sincalide and Inflammation

This study aimed at exploring the expression level of LTBP1 in the mouse model of epilepsy. The mechanism of LTBP1 in epileptic cerebral neural stem cells was deeply investigated to control the occurrence of epilepsy with neuroprotection.. qRT-PCR was conducted for the expression levels of LTBP1 in clinical human epileptic tissues and neural stem cells, as well as normal cerebral tissues and neural stem cells. The mouse model of postischemic stroke epilepsy (PSE) was established by the middle cerebral artery occlusion (MCAO). Then, qRT-PCR was conducted again for the expression levels of LTBP1 in mouse epileptic tissues and neural stem cells as well as normal cerebral tissues and neural stem cells. The activation and inhibitory vectors of LTBP1 were constructed to detect the effects of LTBP1 on the proliferation of cerebral neural stem cells in the PSE model combined with CCK-8. Finally, Western blot was conducted for the specific mechanism of LTBP1 affecting the development of epileptic cells.. Racine score and epilepsy index of 15 mice showed epilepsy symptoms after the determination with MCAO, showing a successful establishment of the PSE model. LTBP1 expression in both diseased epileptic tissues and cells was higher than that in normal clinical epileptic tissues and cells. Meanwhile, qRT-PCR showed higher LTBP1 expression in both mouse epileptic tissues and their neural stem cells compared to that in normal tissues and cells. CCK-8 showed that the activation of LTBP1 stimulated the increased proliferative capacity of epileptic cells, while the inhibition of LTBP1 expression controlled the proliferation of epileptic cells. Western blot showed an elevated expression of TGFβ/SMAD signaling pathway-associated protein SMAD1/5/8 after activating LTBP1. The expression of molecular MMP-13 associated with the occurrence of inflammation was also activated.. LTBP1 can affect the changes in inflammation-related pathways by activating the TGFβ/SMAD signaling pathway and stimulate the development of epilepsy, and the inhibition of LTBP1 expression can control the occurrence of epilepsy with neuroprotection.

Topics: Animals; Cerebral Cortex; Disease Models, Animal; Epilepsy; Gene Expression; Humans; Inflammation; Latent TGF-beta Binding Proteins; Mice; Neuroprotection; Sincalide; Stroke; Transforming Growth Factor beta

Rheumatoid arthritis (RA) is a systemic autoimmune disease, which has been reported closely associated with the dysfunction of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. This study aims to explore the potential therapeutic effect of Tofacitinib, a putative JAK/STAT inhibitor, in RA. Tofacitinib suppressed proliferation and accelerated apoptosis of rheumatoid arthritis synovial fibroblasts (RA-FLS) as confirmed by CCK-8, EdU and Western blot assays. Tofacitinib significantly inhibited expression of pro-inflammatory factors including tumor necrosis factor-α (TNF-α), vascular endothelial growth factor A, matrix metalloproteinase 1, matrix metalloproteinase 3, interleukin-6 and interferon gamma in RA-FLS cells. mechanistically, tofacitinib decreased signal transducer and activator of transcription 6 (STAT6), which transcriptionally activates miR-425-5p, and thus increased insulin like growth factor 1 (IGF1) expression, a target of miR-425-5p in RA-FLS. Overexpression of STAT6 restored the expression of pro-inflammatory factors and proliferation inhibited by Tofacitinib in RA-FLS. Overall, Tofacitinib exerted inhibitory effect on proliferation and inflammation of RA-FLS through modulating STAT6/miR-425-5p/IGF1 signal axis. These findings shed light on the novel strategies for improving RA.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Fibroblasts; Humans; Inflammation; Insulin-Like Growth Factor I; Interferon-gamma; Interleukin-6; Janus Kinases; Matrix Metalloproteinase 1; MicroRNAs; Piperidines; Pyrimidines; Sincalide; STAT6 Transcription Factor; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Asthma is a chronic airway disease involving airway inflammation and remodeling. Studies showed that tripartite motif-containing protein 33 (TRIM33) regulated natural immunity, inflammation, and pulmonary fibrosis. However, the role and regulatory mechanism of TRIM33 in children's asthma are unclear. In this study, the TRIM33 expressions in serum samples and platelet-derived growth factor BB (PDGF-BB)-induced airway smooth-muscle cells (ASMCs) were evaluated. A gain-of-function experiment was performed, and cell proliferation and migration were detected using CCK-8 and wound healing assays. Besides, the protein levels of EMT biomarkers and airway-remodeling markers were determined by Western blot assay. ELISA analyzed the contents of IL-1β, IL-6, and TNF-α in the supernatant. The modulation of Smad4 expression and subsequent activation of Wnt/β-catenin by TRIM33 were also assessed. We found that TRIM33 was downregulated in the serum from children who were asthma patients and PDGF-BB-induced ASMCs. TRIM33 overexpression showed decrease of PDGF-BB-induced ASMC proliferation and migration. Moreover, the augment of TRIM33 reduced the PDGF-BB-induced cell EMT and airway-remodeling marker levels and suppressed the secretions of inflammatory cytokines in PDGF-BB-induced ASMCs. Additionally, TRIM33 overexpression inhibited activation of Wnt/β-catenin via reducing Smad4 expression to regulate asthma inflammation and airway remodeling. All in all, our study revealed that TRIM33 expression was downregulated in children who were asthma patients and PDGF-BB-induced ASMCs. TRIM33 modulated PDGF-BB-induced inflammation and airway remodeling of ASMCs by the Wnt/β-catenin pathway via regulating Smad4, which may provide a new treatment direction for asthma.

