Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct.

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

Tetrahedral Framework Nucleic Acids Can Alleviate Taurocholate-Induced Severe Acute Pancreatitis and Its Subsequent Multiorgan Injury in Mice.

Severe acute pancreatitis (SAP) is an inflammatory disease of the pancreas accompanied by tissue injury and necrosis. It not only affects the pancreas but also triggers a systemic inflammatory response that leads to multiorgan failure or even death. Moreover, there is no effective treatment currently that can reverse the disease progression. In this study, tetrahedral framework nucleic acids (tFNAs) were utilized to treat SAP in mice for the first time and proved to be effective in suppressing inflammation and preventing pathological cell death. Serum levels of pancreatitis-related biomarkers witnessed significant changes after tFNAs treatment. Reduction in the expression of certain cytokines involved in local and systemic inflammatory response were observed, together with alteration in proteins related to cell death and apoptosis. Collectively, our results demonstrate that tFNAs could both alleviate SAP and its subsequent multiorgan injury in mice, thus offering a novel and effective option to deal with SAP in the future.

Release of endogenous hydrogen sulfide in enteric nerve cells suppresses intestinal motility during severe acute pancreatitis.

Previous studies have shown that during severe acute pancreatitis (SAP) attacks, hydrogen sulfide (H2S) is released in the colon. However, the roles played by H2S in regulating enteric nerves remain unclear. In this study, we examined the association between SAP-induced H2S release and loss of intestinal motility, and also explored the relevant mechanism in enteric nerve cells. A rat SAP model was constructed and enteric nerve cells were prepared. Intestinal mobility was evaluated by measuring the number of bowel movements at indicated time points and by performing intestinal propulsion tests. The production of inflammatory cytokines during a SAP attack was quantified by ELISA, and the levels of cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS) were examined by immunohistochemistry and western blot analysis. In vivo studies showed that PI3K/Akt/Sp1 signaling in enteric nerve cells was blocked, confirming the mechanism of endogenous H2S formation by western blot analysis and immunofluorescence. Our results also showed that rats with SAP symptoms had reduced intestinal motility. Furthermore, PI3K/Akt/Sp1 signaling was triggered and CSE expression was up-regulated, and these changes were associated with H2S formation in the colon. In addition, propargylglycine reduced the levels of inflammatory cytokines and suppressed the release of H2S. Enteric nerve cells that were incubated with LY294002 and transfected with a Sp1-knockdown vector displayed decreased levels of CSE production, which led to a decrease in H2S production. These results suggest that SAP symptoms suppressed the intestinal motility of rats via the release of H2S in enteric nerve cells, which was dependent on the inflammation-induced PI3K/Akt/Sp1 signaling pathway.

Acute pancreatitis promotes the generation of two different exosome populations.

Exosomes are small extracellular vesicles that act as intercellular messengers. Previous studies revealed that, during acute pancreatitis, circulating exosomes could reach the alveolar compartment and activate macrophages. However, proteomic analysis suggested that the most likely origin of these exosomes could be the liver instead of the pancreas. The present study aimed to characterize the exosomes released by pancreas to pancreatitis-associated ascitic fluid (PAAF) as well as those circulating in plasma in an experimental model of taurocholate-induced acute pancreatitis in rats. We provide evidence that during acute pancreatitis two different populations of exosomes are generated with relevant differences in cell distribution, protein and microRNA content as well as different implications in their physiological effects. During pancreatitis plasma exosomes, but not PAAF exosomes, are enriched in the inflammatory miR-155 and show low levels of miR-21 and miR-122. Mass spectrometry-based proteomic analysis showed that PAAF exosomes contains 10-30 fold higher loading of histones and ribosomal proteins compared to plasma exosomes. Finally, plasma exosomes have higher pro-inflammatory activity on macrophages than PAAF exosomes. These results confirm the generation of two different populations of exosomes during acute pancreatitis. Deep understanding of their specific functions will be necessary to use them as therapeutic targets at different stages of the disease.

Effect of SOCS3 on lung injury in rats with severe acute pancreatitis through regulating JAK2/STAT3 signaling pathway.

