ceruletide and Inflammation

Develop a novel murine models of malignant pancreatitis.. Although patients with chronic pancreatitis are at a greater risk of developing pancreatic cancer, there is no definitive mouse model that currently develops chronic pancreatitis-induced pancreatic cancer.. Characterization of eosinophilic inflammation-mediated malignant pancreatitis in novel murine model.. We developed a murine model of chronic eosinophilic inflammation associated with pancreatitis that also shows characteristic features of pancreatic malignancy. The mouse received cerulein and azoxymethane via intraperitoneal administration developed pathological malignant phenotype, as well as concomitant lung inflammation.. We discovered pathological alterations in the pancreas that were associated with chronic pancreatitis, including a buildup of eosinophilic inflammation. Eosinophil degranulation was reported nearby in the pancreas tissue sections that show acinar-to-ductal metaplasia and acinar cell atrophy, both of which are characteristic of pancreatic malignancies. Additionally, we also observed the formation of PanIN lesions after three initial doses of AOM and eight weeks of cerulein with the AOM treatment regimen. We discovered that persistent pancreatic eosinophilic inflammation linked with a pancreatic malignant phenotype contributes to pulmonary damage. The RNA seq analysis also confirmed the induction of fibro-inflammatory and oncogenic proteins in pancreas and lung tissues. Further, in the current manuscript, we now report the stepwise kinetically time-dependent cellular inflammation, genes and proteins involved in the development of pancreatitis malignancy and associated acute lung injury by analyzing the mice of 3 AOM with 3, 8, and 12 weeks of the cerulein challenged protocol regime.. We first show that sustained long-term eosinophilic inflammation induces time-dependent proinflammatory, profibrotic and malignancy-associated genes that promote pancreatic malignancy and acute lung injury in mice.

Topics: Animals; Ceruletide; Disease Models, Animal; Inflammation; Mice; Pancreatic Neoplasms; Pancreatitis, Chronic

Xiao Chai Hu Tang (XCHT) derived from the classic medical book Shang Han Lun (Treatise on Febrile Diseases) in the Eastern Han Dynasty, which has been widely used in China and other Asian countries for the treatment of inflammation and fibrosis of chronic pancreatitis (CP), but the therapeutic mechanism of XCHT in pancreatic fibrosis remains unclear.. This study aimed to evaluate the intervention effects and explore pharmacological mechanism of XCHT on inflammation and fibrosis in cerulein-induced CP model.. Fifty male C57BL/6 mice were randomly divided into five main groups, 10 animals in each: Control, CP model (50 μg/kg cerulein), high dose XCHT-treated CP group (60 g/kg XCHT), medium dose XCHT-treated CP group (30 g/kg XCHT) and low dose XCHT-treated CP group (15 g/kg XCHT). Different doses of XCHT were given to mice by gavage twice a day for 2 weeks after the CP model induction. Pancreatic tissues were harvested and the pancreatic inflammation and fibrosis were evaluated by histological score, Sirius red staining, and alpha-smooth muscle actin (α-SMA) immunohistochemical staining. ELISA, IHC and RT-qPCR were performed to detect the expression of Vitamin D. The pathohistological results demonstrated that XCHT markedly inhibited the fibrosis and chronic inflammation of cerulein-induced CP, indicated by reduction of collagen I, collagen III, α-SMA, and NLRP3 expressions. XCHT significantly increased VD

Topics: Actins; Animals; Ceruletide; Collagen; Disease Models, Animal; Fibrosis; Inflammasomes; Inflammation; Interleukin-6; Male; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatitis, Chronic; Receptors, Calcitriol; Signal Transduction; Tumor Necrosis Factor-alpha; Vitamin D

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

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

Chronic pancreatitis (CP) is an irreversible and progressive inflammatory disease. Knowledge on the development and progression of CP is limited. The goal of the study was to define the serum profile of pro-inflammatory cytokines and the cell antioxidant defense system (superoxidase dismutase-SOD, and reduced glutathione-GSH) over time in a cerulein-induced CP model and explore the impact of these changes on selected cytokines in the intestinal mucosa and pancreatic tissue, as well as on selected serum biochemical parameters. The mRNA expression of

Topics: Animals; Ceruletide; Cytokines; Disease Models, Animal; Inflammation; Interleukin-6; Pancreas; Pancreatitis, Chronic; Pilot Projects; RNA, Messenger; Superoxide Dismutase; Swine; Tumor Necrosis Factor-alpha

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 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