Topics: Airway Remodeling; Asthma; Becaplermin; beta Catenin; Cell Movement; Cell Proliferation; Cells, Cultured; Child; Humans; Inflammation; Interleukin-6; Myocytes, Smooth Muscle; Sincalide; Transcription Factors; Tumor Necrosis Factor-alpha; Wnt Signaling Pathway

Diabetic retinopathy (DR), the major complication of diabetes, is the leading cause of vision loss and blindness globally. Altered circular RNAs (circRNAs) expression has been found to be involved in DR process. Hence, this work aimed to explore the role and mechanism of circCOL1A2 in DR.. Human retinal microvascular endothelial cells (RMECs) treated with high glucose (HG) were used for functional analysis. Levels of genes and proteins were detected using quantitative real-time polymerase chain reaction and western blotting. In vitro experiments were conducted by transwell, tube formation, CCK-8 assays and ELISA, respectively. The binding interaction between miR-646 and circCOL1A2 or FGF7 (Fibroblast Growth Factor 7) was confirmed using dual-luciferase reporter and RNA immunoprecipitation assays.. CircCOL1A2 was highly expressed in retinal tissues of DR patients and HG-induced RMECs. Then RMECs were exposed to HG treatment to mimic the diabetic conditions in vitro. Functionally, circCOL1A2 knockdown attenuated HG-evoked RMEC migration, proliferation, angiogenesis, blood-retina barrier (BRB) injury and inflammation. Mechanistically, circCOL1A2 functioned as a sponge for miR-646, and miR-646 directly targeted FGF7. Further rescue experiments showed that miR-646 inhibition abated the protective effects of circCOL1A2 knockdown on RMEC function under HG treatment. Besides that, miR-646 was decreased in HG-induced RMECs, re-expression of miR-646 reversed HG-evoked RMEC dysfunction, which was rescued by FGF7 overexpression.. CircCOL1A2 silencing can suppress HG-induced migration, proliferation, angiogenesis, BRB injury and inflammation in RMECs through miR-646/FGF7 axis, suggesting the potential involvement of circCOL1A2 in DR process.

Topics: Cell Proliferation; Diabetes Mellitus; Diabetic Retinopathy; Down-Regulation; Endothelial Cells; Fibroblast Growth Factor 7; Glucose; Humans; Inflammation; MicroRNAs; Neovascularization, Pathologic; Retina; RNA, Circular; Sincalide

Topics: Animals; Cytokines; Dexmedetomidine; Glycogen Synthase Kinase 3 beta; Hydrogen Peroxide; Inflammation; Intracranial Aneurysm; Muscle, Smooth, Vascular; NF-E2-Related Factor 2; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Adrenergic, alpha-2; Sincalide