OBJECTIVE: To explore the effect of suppressor of cytokine signaling 3 (SOCS3) on the lung injury in rats with severe acute pancreatitis (SAP) by regulating the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. MATERIALS AND METHODS: Sprague-Dawley rats were divided into control group (n=20) and SAP model group (established via injection of 5% sodium taurocholate, n=40). Then, SOCS3 was overexpressed using the adenovirus in 20 rats in SAP model group. The serum amylase (AMY) was detected, whether the transfection was successful was verified via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), the hepatic function indexes were detected, the pathological changes were observed using hematoxylin-eosin (HE) staining, and the wet/dry weight ratio (W/D) was calculated. Moreover, the content of serum inflammatory factors was detected via enzyme-linked immunosorbent assay (ELISA) and the expression levels of JAK2/STAT3 signaling pathway genes and proteins were detected through RT-PCR and Western blotting. RESULTS: The content of AMY in SAP model group was significantly increased, indicating the successful modeling. SOCS3 was significantly increased in transfection group, suggesting that the transfection efficiency was significant. The content of alanine aminotransferase (ALT),  and aspartate aminotransferase (AST) in SOCS3 transfection group was significantly lower than in model group. According to the histopathological observation, there were lung injury, pulmonary edema, hemorrhage, severe inflammatory response, and alveolar congestion in SAP model group. There were almost no pathological changes in SOCS3 transfection group. In SOCS3 transfection group, the content of serum interleukin-6 (IL-6), IL-18 and tumor necrosis factor-α (TNF-α), the mRNA and protein expressions of IL-6, JAK2, and STAT3 were all remarkably declined. CONCLUSIONS: SOCS3 inhibits the activation of the JAK2/STAT3 pathway and the increase of inflammatory factors, promoting the repair of lung injury in SAP rats.

c-Abl kinase regulates neutrophil extracellular trap formation, inflammation, and tissue damage in severe acute pancreatitis.

Neutrophil extracellular traps (NETs) are involved in acute pancreatitis (AP) but mechanisms controlling NET expulsion in AP are incompletely understood. Herein, we examined the role of c-Abelson (c-Abl) kinase in NET formation and tissue damage in severe AP. AP was induced by taurocholate infusion into pancreatic duct or intraperitoneal administration of l-arginine in mice. Pancreatic, lung, and blood samples were collected and levels of phosphorylated c-Abl kinase, citrullinated histone 3, DNA-histone complexes, myeloperoxidase, amylase, cytokines, and CXC chemokines were quantified. Citrullinated histone 3, reactive oxygen species (ROS), and NET formation were determined in bone marrow neutrophils. Taurocholate challenge increased phosphorylation of c-Abl kinase and levels of citrullinated histone 3 in the pancreas as well as DNA-histone complexes in the plasma. Administration of the c-Abl kinase inhibitor GZD824 not only abolished activation of c-Abl kinase but also decreased levels of citrullinated histone 3 in the pancreas and DNA-histone complexes in the plasma of animals with AP. Moreover, GZD824 decreased plasma levels of amylase, IL-6, and MMP-9 as well as edema, acinar cell necrosis, hemorrhage, CXC chemokine formation, and neutrophil infiltration in the inflamed pancreas. A beneficial effect of c-Abl kinase inhibition was confirmed in l-arginine-induced pancreatitis. In vitro, inhibition of c-Abl kinase reduced TNF-α-induced formation of ROS, histone 3 citrullination, and NETs in isolated bone marrow neutrophils. Our findings demonstrate that c-Abl kinase regulates NET formation in the inflamed pancreas. In addition, inhibition of c-Abl kinase reduced pancreatic tissue inflammation, and damage in AP. Thus, targeting c-Abl kinase might be a useful way to protect the pancreas in severe AP.

Host factors influence Barrett's carcinogenesis: findings from a mouse gastroduodenal reflux model.

