-10; Lipase; Male; Pancreas; Pancreatitis; Peptide Hormones; Rats; Rats, Wistar; Regional Blood Flow; Vacuoles

Insulin-like growth factor-1 (IGF-1) and other growth factors overexpression was reported in acute pancreatitis. Previous studies have shown the protective effect of epidermal growth factor (EGF), Hepatocyte Growth Factor (HGF) and Fibroblast Growth Factor (FGF) in the course of experimental acute pancreatitis. The aim of our studies was to determine the effect of IGF-1 administration on the development of caerulein-induced pancreatitis.. Acute pancreatitis was induced by infusion of caerulein (10 micro/kg/h) for 5 h. IGF-1 was administrated twice at the doses: 2, 10, 50, or 100 micro/kg s.c.. Administration of IGF-1 without induction of pancreatitis increased plasma interleukin-10 (IL-10). Infusion of caerulein led to development of acute edematous pancreatitis. Histological examination showed pancreatic edema, leukocyte infiltration and vacuolization of acinar cells. Also, acute pancreatitis led to an increase in plasma lipase and interleukin 1beta (IL-1beta) level, whereas pancreatic DNA synthesis and pancreatic blood flow were decreased. Treatment with IGF-1, during induction of pancreatitis, increased plasma IL-10 and attenuated the pancreatic damage, what was manifested by histological improvement of pancreatic integrity, the partial reversion of the drop in pancreatic DNA synthesis and pancreatic blood flow, and the reduction in pancreatitis-evoked increase in plasma amylase, lipase and IL-1beta level. Protective effect of IGF-1 administration was dose-dependent. Similar strong protective effect was observed after IGF-1 at the dose 2 x 50 and 2 x 100 microg/kg.. (1) Administration of IGF-1 attenuates pancreatic damage in caerulein-induced pancreatitis; (2) This effect is related, at least in part, to the increase in IL-10 production, the reduction in liberation of IL-1beta and the improvement of pancreatic blood flow.

Topics: Animals; Ceruletide; DNA; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Therapy, Combination; Injections, Subcutaneous; Insulin-Like Growth Factor I; Interleukin-1; Interleukin-10; Lipase; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Regional Blood Flow; Signal Transduction; Vacuoles

The aim of our study was to evaluate the possible influence of adenosine receptors agonists and antagonists on pancreatic blood flow (PBF) and the development of acute pancreatitis (AP) in rats.. Ninety male Wistar rats were subdivided into ten equal groups, nine rats in each. The study was carried out in two stages. In the first one the first group (control) received i.v. saline infusion for 12 hours. The groups 2-5 (the first stage) received i.v. caerulein infusion as in the first group, but with pretreatment with: in the second group--DPCPX (A1 receptor antagonist), in the third group--CGS 21680 (A2 receptor agonist), in the fourth group--ZM 241385 (A2 receptor antagonist), in the fifth group--IB-MECA (A3 receptor agonist). In the second stage the first group received i.v. caerulein infusion at the dose of 5 micrograms/kg/h for 12 hours. The groups 2-5 (the second stage) received i.v. caerulein infusion as in the first group, but with pretreatment with: in the second group--DPCPX (A1 receptor antagonist), in the third group--CGS 21660 (A2 receptor agonist), in the fourth group--ZM 241385 (A2 receptor antagonist), in the fifth group--IB-MECA (A3 receptor agonist). Pancreatic blood flow was measured by laser Doppler flowmetry. Pancreatic inflammation was evaluated by serum alpha-amylase activity, pancreatic weight and histological changes in the pancreatic tissue.. We observed a significant attenuation of serum alpha-amylase activity increase (19.1 +/- 2.8 kIU/L vs 30.12 +/- 2.64 kIU/L), pancreatic weight (expressed as percentage of rat's body weight--0.85 +/- 0.16% vs 1.25 +/- 0.14%), and improvement of PBF (79.8 +/- 6.1% vs 60.1 +/- 3.6%), a reduced degree of pancreatic tissue damage (oedema, leukocyte infiltration, vacuolisation of acinar cells) in the third group (CGS 21680 + caerulein) compared with the first group in the second stage (only caerulein infusion). Neither agonists nor antagonists exerterd any appreciable effects on measured parameters in healthy rats.. Pretreatment with A2 receptors agonist seems to be protective against the damage to the pancreas during the course of caerulein-induced acute pancreatitis in rats. This effect could be due to improvement of pancreatic blood flow. This finding could have some therapeutic implications.

Topics: Acute Disease; Animals; Ceruletide; Gastrointestinal Agents; Male; Models, Animal; Pancreas; Pancreatitis; Rats; Rats, Wistar; Receptors, Purinergic P1

Increase in intracellular chymotrypsin activity was reported during acute pancreatitis. Beside chymotrypsin, there are at least two enzymes with chymotrypsin-like activity: proteasome and lysosomal cathepsin A. Until now it is not known whether and to what extent they contribute to increases in chymotrypsin activity in acute pancreatitis. Our aim was to study organ chymotrypsin-like activities during experimental acute pancreatitis.. Rat cerulein model of acute pancreatitis was used. The chymotrypsin-like activities were assessed in pancreas, liver, lung, heart, spleen and kidney using highly selective synthetic substrates of the proteasome and the cathepsin A, at neutral and acidic pH. Determinations after addition of selective inhibitor were also performed.. During acute pancreatitis we found in the pancreas an increase only in neutral chymotrypsin-like activity, as compared to the control animals. In other organs neutral chymotrypsin-like activity did not increase, and in kidney it even decreased. There were no changes in acidic chymotrypsin-like activity in any of organs studied. The studies using the inhibitor of the proteasome showed that the neutral chymotrypsin-like activity in the pancreas of the rats with acute pancreatitis should not be attributed to the proteasome activity, but rather to the chymotrypsin.. Our results did not confirm any significant contribution of proteasome or cathepsin A to increased chymotrypsin-like activity in acute pancreatitis. We showed a decrease in neutral chymotrypsin-like activity of proteasome in the kidney, but the significance of this finding remains to be established.

Topics: Acute Disease; Animals; Ceruletide; Chymotrypsin; Gastrointestinal Agents; Hydrogen-Ion Concentration; Models, Animal; Pancreatitis; Rats; Rats, Wistar

Elevated Ca(2+) concentrations within the pancreatic acinar cells represent a risk factor for the development of acute pancreatitis. Apoptosis is an important characteristic of pancreatitis, with induction of apoptotic genes and intraceullar increase of calcium, endonucleases, and protease. The present study, which aims to investigate whether (1) cerulein induces apoptotic gene expression (bax, bid, p53) in pancreatic acinar AR42J cells and (2) cerulein-induced gene expression is mediated by intracellular Ca(2+), monitored the gene expression profile in the cells treated with the Ca(2+) chelator BAPTA-AM. Results showed that cerulein (10(-7) M) evoked an initial peak Ca(2+) signal; a further Ca(2+) signal was induced with second treatment of cerulein. Cerulein-induced Ca(2+) signal could not be detected in the cells treated with the Ca(2+) chelator BAPTA-AM. Cerulein dose-dependently induced apoptosis, determined by DNA fragmentation and pro-apoptotic bid expression in AR42J cells. Cerulein induced bid, bax, and p53 mRNA expression, which was inhibited in the cells treated with cerulein and cultured in the presence of BAPTA-AM. The present results suggest that increase in the free cytosolic Ca(2+) may be the upstream event of apoptotic gene (bax, bid, p53) expression, which contribute to cerulein-induced apoptosis in pancreatic acinar cells.

Topics: Acute Disease; Apoptosis; Calcium; Calcium Signaling; Cell Line; Ceruletide; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Pancreatitis

experimental pancreatitis and explore the mechanism of bacterial translocation at the molecular level in mice with severe acute pancreatitis.. Thirty-six ICR mice were randomly divided into 6 groups with 6 mice in each group. The animals in the experimental groups (from A to E) received seven intraperitoneal injections of caerulein (50 microg/kg body weight) at hourly intervals over 6 hours, and were killed at 9, 18, 24, 48 and 72 hours after the first injection. The control group (F) received intraperitoneal injection of the same volume of saline, and the animals were killed at the 18th hour after the first injection. Blood and pancreatic tissue samples were obtained after the animals were killed. Amylase and pancreatic pathological alterations were observed. The ileum sequential segments were removed from each mouse. The amplification products of RT-PCR were electrophoresed and the images were analyzed by UVI software.. Acute necrotizing pancreatitis in ICR mice were induced by the intraperitoneal injection of caerulein at large doses. Markedly pathological lesions were observed at the 18th hour after the first injection of caerulein. And, intestinal cryptdin-4 mRNA expression was down regulated slightly at the 9th hour and most markedly at the 18th hour (P < 0.05). After 24 hours, the cryptdin-4 mRNA expression recovered to the normal level gradually.. Acquired cryptdin-4 deficiency may play an important role in the pathogenesis of bacterial tanslocation in acute necrotizing pancreatits.

Topics: Acute Disease; alpha-Defensins; Animals; Base Sequence; Ceruletide; Intestine, Small; Male; Mice; Mice, Inbred ICR; Molecular Sequence Data; Pancreatitis; Random Allocation; RNA, Messenger

Pancreatic stone protein (PSP/reg) is a constitutively secreted protein in pancreatic juice. Pancreatitis-associated protein (PAP) belongs to the same family of proteins. PAP is highly increased during acute pancreatitis, while no exact data exist regarding PSP/reg protein synthesis and secretion. Recently, an attempt to determine PSP/reg and PAP levels in sera of rats with acute pancreatitis showed a significant increase in PAP but failed to demonstrate changes in PSP/reg. Others reported that surgical manipulation of the pancreas, including sham controls, affected mRNA levels of PSP/reg. Neither report determined protein levels of PSP/reg.. Rats were treated intraperitoneally with a supramaximal dose of caerulein to induce pancreatitis, a physiological dose of caerulein, or a saline injection. Pancreata were analyzed for PAP and PSP/reg using ELISAs. RNA was extracted for Northern blot analysis of PAP I, II, and III and PSP/reg mRNA.. Experimental induction of acute pancreatitis caused a coordinate increase in both PSP/reg and PAP. PAP showed an acute response and returned to low levels within 48 h while PSP/reg exhibited a more sustained response. Intraperitoneal application of a physiological dose of caerulein and even a saline injection caused an increase in PSP/reg.. PSP/reg and PAP levels are increased through similar mechanisms by physiological and supramaximal doses of caerulein. However, PSP/reg regulation appears to sustain high levels while PAP levels are more transient. Since the regulation of this protein family is affected even under mild stress, we define them as secretory stress proteins.

Topics: Acute Disease; Acute-Phase Proteins; Animals; Antigens, Neoplasm; Biomarkers, Tumor; Calcium-Binding Proteins; Ceruletide; Lectins, C-Type; Lithostathine; Nerve Tissue Proteins; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Protein Isoforms; Rats; RNA, Messenger; Tissue Distribution

Recent identification of specific leptin receptors in the pancreas suggests that this peptide may also play some role in this gland.. To examine the effect of intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of leptin in rats on caerulein-induced pancreatitis (CIP), pancreatic gene expression of leptin and inflammatory cytokine production.. Caerulein (25 micrograms/kg) was infused subcutaneously into conscious rats over 5 h to produce CIP. Leptin (1, 5, or 10 micrograms/kg) was injected i.p. or i.c.v. 30 min prior to the CIP induction. The plasma level of TNF alpha and IL-4 was determined by ELISA, while plasma leptin was measured by RIA and leptin gene expression in pancreas by RT-PCR.. CIP was characterized by the usual pancreatic edema, reduction in pancreatic blood flow (PBF) and an increase in serum levels of amylase, TNF alpha and IL-4. Pretreatment with i.p. or i.c.v. leptin of the CIP rats partially reversed the harmful effects of CIP on the pancreas, and reduced pancreatic inflammation and the fall in PBF. This was accompanied by a dose-dependent reduction in serum levels of amylase and TNF alpha, while serum IL-4 in the CIP rats pretreated with leptin rose dose-dependently as compared to control rats with CIP alone. Pretreatment with leptin resulted in the dose-dependent rise in plasma leptin level over that observed in vehicle-treated controls. Leptin mRNA expression in the pancreas was dose-dependently increased after infusion of caerulein. Leptin content in isolated pancreatic acini was also increased dose-dependently by caerulein added to the incubation medium bathing these acini.. (1) Exogenous leptin protects the pancreas against damage by CIP; (2) endogenous leptin seems to limit the extend of pancreatic damage, and (3) these protective effects of leptin could be attributed to the reduction in TNF alpha and to the increase in IL-4 production.

Topics: Animals; Ceruletide; Cytokines; Gene Expression; In Vitro Techniques; Injections, Intraperitoneal; Injections, Intraventricular; Interleukin-4; Leptin; Male; Osmolar Concentration; Pancreas; Pancreatitis; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Leptin is a pleiotropic hormone that is involved in the regulation of food intake and body weight. Recent findings demonstrated that leptin receptors are present in the pancreas but the involvement of leptin in pancreatitis remains unknown. The aim of the present study was: (1) to assess plasma leptin levels in rats with caerulein-induced pancreatitis (CIP) and humans with acute pancreatitis; and (2) to determine the effects of exogenous leptin on the course of acute CIP in rats.. CIP was produced in Wistar rats by s.c. infusion of 5 microg of caerulein for 5 h. Plasma leptin was measured by specific RIA and leptin expression in the pancreas was determined at the transcriptional and protein levels. In addition, the effects of exogenous leptin at the doses of 1 or 10 microg/kg i.p. on the course of CIP and the plasma levels and mRNA expression in pancreas of cytokines TNFalpha and IL-4 were studied. Furthermore, pancreatic cNOS and iNOS expression at mRNA level were measured in rats with CIP and pretreated with leptin. Parallel to these studies, the plasma levels of leptin were measured in 15 patients with acute edematous pancreatitis and in 30 healthy controls of comparable age and body mass index.. In rats, plasma leptin rose significantly from the median of 0.14 (0.03-0.3 ng/ml) in the control group to 0.56 (0.2-3.2 ng/ml) in rats with CIP. The CIP was associated with an upregulation of mRNA and protein for leptin in the pancreas. The administration of exogenous leptin significantly reduced the weight of pancreas, histological manifestations of pancreatitis, plasma TNFalpha and mRNA expression for iNOS in the pancreatic tissue. The assessment of leptin plasma level in humans demonstrated significantly higher median values of plasma leptin in patients with acute pancreatitis [7.5 (4.3-18.4 ng/ml)] than in healthy controls [2.1 (1.0-11.8 ng/ml)].. (1) Acute pancreatitis in rats and in humans is associated with a marked increase in the plasma level of leptin. (2) The transcriptional upregulation of leptin in the pancreas after induction of pancreatitis indicates that the inflamed pancreas could be the source of local production of leptin. (3) Exogenous leptin protects the pancreas against development of acute CIP in rats and one possible mechanism of action of leptin might be attributed to the activation of nitric oxide pathway.

Topics: Acute Disease; Animals; Ceruletide; Female; Humans; Interleukin-1; Interleukin-4; Leptin; Male; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pancreatitis; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

Interleukin-6 (IL-6) exerts a wide spectrum of regulatory activities during immune and inflammatory responses. The aim of this study was to investigate the role of endogenous IL-6 in the inflammatory response associated with acute pancreatitis. Acute pancreatitis was induced by hourly (x5) i.p. injections of cerulein (50 microg/kg, suspended in saline solution) in IL-6 deficient mice (IL-6-KO) and wild-type (IL-6WT) littermates. IL-6KO mice exhibited a more severe tissue injury and a higher rate of mortality and when compared to IL-6WT mice. Acute pancreatitis was characterized by edema, neutrophil infiltration, tissue hemorrhage and cell necrosis, upregulation of P-selectin and intercellular adhesion molecule-1 (ICAM-1), as well as increases in the serum levels of amylase and lipase. The degree of oxidative and nitrosative tissue damage was significantly greater in IL-6KO mice than in wild-type littermates, as indicated by higher tissue levels of malondialdehyde and nitrosylated proteins. Plasma levels of the inflammatory cytokines tumour necrosis factor-alpha and interleukin-1beta were also greatly enhanced in IL-6KO mice when compared to wild-type mice. These events were correlated with an increase in the staining (immunoreactivity) for poly (ADP-ribose) polymerase (PARP) in the pancreas of cerulein-treated IL-6WT. The staining for PARP was more pronounced in IL-6KO mice subjected to acute pancreatitis than in the corresponding WT mice. These data demonstrate that endogenous IL-6 exerts an anti-inflammatory role during acute pancreatitis, possibly by regulating the expression of adhesion molecules, the subsequent adhesion and activation of neutrophils and finally the generation of cytokine and reactive oxygen or nitrogen species.

Topics: Amylases; Animals; Ceruletide; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-6; Lipase; Lipid Peroxidation; Male; Mice; Mice, Knockout; Microscopy, Fluorescence; P-Selectin; Pancreas; Pancreatitis; Peroxidase; Phenotype; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

Severe pancreatitis is frequently associated with acute lung injury (ALI) and the respiratory distress syndrome. The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mediating the ALI associated with secretagogue-induced experimental pancreatitis was evaluated with GM-CSF knockout mice (GM-CSF -/-). Pancreatitis was induced by hourly (12x) intraperitoneal injection of a supramaximally stimulating dose of the cholecystokinin analog caerulein. The resulting pancreatitis was similar in GM-CSF-sufficient (GM-CSF +/+) control animals and GM-CSF -/- mice. Lung injury, quantitated by measuring lung myeloperoxidase activity (an indicator of neutrophil sequestration), alveolar-capillary permeability, and alveolar membrane thickness was less severe in GM-CSF -/- than in GM-CSF +/+ mice. In GM-CSF +/+ mice, pancreas, lung and serum GM-CSF levels increase during pancreatitis. Lung levels of macrophage inflammatory protein (MIP)-2 are also increased during pancreatitis, but, in this case, the rise is less profound in GM-CSF -/- mice than in GM-CSF +/+ controls. Administration of anti-MIP-2 antibodies was found to reduce the severity of pancreatitis-associated ALI. Our findings indicate that GM-CSF plays a critical role in coupling pancreatitis to ALI and suggest that GM-CSF may act indirectly by regulating the release of other proinflammatory factors including MIP-2.

Topics: Acute Disease; Animals; Antibodies; Ceruletide; Chemokine CXCL2; Chemokines; Granulocyte-Macrophage Colony-Stimulating Factor; Leukocyte Count; Lung; Lung Diseases; Mice; Mice, Knockout; Pancreas; Pancreatitis; Reference Values; Severity of Illness Index

Intrapancreatic activation of trypsinogen is believed to play a critical role in the initiation of acute pancreatitis, but mechanisms responsible for intrapancreatic trypsinogen activation during pancreatitis have not been clearly defined. In previous in vitro studies, we have shown that intra-acinar cell activation of trypsinogen and acinar cell injury in response to supramaximal secretagogue stimulation could be prevented by the cell permeant cathepsin B inhibitor E64d (Saluja A, Donovan EA, Yamanaka K, Yamaguchi Y, Hofbauer B, and Steer ML. Gastroenterology 113: 304-310, 1997). The present studies evaluated the role of intrapancreatic trypsinogen activation, this time under in vivo conditions, in two models of pancreatitis by using another highly soluble cell permeant cathepsin B inhibitor, L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-L-isoleucyl-L-proline methyl ester (CA-074me). Intravenous administration of CA-074me (10 mg/kg) before induction of either secretagogue-elicited pancreatitis in mice or duct infusion-elicited pancreatitis in rats markedly reduced the extent of intrapancreatic trypsinogen activation and substantially reduced the severity of both pancreatitis models. These observations support the hypothesis that, during the early stages of pancreatitis, trypsinogen activation in the pancreas is mediated by the lysosomal enzyme cathepsin B. Our findings also suggest that pharmacological interventions that inhibit cathepsin B may prove useful in preventing acute pancreatitis or reducing its severity.