Inflammation of RPE cells led to different kinds of eye diseases and affected the normal function of the retina. Furthermore, higher levels of ROCK1 and ROCK2 induced injury of endothelial cells and many inflammatory diseases of the eyes. Ripasudil, which was used for the treatment of glaucoma, was one kind of the inhibitor of ROCK1 and ROCK2, but whether ripasudil could relieve the LPS-induced inflammation and damage of RPE cells was not clear.. We used LPS to stimulate ARPE-19 cells, the RPE cell line. After that, we detected the levels of ROCK1 and ROCK2 by western-blotting after the stimulation of LPS and treatment of ripasudil. Then luciferase reporter assays were used to confirm the targeting effect of miR-136-5p on ROCK1 and ROCK2. At last, the levels of NLRP3, ASC, caspase1, IL-1β and IL-18 were detected with the western-blotting after the knockdown of miR-136-5p.. The levels of ROCK1, ROCK2 and miR-136-5p in ARPE-19 cells were promoted after the stimulation of LPS. After the treatment of ripasudil, the expression levels of ROCK1, ROCK2 and miR-136-5p were suppressed. The expression of ROCK1 and ROCK2 was targeted and inhibited by the miR-136-5p. The levels of inflammation related proteins NLRP3, ASC, caspase1, IL-1β and IL-18 was also inhibited after the treatment of ripasudil. However, the expression of these proteins was rescued after the knockdown of miR-136-5p.. Ripasudil relieved the inflammatory injury of RPE cells by upregulating miR-136-5p, therefore inhibiting the expression of ROCK1, ROCK2, NLRP3, ASC, caspase1, IL-1β and IL-18.

Topics: Apoptosis; Blotting, Western; Cell Line; Drug Delivery Systems; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Humans; Inflammation; Isoquinolines; MicroRNAs; NLR Family, Pyrin Domain-Containing 3 Protein; Real-Time Polymerase Chain Reaction; Retinal Pigment Epithelium; rho-Associated Kinases; Sincalide; Sulfonamides; Up-Regulation

Pain is associated with negative emotions such as anxiety, but the underlying neurocircuitry and modulators of the association of pain and anxiety remain unclear. The neuropeptide cholecystokinin (CCK) has both pronociceptive and anxiogenic properties, so we explored the role of CCK in anxiety and nociception in the central amygdala (CeA), a key area in control of emotions and descending pain pathways. Local infusion of CCK into the CeA of control rats increased anxiety, as measured in the light-dark box test, but had no effect on mechanical sensitivity. By contrast, intra-CeA CCK infusion 4 days after Complete Freund's Adjuvant (CFA) injection into the hindpaw resulted in analgesia, but also in loss of its anxiogenic capacity. Inflammatory conditions induced changes in the CeA CCK signaling system with an increase of CCK immunoreactivity and a decrease in CCK1, but not CCK2, receptor mRNA. In CFA rats, patch-clamp experiments revealed that CCK infusion increased CeA neuron excitability. It also partially blocked the discharge of wide dynamic range neurons in the dorsal spinal cord. These effects of CCK on CeA and spinal neurons in CFA rats were mimicked by the specific CCK2 receptor agonist, gastrin. This analgesic effect was likely mediated by identified CeA neurons projecting to the periaqueductal gray matter that express CCK receptors. Together, our data demonstrate that intra-CeA CCK infusion activated a descending CCK2 receptor-dependent pathway that inhibited spinal neuron discharge. Thus, persistent pain induces a functional switch to a newly identified analgesic capacity of CCK in the amygdala, indicating central emotion-related circuit controls pain transmission in spinal cord.

Topics: Amygdala; Animals; Cholecystokinin; Dark Adaptation; Disease Models, Animal; Exploratory Behavior; Freund's Adjuvant; Gastrins; Glutamate Decarboxylase; Inflammation; Male; Neurons; Nociception; Pain; Pain Threshold; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptor, Cholecystokinin B; Signal Transduction; Sincalide; Tetragastrin

Excessive extracellular matrix degradation and chondrocyte apoptosis are the pathological features of osteoarthritis (OA). The ability of flavonoid compounds isolated from Chinese hawthorn leaves to exert protective effects on several diseases, via inhibition of oxidative stress and inflammation, has been demonstrated in several studies. This study explored the effects of vitexin on chondrocytes, and the underlying mechanisms thereof. Vitexin, an active ingredient in hawthorn leaf extracts, was shown to exert protective effects on chondrocytes, by inhibiting the expression of GRP78 and PDI, and an apoptotic protein (CHOP) induced by interleukin-1β. It also modulated thapsigargin-induced upregulation of GRP78 and PDI and subsequently an apoptotic protein (CHOP). Among rat chondrocytes, both the ER stress-activated nuclear factor kappa B (NF-κB) pathway and the induced expression of inflammatory cytokines (IL-6 and TNF-α) were significantly inhibited by vitexin. Finally, vitexin attenuated the progression of OA in vivo in rats. Taken together, all data demonstrate the relationship of ER stress and inflammation in the progression of OA, the ability of vitexin to protect chondrocytes and thus its therapeutic potential in patients with OA.