BACKGROUND: Rat gastroduodenal reflux models have been used for analyzing Barrett's carcinogenesis. Mice seem to be more useful than rats for studies targeting genes. METHODS: We induced gastroduodenal contents reflux by esophagojejunostomy using C57BL/6J mice. Mice were divided into a standard diet and high-fat diet groups and kept for 60 weeks. Bile was sampled from the gallbladder to analyze bile acid fractions, and the esophagus was removed for a histological investigation. Human esophagogastric junction adenocarcinoma cells (OE19) were exposed to taurocholic acid (TCA), after which cell proliferative activity was measured. Rat esophageal cancer cell lines, ESCC-DR and ESCC-DRtca with higher malignant potential induced by continuous TCA exposure, were used to perform comprehensive genetic analysis (CGH). RESULTS: Barrett's epithelium onset occurred in all mice, and no differences in histological changes were noted between the standard diet and high-fat diet groups. However, no development of adenocarcinoma was noted. Most of the mouse bile acid was taurine conjugates. In the experiment using OE-19 cells, TCA promotes cell proliferation in a dose-dependent manner. Array CGH analysis revealed a large number of chromosomal abnormalities in the ESCC-DR, in addition to genetic abnormalities such as in the UGT2B gene, the substrate of which is bile acid. TCA administration resulted in more chromosomal abnormalities being detected. CONCLUSIONS: We showed the effects of TCA in cancer progression in vitro. However, Barrett's adenocarcinoma onset rates differ between mice and rats despite undergoing similar reflux stimulation including taurine-conjugated bile acids being detected in mouse bile juice. These results suggest that host factors seem to influence Barrett's carcinogenesis.

Effects of Panax notoginseng saponins on severe acute pancreatitis through the regulation of mTOR/Akt and caspase-3 signaling pathway by upregulating miR-181b expression in rats.

BACKGROUND: In China, Panax notoginseng has been used to treat oxidative stress-related diseases for a long time. Panax notoginseng saponins is an extract from Panax notoginseng Ledeb. Its therapeutic potential is related to antioxidant activity, but related mechanisms are still unclear. The study aims to assess the protection effects of Panax notoginseng saponins in the taurocholate-induced rat model of acute pancreatitis (AP) and explore underlying mechanisms. METHODS: A rat model of severe acute pancreatitis (SAP) was established in rats induced with taurocholate. Panax notoginseng saponins was firstly administered in the treatment group via intravenous injection. After 2 h, taurocholate administration was performed. After 24 h, the expression levels of miR-181b, Beclin1, LC3-II, Akt and mTOR from pancreas tissues were measured by Western Blotting and RT-PCR. Then the expression levels of Caspase-3 and Blc-2 were determined by immunohistochemistry. Apoptosis was assessed by the TUNEL assay. Amylase and lipase in serum were determined by ELISA and pancreatic water contents in pancreatic tissue were measured. After eosin and hematoxylin staining, the histologic analysis was performed. RESULTS: After SAP induction by taurocholate and the treatment with Panax notoginseng saponins for 24 h, we detected the up-regulated miR-181b, the reduced Bcl-2 expression, the increased activity of mTOR/Akt, the blocked Beclin1 and LC3-II expressions, and the enhanced Caspase-3 expression. Serum lipase and amylase levels were significantly decreased in the treatment group of Panax notoginseng saponins compared to the control group. Histological analysis results verified the attenuation effects of Panax notoginseng saponins on taurocholate-induced pancreas injury, apoptosis, and autophagy. CONCLUSION: By up-regulating the miR-181b expression level, Panax notoginseng saponins significantly reduced taurocholate-induced pancreas injury and autophagy and increased apoptosis. The significant protection effects of Panax notoginseng saponins suggested its potential in treating taurocholate induced-acute pancreatitis.

Melatonin attenuated inflammatory reaction by inhibiting the activation of p38 and NF­κB in taurocholate­induced acute pancreatitis.