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

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

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

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

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

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

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

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

Pancreatitis occurs in two forms defined by its chronicity. Acute pancreatitis (AP) occurs suddenly and only lasts for several days. Consequently, most patients with AP recover without permanent damage to the pancreas, and about 20% of patients with AP have severe disease. In contrast, chronic pancreatitis (CP) is a long-lasting inflammation that causes permanent damage to pancreatic tissue; consequently, this form is marked by the emergence of persistent endocrine and exocrine pancreatic insufficiency. Despite these differences, AP and CP share central mechanisms of disease: in both forms, inflammation is initiated and/or sustained by the intrapancreatic activation of pancreatic digestive enzymes followed by the autodigestion of pancreatic tissues. In addition, in both forms enzymatic damage is accompanied by changes in intestinal permeability and entry of commensal organisms into the pancreas where they elicit innate immune responses that ultimately dominate and define pancreatic inflammation. In the murine models of AP and CP described here, both of these elements of pancreatitis pathogenesis are taken into account. Thus, in one approach mice are administered high doses of cerulein, a cholecystokinin analog with the ability at this dose to induce excessive activation of the cholecystokinin receptor expressed in pancreatic acinar cells and the release of active trypsin that causes both direct and indirect acinar damages due to entry of commensal organisms and stimulation of innate immune responses. In a second approach mice are administered low doses of cerulein, which causes little or no damage to the pancreas unless given along with nucleotide-binding oligomerization domain 1 (NOD1) ligand, which in the presence of low-dose cerulein administration induces a pathologic innate immune response mediated by NOD1. These approaches are adopted to produce AP when cerulein or cerulein plus NOD1 ligand is applied only once or to produce CP when a similar regimen is applied multiple times. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cerulein-induced acute pancreatitis Alternate Protocol 1: Acute pancreatitis induced by cerulein and NOD1 ligand Basic Protocol 2: Cerulein-induced chronic pancreatitis Alternate Protocol 2: Chronic pancreatitis induced by cerulein and NOD1 ligand Support Protocol: Isolation of pancreatic mononuclear cells.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Humans; Inflammation; Ligands; Mice; Pancreatitis, Chronic

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

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

Topics: Acinar Cells; Adenosine Triphosphatases; Animals; Ceruletide; Histones; Inflammation; Lysophosphatidylcholines; Macrophages; Mice; Pancreatitis, Chronic; Phospholipid Transfer Proteins; Transcription Factors

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 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

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 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

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

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

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

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

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

(AP) is a kind of inflammatory misorder existing in pancreas. Non-coding RNAs (ncRNAs) have been reported to play important roles in development of AP. The current study was designed to explore the role of circular RNA zinc finger protein 644 (circRNA circ_ZFP644) in caerulein-induced AR42J cells. AP model in vitro was established by exposure of rat pancreatic acinar AR42J cells to caerulein. Amylase activity was measured using a kit. Enzyme-linked immunosorbent assay (ELISA) was performed to examine the levels of several inflammatory factors. The expression of circ_ZFP644, microRNA (miR)-106b and protein inhibitor of activated STAT 3 (Pias3) was detected by quantitative real-time PCR (qRT-PCR) or western blot assay. And flow cytometry was employed to monitor cell apoptosis. Western blot assay was also conducted to analyze the expression of apoptosis-related proteins. The association among circ_ZFP644, miR-106b and Pias3 was validated by dual-luciferase reporter assay. Caerulein treatment activated amylase activity and promoted the secretion of inflammatory cytokines in AR42J cells. Circ_ZFP644 and Pias3 were downregulated, but miR-106b was upregulated in caerulein-induced AR42J cells. Enforced expression of circ_ZFP644 or miR-106b inhibition could reduce amylase activity and inflammatory cytokine secretion, while promote apoptosis in caerulein-induced AR42J cells, which was almost reversed by Pias3 knockdown. Circ_ZFP644 targeted miR-106b to upregulate Pias3 expression. Circ_ZFP644 might exert its anti-inflammation and pro-apoptosis roles in caerulein-induced AR42J cells by regulating miR-106b/Pias3 axis.

Topics: Acinar Cells; Animals; Ceruletide; Gene Expression Regulation; Inflammation; MicroRNAs; Molecular Chaperones; Pancreas; Protein Inhibitors of Activated STAT; Rats; RNA, Circular

Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis.. Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18 weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes.. Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFβR2 gene expression by 44, 38, and 25%, respectively, compared to control mice.. New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.

Topics: Animals; Ceruletide; Chronic Disease; Disease Models, Animal; Fibrosis; Inflammation; Lipase; Mice; Pancreas; Pancreatitis, Chronic; Proglumide; Receptors, Cholecystokinin

