Protection Of Astragalus Polysacharin On Intestinal Barrier Dysfunction In Rats With Severe Acute Pancreatitis

Objective:Acute pancreatitis(AP)is a common acute abdomen,which can be divided into mild acute pancreatitis(MAP),moderately severe acute pancreatitis(MSAP)and severe acute pancreatitis(SAP).SAP is extremely dangerous and its mortality is high.It is one of the most difficult diseases to treat in the acute abdomen.SAP has two peak time points in high mortality,the first one occurs due to early multiple organ dysfunction syndrome(MODS)that is usually caused by systemic inflammatory response syndrome(SIRS),the second one occurs due to late MODS that is usually caused by necrotic pancreas and sepsis.Intestine is a vital organ not only for food digestion but also for nutrition absorption,at the same time it is the largest organ that contains bacteria.Depending on the intestinal barrier,intestine takes in necessary nutrition selectively but bacteria and toxin.However,SAP is one of the most vital pathogenesis that lead to intestinal barrier dysfunction(IBD).IBD plays a key role in the happening of SIRS during early stage of SAP and sepsis during advanced stage of SAP.Therefore,it is very important to protect intestine barrier during the treatment of SAP.Astragalus polysacharin(APS)is an important component of astragalus.Many studies have confirmed that APS can regulate immune system,inhibit inflammation and virus,delay senility,decrease blood sugar.In this study,we established the model of SAP via retrograde injection of sodium taurocholate hydrate through biliopancreatic duct,APS was applied in the treatment groups.Our aims are to investigate the protective effect of astragalus polysacharin against IBD in rats with SAP,and attempt to find a new way to treat SAP.Methods:Seventy five healthy SD rats were randomly divided into group SO(sham operation),group SAP(SAP model),group APS1(SAP model with low dose of APS intervention),group APS2(SAP model with moderate dose of APS intervention),group APS3(SAP model with high dose of APS intervention).The model of SAP was established via retrograde injection of 5% sodium taurocholate hydrate(1 ml/kg)through biliopancreatic duct?APS was applied(100 mg/kg in group APS1,200 mg/kg in group APS2,300 mg/kg in group APS3)via intraperitoneal injection after establishment of the models.0.5ml saline was applied via intraperitoneal injection instead in group SO and group SAP.All the rats were sacrificed 24 h later,blood was collected.TNF-?,IL-6,D-Lactic acid,DAO were tested using ELISA.Pancreatic tissues and intestinal tissues were subjected to H&E staining for observing morphologic changes.Intestinal tissues were also subjected to immunofluorescence and western blot for detecting the expression of occludin,ZO-1,TLR4.sIgA in intestinal mucus was detected using ELISA.SPSS 17.0 was used to perform statistical analysis,P-value<0.05 was considered statistically significant.Results:1.The presentation of rats from group SAPThe rats from group SAP were bad in general condition and spirit.Lots of hemorrhagic and inflammatory ascites existed in their abdominal cavity.Different rank of ischemia and necrosis was seen in pancreatic gross specimen.Saponification spot could be also seen on the surface of pancreas and omentum.H&E staining of the pancreatic tissue revealed the occurrence of hemorrhage and necrosis.Moreover,destruction of pancreatic lobules,vascular congestion and infiltration of neutrophils and monocytes were also observed.2.Histopathological changes of intestineIn group SO,intestinal mucosal villis were neatly arranged.The intestinal epithelial cell layer was intact,and the brush border was smooth with a thin layer of mucus.In group SAP,intestinal epithelial cells degenerated and necrosed.The arrangement of villus was disordered and some villus sheded.Compared with group SAP,the rank of damaged epithelial cells and disordered villus were attenuated in group APS1,APS2 and APS3.It was negatively correlated with the dose of APS.3.The expression of protein occluding,ZO-1 and TLR4The results of immunofluorescence revealed that occludin and ZO-1pitch on the surface of villus.In group SO,the line was continuous and the density was high.While in group SAP,protein occludin and ZO-1expression declined significantly,the distribution was scattered.The protein expression level of occludin was also lower in group APS1 and APS2,but higher in group APS3.The protein expression level of ZO-1 was also lower in group APS1,but higher in group APS2 and APS3.Protein TLR4 was expressed in each group.Compared with group SO,the expression level of TLR4 increased in group SAP;compared with group SAP,the expression level of TLR4 decreased in group APS2 and APS3.The results of western blot revealed that the expression level of occludin and ZO-1 decreased in group SAP compared with those in group SO(P<0.01).It significantly increased in group APS2(P<0.01)and group APS3(P<0.01)compared with group SAP.The results simultaneously revealed that the expression level of TLR4 increased in group SAP compared with those in group SO(P<0.01).It significantly decreased in group APS2(P<0.05)and group APS3(P<0.01)compared with those in group SAP.4.The changes of TNF-?,IL-6,D-Lactic acid,DAO and sIgACompared with group SO,the level of TNF-?,IL-6,D-Lactic acid and DAO increased in group SAP(P<0.01).Compared with group SAP,the level of TNF-?,IL-6 decreased significantly in group APS1(P<0.01),APS2(P<0.01)and APS3(P<0.01);the level of D-Lactic acid decreased significantly in group APS2(P<0.05)and APS3(P<0.05);the level of DAO decreased significantly in group APS1(P<0.05),group APS2(P<0.01)and APS3(P<0.01).Compared with group SO,the level of sIgA decreased in group SAP(P<0.01).Compared with group SAP,the level of sIgA increased significantly in group APS1(P<0.05),group APS2(P<0.01)and APS3(P<0.01).Conclusion:1.To establish the model of SAP via retrograde injection of sodium taurocholate hydrate through biliopancreatic duct is ideal and stable.2.SAP can cause IBD that include mechanical barrier dysfunction and immune barrier dysfunction,which can present with necrosed intestinal epithelial cells and sheded villus;with decreased expression level of occludin,ZO-1 and increased expression level of TLR4 in intestinal tissues;with high level of D-Lactic acid,DAO in blood and low level of sIgA in intestinal mucus.3.APS can protect intestinal barrier of SAP rats by up regulating occludin and ZO-1,down regulating TLR4 and promoting the secretion of sIgA.APS is considered to be a hopeful way to treat intestine injury in the future.