Topics: Animals; Cathepsin B; Ceruletide; Cysteine Proteinase Inhibitors; Dipeptides; Infusions, Parenteral; Male; Mice; Mice, Inbred Strains; Pancreatic Ducts; Pancreatitis; Rats; Rats, Wistar; Severity of Illness Index; Taurocholic Acid; Trypsinogen

Prognosis of acute pancreatitis is related mainly to systemic involvement. The establishment of this systemic inflammation is mediated by proinflammatory cytokines. Our aim is to study serum levels of some proinflammatory cytokines and the associated damage of the lung in a model of experimental acute pancreatitis.. Eighty seven male Wistar rats were divided into two groups: group A (control) with saline solution administration; group B with acute pancreatitis induced by intraperitoneal caerulein (50 mg/kg every hour, 4 doses). The animals were killed at 0, 2, 6 and 24 hours of the last dose of caerulein or saline solution. Pancreatic and pulmonary histology were examined, and serum levels of IL-1 beta, TNF-alpha and IL-6 were evaluated, as well as some laboratory parameters as indicators of systemic involvement.. The administration of caerulein induced an acute edematous pancreatitis without mortality and with a trend towards resolution in 24 hours. IL-1 beta in animals with acute pancreatitis showed significantly higher levels than in the control group at 6 hours. Serum transaminases, urea and creatinine were also significantly higher at 2 and 6 h. The group with acute pancreatitis showed histological lung damage all over the study.. In our model of acute pancreatitis we observed systemic involvement as judged by alterations of serum transaminases and parameters of renal function, as well as histological lung damage, that correlated with an increase in serum levels of IL-1b.

Topics: Acute Disease; Animals; Ceruletide; Cytokines; Gastrointestinal Agents; Lung; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar

We previously demonstrated that the immunostaining of the gap-junction protein, connexin 32 (Cx 32), in the pancreas was markedly reduced in caerulein (Cn)-induced acute pancreatitis. The expression of Cx 32 in the pancreas during the course of acute pancreatitis is unclear. To address this, we examined Cx 32 mRNA and protein expression in the pancreas.. Cx 32 mRNA and protein expression in the pancreas was examined by Northern blot analysis and Western blot analysis, respectively, 1, 4, 7, and 14 days after the induction of acute pancreatitis.. Cx 32 mRNA was identified in normal rat pancreas, and the value for the relative intensity against 18S rRNA was 0.57 +/- 0.15 (mean +/- SD). After the induction of acute pancreatitis by caerulein, the Cx 32 mRNA expression levels were increased on day 1, day 4, day 7, and day 14 compared with levels in the normal pancreas (1.63-fold, 1.61-fold, 1.49-fold, and 1.35-fold, respectively). A significant increase in Cx 32 protein expression was detected on day 1 and day 4 (1.67 +/- 0.15-fold and 1.72 +/- 0.2-fold, respectively), while Cx 32-positive spots, determined by immunohistochemical analysis, were markedly decreased on day 1 and had returned to normal by day 14.. These results show that the expression of Cx 32 increases early on after the induction of pancreatitis by Cn, and that the normalization of Cx 32-immunostained spots in Cn-induced acute pancreatitis occurs after the increase in Cx 32 mRNA and protein expression.

Topics: Acute Disease; Animals; Blotting, Northern; Blotting, Western; Ceruletide; Connexins; Gap Junction beta-1 Protein; Immunohistochemistry; Male; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley

We hypothesized that neurogenic inflammation is a common final pathway for parenchymal inflammation in pancreatitis and evaluated the role of primary sensory neurons in secretagogue-induced and obstructive pancreatitis. Neonatal rats received either the primary sensory neuron-denervating agent capsaicin (50 mg/kg s.c.) or vehicle. At 8 wk of age, pancreatitis was produced by six hourly injections of caerulein (50 microg/kg i.p.) or by common pancreaticobiliary duct ligation (CPBDL). The severity of pancreatitis was assessed by serum amylase, pancreatic myeloperoxidase (MPO) activity, histological grading, pancreatic plasma extravasation, and wet-to-dry weight ratio. Caerulein significantly increased MPO activity and wet-to-dry weight ratio, produced histological evidence of edematous pancreatitis, induced plasma extravasation, and caused hyperamylasemia. CPBDL increased MPO activity and produced histological evidence of pancreatitis. Neonatal capsaicin administration significantly reduced tissue MPO levels, histological severity scores, and wet-to-dry weight ratio and abolished plasma extravasation. These results demonstrate that primary sensory neurons play a significant role in the inflammatory cascade in experimental pancreatitis and appear to constitute a common final pathway for pancreatic parenchymal inflammation.

Topics: Amylases; Animals; Animals, Newborn; Bile Ducts; Capsaicin; Ceruletide; Denervation; Ligation; Male; Neurons, Afferent; Pancreas; Pancreatic Ducts; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley

Both cerulein and cholecystokinin activate mitogen-activated protein (MAP) kinase (ERK1/2) in vivo and in isolated pancreatic acini.. ERK1/2 in pancreas homogenates was activated in rats rendered pancreatitic by subcutaneous injections of cerulein (5 microg/kg per hour). To determine if blocking ERK1/2 activity might rescue cerulein-induced acute pancreatitis, the "MAP kinase kinase" (also known as MEK1/2) inhibitors PD98059 and U0126 were administered in vivo.. In rats pretreated with PD98059 (10 mg/kg per i.v. injection) or U0126 (5 mg/kg per i.v. injection) 30 minutes before and then together with hourly cerulein injections for 3 hours, pancreatitis was significantly attenuated on the basis of pancreatic wet weight and histology. Serum amylase concentration was significantly reduced when PD98059 was administered intraperitoneally (10 mg/kg per intraperitoneal injection). PD98059 also ameliorated pancreatitis over a 6-hour cerulein time course. The phosphorylation of pancreatic ERK1/2 was attenuated in PD98059- and U0126-treated animals at both 30 minutes and 3 hours after cerulein injection. Rats rendered neutropenic with vinblastine and pretreated with U0126 still showed attenuated manifestations of cerulein-induced acute pancreatitis, a finding suggesting that pancreatic ERK1/2 is mostly responsible for the effect, rather than infiltrating neutrophils.. Inhibition of pancreatic ERK1/2 in vivo affords significant protection against inflammatory sequelae following cerulein-induced acute pancreatitis.

Topics: Acute Disease; Animals; Butadienes; Ceruletide; Enzyme Inhibitors; Flavonoids; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Pancreatitis; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley

Acute pancreatitis (AP) is associated with increased cytokine production, which can ultimately produce deleterious local and systemic effects. The transcription factor NF-kappaB is activated by degradation of its inhibitory factor, IkappaB, and can stimulate various cytokines. The purpose of this study was to determine whether the inhibition of NF-kappaB binding activity with a novel peptide that binds to the NF-kappaB essential modifier binding domain (NBD) could attenuate the severity of AP.. AP was induced in Swiss Webster mice by hourly injections of the cholecystokinin analogue cerulein (50 microg/kg). Mice were injected with either the wild-type or control (mutated) NBD peptide at the time of the first cerulein injection; they were then sacrificed over a time course, and pancreata and lungs were harvested for histologic analysis and scoring. Myeloperoxidase activity was measured to assess neutrophil sequestration as an indicator of inflammation. NF-kappaB binding activity and steady-state levels of IkappaB and NF-kappaB subunits were determined by gel shift and Western blot, respectively.. AP resulted in increased NF-kappaB DNA-binding activity and decreased steady-state levels of IkappaB. Treatment with NBD peptide decreased inflammation in the pancreas, decreased hemorrhage in the lungs, and decreased myeloperoxidase activity in both pancreas and lung.. The marked induction of NF-kappaB binding activity suggests a role for this transcription factor in the early inflammatory changes associated with AP. Treatment with the NBD peptide attenuated the severity of injury associated with AP. Novel compounds that selectively target NF-kappaB may prove to be useful treatment of AP and AP-associated lung injury.

Topics: Acute Disease; Animals; Ceruletide; Cholecystokinin; DNA; Female; I-kappa B Proteins; Mice; NF-kappa B; Pancreas; Pancreatitis; Peptides; Peroxidase; Protein Binding

Infections are frequent complications and determine clinical course and outcome in severe pancreatitis. A novel animal model was used to assess minimal transit time of bacterial translocation (BT) across the gut mucosa in vivo using green fluorescent protein-transfected Escherichia coli and intravital video microscopy.. Three hours after induction of acute pancreatitis by i.p. injection of 40 microg/kg cerulein, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli were injected into the lumen of a small bowel reservoir formed by ligature in anesthetized Wistar rats. Translocation of E. coli was assessed by intravital microscopy. Animals were sacrificed 5 h after induction of pancreatitis.. BT across the mucosa and into the muscularis propria took a mean +/- SD of 36.4 +/- 8 min and 80.9 +/- 9.5 min, respectively, in sham animals. Pancreatitis resulted in a significantly shorter minimal transit time across the mucosa (16.4 +/- 4.9 min, p = 0.007) and into the muscularis propria (47.7 +/- 2.5 min, p = 0.001). E. coli were detected on frozen cross-sections and on bacteriological examination of pancreatic tissue in animals with acute pancreatitis but not in controls.. Intravital microscopy of fluorescent bacteria is a new approach towards studying BT in vivo. Minimal transit time of BT serves as a novel functional aspect of mucosal barrier function during acute pancreatitis. The observation of fluorescent bacteria translocating from the small bowel lumen into the pancreas provides substantial experimental proof for the gut-origin-hypothesis of infectious complications in pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Escherichia coli; Ileum; Intestinal Mucosa; Male; Microscopy, Video; Muscle, Smooth; Necrosis; Pancreatitis; Rats; Rats, Wistar

To study the feature of pancreatic microcirculatory impairment, especially the initial changes, in caerulein-induced experimental acute pancreatitis (AP).. The pancreatic microcirculation of caerulein-induced AP model was studied by intravital fluorescence microscopy with FITC-labeled erythrocytes (FITC-RBC), scanning electron microscopy of vascular corrosion casts, and light microscopy of Chinese ink-injected/cleared tissues.. Animals in caerulein-treated group showed hyperamylemia (X2), pancreatic oedema, infiltration of inflammatory cells in pancreas. Constrictions of intralobular arteriolar sphincters, presence of vacuoles in all layers of sphincter, and gross irregularity in capillary network of acini were found in the AP specimens. The decrease of pancreatic capillary blood flow (0.34+/-0.10 nl x min(-1) vs 0.91+/-0.06 nl x min(-1) of control, P<0.001), reduction of functional capillary density(277+/-13 cm(-1) vs 349+/-8 cm(-1) of control, P<0.001), and irregular intermittent perfusion were observed in caerulein-induced groups.. Impairment and constriction of pancreatic intralobular arteriolar sphincter are the initial microcirculatory lesions in the early phase of acute pancreatitis, and play a key role in the pancreatic ischaemia and pancreatic microvascular failure in acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Arterioles; Ceruletide; Disease Models, Animal; Edema; Erythrocytes; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Male; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Pancreas; Pancreatitis; Rats; Rats, Wistar; Vacuoles

Cyclooxygenase-2 (COX-2), a widely distributed enzyme, plays an important role in inflammation. We have studied the role of COX-2 in acute pancreatitis and pancreatitis-associated lung injury using both the pharmacological inhibition of COX-2 and genetic deletion of COX-2. Pancreatitis was induced in mice by 12 hourly injections of cerulein. The severity of pancreatitis was assessed by measuring serum amylase, pancreatic trypsin activity, intrapancreatic sequestration of neutrophils, and acinar cell necrosis. The severity of lung injury was evaluated by measuring lactate dehydrogenase levels in the bronchoalveolar lavage fluid and by quantitating neutrophil sequestration in the lung. In both the pharmacologically inhibited and genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was reduced compared with the noninhibited strains of COX-2-sufficient mice. This reduction in injury indicates that COX-2 plays an important proinflammatory role in pancreatitis and its associated lung injury. Our findings support the concept that COX-2 inhibitors may play a beneficial role in the prevention of acute pancreatitis or in the reduction of its severity.

Topics: Animals; Celecoxib; Ceruletide; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; HSP70 Heat-Shock Proteins; Isoenzymes; Lung Diseases; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrobenzenes; Pancreatitis; Prostaglandin-Endoperoxide Synthases; Pyrazoles; RNA, Messenger; Severity of Illness Index; Sulfonamides; Trypsinogen

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.

Topics: Amino Acid Sequence; Animals; Ceruletide; Cloning, Molecular; Enteropeptidase; Enzyme Activation; Escherichia coli; Pancreas; Pancreatitis; Protein Isoforms; Rats; Trypsin Inhibitors; Trypsinogen

Adenosine shows protective effects against cellular damage and dysfunction under several adverse conditions such as inflammation and ischemia. In the current study, we examined the effects of 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1,3 )-quinazolinedione hydrochloride (KF24345), an adenosine uptake inhibitor, on cerulein-induced acute pancreatitis in mice to investigate whether inhibition of adenosine uptake could ameliorate the severity of acute pancreatitis.. Acute pancreatitis was induced in mice with six intraperitoneal injections of cerulein (50 microg/kg each) at hourly intervals.. The cerulein injection increased activities of serum amylase and lipase and caused pathologic changes such as interstitial edema, polymorphonuclear cell infiltration, and acinar cell necrosis in the pancreas. KF24345 (10 mg/kg p.o.) ameliorated all these changes observed in mice with acute pancreatitis, and the suppressing effect of KF24345 on the elevation in serum amylase activity was abolished by the treatment with 8-(p-sulfophenyl)theophylline, an adenosine receptor antagonist. In addition, 2-(aminocarbonyl)- -(4-amino-2,6-dichlorophenyl)-4-[5,5-bis-(4-fluorophenyl)pentyl]-1-piperazineacetamide (R75231) and dipyridamole, other adenosine uptake inhibitors, also decreased the elevated serum amylase activity.. These are the first demonstrations that the adenosine uptake inhibitors ameliorate cerulein-induced acute pancreatitis in mice, and these data suggest that adenosine uptake inhibition could ameliorate the severity of acute pancreatitis in vivo.

Topics: Acute Disease; Adenosine; Amylases; Animals; Biological Transport; Ceruletide; Dipyridamole; Female; Lipase; Mice; Mice, Inbred BALB C; Models, Chemical; Pancreatitis; Piperazines; Pyrimidinones; Quinazolines; Theophylline

To explore the changes in hemorheology and expression of neutrophil adhesion molecules CD18 and CD62L in pancreatic microcirculation of Caerulein induced experimental acute pancreatitis (AP).. The Wistar rats (n = 21) were randomized into three groups. The model of AP was established by subcutaneous injection of Caerulein. The changes of apparent viscosity of whole blood were measured by Low- shear 30 rheometer. The expression of adhesion molecules on the surface of neutrophil in duced by shear stress was used with stationary control. CD18 expression was increased on neutrophils treated with shear rate, and andanalyzed using flow cytometry.. Rat treated with Caerulein showed hyperamyleimia (t = 69.029, t = 79.734, P < 0.05). Blood viscosity of two AP groups were significantly elevated (0.512 s(-1): t = 10.725, t = 16.945; 5.96 s(-1): t = 12.781, t = 11.992, P < 0.05). Compared with stationary control, CD18 expression was increased on neutrophil treated with shear rate, and significantly induced with shear rate >/= 94.5 s(-1) (94.5 s(-1): t = 7.403, t = 13.323, t = 16.655; 128.5 s(-1): t = 10.092, t = 28.531, t = 24.563, P < 0.05). The expression of CD62L was less sensitive to low shear rate, and began to be down-regulated significantly when the shear rate >/= 94.5 s(-1) (94.5 s(-1): t = 10.687, t = 19.376, t = 12.848; 128.5 s(-1): t = 26.152, t = 48.402, t = 56.814, P < 0.05).. The changes of apparent viscosity of whole blood, and the effect of fluid shear stress on the expression of neutrophil adhesion molecules CD18, CD62L may play an important role in the pancreatic microcirculatory failure of acute pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Flow Cytometry; Hemorheology; Microcirculation; Neutrophils; Pancreatitis; Rats, Wistar

Many interrelationships exist between the thyroid gland and the gastrointestinal tract. Several past and recent studies have shown that the thyroid gland profoundly influences the structure and function of the exocrine pancreas in the rat. In the present study we investigated the effect of methimazole (METZ), an antithyroid drug, on cerulein induced acute pancreatitis (AP) in rats.. Rats were divided into 3 groups (10-12 weeks age, 200-250 g weight, n: 10). Group B was made hypothyroid with methimazole 5 mg/kg daily for 10 days and the others were untreated euthyroid groups. After 10 days, acute pancreatitis was induced with four doses of 20 microg/kg body weight of cerulein administered s.c at hourly intervals in group A and B while the control group C was given 4 doses of I ml saline. Pancreas wet weight (mg), plasma amylase activity (IU/l) and pancreatic histology were used as endpoints to quantify the severity of the AP.. Plasma tri-iodothyronine (T3) (ng/dl) and thyroxine (T4) (microg/dl) levels were significantly reduced after METZ treatment for 10 days (p < 0.01). METZ pretreatment reduced significantly the cerulein induced increase in pancreatic weight (1,205 +/- 12 mg in METZ treated AP group versus 1,617 +/- 14 mg in AP group, p < 0.05) and the rise in amylase activity (7,078 +/- 816 IU/l in METZ treated AP group versus 8,611 +/- 830 IU/l in AP group p < 0.05).. METZ reduces the severity of cerulein induced AP in rats. This effect might be through its antithyroid property.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Disease Models, Animal; Hypothyroidism; Male; Methimazole; Pancreatitis; Rats; Rats, Wistar; Thyroid Hormones

The present study investigated the involvement of endogenous melatonin in the prevention of pancreatic damage provoked by caerulein-induced pancreatitis (CIP) by using the luzindole, the antagonist of melatonin MT2 receptors. CIP was produced by subcutaneous infusion of caerulein to conscious rats (25 microg/kg). Luzindole (1, 2 or 4 mg/kg) was given as an intraperitoneal bolus injection 30 min prior to the start of CIP. Lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were measured in the pancreas by LPO-584 commercial kit. CIP was confirmed by histological examination and manifested by significant increases of plasma activities of amylase, lipase and tumor necrosis factor alpha (TNFalpha) (by 500%, 1000% and 600%, respectively) comparing to the control values. This was accompanied by a 40% limitation in pancreatic blood flow (PBF) and by 200% increase of MDA+4-HNE in the pancreas of CIP rats. Administration of luzindole to the CIP rats reduced PBF, aggravated the histological manifestations of pancreatitis, resulted in the significant augmentation of pancreatic MDA + 4-HNE content, and produced the marked increases of plasma levels of lipase, amylase and TNFalpha, comparing to the values observes in the rats with CIP alone. These results suggest that endogenous melatonin through its receptor MT2 plays an important role in the attenuation of pancreatic damage produced by overstimulation with caerulein.