Topics: Animals; Apigenin; Apoptosis; Cartilage; Caspase 3; Cell Survival; Chondrocytes; Disease Models, Animal; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Inflammation; Interleukin-1beta; Male; NF-kappa B; Osteoarthritis; Rats; Rats, Sprague-Dawley; Sincalide; Thapsigargin; Transcription Factor CHOP; Tumor Necrosis Factor-alpha; Up-Regulation

Acute lung injury (ALI) is mainly characterized by diffusive injuries to lung epithelium and increased permeability of alveolar-capillary membranes caused by various factors, which leads to pulmonary edema and pulmonary closure. Lipopolysaccharide (LPS), which is the main component of the cell wall of gram-negative bacteria, is one of the most important factors causing pulmonary infection and ALI. More and more reports have indicated that hydrogen sulfide (H

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Cystathionine gamma-Lyase; Humans; Hydrogen Sulfide; Inflammation; Lipopolysaccharides; Rats; Shock, Septic; Sincalide

Inflammatory response and oxidative stress are considered to play an important role in the development of acute liver injury induced by carbon tetrachloride (CCl4) and galactosamine (GalN)/lipopolysaccharides (LPS). Esculentoside A (EsA), isolated from the Chinese herb phytolacca esculenta, has the effect of modulating immune response, cell proliferation and apoptosis as well as anti-inflammatory effects. The present study is to evaluate the protective effect of EsA on CCl4 and GalN/LPS-induced acute liver injury. In vitro, CCK-8 assays showed that EsA had no cytotoxicity, while it significantly reduced levels of TNF-α and cell death rate challenged by CCl4. Moreover, EsA treatment up-regulated PPAR-γ expression of LO2 cells and reduced levels of reactive oxygen species (ROS) challenged by CCl4. In vivo, EsA prevented mice from CCl4-induced liver histopathological damage. In addition, levels of AST and ALT were significantly decreased by EsA treatment. Furthermore, the mice treated with EsA had a lower level of TNF-α, Interleukin (IL)-1β and IL-6 in mRNA expression. EsA prevented MDA release and increased GSH-Px activity in liver tissues. Immunohistochemical staining showed that over-expression of F4/80 and CD11b were markedly inhibited by EsA. The western bolt results showed that EsA significantly inhibited CCl4-induced phosphonated IkBalpha (P-IκB) and ERK. Furthermore, EsA treatment also alleviated GalN/LPS-induced acute liver injury on liver enzyme and histopathological damage. Unfortunately, our results exhibited that EsA had no effects on CCl4-induced hepatocyte apoptosis which were showed by TUNEL staining and Bax, Caspase-3 and cleaved Caspase-3 expression. Our results proved that EsA treatment attenuated CCl4 and GalN/LPS-induced acute liver injury in mice and its protective effects might be involved in inhibiting inflammatory response and oxidative stress, but not apoptosis with its underlying mechanism associated with PPAR-γ, NF-κB and ERK signal pathways.

Topics: Animals; Apoptosis; Carbon Tetrachloride; Cell Proliferation; Chemical and Drug Induced Liver Injury; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Interleukin-1beta; Interleukin-6; Mice; Oleanolic Acid; Oxidative Stress; Reactive Oxygen Species; Saponins; Signal Transduction; Sincalide

Inflammatory process is involved in the pathogenesis of diabetic nephropathy. In this article, we show that cholecystokinin (CCK) is expressed in the kidney and exerts renoprotective effects through its anti-inflammatory actions. DNA microarray showed that CCK was upregulated in the kidney of diabetic wild-type (WT) mice but not in diabetic intracellular adhesion molecule-1 knockout mice. We induced diabetes in CCK-1 receptor (CCK-1R) and CCK-2R double-knockout (CCK-1R(-/-),-2R(-/-)) mice, and furthermore, we performed a bone marrow transplantation study using CCK-1R(-/-) mice to determine the role of CCK-1R on macrophages in the diabetic kidney. Diabetic CCK-1R(-/-),-2R(-/-) mice revealed enhanced albuminuria and inflammation in the kidney compared with diabetic WT mice. In addition, diabetic WT mice with CCK-1R(-/-) bone marrow-derived cells developed more albuminuria than diabetic CCK-1R(-/-) mice with WT bone marrow-derived cells. Administration of sulfated cholecystokinin octapeptide (CCK-8S) ameliorated albuminuria, podocyte loss, expression of proinflammatory genes, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, CCK-8S inhibited both expression of tumor necrosis factor-α and chemotaxis in cultured THP-1 cells. These results suggest that CCK suppresses the activation of macrophage and expression of proinflammatory genes in diabetic kidney. Our findings may provide a novel strategy of therapy for the early stage of diabetic nephropathy.