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

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) is an inflammatory disease with high morbidity and mortality. Dysregulation of microRNAs (miRNAs) was involved in human diseases, including AP. However, the effects of miR-92b-3p on AP process and its mechanism remain not been fully clarified. The expression levels of miR-92b-3p and tumor necrosis factor receptor-associated factor-3 (TRAF3) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of TRAF3, tumor necrosis factor α (TNF-α) TNF-α, interleukin-6 (IL-6), phosphorylated mitogen-activated protein kinase kinase 3 (p-MKK3), MKK3, p38 and phosphorylated p38 (p-p38) were detected by western blot. The concentration of TNF-α and IL-6 in the medium was measured using ELISA kits. The possible binding sites of miR-92b-3p and TRAF3 were predicted by TargetScan and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression level of miR-92b-3p was decreased and TRAF3 expression was increased in AR42J cells stimulated with caerulein. Moreover, the protein levels of pro-inflammatory cytokines (TNF-α and IL-6) were markedly elevated, and the expression levels of autophagy-related markers Beclin1 as well as the ratio of LC3-II/I were obviously increased in AR42J cells treated with caerulein. In addition, overexpression of miR-92b-3p or knockdown of TRAF3 significantly suppressed the release of pro-inflammatory cytokines and autophagy in caerulein-induced AR42J cells. Furthermore, TRAF3 was a direct target of miR-92b-3p and its upregulation reversed the effects of miR-92b-3p overexpression on inflammatory response and autophagy. Besides, overexpression of miR-92b-3p inhibited the activation of the MKK3-p38 pathway by affecting TRAF3 expression. In conclusion, miR-92b-3p attenuated inflammatory response and autophagy by downregulating TRAF3 and suppressing MKK3-p38 pathway in caerulein-induced AR42J cells, providing a novel avenue for treatment of AP.

Topics: Acinar Cells; Animals; Autophagy; Cell Line; Ceruletide; Cytokines; Inflammation; MAP Kinase Kinase 3; MicroRNAs; p38 Mitogen-Activated Protein Kinases; Pancreas; Rats; Signal Transduction; TNF Receptor-Associated Factor 3

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

Chronic pancreatitis (CP) is associated with elevated plasma levels of bacterial lipopolysaccharide (LPS) and we have demonstrated reduced acinar cell autophagy in human CP tissue. Therefore, we investigated the role of autophagy in experimental endotoxin-induced pancreatic injury and aimed to identify LPS in human CP tissue. Pancreatic Atg7-deficient mice were injected with a single sub-lethal dose of LPS. Expression of autophagy, apoptosis, necroptosis, and inflammatory markers was determined 3 and 24 h later utilizing immunoblotting and immunofluorescence. The presence of LPS in pancreatic tissue from mice and from patients and healthy controls was determined using immunohistochemistry, immunoblots, and chromogenic assay. Mice lacking pancreatic autophagy exhibited local signs of inflammation and were particularly sensitive to the toxic effect of LPS injection as compared to control mice. In response to LPS, Atg7

Topics: Acinar Cells; Animals; Autophagy; Ceruletide; Endotoxins; Humans; Inflammation; Lipopolysaccharides; Mice, Transgenic; Pancreatitis, Chronic

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

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

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

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

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

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

The study was designed to investigate the JAK2/STAT3 signaling pathway in pancreatitis and its association with inflammation and cell death to provide a potential treatment method for pancreatitis.. The rat pancreatic acinar AR42J cells were used for the study, and they were transfected with JAK2 and STAT3 siRNAs to mimic knockdown condition. Cerulein was used to treat AR42J cells. Western blot and ELISA were employed to detect the expression of related proteins. Flow cytometry was done to analysis the necrosis of AR42J cells.. In this study, we found that cell death and the secretion of IL-6 and TGF-β1 were significantly increased, and the JAK2/STAT3 signaling pathway was activated in cerulein-induced AP. To determine the role of JAK2 and STAT3, JAK2 siRNA and STAT3 siRNA were used to block JAK2 and STAT3, respectively. The levels of IL-6 and TGF-β1 levels in the medium were lower in JAK2 siRNA and STAT3 siRNA-treated cells compared with controls. Flow cytometry analysis showed that the level of cell death, expression of cleaved caspase-3, and the release of LDH were decreased following JAK2 siRNA and STAT3 siRNA treatment.. These findings point to a novel role for the JAK2/STAT3 signaling pathway in the progression of cerulein-induced AP.

Topics: Animals; Cell Death; Cells, Cultured; Ceruletide; Cytokines; Inflammation; Janus Kinase 2; Rats; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor

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

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

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

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

The present study aimed to investigate the effects of angiotensin (Ang) 1-7 on caerulein (CAE)-stimulated nuclear factor (NF)‑κB, Toll‑like receptor (TLR4) and cytokine expression using pancreatic acinar AR42J cells. AR42J cells were treated with 10 nmol/l CAE for various durations. In addition, cells were pretreated with various concentrations of Ang 1‑7 or A779, a specific antagonist of Ang 1‑7, and were stimulated with CAE for 12 h. Control cells were treated with vehicle (F‑12K complete medium with 2% fetal bovine serum, 10 U/ml penicillin and 100 mg/ml streptomycin) alone. The mRNA and protein expression levels of TLR4, NF‑κB, interleukin (IL)‑6, IL‑8, IL‑10 and tumor necrosis factor‑α (TNF‑α) were determined by western blotting, immunofluorescence and reverse transcription‑quantitative polymerase chain reaction. CAE treatment stimulated TLR4 and NF‑κB expression within AR42J cells. Immunofluorescence indicated that TLR4 was expressed on the membranes and in the cytoplasm of AR42J cells, whereas NF‑κB expression accumulated in the cytoplasm and nuclei. CAE‑induced expression of TLR4 and NF‑κB within AR42J cells was abrogated by 10‑5 mmol/l Ang 1‑7; however, TLR4 and NF‑κB expression was enhanced with the addition of A779, particularly 10‑5 mmol/l. In addition, treatment with 10‑6 and 10‑5 mmol/l Ang 1‑7 significantly mitigated CAE‑induced expression of IL‑6, IL‑8 and TNF‑α, whereas it enhanced IL‑10 expression. Conversely, A779 treatment enhanced the CAE‑induced expression of IL‑6, IL‑8 and TNF‑α, and reduced IL‑10 expression in AR42J cells. In conclusion, these results suggested that Ang 1‑7 may attenuate CAE‑induced inflammation by downregulating TLR4, NF‑κB and proinflammatory cytokine expression within AR42J cells. Therefore, Ang 1‑7 may exert protective effects against the pathological progression of AP in a cell model of AP induced by CAE and may be considered in the development of treatments for this disease.