Topics: Amylases; Animals; Ceruletide; Dose-Response Relationship, Drug; Lipase; Lipid Peroxidation; Male; Melatonin; Organ Size; Pancreas; Pancreatitis; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; Tryptamines; Tumor Necrosis Factor-alpha

The goal of our study was to evaluate the influence of NO synthesis modulation on the ultrastructural changes in the pancreatic acinar cells in connection with morphometric assessment of the volume and numerical densities of mitochondria (Vvm, Nvm) and zymogen granules (Vvz, Nvz) in caerulein-induced acute pancreatitis (AP). During AP induction rats were treated with L-arginine--substrate for NO synthesis, N(G)-nitro-L-arginine (L-NNA)--NO synthase inhibitor, gliceryl trinitrate (NTG)--NO donor, L-arginine+L-NNA or saline. This study demonstrated that administration of L-NNA leads to the formation of numerous, large autophagosomes and mitochondria oedema in pancreatic acinar cells. Treatment with L-arginine or NTG during AP induction resulted in a diminution of the ultrastructural changes with a concomitant increase of Vvz. Vvm and Nvm were significantly lower in the L-arginine treated group compared to the untreated AP. The results indicate that: L-NNA enhances damage to acinar cells which may be indicative of a protective role for endogenous NO in oedematous AP. The application of L-arginine or NTG decreases the damage to acinar cells evaluated ultrastructurally, suggesting the morphological changes accompanying the onset of AP in rats after the administration of either substrate for endogenous NO synthesis or exogenous NO donor follow a favourable course.

Topics: Animals; Arginine; Ceruletide; Enzyme Inhibitors; Enzyme Precursors; Male; Microscopy, Electron; Mitochondria; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroglycerin; Pancreas; Pancreatitis; Rats; Rats, Wistar; Secretory Vesicles

Acute pancreatitis leads to hypoxia caused by vasoconstriction and to activation of lysosomal and digestive enzymes resulting in pancreas autodigestion and damage. This causes activation of leucocytes and increased expression of adhesive molecules enabling margination and adhesion of activated leucocytes to the endothelium. Activated leucocytes are the source of proinflammatory cytokins and oxygen-free radicals which intensify the inflammatory response. The reports indicating that adenosine may prevent activation of the above-mentioned processes in ischaemia prompted us to undertake this study. The study was performed in two stages. The first stage was to evaluate the effects of agonists and antagonists of adenosine receptors on normal pancreas while the second one was to determine the influence of these substances on the development of caerulein-induced acute pancreatitis. During the first stage, the animals were injected intraperitoneally with the substances examined: the A1 receptor antagonist--DPCPX, the A2 receptor agonist--CGS 21680, the A2 receptor antagonist--ZM 241385 and the A3 receptor agonist--IB-MECA and then received intravenous saline. The control animals were subjected only to the 12 h intravenous infusion of 0.15 M NaCl. During the second stage, after the intraperitoneal administration of adenosine receptor agonists and antagonists (as in the first stage), acute pancreatitis was induced with the 12 h intravenous infusion of 5 micrograms/kg/h caerulein. Identical acute pancreatitis was induced in the control animals, however no other substances were administered. The pancreatic tissue samples were collected directly after intravenous infusion. The severity of inflammatory processes in the pancreas was evaluated on the basis of the plasma amylase activity, pancreatic weight and enhancement of histopathological changes observed in this organ. In the animals infused with saline alone, no effects of the substances examined on the pancreatic weight, plasma amylase activity and histopathological features were observed. The intravenous caerulein infusion induced acute pancreatitis expressed as bigger pancreatic weight, increased plasma amylase activity and tissue damage (oedema, cell vacuolization, leucocyte infiltration). The A2 receptor agonist administration preceding the induction of acute pancreatitis decreased the pancreas damage caused by caerulein. Lower weight of the pancreas and decreased plasma amylase activities were observed; on hist

Topics: Acute Disease; Adenosine; Animals; Ceruletide; Male; Pancreatitis; Rats; Rats, Wistar; Receptors, Purinergic P1

Adenosine plays important roles in a variety of pathophysiologic conditions through receptor-mediated mechanisms. Recent studies have shown that adenosine exerts potent anti-inflammatory properties that are chiefly brought about through the occupancy of the A2a receptor.. To examine the effect of A2a receptor stimulation or inhibition on the pathologic findings during acute pancreatitis.. Rats were randomized into three groups and received a selective A2a receptor agonist CGS-21680 (CGS), a selective A2a antagonist 3,7-dimethyl-1-[2-propynyl]-xanthine (DMPX), or saline. Thirty minutes after the injection, acute pancreatitis was produced in the rats by seven intraperitoneal injections of caerulein. The severity of acute pancreatitis was evaluated by serum amylase activity, pancreas myeloperoxidase (MPO) activity, Evans blue extravasation, and pathologic changes of the pancreas. In addition, we investigated the effects of CGS on the pathologic findings of caerulein pancreatitis induced in neutrophil-depleted rats.. Administration of caerulein produced hyperamylasemia and morphologic changes of the pancreas including interstitial edema, acinar cell vacuolization, and infiltration of inflammatory cells. In CGS-treated rats, the pancreatic edema and the Evans blue extravasation were aggravated significantly compared with those of saline-treated rats, whereas leukocyte infiltration and MPO activity of the pancreas were decreased. In contrast to CGS, administration of DMPX ameliorated the pancreatic edema and Evans blue extravasation. Treatment with CGS accelerated the pancreatic edema in pancreatitis even after the depletion of neutrophils.. The activation of adenosine A2a receptors modulates the pathology of acute pancreatitis through at least two diverse properties. One is an anti-inflammatory effect involving neutrophils, and the other is a propagating effect for pancreatic edema formation. The actions of the A2a receptor pathways are unique, and they may have an important role in the progression of acute pancreatitis.

Topics: Adenosine; Amylases; Animals; Ceruletide; Edema; Leukocytes; Male; Pancreatitis; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Theobromine

Recent studies have indicated that prior thermal stress causes upregulation of heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue induced pancreatitis. The mechanisms responsible for the protective effect are not known. Similarly, the effects of prior non-thermal stress on HSP70 expression and pancreatitis are not known. The current studies were designed to specifically address these issues.. In the current studies pancreatitis was induced by administration of a supramaximally stimulating dose of caerulein 12 hours after thermal stress and 24 hours after non-thermal (that is, beta adrenergic stimulation) stress.. Both thermal and non-thermal stresses caused pancreatic HSP70 levels to rise and resulted in increased expression of HSP70 in acinar cells. Both forms of stresses protected against caerulein induced pancreatitis and prevented the early intrapancreatic activation of trypsinogen which occurs in this model of pancreatitis.. These results suggest that both thermal and non-thermal stresses protect against pancreatitis by preventing intrapancreatic digestive enzyme activation and that HSP70 may mediate this protective effect.

Topics: Amylases; Analysis of Variance; Animals; Blotting, Western; Ceruletide; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; HSP70 Heat-Shock Proteins; Hyperthermia, Induced; Luminescent Measurements; Male; Pancreatitis; Peroxidase; Rats; Rats, Wistar; Stress, Physiological; Trypsinogen

Prior thermal stress induces heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue-induced pancreatitis, but it is not clear that this thermal stress-induced protection is actually mediated by HSP70 since thermal stress may have other, non-HSP related, effects.. In the present study, we have administered antisense (AS) oligonucleotides, which prevent pancreatic expression of HSP70 to rats, in vivo, to evaluate this issue. In a separate series of experiments, designed to examine the role of pancreatitis-induced HSP70 expression in modulating the severity of pancreatitis, rats not subjected to prior thermal stress were given AS-HSP70 before cerulein administration, and trypsinogen activation as well as the severity of pancreatitis were evaluated.. Hyperthermia induced HSP70 expression, prevented intrapancreatic trypsinogen activation, and protected against cerulein-induced pancreatitis. Administration of AS-HSP70 but not sense-HSP70 reduced the thermal stress-induced HSP70 expression, restored the ability of supramaximal cerulein stimulation to cause intrapancreatic trypsinogen activation, and abolished the protective effect of prior thermal stress against pancreatitis. In non-thermally stressed animals, pretreatment with AS-HSP70 before the induction of pancreatitis exacerbated all the parameters associated with pancreatitis.. These findings lead us to conclude that HSP70 induction, rather than some other thermal stress-related phenomenon, mediates the thermal stress-induced protection against pancreatitis and that it protects against pancreatitis by preventing intrapancreatic activation of trypsinogen. The worsening of pancreatitis, which occurs when non-thermally stressed animals are given AS-HSP70 before cerulein, suggests that cerulein-induced HSP70 expression in nontreated animals acts to limit the severity of pancreatitis.

Topics: Acute Disease; Animals; Ceruletide; Gene Expression Regulation; Heat Stress Disorders; HSP70 Heat-Shock Proteins; Oligonucleotides, Antisense; Pancreatitis; Rats; Rats, Wistar; Stress, Physiological; Trypsinogen

A premature and intracellular activation of digestive zymogens is thought to be responsible for the onset of pancreatitis. Because trypsin has a critical role in initiating the activation cascade of digestive enzymes in the gut, it has been assumed that trypsin also initiates intracellular zymogen activation in the pancreas. We have tested this hypothesis in isolated acini and lobules from rat pancreas. Intracellular trypsinogen activation was induced by supramaximal secretagogue stimulation and measured using either specific trypsin substrates or immunoreactivity of the trypsinogen activation peptide (TAP). To prevent a trypsin-induced trypsinogen activation, we used the cell-permeant, highly specific, and reversible inhibitor Nalpha-(2-naphthylsulfonyl)-3-amidinophenylalanine-carboxymethylpiperazide (S124), and to prevent cathepsin-induced trypsinogen activation, we used the cysteine protease inhibitor E-64d. Incubation of acini or lobules in the presence of S124 completely prevented the generation of trypsin activity in response to supramaximal caerulein but had no effect whatsoever on the generation of TAP. Conversely, when trypsin activity was recovered at the end of the experiment by either washout of S124 from acini or extensive dilution of lobule homogenates, it was up to 400% higher than after caerulein alone and corresponded, in molar terms, to the generation of TAP. Both trypsin activity and TAP release were inhibited in parallel by E-64d. We conclude that caerulein-induced trypsinogen activation in the pancreas is caused by an E-64d-inhibitable mechanism such as cathepsin-induced trypsinogen activation, and neither involves nor requires intracellular trypsin activity. Specific trypsin inhibition, on the other hand, prevents 80% of trypsin inactivation or autodegradation in the pancreas.

Topics: Acute Disease; Animals; Cathepsin B; Ceruletide; Enzyme Activation; Male; Naphthalenes; Pancreas; Pancreatitis; Piperazines; Rats; Rats, Wistar; Trypsin; Trypsin Inhibitors; Trypsinogen

Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause zymogen activation and pancreatitis. The effects of the CCK analog, caerulein, and alcohol on trypsin and chymotrypsin activation in isolated pancreatic acini were examined. Caerulein increased markers of zymogen activation in a time- and concentration-dependent manner. Notably, trypsin activity reached a peak value within 30 min, then diminished with time, whereas chymotrypsin activity increased with time. Ethanol (35 mM) sensitized the acinar cells to the effects of caerulein (10(-10) to 10(-7) M) on zymogen activation but had no effect alone. The effects of ethanol were concentration dependent. Alcohols with a chain length of >or=2 also sensitized the acinar cell to caerulein; the most potent was butanol. Branched alcohols (2-propanol and 2-butanol) were less potent than aliphatic alcohols (1-propanol and 1-butanol). The structure of an alcohol is related to its ability to sensitize acinar cells to the effects of caerulein on zymogen activation.

Topics: 2-Propanol; Acute Disease; Alcohols; Animals; Butanols; Ceruletide; Chymotrypsin; Enzyme Activation; Enzyme Precursors; Ethanol; Kinetics; Male; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Trypsin

Topics: Animals; Cathepsin B; Ceruletide; Disease Models, Animal; Gene Expression; Pancreatitis; Rats; RNA, Messenger

To compare the effect of different vasoactive mediator antagonists in the same model of severe acute pancreatitis (AP) and to evaluate whether combinations of the agents exhibit synergistic effects.. Prospective experimental study.. Microcirculation and pancreas research laboratory at an university hospital.. Hundred eighty anesthetized male Sprague-Dawley rats.. Six hours after inducing AP by intra-ductal bile salt infusion and i.v. cerulein in 168 rats, these were randomized for therapy with (1) saline, (2) endothelin receptor antagonist (ET-RA), (3) platelet activating factor receptor antagonist (PAF-RA), (4) intercellular adhesion molecule-1 antibody (ICAM-1-AB) or different combinations (5-7). After 24 h the animals underwent a second laparotomy for intra-vital microscopic determination of pancreatic and colonic capillary permeability, blood flow and leukocyte-endothelial interaction.. AP induction decreased capillary blood flow and increased permeability and leukocyte rolling. ET-RA, PAF-RA and ICAM-1-AB decreased capillary permeability, increased blood flow and reduced leukocyte rolling. ET-RA was most effective in decreasing capillary permeability in both organs as well as in increasing pancreatic capillary blood flow. Combining vasoactive mediator blockers did not further improve target parameters.. This study supports previous observations that ET-RA, PAF-RA and ICAM-1-AB improve microcirculation in AP and that ET-RA is more effective than PAF-RA or ICAM-1-AB, especially in counteracting capillary leakage. Although this may suggest that they act through different mechanisms, antagonist combinations failed to improve microcirculation further. We conclude that ET-RA is the most promising candidate for a clinical trial to reduce capillary leakage in patients with AP.

Topics: Acute Disease; Animals; Antibodies; Bile Acids and Salts; Capillary Leak Syndrome; Ceruletide; Endothelin Receptor Antagonists; Intercellular Adhesion Molecule-1; Male; Microcirculation; Pancreatitis; Platelet Membrane Glycoproteins; Prospective Studies; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Vascular Diseases

Central nervous system affects pancreatic secretion of enzymes however, the neural modulation of acute pancreatitis has not been investigated. Leptin and melatonin have been recently reported to affect the inflammatory response of various tissues. The identification of specific receptors for both peptides in the pancreas suggests that leptin and melatonin could contribute to the pancreatic protection against inflammation. The aim of this study was: 1/ to compare the effect of intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) administration of leptin or melatonin on the course of caerulein-induced pancreatitis (CIP) in the rat, 2/ to examine the involvement of sensory nerves (SN) and calcitonin gene-related peptide (CGRP) in pancreatic protection afforded by leptin or melatonin, 3/ to assess the effect of tested peptides on lipid peroxidation products (MDA + 4-HNE) in the pancreas of CIP rats, 4/ to investigate the influence of leptin or melatonin on nitric oxide (NO) release from isolated pancreatic acini and 5/ to determine the effects of caerulein and leptin on leptin receptor gene expression in these acini by RT-PCR. CIP was induced by subcutaneous (s.c.) infusion of caerulein (25 microg/kg) to the conscious rats, confirmed by the significant increases of pancreatic weight and plasma amylase and by histological examination. This was accompanied in marked reduction of pancreatic blood flow and significant rise of MDA + 4-HNE in the pancreas. Leptin or melatonin were administered i.p. or i.c.v. 30 min prior to the start of CIP. Deactivation of SN was produced by s.c. capsaicin (100 mg/kg). An antagonist of CGRP, CGRP 8-37 (100 microg/kg i.p.), was given together with leptin or melatonin to the CIP rats. MDA + 4-HNE was measured using LPO commercial kit. NO was determined using the Griess reaction. Pretreatment of CIP rats with i.p. leptin (2 or 10 microg/kg) or melatonin (10 or 50 mg/kg) significantly attenuated the severity of CIP. Similar protective effects were observed following i.c.v. application of leptin (0.4 or 2 microg/rat) but not melatonin (10 or 40 microg/rat) to the CIP rats. Capsaicin deactivation of SN oradministration of CGRP 8-37 abolished above beneficial effects of leptin on CIP, whereas melatonin-induced protection of pancreas was unaffected. Pretreatment with i.p. melatonin (10 or 50 mg/kg), but not leptin, significantly reduced MDA + 4-HNE in the pancreas of CIP rats. Leptin (10(-10) - 10(-6) M) but not melatonin (10(-8) -

Topics: Amylases; Animals; Antioxidants; Calcitonin Gene-Related Peptide; Carrier Proteins; Central Nervous System; Ceruletide; Free Radicals; Injections, Intraperitoneal; Injections, Intraventricular; Leptin; Male; Melatonin; Neurons, Afferent; Organ Size; Pancreas; Pancreatitis; Peripheral Nervous System; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Leptin; Regional Blood Flow

Lipopolysaccharides (LPS) are responsible for septic shock but low doses of LPS reduce pancreatic damage produced by caerulein-induced pancreatitis (CIP) in rats. Leptin, produced by adipocytes attenuates the severity of CIP. The aim of this study was to evaluate the effect of intracerebroventricular (i.c.v.) administration of LPS on CIP and plasma leptin level and to investigate the involvement of sensory nerves (SN) in the effects of LPS on CIP.. CIP was produced by subcutaneous (s.c.) infusion of caerulein (25 Kg/kg) to conscious rats. SN were deactivated with capsaicin (100 mg/kg s.c.). LPS (0.2, 2, or 20 Kg/rat) were applied to the right cerebral ventricle 30 min prior to CIP.. CIP was manifested by an increase in plasma levels of amylase, lipase, leptin and an anti-inflammatory interleukin 10 (IL-10), (by 400%, 1000%, 700% and 50%, respectively), confirmed by histological examination and accompanied by 40% reduction in pancreatic blood flow. Pretreatment of CIP rats with i.c.v. LPS resulted in significant reduction of CIP accompanied by dose-dependent increase in plasma levels of leptin and IL-10. Deactivation of SN, which by itself failed to affect CIP, completely reversed the beneficial effects of i.c.v. administration of LPS on CIP and reduced plasma leptin and IL-10 concentrations.. Pretreatment with LPS given i.c.v. prevents the development of caerulein-induced pancreatitis through the activation of SN and though the release of leptin.

Topics: Acute Disease; Adipocytes; Afferent Pathways; Amylases; Animals; Capsaicin; Ceruletide; Escherichia coli; Injections, Intraventricular; Interleukin-10; Leptin; Lipase; Lipopolysaccharides; Neurons, Afferent; Pancreatitis; Rats; Rats, Wistar; Sympathectomy, Chemical

We studied the effect of iron chelators, 2,2'-dipyridyl and desferrioxamine, on cerulein-induced pancreatitis in rats. Acute pancreatitis was induced by a single subcutaneous injection of 100 micrograms/kg body weight of cerulein, which caused hyperamylasemia and edematous pancreatitis with neutrophilic infiltration. Blood samples were collected for determination of serum amylase values and the pancreas was removed for the histological examination 6 h after the cerulein injection. Intraperitoneal administration of a ferrous iron chelator, 2,2'-dipyridyl, prior to the cerulein injection resulted in amelioration of hyperamylasemia and histological abnormalities such as edema and inflammation but not of acinar cell vacuolization. In contrast, administration of a ferric iron chelator, desferrioxamine, did not show any beneficial effects. These results indicate that administration of 2,2'-dipyridyl ameliorates the pancreatitis induced by the supramaximal dose of cerulein.