The aim of the present study was to investigate the protective mechanism underlying of melatonin in severe acute pancreatitis (SAP). A total of 64 male Sprague­Dawley rats were randomly divided into four groups: The sham operation (SO) group, SAP group, melatonin treatment (MLT) group and p38 inhibitor (SB203580) treatment (SB) group. Acute pancreatitis was induced by 5% taurocholate through retrograde infusion into the biliopancreatic ducts. The melatonin and SB203580 treatment groups were administered with MLT and SB 30 min before operation the induction of SAP. Rats in each group were euthanized at 6 and 12 h following SAP induction. Blood and pancreatic tissues were removed for inflammatory examination. Peripheral blood mononuclear cells (PBMCs) were isolated following sacrifice to measure the phosphorylation of p38 and nuclear factor­κB (NF­κB was measured as p65 and phosphorylation of p65). The pretreatment of melatonin significantly attenuated the severity of pancreatitis. In addition, melatonin also reduced serum amylase and proinflammatory cytokine levels, including tumor necrosis factor­α, interleukin (IL)­1 and IL­6. The mean pathological scores for pancreatic tissues in the MLT group were higher than those for samples in the SO group, but were lower than those for samples in the SAP group at each time-point. Phosphorylation of p38 and p65 levels in the melatonin treatment group were lower than that in the SAP group, and higher in the SAP group than in the SO group, and the SB203580 treatment group. Furthermore, melatonin significantly inhibited the activation of p38 and NF­κB in PBMCs. The authors revealed that melatonin may attenuate inflammatory reactions by inhibiting the activation of p38 MAPK and NF­κB in both acute pancreatitis rats and PBMCs. SAP is a severe disease with a high risk of morbidity and mortality. It is important to attenuated inflammatory reaction in acute pancreatitis. Thus, the authors studied melatonin, which is synthesized by the pineal gland and released into the blood. Previous studies have shown that melatonin serves a protective role in the early course of human acute pancreatitis, and melatonin concentration variations are closely related to the severity of acute pancreatitis. It may be concluded that melatonin may attenuates inflammatory reactions by inhibiting the activation of p38 MAPK and NF­κB in both acute pancreatitis rats and PBMCs.

Diabetes development increased concentrations of the conjugated bile acid, taurocholic acid in serum, while treatment with microencapsulated-taurocholic acid exerted no hypoglycaemic effects.

CONTEXT: The bile acid taurocholic acid (TCA) is endogenously produced, and has shown formulation-stabilising effects when incorporated into microcapsules containing potential antidiabetic drugs. This study aimed to develop and characterise TCA-microcapsules, and test their antidiabetic effects, in an animal model of Type 1 diabetes (T1D). METHODS: Using the polymer sodium alginate (SA), SA-microcapsules (control) and TCA-microcapsules (test) were prepared, and assessed for morphology, surface composition, chemical and thermal stability, swelling, buoyancy, mechanical, release and rheological properties. TCA-microcapsules were gavaged as a single dose (1.2mg/300g) to alloxan-induced diabetic rats, and blood glucose and TCA concentrations in serum, tissues (ileum, liver and pancreas) and faeces, were measured. One healthy and one diabetic group were used as control and gavaged SA-microcapsules. RESULTS: TCA-microcapsules showed consistent size, TCA presence on surface and all layers of microcapsules, chemical and thermal stability, enhanced swelling, buoyancy and targeted-release properties and rheological analysis showed Non-Newtonian flow properties. TCA serum concentrations were lower in the healthy group, compared with the diabetic and diabetic-treated groups, but there was no significant difference between diabetic control and diabetic treated groups, in terms of TCA levels, and blood glucose concentrations. CONCLUSIONS: The developed TCA-microcapsules showed good stability and release properties, but did not lower blood glucose levels in T1D, which suggests absence of insulin-mimetic effects, when using a single 1.2mg/rat oral dose.

Sex differences and effects of oestrogen in rat gastric mucosal defence.