Topics: Acinar Cells; Angiotensin I; Angiotensin II; Animals; Cell Line; Ceruletide; Down-Regulation; Inflammation; Interleukin-10; Interleukin-6; Microscopy, Fluorescence; NF-kappa B; Peptide Fragments; Rats; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

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

Adult pancreatic acinar cells have the ability to re-enter the cell cycle and proliferate upon injury or tissue loss. Despite this mitotic ability, the extent of acinar proliferation is often limited and unable to completely regenerate the injured tissue or restore the initial volume of the organ, thus leading to pancreatic dysfunction. Identifying molecular determinants of enhanced proliferation is critical to overcome this issue. In this study, we discovered that Murphy Roths Large (MRL/MpJ) mice can be exploited to identify molecular effectors promoting acinar proliferation upon injury, with the ultimate goal to develop therapeutic regimens to boost pancreatic regeneration. Our results show that, upon cerulein-induced acinar injury, cell proliferation was enhanced and cell cycle components up-regulated in the pancreas of MRL/MpJ mice compared to the control strain C57BL/6. Initial damage of acinar cells was exacerbated in these mice, manifested by increased serum levels of pancreatic enzymes, intra-pancreatic trypsinogen activation and acinar cell apoptosis. In addition, MRL/MpJ pancreata presented enhanced inflammation, de-differentiation of acinar cells and acinar-to-ductal metaplasia. Manipulation of inflammatory levels and mitogenic stimulation with the thyroid hormone 5,3-L-tri-iodothyronine revealed that factors derived from initial acinar injury rather than inflammatory injury promote the replicative advantage in MRL/MpJ mice.

Topics: Acinar Cells; Animals; Cell Proliferation; Ceruletide; Inflammation; Male; Mice; Mice, Inbred C57BL; Pancreas

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

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

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

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

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

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

Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure with considerable mortality. Recently, resolvin D1 (RvD1) as an endogenous anti-inflammatory lipid mediator has been confirmed to protect against many inflammatory diseases. This study was designed to investigate the effects of RvD1 in acute pancreatitis and associated lung injury. Acute pancreatitis varying from mild to severe was induced by cerulein or cerulein combined with LPS, respectively. Mice were pretreated with RvD1 at a dose of 300 ng/mouse 30 min before the first injection of cerulein. Severity of AP was assessed by biochemical markers and histology. Serum cytokines and myeloperoxidase (MPO) levels in pancreas and lung were determined for assessing the extent of inflammatory response. NF-κB activation was determined by Western blotting. The injection of cerulein or cerulein combined with LPS resulted in local injury in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the cerulein and LPS group. Pretreated RvD1 significantly reduced the degree of amylase, lipase, TNF-α, and IL-6 serum levels; the MPO activities in the pancreas and the lungs; the pancreatic NF-κB activation; and the severity of pancreatic injury and associated lung injury, especially in the severe acute pancreatitis model. These results suggest that RvD1 is capable of improving injury of pancreas and lung and exerting anti-inflammatory effects through the inhibition of NF-κB activation in experimental acute pancreatitis, with more notable protective effect in severe acute pancreatitis. These findings indicate that RvD1 may constitute a novel therapeutic strategy in the management of severe acute pancreatitis.

Topics: Animals; Anti-Inflammatory Agents; Ceruletide; Disease Models, Animal; Docosahexaenoic Acids; Gastrointestinal Agents; Inflammation; Interleukin-6; Lung; Lung Injury; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreas; Pancreatitis, Acute Necrotizing; Peroxidase; Protective Agents; Signal Transduction

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

Topics: Acinar Cells; Animals; Cell Transformation, Neoplastic; Ceruletide; Heterozygote; Homeodomain Proteins; Humans; Inflammation; Metaplasia; Mice; Pancreas; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Tumor Suppressor Proteins

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

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

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis - known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Astrocytes; Cell Line, Tumor; Ceruletide; Chondrocytes; Disease Models, Animal; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Nociception; Pain; Pancreatitis, Acute Necrotizing; Primary Cell Culture; Rats; Swine; Thiazoles; Trigeminal Ganglion; TRPA1 Cation Channel; TRPV Cation Channels

Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury.