Topics: 2,2'-Dipyridyl; Acute Disease; Animals; Ceruletide; Deferoxamine; Edema; Injections, Intraperitoneal; Iron Chelating Agents; Male; Neutrophil Infiltration; Pancreatitis; Rats; Rats, Wistar

The early events in pancreatic fibrosis are poorly understood. We examined the production of collagen and matrix metalloproteinases as well as the activation of pancreatic stellate cells in a rodent model of pancreatic fibrosis.. Pancreatitis was induced in rats by hyperstimulation with cerulein (50 microg/kg/day ip) and concurrent pancreatic duct obstruction (SHOP model) for 96 h (n = 48). Sham animals were injected with saline and underwent laparotomy and manipulation of the pancreas with no duct obstruction (n = 28). Rats were sacrificed daily for 18 days. Serial pancreatic sections were stained with H&E [histology], trichrome [collagen], and alpha smooth muscle actin (alpha-SMA) antibodies [activated stellate cells]. Total pancreatic matrix metalloproteinase (MMP)-2 and 9 were determined by gelatin zymography. MMP-1 production was examined using Western blotting.. There were occasional alpha-SMA-positive cells in the pancreatic parenchyma of normal and sham animals. Within 48 h of pancreatitis induction in SHOP animals, histologic evidence of pancreatic inflammation was present, and stellate cells (alpha-SMA-positive cells) appeared surrounding pancreatic acini. The appearance of these cells was followed by collagen deposition in the same area. MMP-1 and 2 proteins increased significantly during pancreatitis while MMP-9 did not. The pancreatic architecture returned to normal by 18 days after the induction of pancreatitis.. Acute pancreatic inflammation results in stellate cell activation and collagen deposition in the same area. Collagen is then resorbed at a time when MMP-1 and 2 peak. The fibrosis of acute pancreatic inflammation in this model completely resolves with restoration of normal architecture.

Topics: Actins; Animals; Blotting, Western; Ceruletide; Collagen; Constriction; Edema; Fibrosis; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Muscle, Smooth; Pancreas; Pancreatic Ducts; Pancreatitis; Rats; Rats, Sprague-Dawley; Tissue Distribution

Oxidative stress plays an important role in the early stage of acute pancreatitis as well as the associated multiple organ injury. Here we compare the degree of pancreatitis caused by cerulein in mice lacking the inducible (or type 2) nitric oxide synthase (iNOS) and in the corresponding wild-type mice. Intraperitoneal injection of cerulein resulted in wild-type mice in a severe, acute pancreatitis, which was characterized by edema, neutrophil infiltration, tissue hemorrhage and cell necrosis as well as increases in the serum levels of amylase and/or lipase. The infiltration of the pancreatic tissue of these animals with neutrophils (measured as increase in myeloperoxidase activity) was associated with up-regulation/expression of the adhesion molecules ICAM-1 and P-selectin as well as signs of enhanced lipid peroxidation (e.g., increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for nitrotyrosine and poly (ADP-ribose) synthetase (PARS) in the pancreas of cerulein-treated iNOS wild-type mice. In contrast, the degree of pancreatic inflammation and tissue injury (histological score), upregulation/expression of P-selectin and ICAM-1, the staining for nitrotyrosine and PARS, and lipid peroxidation was markedly reduced in pancreatic tissue sections obtained from cerulein-treated iNOS-deficient mice. These findings support the view that iNOS plays an important, pro-inflammatory role in the acute pancreatitis caused by cerulein in mice.

Topics: Amylases; Animals; Ceruletide; DNA-Binding Proteins; I-kappa B Proteins; Intercellular Adhesion Molecule-1; Lipase; Lipid Peroxidation; Male; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Mortality; Neutrophil Infiltration; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; P-Selectin; Pancreatitis; Peroxynitrous Acid; Poly(ADP-ribose) Polymerases; Tyrosine

To explore the pathogenesis of acute pancreatitis (AP) and evaluate the effect of a Chinese herb WPY on the course of AP.. Intravital fluorescence microscopy was used. The pancreatic microvascular diameter, RBC velocities and functional capillary density (FCD) were estimated following intracutaneous injection of caerulein alone or with gastropipe WPY.. Caerulein mediated a significant decrease in functional capillary density (FCD), RBC velocities and diameter of interlobular arteriole (P < 0.01). Compared with AP group, WPY was effective in restoring functional capillary density, interlobular arteriole diameter and RBC velocity.. Impairment of pancreatic microcirculation in the early phase of acute pancreatitis may play a key role in the progression of this disease. Possible contributory mechanisms include reduced blood flow and functional capillary density, interlobular arteriole spasm, and leukocyte-endothelial cell interaction. WPY has a beneficial effect on the course of acute pancreatitis. Possible causes include attenuating microcirculatory failure.

Topics: Acute Disease; Animals; Blood Flow Velocity; Ceruletide; Drug Combinations; Drugs, Chinese Herbal; Mice; Microcirculation; Microscopy, Fluorescence; Pancreas; Pancreatitis

We demonstrated that the dynamic aspects of cytokine production in rat acute pancreatitis, which was induced by cerulein and aggravated by subsequent lipopolysaccharide (LPS) injection. A priming effect by induction of mild pancreatitis with cerulein enhanced the subsequent cytokine production by LPS injection. Alternatively, after induction of severe pancreatitis with cerulein and LPS, cytokine production was markedly suppressed for > or = 90 hours. Production of interleukin-2 (IL-2) by splenocytes decreased, and mortality rate after cecal ligation and puncture (CLP) increased significantly after induction of severe acute pancreatitis. These results suggest that the suppression of a cytokine response in severe acute pancreatitis may alter the defense system and, as a result, increase mortality after CLP.

Topics: Acute Disease; Animals; Cells, Cultured; Ceruletide; Cytokines; Endotoxemia; Escherichia coli; Lipopolysaccharides; Male; Pancreatitis; Peritonitis; Rats; Rats, Wistar; Spleen

Reactive oxygen species have been implicated in the pathogenesis of acute pancreatitis. Few studies have focused on the loss of endogenous antioxidants and molecular oxidative damage. Two acute pancreatitis models in rats; taurocholate (3% intraductal infusion) and cerulein (10 microg/kg/h), were used to study markers of oxidative stress: Glutathione, ascorbic acid, and their oxidized forms (glutathione disulfide and dehydroascorbic acid), malondialdehyde, and 4-hydroxynoneal in plasma and pancreas, as well as 7-hydro-8-oxo-2'-deoxyguanosine in pancreas. In both models, pancreatic glutathione depleted by 36-46% and pancreatic ascorbic acid depleted by 36-40% (p <.05). In the taurocholate model, plasma glutathione was depleted by 34% (p <.05), but there were no significant changes in plasma ascorbic acid or in plasma and pancreas dehydroascorbic acid, malondialdehyde, and 4-hydroxynoneal, and no significant changes in the pancreas glutathione disulfide/glutathione ratio. While pancreas glutathione disulfide/glutathione ratio increased in the cerulein model, there were no significant changes in plasma glutathione, plasma, or pancreas ascorbic acid, dehydroascorbic acid, 4-hydroxynoneal, and malondialdehyde, or in pancreas 7-hydro-8-oxo-2'-deoxyguanosine. Reactive oxygen species have a minor role in the intermediate stages of pancreatitis models.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Acute Disease; Aldehydes; Animals; Ascorbic Acid; Biomarkers; Ceruletide; Dehydroascorbic Acid; Deoxyguanosine; Glutathione; Glutathione Disulfide; Male; Malondialdehyde; Oxidation-Reduction; Oxidative Stress; Pancreatitis; Rats; Rats, Wistar; Reactive Oxygen Species; Taurocholic Acid

It is well known that endotoxemia, which is caused by a bacterial infection, can exacerbate acute pancreatitis, whereas pancreatitis-associated protein (PAP) has the ability to induce bacterial aggregation. Pancreatitis-associated protein is supposed to protect the tissue from infection during inflammation. In order to clarify the relationship between PAP mRNA expression and endotoxemia during acute pancreatitis, the kinetic patterns of PAP-I mRNA in mouse pancreas treated with either cerulein or lipopolysaccharide (LPS) or both were investigated in this study.. The administration of LPS (5 mg/kg) intraperitoneally resulted in a dramatic upregulation of PAP-I mRNA expression, increasing 18.61-fold to a maximum at 12 h, then decreasing, but still sustaining at a high level and reaching baseline on day five. These changes were accompanied by the upregulation of tumor necrosis factor (TNF)-alpha, interleukin-1beta (IL-1beta), interleukin 6 (IL-6) and interferon gamma (IFNgamma) mRNA expressions in the pancreas, but not by marked alterations of serum amylase, lactic dehydrogenase (LDH) and histology. Cerulein also increased PAP-I mRNA expression. However, the combination of cerulein and LPS was not able to enhance PAP-I mRNA expression further, although more prominent pancreatitis based on significant changes of serum amylase, LDH and histology were observed.. These results suggest that PAP-I mRNA might be modulated by endotoxemia, independent of cerulein-pancreatitis. There were no strong correlations between PAP-I mRNA expression and the severity of pancreatitis.

Topics: Acute Disease; Acute-Phase Proteins; Animals; Antigens, Neoplasm; Biomarkers, Tumor; Blotting, Northern; Ceruletide; Cytokines; Immunohistochemistry; Lectins, C-Type; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Pancreatitis; Pancreatitis-Associated Proteins; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

Endothelin-1 has been shown to reduce pancreatic blood flow and cause focal acinar cell necrosis similar to those seen in acute pancreatitis (AP), whereas therapy with endothelin receptor antagonists enhanced pancreatic capillary blood flow (PCBF) and decreased mortality rates. The current study evaluated the role of endothelin in the development of severe AP. Trypsinogen activation peptides, acinar cell necrosis, and PCBF were used as local indicators of disease severity, fluid sequestration, cardiorespiratory and renal parameters, and colonic capillary blood flow as systemic disease indicators. The following groups of animals were examined: 1) rats with mild edematous AP and 2) severe necrotizing AP treated with and without endothelin, 3) transgenic rats overexpressing endothelin with severe AP, and 4) rats with severe AP prophylactically treated with endothelin receptor antagonists. The following observations were made: endothelin superimposed on mild AP caused hemoconcentration, a decrease in PCBF, and necrosis and ascites not seen in this model without endothelin exposure. Endothelin superimposed on severe AP had no significant effects. After induction of severe AP, less PCBF and more acinar cell necrosis were observed in transgenic rats than in their normal littermates. Prophylactic endothelin receptor antagonists improved local (acinar necrosis, PCBF) and systemic parameters (ascites, urine production, colonic capillary blood flow) of disease severity in animals with severe AP. These observations underscore the role of endothelin as a mediator of disease severity in AP and suggest that endothelin receptor blockade may become a promising therapeutic tool in this disease.

Topics: Acute Disease; Animals; Animals, Genetically Modified; Blood Pressure; Capillaries; Ceruletide; Edema; Endothelin-1; Gene Expression; Hematocrit; Male; Pancreas; Pancreatitis; Pancreatitis, Acute Necrotizing; Rats; Rats, Sprague-Dawley

Complement factor C5a acting via C5a receptors (C5aR) is recognized as an anaphylotoxin and chemoattractant that exerts proinflammatory effects in many pathological states. The effects of C5a and C5aR in acute pancreatitis and in pancreatitis-associated lung injury were evaluated using genetically altered mice that either lack C5aR or do not express C5. Pancreatitis was induced by administration of 12 hourly injections of cerulein (50 microg/kg ip). The severity of pancreatitis was determined by measuring serum amylase, neutrophil sequestration in the pancreas, and acinar cell necrosis. The severity of lung injury was evaluated by measuring neutrophil sequestration in the lung and pulmonary microvascular permeability. In both strains of genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was greater than that noted in the comparison wild-type strains of C5aR- and C5-sufficient animals. This exacerbation of injury in the absence of C5a function indicates that, in pancreatitis, C5a exerts an anti-inflammatory effect. Potentially, C5a and its receptor are capable of both promoting and reducing the extent of acute inflammation.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Antigens, CD; Capillaries; Ceruletide; Complement C5a; Crosses, Genetic; Inflammation; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis; Peroxidase; Receptor, Anaphylatoxin C5a; Receptors, Complement

Prostaglandins (PG), the products of arachidonate metabolism through cyclooxygenase (COX) pathway, protect the pancreas from the acute damage. The existence of two isoforms of COX was documented including: COX-1, present in normal tissues and COX-2, expressed at the site of inflammation, such as induced by bacterial lipopolysaccharide (LPS). Pretreatment with low dose of LPS and activation of nitric oxide (NO) synthase (NOS) has been shown to prevent the injury caused by caerulein-induced pancreatitis (CIP) in the rat. The aim of this study was to investigate the role of COX-1 and COX-2 in the LPS-induced protection of the pancreas against CIP and the involvement of NOS in the activation of COX-PG system in the rats with CIP. CIP was produced by subcutaneous (s.c.) infusion of caerulein (5 microg/kg-h for 5 h) to the conscious rats. Protective dose of LPS, from Escherichia coli, (1 mg/kg) was given intraperitoneally (i.p.) 15 min prior to the start of CIP. Nonselective inhibitor of COX; indomethacin (5 or 10 mg/kg), selective inhibitor of COX-1: resveratrol, or a highly selective inhibitors of COX-2: rofecoxib or NS-398 (2 or 10 mg/kg) were injected i.p. 15 min prior to the administration of LPS. COX-1 or COX-2 mRNA was determined by reverse transcription-polimerase chain reaction (RT-PCR) in the pancreatic tissue. Pancreatic blood flow (PBF) was measured by a laser Doppler flowmetry. PGE2 content in the pancreas was measured by radioimmunoassay. CIP was manifested by an increase of pancreatic weight and plasma amylase activity (by 500% and 700%, respectively) and it was confirmed by histological examination. CIP slightly increased pancreatic PGE2 generation (by 12%) and diminished PBF (by about 40%). LPS (1 mg/kg i.p.), given prior to the start of CIP, increased PGE2 generation in the pancreas (by 45%), reversed the histological manifestations of pancreatitis, reduced the rise in amylase blood level and improved PBF. Administration of nonselective inhibitor of COX; indomethacin (5 or 10 mg/kg i.p.) prior to the injection of LPS abolished its protective effects on CIP and reduced pancreatic PGE2 generation. Selective inhibitor of COX-1; resveratrol (10 mg/kg i.p.) given prior to the injection of LPS reversed its protective effects against CIP. Pretreatment with a selective inhibitors of COX-2: rofecoxib or NS-398 (10 mg/kg) attenuated LPS-induced pancreatic protection in the CIP rats. COX-1 expression was detected in the intact pancreas and was not signif

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Ceruletide; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Inhibitors; Indomethacin; Isoenzymes; Lactones; Lipopolysaccharides; Male; Membrane Proteins; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Nitrobenzenes; Organ Size; Pancreas; Pancreatitis; Penicillamine; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Resveratrol; S-Nitroso-N-Acetylpenicillamine; Stilbenes; Sulfonamides; Sulfones

The molecular mechanisms that lead from acute pancreatitis (AP) to multiple organ failure remain to be clarified. We previously reported that ascitic fluids from a rat model of severe acute pancreatitis (pancreatitis-associated ascitic fluids, PAAF) transcriptionally activated endothelial cells and leukocytes in vitro. To clarify the role of ascitic fluids on the development of multiple organ failure in AP, we examined the effects of PAAF on the prognosis and immunohistologic findings in cerulein pancreatitis, an experimental model of mild pancreatitis in vivo. Intraperitoneal injection of PAAF decreased the survival rates in a dose-dependent manner. Histologically, destruction of vessels, alveolar septal thickening, interstitial hypertrophy, and infiltration of inflammatory cells were prominent in the lung of PAAF-injected rats. Transcription factor, nuclear factor KB (NF-kappaB) was activated and the mRNA levels of tumor necrosis factor-alpha and interleukin-1beta were increased in the lung of the PAAF-injected rats. The permeability index assessed by Evans blue assay and the lung myeloperoxidase activity levels were significantly higher in the PAAF-injected rats than in controls. Inhibition of NF-kappaB ameliorated the histologic findings and improved the survival rates. Our results suggest that PAAF play a role in the pathogenesis of lung injury in severe AP, at least in part through the activation of NF-kappaB.

Topics: Acute Disease; Animals; Ascites; Ascitic Fluid; Ceruletide; Disease Models, Animal; Evans Blue; Immunohistochemistry; Interleukin-1; Lung; Lung Diseases; Male; NF-kappa B; Pancreatitis; Peroxidase; Prognosis; Rats; Rats, Wistar; RNA, Messenger; Survival Rate; Tumor Necrosis Factor-alpha

Although the pancreatic heat shock response has already been reported to confer protective effects during experimental pancreatitis, the mechanism of action remains unknown. We investigated the effects of hyperthermia in cerulein-induced pancreatitis. Heat shock protein 70 (HSP70) expression in rats was induced by a 20-min period of water immersion (42 degrees C). The severity of pancreatitis as well as the pancreatic expression of cytokines, nuclear factor-kappaB (NF-kappaB), and inhibitory factor kappaB-alpha (IkappaB-alpha) were evaluated in the presence and absence of hyperthermia. We found that hyperthermia resulted in time-dependent expression of HSP70 within the pancreas associated with a reduction in the severity of acute pancreatitis. Tumor necrosis factor-alpha and intercellular adhesion molecule-1 expression was significantly reduced in the presence of hyperthermia. Moreover, NF-kappaB activity was delayed in the presence of hyperthermia whereas IkappaB-alpha was stabilized in the cytoplasm. These results suggest that hyperthermia decreases the severity of cerulein-induced pancreatitis by decreasing cytokine expression in the pancreas through the modulation of NF-kappaB activity.

Topics: Acute Disease; Animals; Ceruletide; Fever; HSP70 Heat-Shock Proteins; Intercellular Adhesion Molecule-1; Male; NF-kappa B; Osmolar Concentration; Pancreatitis; Rats; Rats, Wistar; Reference Values; Tumor Necrosis Factor-alpha

Taurine, or 2-aminoethane sulfonic acid, is an intracellular amino acid and has been suggested to have a function in protecting biological systems from oxidative tissue damage. The aim of this study was to determine the effect of taurine against cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by administering three subcutaneous injections of cerulein (40 microg/kg body weight) at 1-hour intervals, while taurine was administered intravenously at graded doses (30, 100, or 300 mg/kg, respectively) following the first cerulein injection. The severities of pancreatitis and lung injury were determined by measuring biochemical parameters, tissue myeloperoxidase (MPO), and histological changes. To clarify the mechanism of taurine, serum IL-1beta and TNF-alpha levels and tissue concentrations of malondialdehyde (MDA) were evaluated. In cerulein-induced acute edematous pancreatitis, treatment with taurine significantly decreased hyperamylasemia, tissue MPO, pancreatic edema, and the extent of pancreatic and pulmonary injury. Taurine decreased MDA concentration in the pancreas and lung, but not the serum cytokine concentration. We would conclude that taurine has beneficial effects in cerulein-induced acute pancreatitis and lung injuries by preventing the production of oxygen free radicals.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Disease Models, Animal; Interleukin-1; Lung Diseases; Male; Malondialdehyde; Organ Size; Oxidative Stress; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley; Taurine; Tumor Necrosis Factor-alpha

To investigate the effects of somatostatin analogs (SSa) on apoptosis of pancreatic acinar cells and apoptosis-regulated gene bax, and p53 in treating acute pancreatitis in mice.. In cerulein-induced pancreatitis, with or without treatment of somatostatin, analogs (Octreotide) in CD-1 (BALB/c x DBetaAlpha/1) mice, apoptosis of pancreatic acinar cells was detected by using the TdT-mediated dUTP nick-end labeling (TUNEL) method, and the expression of apoptosis-regulated gene bax and p53 was determined by using the streptavidin-peroxidase immunohistochemical technique and the RT-PCR method, respectively.. On HE staining, acinar cells in the pancreas showed pyknotic nuclei and the formation of apoptotic bodies, which are the typical morphological features of apoptosis. Regarding TUNEL use, the apoptotic index of pancreatic acinar cells in the non-treated group at 5 and 14 h after induction of acute pancreatitis was significantly lower than those of the SSa-treated group, respectively (P < 0.01). On immunohistochemistry and RT-PCR, there was an expression of neither bax nor p53 in normal pancreatic tissues. The expression of bax in the SSa-treated group at 5 and 14 h after treatment of SSa was markedly higher than those of the non-treated group, respectively (P < 0.01), but there was no significant difference in the expression of p53 between the SSa-treated group and the non-treated group.. The induction of apoptosis in pancreatic acinar cells injury to reduce inflammatory reaction might be one of the mechanisms of SSa in treating acute pancreatitis in mice, and the mechanisms of apoptosis probably correlated with the expression of apoptosis-regulated gene bax, but have no relationship with the expression of p53.