AIM: To evaluate sex differences and the effects of oestrogen administration in rat gastric mucosal defence. METHODS: Sex differences in gastric mucus thickness and accumulation rate, absolute gastric mucosal blood flow using microspheres, the integrity of the gastric mucosal epithelium in response to a chemical irritant and the effects of oestrogen administration on relative gastric mucosal blood flow in an acute setting was assessed in an in vivo rat experimental model. Subsequently, sex differences in the distribution of oestrogen receptors and calcitonin gene related peptide in the gastric mucosa of animals exposed to oestrogen in the above experiments was evaluated using immunohistochemistry. RESULTS: The absolute blood flow in the GI-tract was generally higher in males, but only significantly different in the corpus part of the stomach (1.12 ± 0.12 mL/min•g in males and 0.51 ± 0.03 mL/min•g in females) (P = 0.002). After removal of the loosely adherent mucus layer the thickness of the firmly adherent mucus layer in males and females was 79 ± 1 µm and 80 ± 3 µm respectively. After 60 min the mucus thickness increased to 113 ± 3 µm in males and 121 ± 3 µm in females with no statistically significant difference seen between the sexes. Following oestrogen administration (0.1 followed by 1 µg/kg•min), mean blood flow in the gastric mucosa decreased by 31% [68 ± 13 perfusion units (PFU)] in males which was significantly different compared to baseline (P = 0.02). In females however, mean blood flow remained largely unchanged with a 4% (5 ± 33 PFU) reduction. The permeability of the gastric mucosa increased to a higher level in females than in males (P = 0.01) after taurocholate challenge. However, the calculated mean clearance increase did not significantly differ between the sexes [0.1 ± 0.04 to 1.1 ± 0.1 mL/min•100 g in males and 0.4 ± 0.3 to 2.1 ± 0.3 mL/min•100 g in females (P = 0.065)]. There were no significant differences between 17ß-Estradiol treated males (mean ratio of positive staining ± SEM) (0.06 ± 0.07) and females (0.11 ± 0.11) in the staining of ERα (P = 0.24). Also, there were no significant differences between 17ß-Estradiol treated males (0.18 ± 0.21) and females (0.06 ± 0.12) in the staining of ERß (P = 0.11). Finally, there were no significant differences between 17ß-Estradiol treated males (0.04 ± 0.05) and females (0.11 ± 0.10) in the staining of CGRP (P = 0.14). CONCLUSION: Gastric mucosal blood flow is higher in male than in female rats and is reduced in male rats by oestrogen administration.

Experimental Models in Syrian Golden Hamster Replicate Human Acute Pancreatitis.

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

Protective Effects of Hydrogen Gas on Experimental Acute Pancreatitis.

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

Safety studies on intravenous infusion of a potent angiogenesis inhibitor: taurocholate-conjugated low molecular weight heparin derivative LHT7 in preclinical models.

CONTEXT: As a class of angiogenesis inhibitors, heparin conjugates have shown significant effectiveness in several studies. OBJECTIVES: The purpose of our current study is to evaluate the effectiveness and safety of infusing the conjugate of low molecular weight heparin and taurocholate (LHT7), which has been developed as a potent angiogenesis inhibitor. METHODS: To evaluate its safety, the method of intravenous infusion was compared with its i.v. bolus administration. Intravenous infusion was administered at a rate of 400 µl/min/kg of body weight for 30 min. Pharmacokinetic (PK) analysis, organ accumulation, and plasma concentration profiles of LHT7 were measured. The anticancer effect of LHT7 was evaluated in murine and human xenograft models, and preclinical studies were performed in SD rats and beagle dogs. RESULTS: The results of the PK studies showed reduced organ accumulation in mice and the AUC(0-96 h) (area under the curve) was increased up to 1485 ± 125 h × µg/ml. The efficacy, at dose 1 mg/kg/2 d was higher for i.v. infusion than for i.v. bolus administration in both murine and human cancer models. The preclinical studies showed the safety dose of LHT7 is less than 20 mg/kg in SD rats and in the next safety analysis in beagle dogs showed that there were no organ-specific adverse effects in higher doses, such as, 12 mg/kg. LHT7 showed sustained effects with minimized adverse events when administered through i.v. infusion. CONCLUSIONS: LHT7 (i.v. infusion) could be safely used for further clinical development as a multi-targeting anti-angiogenic agent.

Effect of Chaiqinchengqi decoction on inositol requiring enzyme 1α in alveolar macrophages of dogs with acute necrotising pancreatitis induced by sodium taurocholate.

