| Part 1: The role of endoplasmic reticulum stress-mediated intestinal epithelial cell apoptosis in rats with severe acute pancreatitisObjective:The present study aimed to determine whether intestinal epithelial cell(IECs)could be damaged by endoplasmic reticulum stress(ERS)-associated apoptosis in acute necrotizing pancreatitis(ANP).Methods: 18 male Sprague-Dawley rats were randomly divided into two groups:The sham operation(SO)group,the ANP group(Acute necrotizing pancreatitis,ANP group).A rat model of ANP was induced by retrograde injection of 5% sodium taurocholate(STC)into the biliopancreatic duct.The levels of serum AMY,LIPA were measured by automatic biochemical analyzer,Serum DAO level,Serum Endotoxin level were measured by DAO ELISA kit and EKT-5 M set dynamic Gram-negative bacteria test kit.Rats were sacrificed at 12 h post-STC infusion with prolonged anesthesia,the head of the pancreas,and ileal tissues(5cm from terminal ileac segments)adjacent to the cecum,were harvested and fixed in 4% PBS-buffered formaldehyde for histopathology.The expression of GRP78 、 Caspase-12 in the intestinal were detected by immunohistochemistry.The expression of GRP78 、Caspase-12、Caspase-3 in the intestinal was detected by Western Blot.The apoptosis index was detected by TUNEL.Results:Compared with the SO group,serum AMY and LIPA levels were significantly increased at 12 h in SAP group(P<0.05).In addition,rats subjected to ANP exhibited a significant increase in serum DAO and Endotoxin levels(P<0.05).No morphological evidence of pancreatic injury was detected in the SO group.Interstitial edema,acinar cell necrosis and infiltration of inflammatory cells were observed in the SAP group(P <0.05).The expression level of GRP78、Caspase-12 in the intestinal were significantly higher than that in the SO group(P <0.05).Compared with the SO group,the levels of GRP78、Caspase-12 、Caspase-3 in the intestinal of ANP group were significantly higher(P <0.05).The apoptosis of IEC level was significantly higher than those of SO group(P <0.05).Conclusion:1.ANP-associated intestinla injury was successfully induced in the rats after 12h of modeling.2.Endoplasmic reticulum stress-mediated cell apoptosis may be involved in the development of ANP-associated intestinla injury.Part 2: 4-BA attenuates endoplasmic reticulum stress-mediated intestinal epithelial cell apoptosis in rats with acute necrotizing pancreatitisObjective:To investigate the role of chemical chaperone 4-phenylbutyric acid(4-PBA)in ANP-associated intestinal barrier injury and its possible mechanism by using 4-PBA to treat the rat of ANP.Methods:A total of 24 male rats were randomly assigned into three groups(n=8per group): The sham operation(SO)group,the ANP group,and the ANP plus 4-PBA treatment(4-PBA)group.In the SO group,rats received a sham surgery where the pancreas and duodenum were flipped a number of times instead of STC infusion.In the 4-PBA group,rats received 4-PBA solution via intraperitoneal injection at a dose of 50 mg/kg per day for 3 days,which was prepared as following steps: equimolar amounts of 4-PBA and sodium hydroxide were titrated to PH 7.4 and dissolved in physiological saline.Serum amylase(AMY)and lipase(LIPA)levels were measured using an automatic biochemistry analyzer with standard techniques,Serum DAO level,Serum Endotoxin level were measured by DAO ELISA kit and EKT-5 M set dynamic Gram-negative bacteria test kit.Paraffin-embedded samples from the pancreas and ileum were sectioned(5 μm)and stained with hematoxylin and eosin(H&E),All tissue sections were examined under optical microscope.Serum levels of tumor necrosis factor(TNF)-α-,interleukin(IL)-1βand IL-6(Ebioscience Inc.were quantified using specific ELISA kits,according to the manufacturer’s protocol.The expression of GRP78,PERK,IRE1α,ATF6 in the intestinaly were detected by immunohistochemistry and RT-PCR.The expression of Caspase-12 、 Caspase-3 、CHOP in the intestinal were detected by Western Blot.The apoptosis index was detected by TUNEL.The changes to IECs and microvilli were examined under a HT7700 transmission electron microscope.Results:1.Compared with SO group,in ANP group,the levels of serum AMY,LIPA,Serum DAO level,Serum Endotoxin level were significantly higher than those in SO group(P <0.05).Concordant with the pathological damage incited by SAP,serum levels of TNF-α,IL-6 and IL-1β were significantly increased in the SAP group compared with in the SO group(P <0.05).No morphological evidence of pancreatic injury was detected in the SO group.Interstitial edema,acinar cell necrosis and infiltration of inflammatory cells were observed in the ANP group(P <0.05).In ANP group,the expression of GRP78,PERK,ATF6 were significantly higher than that in SO group(P <0.05).The level of Caspase-12、Caspase-3、CHOP in ANP group were significantly higher than that in SO group,the difference was statistically significant(P <0.05).2.After 4-PBA treatment,The results indicated that 4-PBA attenuated the following:(1)pancreas and intestinal pathological injuries,(2)Serum TNF-α,IL-1βand IL-6,(3)Serum DAO level,Serum Endotoxin level,(4)the apoptosis of IECs,(5)ER stress markers(Caspase-12,CHOP,GRP78,PERK,IRE1α,ATF6)and Caspase-3 expression in intestinal.The results were significantly lower than those in ANP group(P <0.05).3.TEM analysis of the intestinal epithelium demonstrated that the integrity of the intestinal villi and IECs of rats in the SO group was maintained throughout the course of the experiment.The mitochondria,ER,ribosomes and other cellular organelles were normal following examination at a magnification of x2,500.The IECs in rats following ANP exhibited extensively irregular,dilated ER.In addition,moderate mitochondrial swelling was observed and the cristae were disintegrated in parts.The microvilli and IECs in the ANP group also presented with a loss of integrity.Conversely,microvilli and IECs integrity was significantly improved in rats from the4-PBA group.Conclusion:4-Phenylbutyric acid attenuates endoplasmic reticulum stress-mediated intestinal epithelial cell apoptosis in rats with severe acute pancreatitis... |
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