Topics: Acinar Cells; Acute Disease; Alcohol Drinking; Animals; Ceruletide; Chronic Disease; Collagen; Disease Models, Animal; Gene Deletion; Humans; Inflammation; Macrophages; Mice, Inbred C57BL; Pancreas; Pancreatitis, Chronic; Proto-Oncogene Proteins c-met; Regeneration; Wound Healing

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

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

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies, with a poor long-term prognosis, and effective therapeutic options are lacking. Observing the dynamics of the pathogenesis of pancreatic intraepithelial neoplasia (PanIN) and PDAC in tumor models can facilitate understanding of the molecular mechanisms involved in early PDAC. Furthermore, it can compensate for the research limitations associated with analyzing clinical specimens of late-stage PDAC. In this study, we orthotopically treated the pancreas with dimethylbenzanthracene (DMBA) combined with caerulein in wild-type C57BL/6 J mice to induce inflammation-related pancreatic carcinogenesis. We observed that DMBA and caerulein treatment induced a chronic consumptive disease, which caused a decrease in the relative body and pancreas weights, diminishing the health status of the mice and enhancing the inflammation-related histological changes. Moreover, mid-dose and high-frequency treatment with caerulein caused prolonged inflammatory damage to the pancreas and contributed to a permissive environment for the development of PDAC. CXCL12/CXCR4, CCL2/CCR2, and several cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were upregulated in the tumor tissue of DMBA and caerulein-induced PDAC mice. This orthotopic mouse pancreatic carcinogenesis model mimic human disease because it reproduces a spectrum of pathological changes observed in human PDAC, ranging from inflammatory lesions to pancreatic intraepithelial neoplasia. Thus, this mouse model may improve the understanding of molecular mechanisms underlying the injury-inflammation-cancer pathway in the early stages of pancreatic carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogenesis; Carcinoma, Pancreatic Ductal; Ceruletide; Female; Inflammation; Interleukin-1beta; Interleukin-6; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Tumor Necrosis Factor-alpha; Up-Regulation

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

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

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

Normal tissue architecture is disrupted following injury, as resident tissue cells become damaged and immune cells are recruited to the site of injury. While injury and inflammation are critical to tissue remodeling, the inability to resolve this response can lead to the destructive complications of chronic inflammation. In the pancreas, acinar cells of the exocrine compartment respond to injury by transiently adopting characteristics of progenitor cells present during embryonic development. This process of de-differentiation creates a window where a mature and stable cell gains flexibility and is potentially permissive to changes in cellular fate. How de-differentiation can turn an acinar cell into another cell type (such as a pancreatic β-cell), or a cell with cancerous potential (as in cases of deregulated Kras activity) is of interest to both the regenerative medicine and cancer communities. While it is known that inflammation and acinar de-differentiation increase following pancreatic injury, it remains unclear which immune cells are involved in this process. We used a combination of genetically modified mice, immunological blockade and cellular characterization to identify the immune cells that impact pancreatic regeneration in an in vivo model of pancreatitis. We identified the innate inflammatory response of macrophages and neutrophils as regulators of pancreatic regeneration. Under normal conditions, mild innate inflammation prompts a transient de-differentiation of acinar cells that readily dissipates to allow normal regeneration. However, non-resolving inflammation developed when elevated pancreatic levels of neutrophils producing interferon-γ increased iNOS levels and the pro-inflammatory response of macrophages. Pancreatic injury improved following in vivo macrophage depletion, iNOS inhibition as well as suppression of iNOS levels in macrophages via interferon-γ blockade, supporting the impairment in regeneration and the development of chronic inflammation arises from aberrant activation of the innate inflammatory response. Collectively these studies identify targetable inflammatory factors that can be used to influence the development of non-resolving inflammation and pancreatic regeneration following injury.

Topics: Acinar Cells; Animals; B-Lymphocytes; Cell Dedifferentiation; Ceruletide; Homeodomain Proteins; Immunity, Innate; Inflammation; Interferon-gamma; Macrophage Activation; Macrophages; Mice, Inbred C57BL; Neutrophils; Nitric Oxide Synthase Type II; Pancreas; Regeneration; T-Lymphocytes

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

In the present study, we investigated the protective effects of sivelestat on acute pancreatitis (AP) in a rat model. Sivelestat is a specific neutrophil elastase inhibitor, which has been developed in Japan in 1991. Varying doses of sivelestat in normal saline were infused continuously in sivelestat-treated groups through osmotic pumps. Blood and pancreas samples were collected for serological and histopathological studies, and ten rats in each group were taken for survival observation. Increasing doses of sivelestat inhibits the expression of lipase, amylase, corticosterone, IL-1β, TNF-α, and nuclear factor-κB. Furthermore, sivelestat reduces the inflammatory cells infiltration, histological damage, and mortality rate. Meanwhile, the total antioxidant power and serum level of IL-4 in high-dose sivelestat-treated groups were increased. Our findings suggest that the increasing doses of sivelestat protect against caerulein-induced AP in rats, and this protection is possibly associated with the anti-inflammatory ability of sivelestat.