OBJECTIVE: To investigate the effect of Chaiqinchengqi decoction (CQCQD) on inositol requiring enzyme lα (IRElα) in alveolar macrophages (AMs) of the dog model of acute necrotising pancreatitis (ANP) induced by sodium taurocholate. METHODS: Fifteen beagle dogs were randomised into a control group, ANP group and CQCQD group (n = 5 per group). ANP was induced by a retrograde duct injection of 50 mg/kg of 5% sodium taurocholate. The dogs in the control group received injections of the same volume of saline as the sodium taurocholate. After the models were induced, the dogs in the CQCQD group were administered 10 mL/kg CQCQD every 2 h for 6 h. Two hours after the last administration of either CQCQD or saline, they were sacrificed by anaesthesia. AMs were collected to determine the IRElα and interleukin-1ß (IL-1ß) mRNA and protein expression, and pancreatic tissues were collected for histopathology analysis. RESULTS: Compared with the ANP group, the mRNA and protein expression of IREl a and the protein expression of IL-1ß of AMs in the CQCQD group were significantly down-regulated, and the pancreatic histopathology score of the CQCQD group also was lower. There was no significant difference in the mRNA expression of IL-1ß of AMs between the two groups. CONCLUSION: The CQCQD-induced down-regulation of the IL-1ß protein expression may involve the down-regulation of the mRNA and protein expression of IRElα in AMs.

Rosmarinic Acid Attenuates Sodium Taurocholate-Induced Acute Pancreatitis in Rats by Inhibiting Nuclear Factor-κB Activation.

Rosmarinic Acid (RA), a caffeic acid ester, has been shown to exert anti-inflammation, anti-oxidant and antiallergic effects. Our study aimed to investigate the effect of RA in sodium taurocholate ( NaTC )-induced acute pancreatitis, both in vivo and in vitro. In vivo, RA (50 mg/kg) was administered intraperitoneally 2 h before sodium taurocholate injection. Rats were sacrificed 12 h, 24 h or 48 h after sodium taurocholate injection. Pretreatment with RA significantly ameliorated pancreas histopathological changes, decreased amylase and lipase activities in serum, lowered myeloperoxidase activity in the pancreas, reduced systematic and pancreatic interleukin-1 ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) levels, and inhibited NF-κB translocation in pancreas. In vitro, pretreating the fresh rat pancreatic acinar cells with 80 µ mol/L RA 2 h before 3750 nmol/L sodium taurocholate or 10 ng/L TNF-α administration significantly attenuated the reduction of isolated pancreatic acinar cell viability and inhibited the nuclear activation and translocation of NF-κB. Based on our findings, RA appears to attenuate damage in sodium taurocholate-induced acute pancreatitis and reduce the release of inflammatory cytokines by inhibiting the activation of NF-κB. These findings might provide a basis for investigating the therapeutic role of RA in managing acute pancreatits.

Effect of gentiopicroside on experimental acute pancreatitis induced by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats.

Gentiopicroside (otherwise known as Gentiopicrin), one of the main active ingredients from the traditional Chinese herb medicine Gentiana manshurica Kitag, presents the effect of attenuating acute pancreatitis in rats. The experimental acute pancreatitis was made by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats. Gentiopicroside was given orally and it markedly reduced the pancreatitis-evoked increase of serum amylase and lipase activity, decreased the pancreas mass/body mass index, tissue water content, TNF-α and IL-1ß concentrations, and attenuated the histopathological changes and NF-κB p65 protein expression in pancreatic tissue. The results indicate that the function of gentiopicroside on acute pancreatitis may be related to inhibiting the release of inflammatory mediators and NF-κB p65 protein expression.

Protective effects of daphnetin on sodium taurocholate­induced severe acute pancreatitis in rats.

Severe acute pancreatitis (SAP) is the sudden onset of pancreatic inflammation, which is characterized by edema, acinar cell necrosis, hemorrhage and severe inflammation of the pancreas and is associated with a high mortality rate. Daphnetin has been shown to alleviate organ injury in a variety of preclinical animal models of coagulation disorders. The aim of the present study was to investigate the protective effects of daphnetin on severe acute pancreatitis in a rat model. Severe acute pancreatitis in the rat model was induced by retrograde infusion of 5% sodium taurocholate (1 ml/kg) into the bile-pancreatic duct. Daphnetin (4 mg/kg) was administered intraperitoneally at 30 min prior to the infusion of sodium taurocholate. The severity of pancreatitis was evaluated by various analyses of serum amylase and lipase, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels, myeloperoxidase (MPO) activity and malondialdehyde (MDA) content, as well as by histological grading. The levels of TNF-α and IL-1ß in the serum were measured by ELISA. The results revealed that the daphnetin-treated SAP rat group (SAP-D) exhibited a lower pathological score of the pancreas compared with the SAP group (SAP). Further analyses demonstrated that the SAP-D group had lower levels of serum amylase, lipase and pro-inflammatory cytokines, including TNF-α and IL-1ß, and a decreased MPO activity and MDA content 3, 6 and 12 h subsequent to the infusion of sodium taurocholate compared with the SAP group (SAP). These findings indicated that daphnetin exerted a protective function in the SAP rat model. Therefore, daphnetin may be considered as a potential compound for the therapy and prevention of acute pancreatitis.