Topics: Acute Disease; Animals; Apoptosis; bcl-2-Associated X Protein; Ceruletide; Female; Genes, p53; Hormones; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Octreotide; Pancreatitis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Somatostatin

The interactions between inflammatory cells and their mediators play important roles in many inflammatory processes, but their importance during acute experimental pancreatitis and pancreatitis-associated lung injury is unclear. To address the role of the interaction between CD40 and its ligand CD40L, molecules that mediate major immunoregulatory functions, pancreatitis was induced by administering supramaximal doses of cerulein in mice that do not express CD40L.. The severity of pancreatitis was measured by serum amylase activity, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity (an indicator of neutrophil infiltration). Lung injury was quantitated by evaluating lung microvascular permeability and lung myeloperoxidase activity.. In pancreatic tissue from control mice and cerulein-treated mice, the expression of both CD40 and CD40L was detected. Immunohistochemical analysis performed in isolated acini from wild-type pancreata showed that both CD40 and CD40L were expressed on the acinar cell surface. Interestingly, pancreatitis and pancreatitis-associated lung injury were markedly decreased in mice deficient in CD40L compared with wild-types.. These observations indicate that CD40L plays an important proinflammatory role in pancreatitis and pancreatitis-associated lung injury.

Topics: Acute Disease; Amylases; Animals; CD40 Ligand; Ceruletide; Lung Diseases; Male; Mice; Pancreatitis; Tumor Necrosis Factor-alpha

The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.

Topics: Animals; Cell Division; Cells, Cultured; Ceruletide; Extracellular Matrix; Growth Substances; Male; Pancreas; Pancreatitis; Phenotype; Rats; Rats, Wistar; Reference Values

In experimental models of acute pancreatitis (AP), acinar cell death occurs by both necrosis and programmed cell death or apoptosis. Apoptosis is an active form of cell death associated with a tightly regulated expression of gene products that are either pro- or antiapoptotic. The aim of this study was to characterize pancreatic mRNA levels by Northern blotting analysis of apoptosis-associated genes used during the course of cerulein-induced AP in mice. Histone H3 mRNA levels were also examined as an indicator of cell proliferation. Acinar cell apoptosis was confirmed histologically. The findings show that AP modifies pancreatic mRNA levels of both pro- and antiapoptotic genes simultaneously. Pancreatic bclXL, bax, and p53 mRNA levels increased significantly in a temporal fashion during induction of AP. Pancreatic bcl-2 mRNA levels were unchanged during AP. Pancreatic mRNA levels of insulin-like growth factor-1 (IGF-1), a mitogen and cell survival factor, and its receptor (IGF-1R) also increased in a temporal fashion during induction of AP. In summary, this study indicates that acinar cell death during cerulein-induced AP in mice can occur by the apoptotic pathway. Since factors promoting and antagonistic for cell survival are activated simultaneously, regulation of acinar cell survival appears complex and dynamic during AP.

Topics: Acute Disease; Amylases; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Northern; Blotting, Western; Cell Survival; Ceruletide; Female; Genes, p53; Histones; Insulin-Like Growth Factor I; Mice; Pancreatitis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger

Acute severe (necrotizing) pancreatitis is often associated with pancreatic or peripancreatic infection. Decreased bacterial clearance due to impaired immune defense may cause local infection. We investigated expressions of surface opsonin receptors (CD11b, complement receptor 3; CD32/CD16, immunoglobulin G Fc receptor) on local and circulatory neutrophils, in murine acute pancreatitis. The mild and severe forms of acute pancreatitis were induced by seven and 13 subcutaneous injections of caerulein, respectively. Peritoneal exudative and circulatory neutrophils were counted and assayed for receptor expressions by flow cytometry, serially at 1-72 hours after pancreatitis induction. Histologically, mild and severe forms showed edematous and necrotizing pancreatitis, respectively. The peritoneal exudative neutrophil count was greater in mild than in severe pancreatitis. Expressions of CD11b and CD32/CD16 on local neutrophils were upregulated early in mild pancreatitis. This upregulation was attenuated in severe pancreatitis. The circulatory neutrophil count was elevated in severe pancreatitis but was unchanged in mild pancreatitis. Opsonin receptor expression on circulatory neutrophils showed a transient, modest upregulation in the early phase of mild pancreatitis. Receptor-positive circulatory neutrophils showed a marked elevation that persisted throughout the course of severe pancreatitis. In conclusion, severe (necrotizing) pancreatitis is associated with reduced opsonin receptor expression on local neutrophils and enhanced expression on circulatory neutrophils, as compared with mild (edematous) pancreatitis. These changes may contribute to local infectious complications and multiple organ failure, in severe pancreatitis.

Topics: Acute Disease; Animals; Ascitic Fluid; Ceruletide; Complement Activation; Disease Progression; Drug Administration Schedule; Edema; Female; Leukocyte Count; Macrophage-1 Antigen; Mice; Mice, Inbred BALB C; Neutrophils; Pancreatitis; Pancreatitis, Acute Necrotizing; Phagocytosis; Receptors, Fc; Receptors, IgG; Receptors, Immunologic

Investigation of pancreatic interstitial fibroblasts has proven difficult in situ. We have established a method for the isolation of pancreatic fibroblastoid/stellate cells by outgrowth from pancreatic tissue explanted into culture dishes. This technique gives a high yield of viable cells from small tissue samples. Outgrown fibroblastoid cells were established as a primary cell line and characterized during long-term culture. We investigated the development of stellate cell markers, i.e. fat storage, expression of desmin, and alpha-smooth muscle actin (alphaSMA), over weeks in culture. AlphaSMA, investigated by indirect immunofluorescence staining and Western blot analysis, revealed a constant rise in expression during routine culture. After 13 passages. approximately 100% of cells were positive for alphaSMA expression, indicating a myofibroblast type of differentiation in vitro.

Topics: Actins; Animals; Azo Compounds; Biomarkers; Blotting, Western; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Separation; Ceruletide; Coloring Agents; Contact Inhibition; Culture Media; Desmin; Fibroblasts; Fluorescent Antibody Technique, Indirect; Lipids; Male; Muscle, Smooth; Pancreas; Pancreatitis; Rats; Rats, Wistar; Staining and Labeling

Production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been proposed as a pathogenic factor in acute pancreatitis, but its role has still not been fully examined. The present study explored the role of iNOS in cerulein-induced acute pancreatitis using iNOS-deficient mice. Twelve- to 14-week-old male mice (C57B1/6 and iNOS-deficient) were administered cerulein by intraperitoneal (i.p.) injection at hourly intervals for 7 hours and killed 24 hours later after the first dose. Pancreatic wet weight, pancreatic myeloperoxidase (MPO) activity, and levels of plasma nitrite and serum amylase were measured. In another experiment isosorbide dinitrate (an NO donor) was given by oral gavage every 6 hours for 24 hours beginning simultaneously with cerulein injections in iNOS-deficient mice. Cerulein administration dose-dependently increased pancreatic wet weight, myeloperoxidase activity, and levels of nitrite and amylase in C57B1/6 mice. These parameters (except nitrite levels) were significantly intensified in iNOS-deficient mice. At the dose employed, cerulein failed to increase nitrite levels in iNOS-deficient mice. The susceptibility to cerulein toxicity in iNOS-deficient mice was abolished by NO donor treatment. NO release from an iNOS source appears to play a protective role in cerulein-induced pancreatitis. At least in part, NO may prevent neutrophil accumulation after cerulein administration.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Genetic Predisposition to Disease; Injections, Intraperitoneal; Isosorbide Dinitrate; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Pancreatitis; Peroxidase

An in vivo microscopic technique was used to clarify the increase in microvascular permeability and enhanced leukocyte-endothelium interaction of pancreatic microcirculation in experimental pancreatitis of differing severity. Using bovine albumin fluorescein isothiocyanate (FITC) and carboxyfluorescein diacetate succinimidyl ester (CFDASE) as tracers, the change in permeability and the behavior of leukocytes in the acinar microcirculation were quantified during the initial 1, 2, 6, and 12h after the induction of caerulein pancreatitis in mice. Cold stress was added to produce the severe model. It was revealed that the early microcirculatory changes in the pancreas of caerulein pancreatitis included the increased permeability of endothelial lining and an accumulation of extravasated fluid in the perilobular space, which were more severe if cold stress was added. A decrease in flow velocity was also noted 2h after the onset of severe pancreatitis. Leukocyte adherence to the endothelial cells was not observed during the first 12h in either model of severity. In contrast, observation of the hepatic microcirculation revealed a significant number of adherent leukocytes 2h after the induction of severe pancreatitis. These results suggest that during the early course of acute pancreatitis, leukocyte adherence in the pancreatic microcirculation is a secondary event following the increase in pancreatic vascular permeability.

Topics: Acute Disease; Amylases; Animals; Aspartate Aminotransferases; Blood Flow Velocity; Capillary Permeability; Cell Adhesion; Ceruletide; Disease Models, Animal; Endothelium, Vascular; Hemorheology; Leukocytes; Lipase; Male; Mice; Mice, Inbred ICR; Microcirculation; Microscopy, Fluorescence; Pancreas; Pancreatitis; Signal Processing, Computer-Assisted; Time Factors

Intra-acinar cell activation of digestive enzyme zymogens including trypsinogen is generally believed to be an early and critical event in acute pancreatitis. We have found that the phosphatidylinositol 3-kinase inhibitor wortmannin can reduce the intrapancreatic activation of trypsinogen that occurs during two dissimilar experimental models of rodent acute pancreatitis, secretagogue- and duct injection-induced pancreatitis. The severity of both models was also reduced by wortmannin administration. In contrast, the NF-kappa B activation that occurs during the early stages of secretagogue-induced pancreatitis is not altered by administration of wortmannin. Ex vivo, caerulein-induced trypsinogen activation is inhibited by wortmannin and LY294002. However, the cytoskeletal changes induced by caerulein were not affected by wortmannin. Concentrations of caerulein that induced ex vivo trypsinogen activation do not significantly increase phosphatidylinositol-3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate levels or induce phosphorylation of Akt/PKB, suggesting that class I phosphatidylinositol 3-kinases are not involved. The concentration of wortmannin that inhibits trypsinogen activation causes a 75% decrease in phosphatidylinositol 3-phosphate, which is implicated in vesicle trafficking and fusion. We conclude that a wortmannin-inhibitable phosphatidylinositol 3-kinase is necessary for intrapancreatic activation of trypsinogen and regulating the severity of acute pancreatitis. Our observations suggest that phosphatidylinositol 3-kinase inhibition might be of benefit in preventing acute pancreatitis.

Topics: Acute Disease; Androstadienes; Animals; Cells, Cultured; Ceruletide; Chromones; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Lysosomes; Male; Mice; Morpholines; Necrosis; NF-kappa B; Pancreatitis; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphorylation; Rats; Time Factors; Trypsinogen; Wortmannin

Hepatocyte growth factor (HGF) overexpression was reported in experimental and clinical acute pancreatitis. These observations prompted us to determine the effect of HGF administration on the development of caerulein-induced pancreatitis in rats. Acute pancreatitis was induced by s.c. infusion of caerulein (10 microg/kg/h) for 5 h. HGF was administrated twice (30 min before caerulein or saline infusion and 3 h later) at the doses: 0.4, 2, 10 or 50 microg/kg s.c. Immediately after cessation of caerulein or saline infusion, the pancreatic blood flow, plasma amylase and lipase activity, plasma cytokines concentration, cell proliferation, and morphological signs of pancreatitis were examined. Caerulein administration induced acute edematous pancreatitis manifested by 41% decrease in DNA synthesis, 53% inhibition of pancreatic blood flow, a significant increase in plasma amylase and lipase activity, plasma interleukin-1beta and interleukin-6 concentration, as well as, the development of the histological signs of pancreatic damage (edema, leukocyte infiltration, and vacuolization). Administration of HGF without induction of pancreatitis increased plasma interleukin-10. Treatment with HGF, during induction of pancreatitis, increased plasma interleukin-10 and attenuated the pancreatic damage, what was manifested by histological improvement of pancreatic integrity, the partial reversion of the drop in DNA synthesis and pancreatic blood flow, and the reduction in pancreatitis evoked increase in plasma amylase, lipase, and interleukin-1beta and interleukin-6 levels. HGF administrated at the dose 2 microg/kg exhibited a similar beneficial effect as administration of HGF at the doses 10 or 50 microg/kg. Treatment with HGF at the dose 0.4 microg/kg was less effective. We conclude that: (1) administration of HGF attenuates pancreatic damage in caerulein-induced pancreatitis; (2) this effect seems to be related to the increase in production of interleukin-10, the reduction in release of interleukin-1beta and interleukin-6, and the improvement of pancreatic blood flow.

Topics: Amylases; Animals; Ceruletide; Disease Models, Animal; Hepatocyte Growth Factor; Infusions, Intravenous; Interleukin-6; Lipase; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Regional Blood Flow; Time Factors

External perfusions of the pancreatic glands of Wistar-rats were done, using a modified Krebs-Ringer-Buffer (KRB). We looked for an elevation of amylase, lipase and lactate-dehydrogenase in the effusion fluid (portal outflow fluid). We investigated a normal perfusion (KRB, 60 minutes), a long term perfusion (KRB, 240 minutes) and a perfusion (60 minutes) including an additive of the detergents triton x-100 or the cholecystokinin analogue ceruletid (10(-8) M).. An isolated external perfusion of a rat pancreas is possible without inducing any increase of parameters of damage such as amylase, lipase or lactate-dehydrogenase in the outflow medium. The perfusion time should be limited to 80 minutes including a 20 minutes equilibration period. A damage of pancreatic parenchyma is indicated by increased levels of pancreatic enzymes in the perfusion medium. Such damage can be induced by various noxious substances like detergents or cerulein, which has a significance in the pathophysiology of experimental acute pancreatitis. A significant increase (p < 0.01) of lactate-dehydrogenase, lipase and amylase was found 10, 20 and 30 minutes after an application of triton x-100. During a perfusion with the cholecystokinin analogue ceruletid (10(-8) M) we found an increase of lipase (p < 0.05) after 30 minutes and an increase of amylase (p < 0.05) after 50 minutes perfusion.. The isolated perfused rat pancreas is a valuable experimental model to investigate the early phase of pathophysiology in acute pancreatitis.

Topics: Acute Disease; Amylases; Analysis of Variance; Animals; Ceruletide; Detergents; Gastrointestinal Agents; In Vitro Techniques; L-Lactate Dehydrogenase; Lipase; Male; Models, Animal; Pancreas; Pancreatitis; Perfusion; Polyethylene Glycols; Rats; Rats, Wistar; Time Factors

The pancreas of 24 male Wistar rats was perfused extracoporally by modified Krebs-Ringer-buffer for 80 minutes (including a 20 minutes equilibration period). To verify any organ damage we measured the activity of pancreatic enzymes like amylase, lipase and lactatdehydrogenase in the portal effluent. Furthermore histological changes were analysed after perfusion. Organ damage was induced by adding cerulein in a physiological dose (10(-10) M, n = 6) and in a supramaximal dose (10(-8) M, n = 6) and by intraductal injection of taurocholate (3.5 %, n = 6).. Already 10 minutes after stimulation with cerulein (10(-8) M) and after intraductal injection of taurocholate increased activities (p < 0.01) of amylase and lipase were measured in the portal effluent compared to the group without any treatment. Lactatdehydrogenase levels did not changed. Apart from marked oedema in both groups considerable zones of necrosis could be noticed especially in the taurocholate group.. Our data suggest that the isolated perfused rat pancreas (IPRP) is a valuable experimental tool to verify pathophysiological changes in the early phase of acute pancreatitis (AP). Various established models of AP such as by cerulein hyperstimulation or intraductal injection of taurocholate, could be applied to the IPRP. We conclude that this method enlarges the spectrum of established experimental models of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Cholagogues and Choleretics; Detergents; Disease Models, Animal; Gastrointestinal Agents; In Vitro Techniques; L-Lactate Dehydrogenase; Lipase; Male; Necrosis; Pancreas; Pancreatitis; Perfusion; Rats; Rats, Wistar; Taurocholic Acid; Time Factors

The function of primary sensory neurons is to receive and transmit information from external environment and these neurons are able to release neuromediators from the activated peripheral endings. The aim of this study was to determine the influence of sensory nerves and administration of their mediator--calcitonin gene related peptide (CGRP) on the course of acute pancreatitis (AP). Ablation of sensory nerves was performed by neurotoxic dose of capsaicin (100 mg/kg). Single or repeated episodes of AP were induced by caerulein infusion (10 microg/kg/h for 5 h). Five repeated AP were performed once a week. Capsaicin at the dose which stimulates sensory nerves (0.5 mg/kg/dose) or CGRP (10 microg/kg/dose) was administrated before and during or after single induction of AP, as well as, after each induction of repeated AP. Rats were killed at the time 0, 3 or 9 h after single induction of AP or two weeks after last induction of repeated AP. Ablation of sensory nerves aggravated pancreatic damage in caerulein-induced AP. Treatment with stimulatory doses of capsaicin or CGRP before and during single induction of AP attenuated the pancreatic damage in morphological examination. This effect was also manifested by partial reversion of AP evoked drop in DNA synthesis and pancreatic blood flow (PBF). Administration of CGRP after single AP induction aggravated histologically manifested pancreatic damage. The further decrease in PBF and DNA synthesis was also observed. Animals with five episodes of AP showed almost full pancreatic recovery two weeks after last induction of AP concerning all parameters tested. In stimulatory doses of capsaicin treated rats, we observed the decrease in pancreatic amylase and fecal chymotrypsin activity, as well as, the drop in DNA synthesis. Similar but less pronounced effects were observed after treatment with CGRP. We conclude that effect of sensory nerves and CGRP on AP is two-phase and time dependent. Stimulation of sensory nerves or the administration of CGRP during development of AP exhibits protective effects against pancreatic damage induced by caerulein overstimulation. After induction of AP, persistent activity of sensory nerves and presence of CGRP aggravate pancreatic damage and lead to functional insufficiency typical for chronic pancreatitis.