Topics: Amylases; Animals; Antioxidants; Ceruletide; Corticosterone; Disease Models, Animal; Glycine; Inflammation; Interleukin-1beta; Interleukin-4; Lipase; Male; Oxidative Stress; Pancreas; Pancreatitis, Acute Necrotizing; Proteinase Inhibitory Proteins, Secretory; Rats; Rats, Sprague-Dawley; Serine Proteinase Inhibitors; Sulfonamides; Survival; Transcription Factor RelA; Tumor Necrosis Factor-alpha

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

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

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

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

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Enzyme Induction; Esters; Feedback, Physiological; Gabexate; Gene Expression Regulation, Neoplastic; Gene Knock-In Techniques; Genes, ras; Guanidines; Humans; I-kappa B Kinase; Inflammation; Inflammation Mediators; Lipopolysaccharides; Mice; Mice, Transgenic; Neoplasm Proteins; NF-kappa B; Pancreas; Pancreatic Neoplasms; Pancreatitis, Chronic; Precancerous Conditions; Proto-Oncogene Proteins p21(ras); Sincalide

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

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

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

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

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

Neutrophils and their chemoattractants, the CXC-ELR chemokines keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2), play a critical role in pancreatitis. While acute pancreatitis is initiated in acinar cells, it is unclear if these are a source of CXC-ELR chemokines. KC and MIP-2 have NF-κB, activator protein-1 (AP-1) sites in their promoter regions. However, previous studies have shown increased basal and reduced caerulein-induced AP-1 activation in harvested pancreatic tissue in vitro, which limits interpreting the caerulein-induced response. Moreover, recent studies suggest that NF-κB silencing in acinar cells alone may not be sufficient to reduce inflammation in acute pancreatitis. Thus the aim of this study was to determine whether acinar cells are a source of KC and MIP-2 and to understand their transcriptional regulation. Primary overnight-cultured murine pancreatic acini were used after confirming their ability to replicate physiological and pathological acinar cell responses. Upstream signaling resulting in KC, MIP-2 upregulation was studied along with activation of the transcription factors NF-κB and AP-1. Cultured acini replicated critical responses to physiological and pathological caerulein concentrations. KC and MIP-2 mRNA levels increased in response to supramaximal but not to physiological caerulein doses. This upregulation was calcium and protein kinase C (PKC), but not cAMP, dependent. NF-κB inhibition completely prevented upregulation of KC but not MIP-2. Complete suppression of MIP-2 upregulation required dual inhibition of NF-κB and AP-1. Acinar cells are a likely source of KC and MIP-2 upregulation during pancreatitis. This upregulation is dependent on calcium and PKC. MIP-2 upregulation requires both NF-κB and AP-1 in these cells. Thus dual inhibition of NF-κB and AP-1 may be a more successful strategy to reduce inflammation in pancreatitis than targeting NF-κB alone.

Topics: Animals; Calcium; Ceruletide; Chemokine CXCL1; Chemokine CXCL2; Inflammation; Mice; NF-kappa B; Pancreas; Prostaglandin D2; Protein Kinase C; RNA, Messenger; Tissue Culture Techniques; Transcription Factor AP-1; Transcription, Genetic; Up-Regulation

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

Oxidative stress is regarded as a major pathogenic factor in acute pancreatitis. Inflammation and apoptosis linked to oxidative stress has been implicated in cerulein-induced pancreatitis as an experimental model of acute pancreatitis. Recently, we found that reactive oxygen species mediate inflammatory cytokine expression and apoptosis of pancreatic acinar cells stimulated with cerulein. Omega-3 fatty acids show antioxidant action in various cells and tissues. In the present study, we investigated whether omega-3 fatty acids inhibit cytokine expression in cerulein-stimulated pancreatic acinar cells and whether omega-3 fatty acids suppress apoptotic cell death in pancreatic acinar cells exposed to hydrogen peroxide. We found that omega-3 fatty acids, such as docosahexaenoic acid (DHA) and alpha-linolenic acid (ALA), suppressed the expression of inflammatory cytokines (IL-1beta, IL-6) and inhibited the activation of transcription factor activator protein-1 in cerulein-stimulated pancreatic acinar cells. DHA and ALA inhibited DNA fragmentation, inhibited the decrease in cell viability, and inhibited the expression of apoptotic genes (p53, Bax, apoptosis-inducing factor) induced by hydrogen peroxide in pancreatic acinar cells. In conclusion, omega-3 fatty acids may be beneficial for preventing oxidative stress-induced pancreatic inflammation and apoptosis by inhibiting inflammatory cytokine and apoptotic gene expression of pancreatic acinar cells.