Continuous taurocholic acid exposure promotes esophageal squamous cell carcinoma progression due to reduced cell loss resulting from enhanced vascular development.

BACKGROUND: Refluxogenic effects of smoking and alcohol abuse may be related to the risk of esophageal squamous cell carcinoma (ESCC). The present study attempts to clarify the effects of continuous taurocholic acid (TCA) exposure, which is neither mutagenic nor genotoxic, on ESCC progression. METHODS: A squamous carcinoma cell line (ESCC-DR) was established from a tumor induced in a rat model of gastroduodenal reflux. ESCC-DR cells were incubated with 2 mM TCA for ≥2 months. The effects of continuous TCA exposure were evaluated in vitro on cell morphology, growth, and invasion and in vivo on xenograft tumor growth in nude mice. Moreover, the mean level of secreted transforming growth factor (TGF)-ß1 and vascular endothelial growth factor (VEGF) proteins in cell culture supernatants and mRNA synthesis of TGF-ß1 and VEGF-A of ESCC cells were measured. The angiogenic potential was further examined by a migration assay using human umbilical vein endothelial cells (HUVECs). RESULTS: Continuous TCA exposure induced marked formation of filopodia in vitro. Expression levels of angiogenic factors were significantly higher in the cells treated with TCA than in control cells. Tumor xenografts derived from cells pre-exposed to TCA were larger and more vascularized than those derived from control cells. In addition, TCA exposure increased HUVEC migration. CONCLUSION: Continuous TCA exposure enhanced ESCC progression due to reduced cell loss in vivo. Cell loss was inhibited by TCA-induced vascular endothelial cell migration, which was mediated by TGF-ß1 and VEGF-A released from ESCC cells.

Pulmonary function changes in rats with taurocholate-induced pancreatitis are attenuated by pretreatment with melatonin.

Melatonin is a free radical scavenger and broad-spectrum antioxidant with immunomodulatory effects. We studied the effects of melatonin on changes in lung function, oxidative/nitrosative stress, and inflammatory cell sequestration in an acute pancreatitis (AP)-associated lung inflammation model. Acute pancreatitis was induced by injection of 5% sodium taurocholate into the pancreatic duct of rats. Animals were randomized into control, AP, and a melatonin pretreatment (10 mg/kg)/AP group. Functional residual capacity (FRC), lung compliance (Cchord), expiratory flow rate at 50% (FEF50), airway resistance index (RI), and peak expiratory flow rate (PEF) were evaluated. White blood cell count (WBC) and hydrogen peroxide, lung lavage fluid WBC, methylguanidine, protein, lactic dehydrogenase (LDH), nitric oxide (NO), and leukotriene B4 (LTB4) levels were determined. Lung wet-to-dry weight ratio, peroxynitrite, and inducible nitric oxide synthase (NOS) mRNA and protein were measured. AP induction resulted in reductions in FRC, Cchord, FEF50, and PEF, and increase in RI and lung wet-to-dry weight ratio. Blood and lung lavage fluid WBC, lavage fluid LDH, protein, and blood hydrogen peroxide also increased. Levels of hydroxyl radicals, nitric oxide, and LTB4 in lung lavage fluid, inducible NOS mRNA, protein expression, and peroxynitrite in lung tissue also were significantly elevated. Pretreatment with melatonin attenuated obstructive and restrictive ventilatory insufficiency induced by AP. Blood and lavage WBC, lavage LDH and protein, lung edema, oxidative/nitrosative stress, and lipoxygenase pathway derivatives were also significantly attenuated by melatonin. We conclude that melatonin decreases AP-induced obstructive and restrictive lung function changes via its antioxidant and anti-inflammatory properties.