Topics: Acute Disease; Amylases; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Ceruletide; DNA; Interleukin-1; Male; Neurons, Afferent; Nitric Oxide; Pancreas; Pancreatitis; Rats; Rats, Wistar

To examine the role of p38 during acute experimental cerulein pancreatitis.. Rats were treated with cerulein with or without a specific JNK inhibitor (CEP1347) and/or a specific p38 inhibitor (SB203580) and pancreatic stress kinase activity was determined. Parameters to assess pancreatitis included trypsin, amylase, lipase, pancreatic weight and histology.. JNK inhibition with CEP1347 ameliorated pancreatitis, reducing pancreatic edema. In contrast, p38 inhibition with SB203580 aggravated pancreatitis with higher trypsin levels and, with induction of acinar necrosis not normally found after cerulein hyperstimulation. Simultaneous treatment with both CEP1347 and SB203580 mutually abolished the effects of either compound on cerulein pancreatitis.. Stress kinases modulate pancreatitis differentially. JNK seems to promote pancreatitis development, possibly by supporting inflammatory reactions such as edema formation while its inhibition ameliorates pancreatitis. In contrast, p38 may help reduce organ destruction while inhibition of p38 during induction of cerulein pancreatitis leads to the occurrence of acinar necrosis.

Topics: Acute Disease; Animals; Carbazoles; Ceruletide; Enzyme Inhibitors; Imidazoles; Indoles; Mitogen-Activated Protein Kinases; Models, Animal; Necrosis; p38 Mitogen-Activated Protein Kinases; Pancreatitis; Pyridines; Rats; Trypsin

The aim of this paper was to evaluate the influence of nifedipine (calcium channel blocker) and Bay-K-8644 (calcium channel agonist) on cerulein acute pancreatitis (AP) in rats. AP was induced according to the Lampel and Kern method (1) by the continuous intravenous infusion of cerulein in the doses of 5 x 10(-6) g/kg/h for 12 hours. There was obtained a statistically significant decrease in serum amylase activity and pancreatic weight in the groups treated with higher doses of nifedipine before infusion of the cerulein compared with rats treated only with cerulein. However, in the group treated with Bay-K-8644 before infusion of cerulein statistically significant increase was obtained in serum amylase activity and pancreatic weight compared with the group treated only with cerulein. The investigations suggest a beneficial effect of higher doses of nifedipine on cerulein induced AP. The inflammatory changes in the pancreas in the groups treated with nifedipine observed under the light microscope were smaller than in the group treated only with cerulein.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Amylases; Animals; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Ceruletide; Cholecystokinin; Male; Nifedipine; Organ Size; Pancreas; Pancreatitis; Rats; Rats, Wistar; Second Messenger Systems

In this study we aimed to clarify the role of mast cells in the development and progression of inflammation in cerulein-induced acute pancreatitis (AP) in rats. We have also examined the effects of ketotifen; a mast-cell stabilizing agent in the treatment of acute pancreatitis and its relation with nitric oxide (NO) synthesis.. In the first part of the study we planned to examine the effects mast cell stabilization in acute pancreatitis, while the second part was focused on examining the relation between NO synthesis and the potential effects of ketotifen in AP. Wistar albino rats were randomly divided into 6 groups (n: 10). In the first part of the study, AP was induced by four subcutaneous (sc) injections of 20 microg/kg body weight of cerulein at hourly intervals in Groups A and B while Group C was treated with saline as the control group. Group B was pretreated with ketotifen 1 mg/kg (ip). In the second part, the study design was similar except for the inhibition of nitric oxide synthesis by N-nitro L-arginine methyl ester (L-NAME) 30 mg/kg (ip) in Groups D, E and F. Group D was treated with L-NAME and cerulein and Group E was treated with ketotifen, L-NAME and cerulein. Group F was treated with L-NAME and saline as the control group. Serum amylase activity and pancreatic myeloperoxidase activity (MPO) were measured. Pancreatic histology and mast-cell count in pancreatic tissue were evaluated.. Mast cell count was found to be increased in the pancreatic tissue in cerulein-induced AP. (2.93+/-0.26 vs 1.98+/-0.26; p<0.001). Ketotifen treatment significantly reduced cerulein induced edema (1.30+/-0.21 vs 0.70+/-0.15; p<0.001), neutrophil infiltration (1.50+/-0.16 vs 0.60+/-0.16; p<0.001) and attenuated the increase in amylase (4394.0+/-149.5 U/L vs 3350.5+/-216.9 U/L; p<0.05) and MPO activity (1.14+/-0.13 U/gr tissue vs 0.54+/-0.08 U/gr tissue; p<0.001). Mast-cell count in pancreatic tissue was also decreased significantly with ketotifen pretreatment (2.93+/-0.26 vs 1.70+/-0.21; p<0.05). Inhibition of NO synthesis with L-NAME treatment decreased the beneficial effects of ketotifen.. It seems likely that mast cell activity may play an important role in the initiation and progression of acute pancreatitis. Ketotifen treatment may reduce the severity of AP in rats. The protective action of ketotifen in cerulein-induced acute pancreatitis is most probably owing to mast cell stabilization and stimulation of NO synthesis.

Topics: Acute Disease; Animals; Anti-Allergic Agents; Ceruletide; Drug Therapy, Combination; Enzyme Inhibitors; Ketotifen; Male; Mast Cells; NG-Nitroarginine Methyl Ester; Pancreatitis; Rats; Rats, Wistar

We previously reported that serum hepatocyte growth factor (HGF) levels are elevated in patients with acute pancreatitis and that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s) inducing apoptosis on Madin-Darby canine kidney (MDCK) cells. In this study, plasma HGF levels and HGF tissue distribution were investigated in rats with experimental acute pancreatitis, and the effects of HGF on the cytotoxic activity and apoptosis-inducing activity of PAAF also were examined. Plasma HGF levels were elevated in rats with two experimental pancreatitis models of different grades of severity. The degree of its elevation was correlated with the severity and the organ dysfunctions. In rats with severe pancreatitis, HGF protein and messenger RNA (mRNA) levels significantly increased in liver, kidney, and lung, which were injured organs. When anti-HGF neutralizing antibody was administered in severe pancreatitis, liver dysfunction worsened, and apoptotic cells increased in kidney. Recombinant HGF inhibited the cytocidal activity of PAAF on MDCK cells in a dose-dependent manner. Moreover, recombinant HGF prevented the apoptotic cell death (DNA fragmentation, nuclear fragmentation, and caspase-3 activation) induced by PAAF. These results suggest that HGF is produced in injured organs and may function as an organotrophic and antiapoptotic factor against the organ injuries in acute pancreatitis.

Topics: Acute Disease; Animals; Apoptosis; Caspase 3; Caspases; Cell Line; Ceruletide; Deoxycholic Acid; DNA Fragmentation; Dogs; Edema; Hepatocyte Growth Factor; Kidney; Liver; Lung; Male; Multiple Organ Failure; Pancreatitis; Pancreatitis, Acute Necrotizing; Rats; Rats, Wistar; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen

Pancreatic caerulein-induced activation of c-Jun NH(2)-terminal kinase (JNK) has been reported, and JNK has been proposed as a mediator during induction of hyperstimulated pancreatitis. CEP-1347 has recently been described as a specific JNK inhibitor. We tested whether CEP-1347 inhibits caerulein-induced pancreatic JNK activation in isolated acini and in vivo. CEP-1347 dose dependently inhibited acinar caerulein-induced JNK activation with nearly complete inhibition at 2 microM but had no effect on digestive enzyme release. For in vivo studies, rats were pretreated with CEP-1347 before caerulein hyperstimulation. For assessment of JNK activation and histological alterations, animals were killed 30 min or 2 and 4 h after caerulein hyperstimulation, respectively. Pancreatic wet weight, serum enzyme levels, and pancreatic activity of p38 and extracellular signal-regulated kinase (ERK) were also determined. Caerulein hyperstimulation strongly activated JNK, p38, and ERK. CEP-1347 pretreatment dose dependently reduced caerulein-induced pancreatic JNK activation without p38 or ERK inhibition. JNK inhibition also reduced pancreatic edema formation and reduced histological severity of pancreatitis. Thus we show that CEP-1347 inhibits JNK activation in vivo and ameliorates caerulein-induced pancreatitis.

Topics: Amylases; Animals; Carbazoles; Ceruletide; Dose-Response Relationship, Drug; Edema; Enzyme Activation; Enzyme Inhibitors; In Vitro Techniques; Indoles; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; Pancreas; Pancreatic Diseases; Pancreatitis; Rats; Rats, Sprague-Dawley

Activation of the receptor c-met stimulates motility, mitosis, morphogenesis, processes involved in organ regeneration, or progression of malignancies. In the present study we investigated the expression of c-met protein in the regenerating pancreas and characterized the influence of cytokines on c-met expression.. Acute pancreatitis was induced in rats by cerulein injection. Rat acini and rat and human pancreatic cancer cells were stimulated with interleukin-1alpha (IL-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). C-met expression was analyzed by means of Western blotting and localization in pancreatic tissue by immunohistochemistry.. C-met protein expression was significantly upregulated in the regenerating pancreas and localized in areas of regenerating tissue. Stimulation with cytokines resulted in a two- to threefold increase of c-met expression in vitro.. Enhanced c-met expression after acute pancreatitis suggests that HGF/met has an important role in pancreatic regeneration, which is probably mediated by cytokines. This regulatory mechanism is also of importance in pancreatic cancer.

Topics: Acute Disease; Animals; Blotting, Western; Cells, Cultured; Ceruletide; Cytokines; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Interleukin-1; Interleukin-6; Male; Pancreas; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins c-met; Rats; Rats, Wistar; Regeneration; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

To elucidate whether pancreatic acinar cell submitted to stress is able to express TNF-alpha, we studied TNF-alpha mRNA expression by Northern blot and in situ hybridization in healthy pancreas, in tissue from caerulein-induced pancreatitis and after lipopolysaccharide (LPS) treatment. In specimens from normal pancreas, TNF-alpha mRNA expression, as judged by both Northern blot and in situ hybridization, was negative, whereas a strong but transient expression was observed in acinar cells from caerulein pancreatitis and LPS treatment. TNF-alpha mRNA appeared as rapidly as 30 min after treatment, and was maximal 6 h after. At this time, there was mild infiltration consisting mostly of polymorphonuclear leukocytes (PMNL) and no signal of TNF-alpha transcript was found in their cytoplasm. Our results strongly indicate that pancreatic acinar cell is the source of TNF-alpha early in the course of acute pancreatitis and LPS treatment, and suggest that the expression of this cytokine is a part of a general response of the acinar cell to aggression.

Topics: Animals; Ceruletide; Gene Expression Regulation; In Situ Hybridization; Lipopolysaccharides; Male; Mice; Pancreas; Pancreatitis; Tumor Necrosis Factor-alpha

Topics: Animals; Ceruletide; Enteropeptidase; Pancreas; Pancreatitis; Rats; Trypsinogen

Pancreatic stellate cells may be a major source of extracellular matrix deposition during injury. This study was undertaken to establish whether pancreatic stellate cells are a source of Type I collagen in vivo and whether they continue to be a source of matrix production in the post-injury fibrotic pancreas. To induce pancreatic fibrogenesis, acute pancreatic injury was induced in mice three times weekly with supraphysiologic doses of cerulein. Animals were treated for 6 weeks and allowed to recover for an additional 6 weeks. Stellate cell activation and pancreatic collagen expression were measured by immunohistochemistry, whole tissue RNA analysis, and in situ hybridization. Histology and digital image analysis demonstrated the development of substantial pancreatic fibrosis after 6 weeks of treatment. During recovery, incomplete resolution of the fibrosis was found. Procollagen alpha1(I) mRNA increased more than 15-fold during treatment and continued to be 5-fold elevated during the post-injury phase. In situ hybridization studies demonstrated that collagen gene expression was colocalized to activated pancreatic stellate cells. Collagen expression and fibrosis persisted in focal areas during recovery. These findings show that pancreatic stellate cells are the major source of collagen during repetitive injury in vivo. Additionally, focal areas of sustained pancreatic fibrogenesis persist after cessation of cerulein treatment, and these areas may contribute to sustained total organ collagen expression in the absence of ongoing injury.

Topics: Acute Disease; Animals; Ceruletide; Female; Immunohistochemistry; In Situ Hybridization; Mice; Pancreas; Pancreatitis; Procollagen; RNA, Messenger

Few data are available on the potential role of T lymphocytes in experimental acute pancreatitis. The aim of this study was to characterize their role in the inflammatory cascade of acute pancreatitis.. To type this issue, acute pancreatitis was induced by repeated injections of cerulein in nude mice and in vivo CD4(+) or CD8(+) T cell-depleted mice. The role of T lymphocyte-costimulatory pathways was evaluated using anti-CD40 ligand or anti-B7-1 and -B7-2 monoclonal blocking antibodies. The role of Fas-Fas ligand was explored using Fas ligand-targeted mutant (generalized lymphoproliferative disease) mice. Severity of acute pancreatitis was assessed by serum hydrolase levels and histology. Intrapancreatic interleukin 12, interferon gamma, Fas ligand, and CD40 ligand messenger RNA were detected by reverse-transcription polymerase chain reaction. Intrapancreatic T lymphocytes were identified by immunohistochemistry.. In control mice, T cells, most of them CD4(+) T cells, are present in the pancreas and are recruited during acute pancreatitis. In nude mice, histological lesions and serum hydrolase levels are significantly decreased. T-lymphocyte transfer into nude mice partially restores the severity of acute pancreatitis and intrapancreatic interferon gamma, interleukin 12, and Fas ligand gene transcription. The severity of pancreatitis is also reduced by in vivo CD4(+) (but not CD8(+)) T-cell depletion and in Fas ligand-targeted mutant mice. Blocking CD40-CD40 ligand or B7-CD28 costimulatory pathways has no effect on the severity of pancreatitis.. T lymphocytes, particularly CD4(+) T cells, play a pivotal role in the development of tissue injury during acute experimental pancreatitis in mice.

Topics: Acute Disease; Animals; B7-1 Antigen; CD28 Antigens; CD4-Positive T-Lymphocytes; CD40 Antigens; Ceruletide; Fas Ligand Protein; Female; Ligands; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Pancreas; Pancreatitis; Reference Values

The role of repetitive acute injury in the pathogenesis of chronic pancreatitis remains unknown. To determine if repetitive injury induced by pancreatic hyperstimulation would reproduce the characteristic features of human chronic pancreatitis, acute reversible pancreatic injury was induced in mice by twice weekly cerulein treatment, 50 microg/kg/hr x 6 hr, for 10 weeks. Procollagen alpha1(I) mRNA was markedly increased by week 2. Sirius red staining of interstitial collagen demonstrated progressive accumulation of extracellular matrix surrounding acinar units and in interlobular spaces. Atrophy, transdifferentiation of acinar units to ductlike tubular complexes, and dilatation of intraacinar lumina also developed. Electron microscopy demonstrated the presence of stromal cells in areas of fibrosis with morphologic characteristics of pancreatic stellate cells. These findings demonstrate that, in a murine model, repetitive acute injury to the pancreas by hyperstimulation can reproduce the major morphological characteristics of human chronic pancreatitis.

Topics: Acute Disease; Animals; Atrophy; Ceruletide; Chronic Disease; Extracellular Matrix; Female; Fibrosis; Gene Expression Regulation; Humans; Mice; Microscopy, Electron; Pancreatitis; Procollagen

Recent observations suggest that immune response is involved in the development of pancreatitis. However, the exact pathogenesis underlying this immune-mediated response is still under debate. TGF-beta has been known to be an important regulating factor in maintaining immune homeostasis. To determine the role of TGF-beta in the initiation or progression of pancreatitis, TGF-beta signaling was inactivated in mouse pancreata by overexpressing a dominant-negative mutant form of TGF-beta type II receptor in the pancreas, under control of the pS2 mouse trefoil peptide promoter. Transgenic mice showed marked increases in MHC class II molecules and matrix metalloproteinase expression in pancreatic acinar cells. These mice also showed increased susceptibility to cerulein-induced pancreatitis. This pancreatitis was characterized by severe pancreatic edema, inflammatory cell infiltration, T- and B-cell hyperactivation, IgG-type autoantibodies against pancreatic acinar cells, and IgM-type autoantibodies against pancreatic ductal epithelial cells. Therefore, TGF-beta signaling seems to be essential either in maintaining the normal immune homeostasis and suppressing autoimmunity or in preserving the integrity of pancreatic acinar cells.

Topics: Animals; Autoantibodies; Autoimmune Diseases; B-Lymphocytes; Ceruletide; Cytokines; Female; Gene Expression; Histocompatibility Antigens Class II; Immunoglobulin G; Immunoglobulin M; Male; Mice; Mice, Transgenic; Mutagenesis; Pancreatitis; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta

Reactive oxygen radicals, nitric oxide, and cytokines have been implicated in the initiation of pancreatic tissue damage and impairment of the pancreatic microcirculation in acute pancreatitis. Pentoxifylline is a methylxanthine derivative with rheologic and marked anti-inflammatory properties and inhibits the production of proinflammatory cytokines. We have examined whether pentoxifylline ameliorates interstitial edema, inflammatory infiltrate, and glutathione depletion associated with cerulein-induced pancreatitis. Cotreatment of animals with pentoxifylline significantly reduced cerulein-induced pancreatic inflammation and edema and attenuated the depletion of pancreatic glutathione and the increase in serum lipase activity, nitrate, and tumor necrosis factor-alpha levels. Pentoxifylline also prevented both mitochondrial swelling and damage to mitochondrial cristae caused by cerulein. Our findings provide an experimental basis for using pentoxifylline to attenuate inflammatory responses within the pancreas in acute pancreatitis and as an adjuvant in the treatment of acute pancreatitis.

Topics: Animals; Antioxidants; Ceruletide; Dose-Response Relationship, Drug; Edema; Gastrointestinal Agents; Glutathione; Lipase; Male; Microscopy, Electron; Nitric Oxide; Oxidation-Reduction; Pancreas; Pancreatitis; Pentoxifylline; Phosphodiesterase Inhibitors; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

We investigated whether the timing of administration of contrast medium after onset of acute pancreatitis is critical in determining the magnitude of microcirculatory derangement.. An acute pancreatitis model in male Sprague-Dawley rats (225-275 g) was established by continuous infusion of cerulein (15 mg/kg per hour). The mean arterial pressure was monitored continuously by means of a femoral artery catheter. Diatrizoate (Hypaque-76), a water-soluble contrast medium, was delivered through a femoral vein catheter at doses corresponding to those given to humans, either 1, 2, or 3 hours after pancreatitis induction. In vivo microscopy and laser-Doppler flowmetry were used to investigate microcirculatory derangement. The water contents of the pancreas and lung, the malondialdehyde levels of the pancreas, and the trypsinogen activation peptide levels in the serum were measured at the end of the experiment (8 hours after infusion of cerulein).. Early administration of contrast medium (1 hour after pancreatitis induction) resulted in significantly greater changes in microcirculation and mean arterial pressure than did late administration (2 or 3 hours after pancreatitis induction). Rats given contrast medium 1 hour after induction also had highest pancreas and lung water contents, the highest pancreas malondialdehyde levels, and the highest serum trypsinogen activation peptide levels.. These results show that a water soluble contrast medium that is often used for computed tomographic imaging of the pancreas can adversely affect the pancreatic microcirculatory parameters, such as tissue perfusion and leukocyte sticking, and hemodynamics in a cerulein-induced model of acute pancreatitis. Early administration seems to cause more severe derangement of the pancreatic microcirculation.