Topics: Animals; Chemokines, CC; Chemotaxis; Cholecystokinin; Diabetes Mellitus; Gene Expression Profiling; Gene Expression Regulation; Inflammation; Intercellular Adhesion Molecule-1; Kidney; Macrophages; Male; Mice; Mice, Knockout; NF-kappa B; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Sincalide; Tumor Necrosis Factor-alpha

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

Animal models of inflammatory pain are characterized by the release of inflammatory mediators such as cytokines and neurotrophic factors, and enhanced analgesic sensitivity to opioids. In this study, we examine the mechanisms underlying this effect, in particular the roles of cholecystokinin (CCK) and nerve growth factor (NGF), in an animal model of central nervous system (CNS) inflammation induced by spinal administration of lipopolysaccharide (LPS). Although spinal administration of LY-225910 (25 ng), a CCK-B antagonist, enhanced morphine analgesia in naïve rats, it was unable to do so in LPS-treated animals. Conversely, spinal CCK-8S administration (1 ng) decreased morphine analgesia in LPS-treated rats, but not in naïve animals. Further, spinal anti-NGF (3 microg) was able to reduce morphine analgesia in LPS-treated rats, but not in naïve animals, an effect that was reversed by spinal administration of LY-225910. While CCK-8S concentration was increased in spinal cord extracts of LPS animals as compared to controls, morphine-induced spinal CCK release in the extracellular space, as measured by in-vivo spinal cord microdialysis was inhibited in LPS animals as compared to controls, and this was reversed by anti-NGF pretreatment. Finally, chronic spinal administration of beta-NGF (7 microg/day) for 7 days enhanced spinal morphine analgesia, possibly by mimicking a CNS inflammatory state. We suggest that in intrathecally LPS-treated rats, spinal CCK release is altered resulting in enhanced morphine analgesia, and that this mechanism may be regulated to an important extent by NGF.

Topics: Analgesics; Animals; Central Nervous System Diseases; Cholecystokinin; Disease Models, Animal; Inflammation; Injections, Spinal; Lipopolysaccharides; Male; Morphine; Nerve Growth Factor; Rats; Rats, Long-Evans; Sincalide; Spinal Cord

To study the anti-inflammatory effects of cholecystokinin-octapeptide (CCK-8) on lipopolysaccharide (LPS)-induced endotoxic shock (ES) and further investigate its signal transduction pathways involving p38 mitogen-activated protein kinase (MAPK) and IkappaB-alpha.. Eighty-four rats were divided randomly into four groups: LPS (8 mg.kg(-1), iv) induced ES; CCK-8 (40 microg.kg(-1), iv) pretreatment 10 min before LPS (8 mg.kg(-1)); CCK-8 (40 microg.kg(-1), iv) or normal saline (control) groups. The inflammatory changes of lung and spleen, phagocytic function of alveolar macrophage, quantification of inflammatory cells in bronchoalveolar lavage (BAL) were investigated in rats by using hematoxylin and eosin (HE) staining, phagocytosis of Candida albicans and differential cell counting. Nitric oxide (NO) production in serum, lung and spleen was measured with the Griess reaction. The mechanism involving p38 MAPK and IkappaB-alpha signal pathways was investigated by Western blot.. Inflammatory changes of lung and spleen induced by LPS were alleviated by CCK-8, the increase of NO induced by LPS in serum, lung and spleen was significantly inhibited and the neutrophil infiltration in BAL was significantly reduced by CCK-8. The number of neutrophils was (52+/-10)X10(6) cells. (-1) in LPS group, while it decreased to (18+/-4)X10(6) cells. (-1) in CCK-8+LPS (P<0.01). The phagocytic rate of CCK-8 group increased to (62.49+/-9.49) %, compared with control group (48.16+/-14.20) %, P<0.05. The phagocytosis rate was (85.14+/-4.64) % in LPS group, which reduced to (59.33+/-3.14) % in CCK-8+LPS group (P<0.01). The results of phagocytosis indexes showed similar changes. CCK-8 may play an important role in increasing the expression of p38 MAPK and decreasing the degradation of IkappaB-alpha in lung and spleen of ES rats.. CCK-8 can result in anti-inflammatory effects, which may be related to activation of p38 MAPK and inhibition on the degradation of IkappaB-alpha.