Topics: alpha-Linolenic Acid; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cell Survival; Ceruletide; Cytokines; DNA Fragmentation; Docosahexaenoic Acids; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Fatty Acids, Omega-3; Gene Expression; Hydrogen Peroxide; Inflammation; Interleukin-1beta; Interleukin-6; Pancreatic Neoplasms; Protein Binding; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor AP-1; Tumor Suppressor Protein p53

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

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 outcome from acute pancreatitis depends on the severity of systemic complications. To be able to investigate mechanisms underlying the development of these systemic complications in acute pancreatitis in both wild-type and genetically engineered animal models, a mouse model of severe necrotizing pancreatitis was developed and characterized.. Pancreatitis was induced by retrograde infusion of sodium taurocholate into the common bile duct in mice. After determining the optimum volume and concentration of taurocholate, the pancreatic damage and systemic inflammatory response were compared with those in cerulein-induced pancreatitis.. Pancreatic damage was higher in taurocholate pancreatitis than hyperstimulation-induced pancreatitis (24 hours: cerulein, 5.8 +/- 0.2 points; taurocholate, 14.8 +/- 0.8 points; P < 0.001) and mortality reached up to 60% within the first 24 hours after taurocholate administration. Pulmonary damage was detected, as measured by an increase in albumin in bronchoalveolar lavage fluid only in taurocholate-induced pancreatitis (12 hours: cerulein, 97.1 +/- 22.83 mg/g of protein; taurocholate, 234.0 +/- 32.7 mg/g of protein; P < 0.001). Furthermore, plasma interleukin 6 concentration was significantly elevated in mice with taurocholate-induced pancreatitis (12 hours: cerulein, 2.6 +/- 6.1 pg/mL; taurocholate, 2168.8 +/- 941.7 microg/mL; P < 0.001) as compared with all other groups.. Taurocholate pancreatitis is a reliable model for severe necrotizing pancreatitis in mice with significantly greater pancreatic damage and systemic inflammatory response in comparison with cerulein-induced pancreatitis.

Topics: Albumins; Amylases; Animals; Bronchoalveolar Lavage Fluid; Ceruletide; Disease Models, Animal; Dose-Response Relationship, Drug; Feasibility Studies; Inflammation; Injections; Interleukin-6; Lipase; Lung Diseases; Male; Mice; Mice, Inbred BALB C; Pancreas; Pancreatitis, Acute Necrotizing; Reproducibility of Results; Severity of Illness Index; Taurocholic Acid; Time Factors

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

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

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

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

Missense mutations in human cationic trypsinogen PRSS1 are frequently detected in patients with hereditary pancreatitis, a rare genetic disease of the pancreas characterized by autodigestive necrosis, chronic inflammation, and fibrosis. To examine the link between PRSS1 mutations and the initiation and progression of hereditary pancreatitis, we have sought to generate a transgenic mouse that carries a missense mutation in the PRSS1 that is most frequently observed in patients.. A transgenic mouse was generated in which the expression of the mouse PRSS1 mutant R122H (R122H_mPRSS1) is targeted to pancreatic acinar cells by fusion to the elastase promoter. The expression of the mutant trypsinogen was assessed by immunohistochemical staining and real-time reverse transcription polymerase chain reaction analysis. The relationship between transgene expression and inflammation was analyzed by morphologic assessment of H&E-stained tissue sections, responsiveness to cerulein-induced pancreatitis, and immunohistochemical identification of cellular and biochemical components of the inflammatory response.. Pancreata from transgenic mice display early-onset acinar cell injury and inflammatory cell infiltration. With progressing age, the transgenic mice develop pancreatic fibrosis and display acinar cell dedifferentiation. Moreover, the expression of R122H_mPRSS1 transgene is associated with enhanced response to cerulein-induced pancreatitis. Finally, cell-specific activation of the inflammation-associated signaling pathways, c-jun-N-terminal kinase and extracellular signal-regulated kinase, was observed in response to expression of R122H_mPRSS1.. These results underscore the importance of PRSS1 mutations as pathogenic mediators of hereditary pancreatitis and indicate that persistent pancreatic injury might be causally linked to chronic pancreatitis.

Topics: Animals; Ceruletide; Disease Models, Animal; Disease Progression; DNA; Gene Expression Regulation; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation, Missense; Pancreas; Pancreatitis, Chronic; Phenotype; Trypsinogen