Topics: Acute Disease; Animals; Blood Pressure; Ceruletide; Contrast Media; Diatrizoate; Laser-Doppler Flowmetry; Male; Malondialdehyde; Microcirculation; Microscopy; Oligopeptides; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Trypsinogen

Pancreatic oedema occurs early in the development of acute pancreatitis, and the overall extent of fluid loss correlates with disease severity. The tachykinin substance P (SP) is released from sensory nerves, binds to the neurokinin-1 receptor (NK1-R) on endothelial cells and induces plasma extravasation, oedema, and neutrophil infiltration, a process termed neurogenic inflammation. We sought to determine the importance of neurogenic mechanisms in acute pancreatitis. Pancreatic plasma extravasation was measured using the intravascular tracers Evans blue and Monastral blue after administration of specific NK1-R agonists/antagonists in rats and NK1-R(+/+)/(-/-) mice. The effects of NK1-R genetic deletion/antagonism on pancreatic plasma extravasation, amylase, myeloperoxidase (MPO), and histology in cerulein-induced pancreatitis were characterized. In rats, both SP and the NK1-R selective agonist [Sar(9) Met(O(2))(11)]SP stimulated pancreatic plasma extravasation, and this response was blocked by the NK1-R antagonist CP 96,345. Selective agonists of the NK-2 or NK-3 receptors had no effect. In rats, cerulein stimulated pancreatic plasma extravasation and serum amylase. These responses were blocked by the NK1-R antagonist CP 96,345. In wildtype mice, SP induced plasma extravasation while SP had no effect in NK1-R knockout mice. In NK1-R knockout mice, the effects of cerulein on pancreatic plasma extravasation and hyperamylasemia were reduced by 60%, and pancreatic MPO by 75%, as compared to wildtype animals. Neurogenic mechanisms of inflammation are important in the development of inflammatory oedema in acute interstitial pancreatitis.

Topics: Acute Disease; Amylases; Animals; Blood Pressure; Ceruletide; Edema; Gastrointestinal Agents; Inflammation; Male; Mice; Neurokinin-1 Receptor Antagonists; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Substance P

Basic fibroblast growth factor (bFGF) is one of the mitogens that facilitate endothelial proliferation and angiogenesis. This study was designed to examine the therapeutic effect of bFGF on experimental pancreatitis in rat. Edematous pancreatitis was induced by intraperitoneal injections of cerulein (50 microg/kg) at hourly intervals. BFGF (70 nmol/kg) was administered intraperitoneally after induction of pancreatitis. DNA synthesis of isolated pancreatic acinar cells of normal rats was determined as the uptake of 5-bromo-2'-deoxyuridine (BrdU) into the cells. Immunohistochemical staining of DNA synthesis in acinar cells during cerulein-induced pancreatitis was also examined with BrdU labeling in vivo technique. Cerulein administration increased serum amylase, lipase level, and wet weight of pancreatic tissue. Treatment with bFGF markedly ameliorated all these parameters. In primary culture system of isolated pancreatic acinar cells of normal rats, bFGF caused a dose-dependent increase in BrdU incorporation into DNA, showing an EC50 value of 0.8 nmol/L and a maximum response of 2.5-fold increase at a concentration of 400 nmol/L. bFGF treatment (70 nmol/kg) markedly increased BrdU labeling in the nucleus of acinar cells of the pancreatitis rats group in immunohistochemical examination when compared with control without bFGF treatment. Treatment with bFGF may represent a promising therapeutic concept for patients with acute pancreatitis.

Topics: Animals; Bromodeoxyuridine; Cell Division; Ceruletide; DNA; Edema; Fibroblast Growth Factor 2; Immunohistochemistry; Male; Pancreatitis; Rats; Rats, Wistar

Topics: Animals; Ceruletide; Enzyme Activation; Pancreas; Pancreatitis; Rats; Trypsinogen

The connective tissue component hyaluronan is accumulated locally in the damaged tissue during various inflammatory conditions. Owing to the strong water-binding capacity of this glycosaminoglycan, increased tissue content of hyaluronan is paralleled by the development of interstitial edema. The aim with the current experiment was to investigate whether hyaluronan is accumulated in acute pancreatitis and if increased levels of hyaluronan can be correlated to the inflammation of the pancreatic tissue.. Acute pancreatitis was induced in Sprague-Dawley rats by the administration of supramaximal doses of the cholecystokinin analogue caerulein. The animals were followed for 5 hours (n = 4), 24 hours (n = 6), or 48 hours (n = 5), and the pancreata were then investigated for hyaluronan and water content, hyaluronan distribution, general morphology and the presence of CD44-positive cells, macrophages, and T lymphocytes.. Hyaluronan accumulated in the edematous interstitium during acute pancreatitis. Twenty-four hours after the induction of pancreatitis, the hyaluronan content of the pancreata had increased by more than 100%. Simultaneously, CD44-positive cells infiltrated the tissue. However, no correlation between hyaluronan and water was seen at any time point.. This study shows that acute pancreatitis is associated with a strong but transient increase in interstitial hyaluronan and an infiltration of CD44-positive cells located mainly in the same region as the accumulated hyaluronan.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Edema; Hyaluronic Acid; Hyaluronoglucosaminidase; Male; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Water

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Edema; Humans; Hyaluronic Acid; Pancreatitis; Rats

We examined the influence of endogenous and exogenous epidermal growth factor (EGF) on pancreatic repair after acute pancreatitis. Caerulein-induced pancreatitis was evoked in rats with intact or removed salivary glands and EGF (10 microg/kg) was administered starting 24 h after cessation of caerulein infusion. The dose of EGF 10 microg/kg was chosen because it was the most effective in preliminary experiments when 1, 10 or 50 microg/kg of EGF was used. Caerulein administration caused acute edematous pancreatitis with biochemical and histological manifestation of pancreatic damage, followed by spontaneous regeneration. The effect of salivectomy on the course of acute pancreatitis was slight, resulting in additional reduction in pancreatic blood flow, DNA synthesis and in an increase in plasma interleukin 1beta level. Treatment with EGF accelerated the healing of pancreatic damage, causing an increase in pancreatic blood flow and DNA synthesis. EGF caused faster normalization of plasma amylase and lipase activity and plasma interleukin 1beta concentration, as well as, this peptide accelerated the restoration of pancreatic amylase activity. On histological examination, EGF caused reduction of pancreatic damage and acceleration of tissue repair. We conclude that EGF reduces the severity of pancreatic damage evoked by caerulein-induced pancreatitis-related pancreatic damage and accelerates tissue repair. The beneficial effects of EGF appear to depend, at least in part, on the improvement of pancreatic blood flow, as well as on an increase of pancreatic cell growth and limitation of the activation cytokine release.

Topics: Amylases; Animals; Ceruletide; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; Interleukin-1; Lipase; Male; Organ Size; Pancreas; Pancreatitis; Rats; Rats, Wistar; Regional Blood Flow; RNA; Time Factors

The exocrine pancreas synthesizes and secretes large amounts of digestive proteases as inactive precursor zymogens. Under physiological conditions a variety of cellular defense mechanisms protect the pancreatic acinar cell against a premature and intracellular activation of these zymogens. When these defenses fail, pancreatic autodigestion is initiated and acute pancreatitis can develop. A number of experimental observations suggest that extra- as well as intracellular calcium concentrations play an important part in the initiation of pancreatic protease activation, but the intracellular signaling events that regulate this process are unknown. Using a model system in which we used pancreatic acini (freshly prepared functional units of living acinar cells), we were able to simulate the conditions found during experimental pancreatitis in rodents. By means of a cell permeant fluorescent trypsin substrate we could demonstrate in these acini that premature protease activation is initiated at the apical acinar cell pole and occurs only in the presence of secretagogue concentrations that exceed those required for a maximum secretory response. By combining this technique with fluorescence ratio imaging for the Ca(2+)-sensitive dye fura-2, we could further show that this protease activation is highly dependent on the spatial as well as the temporal distribution of the corresponding Ca(2+) release from stores within the same subcellular compartment and that it is not propagated to neighboring acinar cells.

Topics: Animals; Calcium; Calcium Signaling; Ceruletide; Chelating Agents; Dose-Response Relationship, Drug; Egtazic Acid; Endopeptidases; Enzyme Activation; Fura-2; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar

Whether acute pancreatitis induced by cerulein was aggravated in human interleukin 6 (IL-6) transgenic mice and whether a specific anti-IL-6 receptor antibody improved pancreatitis were investigated. To induce acute pancreatitis, cerulein (50 microg/kg, seven injections) with or without 1 mg/kg lipopolysaccharides (LPS) was injected intraperitoneally every hour. In some mice, a monoclonal anti-IL-6 receptor antibody was administered before the first cerulein injection. The animals were killed 1 hour after the last injection. The pancreatic wet weight induced by cerulein alone was significantly higher in IL-6 transgenic mice compared with wild-type mice, but pretreatment with a specific anti-IL-6 receptor antibody did not reduce interstitial edema. When cerulein was administered with LPS, the pancreatic wet weight increased much more than when pancreatitis was induced by cerulein alone in both genotypes, and pretreatment with the anti-IL-6 receptor antibody decreased the pancreatic edema only in human-IL-6 transgenic mice. These results suggest that anticytokine antibodies may be effective in improving acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Antibodies, Monoclonal; Ceruletide; Edema; Humans; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Organ Size; Pancreas; Pancreatitis; Receptors, Interleukin-6

Heat shock proteins (Hsps), induced by cell stress, are known to protect against cellular injury. Recent studies have indicated that Hsp60 expression, induced by exposure to water immersion stress, protects against pancreatitis induced by administration of supramaximal doses of cerulein in rats. However, the mechanisms responsible for this protection are not known.. Rats were water-immersed for 3-12 hours. Pancreatitis was induced by cerulein administration.. The results confirm that prior induction of Hsp60 expression by water-immersion stress significantly ameliorates the severity of cerulein-induced pancreatitis as judged by the markedly reduced degree of hyperamylasemia, pancreatic edema, and acinar cell necrosis. Water immersion also prevents the subcellular redistribution of cathepsin B from a lysosome-enriched fraction to a heavier, zymogen granule-enriched fraction that is known to occur in this model of pancreatitis. Intra-acinar cell activation of trypsinogen that occurs shortly after exposure to a supramaximally stimulating dose of cerulein both in vivo and in vitro is prevented by prior water-immersion stress and Hsp60 expression. The protection against pancreatitis that follows water-immersion stress is not caused by alterations of cholecystokinin receptors, because water immersion does not alter the typical biphasic amylase secretory response to stimulation with cerulein.. Water-immersion stress induces Hsp60 expression, ameliorates cerulein-induced pancreatitis, and prevents intra-acinar cell activation of trypsinogen. We suggest that Hsp60 protects against cerulein-induced pancreatitis by preventing trypsinogen activation within acinar cells.

Topics: Amylases; Animals; Ceruletide; Chaperonin 60; Immersion; Immunohistochemistry; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Stress, Physiological; Time Factors; Tissue Distribution; Water

Lipopolysaccharides (LPS), the component of the cell wall of gram-negative bacteria, have been implicated in the pathogenesis of acute pancreatitis, but the mechanism of their action on the pancreas has not been fully explored. The aim of this study was to investigate the effects of various doses of LPS on the integrity of intact pancreas and that involved in acute caerulein-induced pancreatitis (CIP) in the rat and to compare these effects with those of nitric oxide (NO) donor, S-nitrose-acetylpenicillamine (SNAP). The expression of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) mRNA was also examined in the isolated pancreatic acini obtained from the inflamed pancreas of rats treated with LPS. CIP was produced by subcutaneous (s.c.) infusion of caerulein (5 microg/kg.h for 5 h) to conscious rats. Bolus injections of various doses of LPS (0.1, 1, 10, 20 or 40 mg/kg) or SNAP (1.5, 3 or 6 mg/kg) were made intraperitoneally (i.p.) either alone or 30 min prior to s.c. infusion of caerulein to induce CIP. Infusion of caerulein produced acute pancreatitis confirmed by histological examination and manifested by an increase of pancreatic mass (by about 200%). Blood levels of amylase and lipase were augmented by 400 and 800% respectively, whereas the pancreatic blood flow (PBF) was decreased by 50% in rats with CIP. Injection of low doses of LPS (0.1-1 mg/kg i.p.) or SNAP (1.5-3 mg/kg i.p.) 30 min prior to caerulein infusion reversed the harmful effects of pancreatic overstimulation with caerulein and reduced significantly the histological manifestations of CIP such as edema, neutrophil infiltration and vacuolization of the acinar cells. These protective effects of low doses of LPS pretreatment on the pancreas were completely antagonized by the suppression of the activity of NO synthase (NOS) with N(G)-nitro-L-arginine (L-NNA) applied (20 mg/kg i.p.) 15 min prior to the LPS injection. Combination of L-arginine (100 mg/kg i.p.), a substrate for NOS, with L-NNA given prior to low doses of LPS, restored the LPS-induced protection of the pancreas in rats with CIP. In contrast, higher doses of LPS (20-40 mg/kg i.p.) or SNAP (6 mg/kg i.p.), which produced a significant fall of the PBF, did not protect the pancreas against CIP. Administration of various doses of LPS to rats with CIP resulted in significant and dose-dependent stimulation of NO biosynthesis in the isolated acini obtained from the pancreas of these animals. LPS enhanced the expression of

Topics: Animals; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Lipopolysaccharides; Male; Nitric Oxide; Nitric Oxide Synthase; Pancreas; Pancreatitis; Rats; Rats, Wistar

Adenosine has been shown to modulate various pathophysiologic conditions through receptor-mediated mechanisms. However, the role of adenosine in the pathogenesis of acute pancreatitis has not been described. We examined the effect of adenosine-receptor stimulation or inhibition on the pathologic changes of the pancreas.. Rats received intraperitoneal injections of selective agonists of A1, A2a, and A3 adenosine receptors: 2-chloro-N(6)-cyclopentyladenosine (CCPA), CGS-21680 (CGS), or 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-D-ribofuranuronamide (IB-MECA), respectively. Serum amylase activity and pathologic changes of the pancreas were evaluated. The effects of a specific A1-receptor antagonist (FK-838) on the pathologic findings of cerulein- and taurocholate-induced pancreatitis were also examined.. Administration of a selective A1 agonist induced hyperamylasemia and morphologic changes in the pancreas characterized by interstitial edema and leukocyte infiltration; neither A2a nor A3 agonist produced such changes. Treatment with an A1-receptor antagonist significantly attenuated the outcome induced by A1 agonist stimulation. In addition, the A1-receptor antagonist significantly ameliorated pancreatic edema in both pancreatitis models, although it did not improve the acinar cell damage of the pancreas or the increase of serum amylase.. Activation of the adenosine A1-receptor pathway may have an important role in the pathogenesis of acute pancreatitis.

Topics: Acute Disease; Adenosine; Amylases; Animals; Ceruletide; Edema; Leukocytes; Male; Pancreas; Pancreatic Diseases; Pancreatitis; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptor, Adenosine A3; Receptors, Purinergic P1; Taurocholic Acid

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue-induced pancreatitis, the trypsin activity in the pancreas of ctsb(-/-) animals was more than 80% lower than in ctsb(+/+) animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb(-/-) animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Cathepsin B; Ceruletide; Disease Models, Animal; Edema; Enzyme Activation; Gene Deletion; Gene Targeting; Humans; Lipase; Mice; Mice, Knockout; Necrosis; Pancreas; Pancreatitis; Phenotype; Trypsinogen

Neutrophil infiltration into the pancreas is a key event in pancreatitis. Here we show that intercellular adhesion molecule-1 (ICAM-1), which regulates neutrophil adhesion, is present on rat pancreatic acinar cells, is upregulated by a hormone (cerulein) and mediates direct binding of neutrophils to acinar cells. ICAM-1 was upregulated in pancreas of rats with experimental pancreatitis induced by supramaximal doses of cerulein. Furthermore, cerulein time and dose dependently stimulated expression of ICAM-1 mRNA and protein in isolated pancreatic acinar cells. Inhibitory analysis showed that activation of transcription factor nuclear factor-kappaB (NF-kappaB) was involved in ICAM-1 upregulation by cerulein, but NF-kappaB did not mediate basal expression of ICAM-1 mRNA in acinar cells. With an adhesion assay, we found that neutrophils bind to isolated pancreatic acinar cells and that cerulein upregulates the extent of adhesion. Neutralizing ICAM-1 antibody blocked neutrophil binding to both control and cerulein-stimulated acinar cells, suggesting ICAM-1 involvement in this adhesion. Thus the acinar cell is capable of targeting neutrophils to its surface, a process that may be important for inflammatory and cell death responses in pancreatitis and other pancreatic disorders.

Topics: Animals; Cell Adhesion; Cell Nucleus; Cells, Cultured; Ceruletide; Cysteine Proteinase Inhibitors; Gene Expression Regulation; Intercellular Adhesion Molecule-1; Kinetics; Leupeptins; Male; Neutrophils; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

Stimulation of capsaicin sensitive nerves or administration of calcitonin gene-related peptide (CGRP) before induction of acute pancreatitis (AP) attenuates pancreatic damage, whereas CGRP administration after development of AP aggravates lesion of pancreatic tissue. The aim of this study was to determine the effect of prolonged activity of sensory nerves or CGRP administration on the pancreatic repair after repeated episodes of AP. Five episodes of acute caerulein-induced pancreatitis (10 microg/kg/h for 5 h s.c.) were performed at weekly intervals in rats receiving either vehicle or capsaicin at the sensory nerve stimulatory dose (0.5 mg/kg, 3 times daily), or CGRP (10 microg/kg, 3 times daily). Two weeks after the last induction of AP morphological signs of pancreatic damage, pancreatic blood flow (PBF), serum and pancreatic amylase activity, fecal chymotrypsin activity, pancreatic weight, pancreatic RNA and DNA content, as well as, serum interleukin-1beta (Il-1beta ) were assessed. Pancreata of animals receiving vehicle alone showed almost full recovery within two weeks after last episode of pancreatitis induction. In capsaicin-treated group of rats, we observed the increase in PBF by 44% and in serum Il-1beta concentration by 91%. The pancreatic amylase activity, fecal activity of chymotrypsin, pancreatic nucleic acids content and DNA synthesis were decreased. In rats treated with CGRP the alterations in PBF, serum Il-1beta concentration, as well as, in pancreatic and fecal activity of enzymes were similar to capsaicin treated group but less pronounced. We conclude that prolonged activity of capsaicin-sensitive sensory nerves and the presence of their main mediator-CGRP during pancreatic regeneration after AP leads to pancreatic functional insufficiency typical for chronic pancreatitis.

Topics: Acute Disease; Amylases; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Ceruletide; Chymotrypsin; DNA; Interleukin-1; Male; Neurons, Afferent; Pancreas; Pancreatitis; Rats; Rats, Wistar; RNA, Messenger

Topics: Animals; CD4-Positive T-Lymphocytes; Ceruletide; Mice; Mice, Nude; Pancreatitis

Mitogen-activated protein kinase (MAPK) family members such as c-jun N-terminal kinase (JNK) may act as signal transducers early during pancreatitis development and evidence indicates that MAPK phosphatases (MKP) downregulate MAPK. We therefore investigated expression and regulation of pancreatic MKP in vivo. Pancreatic MKP mRNA levels were near or below the detection threshold in unstimulated animals. Cerulein hyperstimulation strongly induced MKP-1, MKP-3, and MKP-5 expression, peaking 30 to 60 min after treatment. Thus, MKP's clearly are early responsive genes during pancreatitis induction. Interestingly, inhibition of MKP-1 expression by Ro-31-8220 maximally induced activation of JNK but not of p38 and ERK in acutely isolated acini. These effects indicate that JNK may indeed be a preferred MKP-1 substrate in vivo.