Topics: Animals; DNA-Binding Proteins; I-kappa B Proteins; Inflammation; Mitogen-Activated Protein Kinases; NF-KappaB Inhibitor alpha; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Phagocytosis; Rats; Rats, Sprague-Dawley; Shock, Septic; Signal Transduction; Sincalide

To elucidate the anti-inflammatory mechanism of an intestinal neuropeptide, sulfated cholecystokinin octapeptide (sCCK-8), the effects of sCCK-8 on lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) mRNA expression and NF-kappaB activity in pulmonary interstitial macrophages (PIMs) were studied.. PIMs from rat were stimulated with LPS (1 mg.L(-1)) in the presence or absence of sCCK-8 (10(-8)-10(-6)mol.L(-1)) or/and CCK receptor antagonist proglumide (2 mg.L(-1)). The expression of TNF-alpha mRNA was assayed by reverse transcription polymerase chain reaction (RT-PCR) at 3h of the stimulation, and nuclear factor-kappaB (NF-kappaB) binding activity was analyzed by electrophoretic mobility shift assay (EMSA) at 1 h of stimulation. The IkappaBalpha protein level in the cytoplasma at 30 min of the stimulation was detected by Western blot.. sCCK-8, at concentrations from 10(-8) mol.L(-1) to 10(-6) mol.L(-1) obviously inhibited LPS-induced TNF-alpha mRNA expression and NF-kappaB binding activity in a dose-dependent manner, P<0.05, P<0.01. Stimulation PIMs with LPS resulted in a reduction of IkappaBalpha protein level, P<0.01, which was elevated by sCCK-8, P<0.05. The effects of sCCK-8 on NF-kappaB activity and IkappaB protein level were attenuated by CCK receptor antagonist proglumide, P<0.01.. sCCK-8 inhibits LPS-induced TNF-alpha mRNA expression by regulating NF-kappaB activity in rat PIMs, which is mediated through CCK receptors and inhibiting IkappaB-alpha degradation. This represents one of the anti-inflammatory mechanisms of sCCK-8.

Topics: Animals; DNA-Binding Proteins; Female; I-kappa B Proteins; In Vitro Techniques; Inflammation; Lipopolysaccharides; Macrophages, Alveolar; NF-kappa B; NF-KappaB Inhibitor alpha; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sincalide; Tumor Necrosis Factor-alpha

Some opioid-resistant pain conditions can be alleviated by voltage-dependent Na(+) channel blockers such as lamotrigine. The mu-opioid-receptor agonist morphine can modulate cation entry into cells to affect overall cellular excitability, an effect which can in turn be endogenously antagonised by the neuropeptide cholecystokinin (CCK). However, lamotrigine may also modulate cellular excitability by non-specifically blocking voltage-dependent ion channels. We have looked for interactions of lamotrigine with the opioid/CCK pathway within the spinal dorsal horn, to rule out the possibility that lamotrigine may attenuate nociceptive responses via actions on this pathway. Both lamotrigine and the mu-opioid agonist DAMGO inhibited mustard oil-evoked cell firing by approximately 50% compared with control levels. Co-application of CCK8S reversed DAMGO-, but not lamotrigine-induced inhibition of cell firing and this reversal was prevented with the selective CCK(B) receptor antagonist PD 135158. Although lamotrigine inhibited both brush- and cold-evoked cell firing in neuropathic animals, lamotrigine inhibition of mustard oil-evoked cell firing in the same animals was not significantly greater than that observed in controls. These results suggest that the antinociceptive properties of lamotrigine within the spinal dorsal horn are unlikely to be mediated via interactions with the opioid/CCK pathway.