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

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

The investigation of the effects of the celecoxib as a cylooxygenase-2 (COX-2) inhibitor on the course of the acute necrotising pancreatitis (ANP) in rats. ANP was induced in 72 rats by standardized intraductal glycodeoxycholic acid infusion and intravenous cerulein infusion. The rats were divided into four groups (six rats in each group): Sham + saline, sham + celecoxib, ANP + saline, ANP + celecoxib. Six hours later after the ANP induction, celecoxib (10 mg/kg) or saline was given i.p. In the 12th hour, routine cardiorespiratuar, renal parameters were monitored to assess the organ function. The serum amylase, alanine amino transferase (ALT), interleukin 6 (IL-6), lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, the serum concentration of the urea, the tissue activity of myeloperoxidase (MPO) and malondialdehyde (MDA) in pancreas and lungs were measured. The pancreas histology was examined. In the second part of the study, 48 rats were studied in four groups similar to the first part. Survival of all the rats after the induction of ANP was observed for 24 h. The induction of the pancreatitis increased the mortality from 0/12, in the sham groups to 4/12 (30%) in the acute pancreatitis with saline group, 5/12 (42%) in the acute pancreatitis with celecoxib group respectively, heart rate, the serum activities of amylase, ALT, the tissue activities of MPO, MDA in the pancreas and lung, and LDH in BAL fluid, the serum concentration of the urea and IL-6, the degree of the pancreatic damage and decreased the blood pressure, the urine production, pO(2) and the serum concentration of calcium. The use of celecoxib did not alter these changes except the serum IL-6 concentration, urine production and MPO, MDA activities in the tissue of the lungs and pancreas. Serum urea concentration and pancreatic damage in ANP + celecoxib group were insignificantly lesser than ANP + saline group. Whereas treatment with celecoxib improves lung and renal functions, the degree of pancreatic damage partially and the serum IL-6 level completely, it does not improve the cardiovascular and liver functions, the mortality rate and the calcium level. Celecoxib may be useful for the support of some organ functions during ANP in rats.

Topics: Animals; Celecoxib; Ceruletide; Cyclooxygenase Inhibitors; Disease Models, Animal; Edema; Glycodeoxycholic Acid; Inflammation; Interleukin-6; Lung; Male; Malondialdehyde; Multiple Organ Failure; Pancreas; Pancreatitis, Acute Necrotizing; Peroxidase; Pyrazoles; Rats; Rats, Sprague-Dawley; Sulfonamides; Survival Analysis; Urea; Urine

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

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

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

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

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

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

This study was done to evaluate the possible preventive effects of reactive oxygen species (ROS) scavenger agent desferrioxamine (DFX) and platelet-activating factor (PAF) antagonist agent ginkgo biloba (GB) in an experimental acute pancreatitis model. Seventy-eight CD-1 mice were divided into six groups consisting of 10-13 mice. Induction of pancreatitis was achieved by cerulein injection in groups 2-5. The first group was control, whereas DFX and GB were used alone or in combinations as preventive agents in groups 3-5. DFX or GB were injected to the mice in groups 6 and 7 to evaluate any toxic effect. The assessment of the pancreatic edema and inflammation, the measurement of the amylase and the pancreatic weight and the measurement of the pancreatic tissue oxidative capacity by chemiluminescence method were the parameters to evaluate pancreatitis. Although the results indicate DFX and GB alone or in combinations have significant preventive roles, this was not a complete prevention.

Topics: Acute Disease; Amylases; Animals; Ceruletide; Deferoxamine; Disease Models, Animal; Edema; Free Radical Scavengers; Ginkgo biloba; Inflammation; Leukocytes; Luminescent Measurements; Male; Mice; Pancreas; Pancreatitis; Phytotherapy; Plants, Medicinal; Platelet Activating Factor

1. The effects of a novel, potent and orally active nonpeptide bradykinin B2 receptor antagonist, FR167344 (N-[N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2 ,4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylamin ocarbonyl) cinnamylamide hydrochloride) were tested in three different in vivo models of inflammation. 2. Oral administration of FR167344 inhibited carrageenin-induced paw oedema in rats (carrageenin: 1%, 0.1 ml per animal, intraplantar), with an ID50 of 2.7 mg kg(-1) at 2 h after carrageenin injection (n=10 or 11). 3. Oral administration of the compound also inhibited kaolin-induced writhing (kaolin: 250 mg kg(-1), i.p.) in mice, with ID50 of 2.8 mg kg(-1) in 10 min writhing and 4.2 mg kg(-1) in 15 min writhing (n=19 or 20). 4. Additionally, oral administration of FR167344 inhibited caerulein-induced pancreatic oedema with an ID50 of 13.8 mg kg(-1) as well as increases in amylase and lipase of blood samples with ID50 of 10.3 and 7.4 mg kg(-1), respectively, in rats (n=10). 5. These results show that FR167344 is an orally active, anti-inflammatory and anti-nociceptive agent in carrageenin-induced paw oedema, kaolin-induced writhing and caerulein-induced pancreatitis. FR167344 may have therapeutic potential against inflammatory diseases by oral administration and it may be a useful tool for studying the involvement of B2 receptors in various in vivo models of inflammation.

Topics: Administration, Oral; Animals; Antidiarrheals; Bradykinin Receptor Antagonists; Carrageenan; Ceruletide; Disease Models, Animal; Excipients; Gastrointestinal Agents; Inflammation; Kaolin; Male; Mice; Mice, Inbred ICR; Pain; Pancreatitis; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; Receptors, Bradykinin