Topics: Acute Disease; Animals; Cell Cycle Proteins; Ceruletide; Dual Specificity Phosphatase 1; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Immediate-Early Proteins; Indoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Pancreatitis; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction

To find out if two immunomodulatory drugs used in organ transplantation (FK506 (tacrolimus) and OKT3 (Orthoclone) would reduce early inflammatory complications in experimental acute pancreatitis.. Laboratory study.. University hospital, Germany.. 36 Balb/c mice.. Pancreatitis induced by 7 intraperitoneal injections of cerulein 50 microg/kg at hourly intervals followed by FK506 0.32 mg/kg, OKT3 0.6 mg/kg, or 0.9% sodium chloride (controls) (n = 12 in each group). 12 hours after induction of pancreatitis the animals were killed.. Serum amylase activity and interleukin-6 (IL-6) concentrations; histological damage to pancreas and lungs, apoptotic cells in pancreas; and myeloperoxidase activity in lungs.. No animal died during the experiment. At 12h serum amylase activity and IL-6 concentrations were increased in all 3 groups, but highest in the OKT3 group. The pancreatic histological score, apoptosis, and inflammatory infiltration were lower in the two experimental groups than controls, but the degree of vacuolisation of acinar cells was similar. Packed cell volume was higher in the control than the experimental groups, and pulmonary damage and myeloperoxidase activity were less in the experimental groups than the controls.. Single therapeutic doses of FK506 and OKT3 reduced the early severity of pancreatitis, pulmonary damage, and haemoconcentration in mice. Single doses of FK506 or OKT3 may therefore be effective in preventing the early complications of pancreatitis.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Ceruletide; Female; Immunosuppressive Agents; Interleukin-6; Lung; Mice; Mice, Inbred BALB C; Muromonab-CD3; Pancreas; Pancreatitis; Peroxidase; Tacrolimus

Heat shock proteins (HSPs) 70 and 27 are stress-responsive proteins that are important for cell survival after injury; the expression of these HSPs is regulated primarily by the transcription factor heat shock factor-1 (HSF-1). The purpose of this study was to determine the effect of acute pancreatitis on pancreatic HSPs (70, 27, 60, and 90) expression and to assess potential mechanisms for HSP induction using a murine model of cerulein-induced pancreatitis. We found an increase of both HSP70 and HSP27 levels with expression noted throughout the pancreas after induction of pancreatitis. HSP60 and HSP90 levels were constitutively expressed in the pancreas and did not significantly change with acute pancreatitis. HSF-1 DNA binding activity increased in accordance with increased HSP expression. We conclude that acute pancreatitis results in a marked increase in the expression of HSP70 and HSP27. Furthermore, the induction of HSP70 and HSP27 expression was associated with a concomitant increase in HSF-1 binding activity. The increased expression of both HSP70 and HSP27 noted with pancreatic inflammation may confer a protective effect for the remaining acini after acute pancreatitis.

Topics: Acute Disease; Animals; Blotting, Western; Ceruletide; DNA; DNA-Binding Proteins; Electrophoresis; Female; Heat Shock Transcription Factors; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Mice; Molecular Chaperones; Neoplasm Proteins; Pancreatitis; Transcription Factors

alpha 1-Acid glycoprotein (AAG), a highly negatively charged glycoprotein, well known for its capillary stabilizing effect, was tested in rat models of acute edematous pancreatitis, acute hemorrhagic-necrotizing pancreatitis, and acute respiratory distress syndrome (ARDS). In cerulein-elicited edematous pancreatitis AAG improved histological alterations at 200 mg/kg i.v. and plasma amylase activity at 1800 or 4200 mg/kg i.v. All other parameters (edema, plasma lipase) were not affected in a biologically relevant manner. In glycodeoxycholic acid-induced hemorrhagic-necrotizing pancreatitis AAG was without effect on parameters measured (plasma amylase, plasma lipase activity, histological scores) at 1800 or 4200 mg/kg i.v. At the extremely high dose of 1500 mg/kg i.v. plasma amylase and lipase levels were decreased. In lipopolysaccharide-mediated ARDS, AAG was tested at 50, 200 or 600 mg/kg i.v. AAG, but also the placebo formulation decreased the myeloperoxidase content in the bronchoalveolar lavage fluid. Histological alterations were improved by AAG, however, not by the placebo formulation. Lung water content was not significantly influenced by AAG, whereas Evans blue extravasation was significantly diminished by all three doses of AAG. It is concluded that the edematous pancreatitis is the first in vivo condition with increased extravascular fluid accumulation, in which AAG is not effective. Based on data presented here and literature data, there is evidence for a beneficial effect of AAG in acute lung injury.

Topics: Acute Disease; Animals; Bronchoalveolar Lavage Fluid; Ceruletide; Edema; Glycodeoxycholic Acid; Hemorrhage; Lipopolysaccharides; Lung Diseases; Male; Orosomucoid; Pancreatitis; Rats; Rats, Sprague-Dawley; Respiratory Tract Diseases

We describe a refined model of intravenous caerulein-induced pancreatitis by using osmotic infusion pumps in the conscious unrestrained Wistar rat. The volume of caerulein required for the 6-h infusion is loaded into PE-55 catheter tubing attached to an Alzet (Alza Corporation, Palo Alto, CA) implantable osmotic pump that has been primed with saline. The technique has reliably induced mild edematous pancreatitis, which we verified histologically. Our refined model has the advantages of unrestrained animals, reduced animal handling and acclimation, and decreased cost.

Topics: Animals; Ceruletide; Disease Models, Animal; Infusions, Intravenous; Male; Osmosis; Pancreatitis; Rats; Rats, Wistar

The regenerative process after acute inflammation of the pancreas is characterized by cell proliferation as well as synthesis and transient deposition of extracellular matrix. Although the regulation of these processes is still unknown, there is growing evidence that the coordinated activity of various growth factors plays an important role in regeneration. Cerulein-induced pancreatitis in the rat was used to analyze whether growth factors and their receptor concentrations are changed in the acute pancreatitis. Messenger RNA hybridization revealed an individual expression pattern for each analyzed growth factor. The mRNA levels of platelet-derived growth factor B (PDGF B), epidermal growth factor (EGF), and insulin-like growth factor 2 (IGF-2) were not altered, whereas fibroblast growth factor-1 (FGF-1) and 2, IGF-1, transforming growth factor-alpha (TGFalpha), and hepatocyte growth factor (HGF) showed markedly increased concentrations with different expression maxima and duration compared with mRNA levels in healthy pancreata. The FGF-2 and IGF-1 expressions were increased between 1 and 3 days after induction of pancreatitis with maxima at day 2. HGF and FGF-1 mRNAs were upregulated between days 3 and 5. In contrast, TGFalpha exhibited the most prolonged overexpression. In the corresponding receptors, only c-met, the HGF-binding protein, showed higher mRNA and protein levels, whereas the expression of the other receptors did not change. Furthermore, in cultured pancreatic epithelial cells, HGF stimulated the expression of its own receptor in an autocrine manner. These results point out that the highly coordinated process of regeneration after pancreatitis may be influenced by a sequential induction and expression of peptide growth factors and their receptors.

Topics: Acute Disease; Animals; Ceruletide; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Gene Expression Regulation; Hepatocyte Growth Factor; Insulin-Like Growth Factor I; Male; Pancreatitis; Rats; Rats, Wistar; Receptors, Growth Factor; RNA, Messenger; Time Factors; Transcription, Genetic; Transforming Growth Factor alpha

Recent evidence suggests that a number of rapid signaling cascades are initiated during secretagogue-induced pancreatitis. However, little is known about the nuclear events. The aim of this study was to explore activation of the transcription factor NF-kappaB/Rel after supramaximal stimulation with the cholecystokinin analogue cerulein in the pancreas.. Nuclear appearance of NF-kappaB/Rel-binding activity was detectable 15 minutes after cerulein injection. The DNA-binding activity consisted of NF-kappaB1 p50, NF-kappaB2 p52, and RelA p65 as judged by supershift assays and Western blot analysis. The onset and termination of NF-kappaB/Rel activation correlated with the degradation and reappearance of IkappaBalpha. Cerulein in supramaximal but not in physiological doses activated NF-kappaB/Rel in vitro. After blocking of NF-kappaB/Rel activation with pyrrolidine dithiocarbamate, the degree of morphological alterations was more pronounced than in controls, serum amylase and lactate dehydrogenase levels were significantly increased, and messenger RNA levels of pancreatitis-associated protein were more strongly induced, reflecting a more severe degree of pancreatitis. Similar results were obtained when N-acetyl-L-cysteine was used as an inhibitor of NF-kappaB activation.. These data show that NF-kappaB/Rel is rapidly activated during cerulein pancreatitis. This activation may induce a self-defending genetic program before the onset of cellular injury, which might prevent higher degrees of damage of pancreatic acinar cells after secretagogue hyperstimulation.

Topics: Acetylcysteine; Amylases; Animals; Antioxidants; Blotting, Northern; Blotting, Western; Ceruletide; DNA Probes; Dose-Response Relationship, Drug; Edema; Free Radical Scavengers; L-Lactate Dehydrogenase; Male; NF-kappa B; Pancreatitis; Pancreatitis-Associated Proteins; Protein Binding; Pyrrolidines; Rats; Rats, Wistar; Thiocarbamates

The priming mechanism of macrophages to secrete cytokines in acute pancreatitis is important for remote organ failure following septic complication. The effects of novel carboxamide derivative, IS-741, on neutrophil chemoattractant production by bronchoalveolar macrophages were studied in rats with cerulein-induced pancreatitis complicated by sepsis.. Pancreatitis was induced by four intramuscular injections of cerulein (50 microg/kg at 1-hour intervals). Pancreatitis rats were injected intraperitoneally with 10 mg/kg of lipopolysaccharide (LPS) 6 h following the first cerulein injection as a septic challenge. Pancreatitis rats received a continuous intravenous injection of IS-741 (3 mg/kg/h) 30 min before the septic challenge.. Intense mononuclear cell infiltration and lung hemorrhage occurred in untreated pancreatitis rats complicated with sepsis, but hemorrhage was not seen in septic pancreatitis rats receiving a continuous intravenous injection of IS-741 shortly before sepsis induction. The IS-741-treated rats had lower serum concentrations of cytokine-induced neutrophil chemoattractant (CINC), as well as fewer the pulmonary neutrophils and infiltrates immunoreactive for CINC or Mac-1 (CD11b/CD18).. The novel carboxamide derivative IS-741 reduced CINC production by bronchoalveolar macrophages and effectively prevented pancreatitis-associated lung injury following the septic challenge.

Topics: Animals; CD18 Antigens; Ceruletide; Chemotaxis; Disease Models, Animal; Enzyme Inhibitors; Injections, Intravenous; Interleukin-8; Macrophage-1 Antigen; Macrophages, Alveolar; Male; Neutrophil Infiltration; Pancreatitis; Pyridines; Rats; Rats, Wistar; Sepsis

This presentation reviews the role of cholecystokinin (CCK) as a contributory factor for the development and progression of acute pancreatitis (AP) and the perspective of CCK receptor antagonists for treatment of AP. High, supraphysiological concentrations of CCK induce AP in various species including man. There is also evidence that physiological increases in plasma CCK deteriorates AP in several animal models. The latter findings support the hypothesis that CCK plays a contributory or permissive role for the development of AP. The majorities of experimental studies show that the prophylactic and therapeutic use of CCK antagonists ameliorates AP. The latter effects were clearly shown for models of biliary AP in which plasma CCK is increased due to a feedback mechanism. However, CCK antagonists also had beneficial effects in models in which plasma CCK is not increased. In animal strains which do not have a CCK-A-receptor due to a genetic abnormality AP induced by a certain noxious factor does not develop to the same severity when compared to animals with a normal CCK-A-receptor. Thus, CCK acts as a permissive or contributory factor for the development and progression of AP. There is also evidence that CCK antagonists have a potential therapeutic benefit. Clinical studies will evaluate their therapeutic potential for patients with AP.

Topics: Acute Disease; Animals; Ceruletide; Cholecystokinin; Devazepide; Disease Models, Animal; Disease Progression; Humans; Indoles; Pancreatitis; Rats; Receptors, Cholecystokinin

Intercellular adhesion molecule 1 (ICAM-1) and neutrophils play important roles in many inflammatory processes, but their importance in both acute pancreatitis and pancreatitis-associated lung injury has not been defined.. To address this issue, mice that do not express ICAM-1 were used and depleted of neutrophils by administration of antineutrophil serum. Pancreatitis was induced by administering either supramaximal doses of the secretagogue cerulein or feeding a choline-deficient, ethionine-supplemented diet. The severity of pancreatitis was evaluated by quantitating serum amylase, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity (i.e., neutrophil content). Lung injury was evaluated by quantitating lung myeloperoxidase activity and pulmonary microvascular permeability. ICAM-1 was quantitated by enzyme-linked immunosorbent assay and was localized by light-microscopic immunohistochemistry.. It was found that serum, pancreas, and lung ICAM-1 levels increase during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in ICAM-1. Neutrophil depletion also reduces the severity of both pancreatitis and lung injury. However, the combination of neutrophil depletion with ICAM-1 deficiency does not reduce the severity of pancreatitis or lung injury to a greater extent than either neutrophil depletion or ICAM-1 deficiency alone. Neither pancreatitis nor pancreatitis-associated lung injury are completely prevented by ICAM-1 deficiency, neutrophil depletion, or combined ICAM-1 deficiency plus neutrophil depletion.. The observations indicate that ICAM-1 plays an important, neutrophil-mediated, proinflammatory role in pancreatitis and pancreatitis-associated lung injury. The studies also indicate that ICAM-1 and neutrophil-independent events also contribute to the evolution of pancreatitis and lung injury in these models.

Topics: Acute Disease; Animals; Capillary Permeability; Ceruletide; Choline Deficiency; Death; Ethionine; Female; Intercellular Adhesion Molecule-1; Lung; Male; Mice; Mice, Knockout; Microcirculation; Neutrophils; Pancreatitis; Peroxidase; Pulmonary Circulation

The therapeutic effects of an intravenously injected carboxamide derivative (IS-741) on lung injury were studied in rats with cerulein-induced pancreatitis complicated by endotoxemia. Pancreatitis was induced by four intramuscular injections of cerulein (50 microg/kg at 1-hr intervals). Pancreatitis rats were injected intraperitoneally with 10 mg/kg of lipopolysaccharide (LPS) 6 hr following the first cerulein injection as a challenge of endotoxemia. Rats were divided into four groups: group I, pancreatitis with LPS; group II, pancreatitis with LPS treated with a continuous intravenous injection of IS-741 at 0.03 mg/kg/hr); group III, pancreatitis with LPS treated with a continuous intravenous injection of IS-741 at 0.3 mg/kg/hr); and group IV, pancreatitis with LPS treated with a continuous intravenous injection of IS-741 at 3 mg/kg/hr). IS-741 was administered 30 min before the endotoxemia challenge. Intense mononuclear cell infiltration and lung hemorrhage occurred in untreated pancreatitis rats with LPS (group I), but hemorrhage was not seen in group IV rats receiving a continuous injection of IS-741 shortly before the induction of endotoxemia. The IS-741-treated rats (groups II, III, and IV) had lower serum concentrations of cytokine-induced neutrophil chemoattractant (CINC), as well as fewer pulmonary infiltrates immunoreactive for CINC or Mac-1 (CD11b/CD18). The number of neutrophils infiltrating the lung in groups II, III, and IV was significantly lower than that of group I. Conversely, CINC production by bronchoalveolar macrophages in vitro were stimulated by LPS but were reduced by the presence of IS-741. The carboxamide derivative IS-741 effectively prevented pancreatitis-associated lung injury following the challenge of endotoxemia.

Topics: Acute Disease; Animals; Bronchoalveolar Lavage Fluid; Cells, Cultured; Ceruletide; Endotoxemia; Enzyme Inhibitors; Hemorrhage; Infusions, Intravenous; Interleukin-16; Lung; Lung Diseases; Macrophages; Male; Neutrophils; Pancreatitis; Phospholipases A; Pyridines; Rats; Rats, Sprague-Dawley

The transforming growth factor-beta (TGFbeta) signaling pathway is one important player in the regulation of extracellular matrix turnover and cell proliferation in epithelial regeneration. We used cerulein-induced pancreatitis in rats as a model to investigate the regulation of TGFbeta receptor type I and type II expression on protein and messenger RNA level during regeneration. In the regenerating pancreas, mRNA levels of TGFbeta receptor I and II were significantly increased with a maximum after 2 days. On protein level, expression of TGFbeta receptor II was significantly increased after three to 3-5 days. This elevated expression could be inhibited by neutralizing the endogenous biological activity of TGFbeta1 with a specific antibody. In cultured pancreatic epithelial cells, TGFbeta1 reduced cell proliferation as measured by [3H]thymidine incorporation. Furthermore the transcript levels of TGFbeta1 as well as mRNA and protein concentrations of type I and type II receptor increased during TGFbeta stimulation in vitro. These results indicate that epithelial pancreatic cells contribute to the enhanced TGFbeta1 synthesis during pancreatic regeneration by an autocrine mechanism. TGFbeta1, furthermore, upregulates the expression of its own receptors during the regenerative process, thereby contributing to the increase of the TGFbeta-induced cellular responses.

Topics: Animals; Ceruletide; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Receptors, Transforming Growth Factor beta; Regeneration; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The aim of this study was to evaluate the controversial specificity of the plasma amino acid (AA)-consumption test in detecting pancreatic diseases by using two different quantitative methods. A total of 55 subjects: 13 healthy subjects, 13 patients with chronic pancreatitis (three mild/moderate, eight severe), 13 patients with pancreatectomy and complete suppression of the exocrine pancreatic secretion, eight patients with chronic liver disease (five with impaired synthetic function), and eight patients with chronic renal failure. Total plasma AAs were quantified by a colorimetric method (p-benzoquinone) in all subjects, at 0, 30, 45, and 60 min during and 30 min after minute 60 of i.v. cerulein infusion (50 ng/kg/h). Either total and individual AAs were quantified by chromatography (high-performance liquid chromatography; HPLC) in 10 healthy subjects, 10 patients with pancreatectomy, and 10 with chronic pancreatitis at 0 and 60 min after the start of the cerulein infusion. For the colorimetric method, healthy subjects had maximal percentage decreases of total AA concentrations not significantly different from those of patients with pancreatectomy and significantly higher than those of patients with chronic pancreatitis (p < 0.0001) or chronic liver disease (p < 0.001). Pancreatic function, as assessed by fecal elastase-1 test, was not significantly correlated to the maximal percentage decrease in total plasma AAs. For the chromatographic method, total AA concentrations were not significantly correlated to those determined by colorimetry. The concentration of each of the individual plasma AAs varied considerably in each group. Fecal elastase-1 values were normal (> or = 200 microg/g) in all patients without pancreatic disease and in only one of 11 patients with chronic pancreatitis and exocrine insufficiency. The type of method used can explain the different results of the AA-consumption test. This test is not very specific for the pancreas.

Topics: Adult; Aged; Amino Acids; Ceruletide; Chromatography, Ion Exchange; Colorimetry; Evaluation Studies as Topic; Feces; Female; Humans; Kidney Failure, Chronic; Liver Diseases; Male; Middle Aged; Pancreatectomy; Pancreatic Diseases; Pancreatic Elastase; Pancreatic Function Tests; Pancreatitis; Time Factors