Topics: Action Potentials; Analgesics, Opioid; Animals; Anti-Anxiety Agents; Calcium Channel Blockers; Cholecystokinin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Indoles; Inflammation; Lamotrigine; Male; Meglumine; Mustard Plant; Neural Pathways; Nociceptors; Opioid Peptides; Pain; Peripheral Nervous System Diseases; Plant Extracts; Plant Oils; Posterior Horn Cells; Rats; Rats, Wistar; Sincalide; Triazines

To compare the common procedure in tensiometry of normalization of the force (in N) produced by a gallbladder tissue strip to units of stress, with normalization of force to the strip content of contractile protein.. A comparison was made in both healthy and in diseased gallbladder tissue strips between two normalization procedures involving anatomical parameters. The contractile response expressed in terms of tissue stress (in N/m2), which entails a normalization to the strip cross-sectional area, was set against normalization to the tissue content of contractile protein (in N/mg actin/g strip wet weight).. Dose-response curves for acetylcholine (ACh) (10(-8) to 10(-3) M) and sulphated cholecystokinin octapeptide (CCK) (10(-12) to 10(-6) M) were assessed in healthy guinea pig (n = 8) and in diseased human gallbladder tissue strips (n = 28). Assuming a tissue density of 1.05 g/cm3, the strip cross-sectional area was calculated from its weight and length. Actin content in homogenized strips was determined by polyacrylamide gel electrophoresis followed by densitometry.. Actin content in human tissue was 19.06 +/- 1.42 mg/g strip wet weight, and 12.84 +/- 0.76 mg/g strip wet weight in guinea pig tissue. No correlation was found between strip cross-sectional area and actin content. In the diseased human tissue, no correlation was found between the inflammation score and either strip cross-sectional area or strip actin content. Maximal force (in mN) exerted in response to either ACh or CCK correlated much more closely in healthy guinea pig gallbladder (r = 0.97) than in diseased human tissue (r = 0.59). Normalization of maximal force to strip cross-sectional area (i.e. to stress) showed considerable more variation (% coefficient of variance) than the normalization to strip actin content in healthy guinea pig tissue, although both strip cross-sectional area and actin content per se showed little variation. Normalization to either parameter did not result in an improved correlation or a decreased variation in the case of diseased human gallbladder tissue.. Normalization of muscle strip force in diseased tissue is questionable, as the assumptions made for healthy tissue are not valid.

Topics: Acetylcholine; Actins; Animals; Cholelithiasis; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gallbladder; Guinea Pigs; Humans; In Vitro Techniques; Inflammation; Muscle Contraction; Sincalide; Stress, Mechanical

1. Cholecystokinin (CCK) has been shown to diminish opioid analgesia. Here we investigate whether changes in the physiological levels of spinal CCK are responsible for the enhanced potency of spinal morphine in animals following carrageenin inflammation, as compared with normal animals. 2. Single dorsal horn nociceptive neurones were recorded in intact halothane-anaesthetized rats in the presence and absence of carrageenin-induced inflammation and comparisons were made between the two groups of animals. Inflammation was induced by the injection of 100 microliters of 2% lambda-carrageenin into the hind paw. 3. The inhibitory effect of intrathecal morphine on the C-fibre-evoked responses of the neurones was enhanced in the carrageenin-treated animals such that the effects of 0.25 microgram and 10 micrograms of morphine in normal animals were comparable to those of 0.01 microgram and 2.5 micrograms in the carrageenin animals. The effect of 0.2 mg kg-1 of the CCKB antagonist, L-365,260, on the antinociceptive potency of intrathecal morphine was examined in both groups of animals. In normal animals, L-365,260 produced a significant enhancement in the effect of morphine indicating a tonic CCK modulation in these animals, but it had no effect on the inhibitions produced by either dose of morphine in the carrageenin animals. 4. The inhibition of the C-fibre-evoked response produced by intrathecal morphine in the presence of 1 microgram of CCK was examined in both groups of animals. CCK attenuated the effects of morphine only in animals with carrageenin inflammation, having no effect on the action of morphine in normal animals. 5. The effects of both CCK and L-365,260 were therefore dependent on the inflammatory state of the animal, with each drug being active in opposite situations.6. We propose that in normal animals, morphine may produce a maximal stimulation of the release of CCK such that exogenous CCK is unable to reduce further the analgesic effects under these conditions.However, the differential effects of the agonist and antagonist in the normal and inflamed rats points to a role of CCK in the enhanced opiate actions. This enhancement of the potency of spinal morphine in inflammation is best explained by a reduction in spinal CCK release by morphine in this state.

Topics: Animals; Benzodiazepinones; Carrageenan; Cholecystokinin; Inflammation; Injections, Spinal; Male; Morphine; Nerve Fibers; Phenylurea Compounds; Rats; Rats, Sprague-Dawley; Sincalide; Spinal Cord