Mechanical Role Of Wortmanin(PI3K/AKT Path Inhibitor) In Severe Acute Pancreatitis Associated Intestinal Injury In Rats

Objective:To investigate the changes of intestinal tissue morphology and to study the changes of PI3K/AKT pathway in the course of the evolution of intestinal tissue in SAP rats.To investigate the dose effect relationship of P13K/AKT inhibitor Wortmanin on SAP induced intestinal injury in rats.To observe the pathological effects of PI3K/AKT pathway on intestinal tissue in SAP rats, and to detect the activation of NF-kappa B in intestinal tissue, and to investigate the mechanism of PI3K/AKT inhibitor Wortmanin on the protective effect of SAP on intestinal injury.Method:Part OnerThe 48 male SPF Wistar rats were established the rat model of SAP by 5% sodium taurocholate retrograde pancreatic duct injection.In the sham operation group (control group), pancreas and duodenum was reversed after a midline incision and sacrificed after 12h.To detect the time courses of AKT and P-AKT activity, rats were randomly divided into control group and group 3,6,12h after SAP model induced. Serum amylase (AMY) and lipase (LPS) were detected.Histology in pancreas and intestinal with H&E (hematoxylin and eosin stain) for light microscopy were analyzed.Part Two The sixty SPF male Wistar rats were randomly allocated into six groups (n=10 for each group):sham+vehicle(sham control group,CON group),SAP+vehicle group (severe acute pancreatitis model group,SAP group),SAP+wortmanin group(wortmanin pretreatment groups at different dosage,0.5mg/kg,lmg/kg,2mg/kg, marked W1、W2、W3 group, respectively)。Pancreatitis model was established by retrograde infusion of 5% sodium taurocholate into the bilio-pancreatic duct。5%DMSO (2ml/kg) was impact injected at 30 min before operation in SAP group and CON group,different doses of Wortmanin by the same amount of 5%DMSO was impact injected at 30 min before operation.The rats were killed at 12h after operation.We measured ascites volume, AMY level,LPS level and observed the pancreatic tissue and pathological changes,then we could determine the optimal dose of Wortmanin.Part Three 100 male wistar rats were randomly divided into experimental group (N=70) and survival rate group (two) (N=30).Rats were randomly divided into three groups: sham operated control groups (CON group, n=10), severe acute pancreatitis model groups (SAP group, n=30), wortmanin preconditioning group (SAP+wortmanin group, n=30) in experimental group.There were 3 time point(3h,6h,12h) in the SAP group and the Wortmanin preconditioning group.5%DMSO (2ml/kg) was impact injected at 30 min before operation in SAP group and CON group,different doses of Wortmanin by the same amount of 5% DMSO was impact injected at 30 min before operation.The rats were killed and the samples were collected at each time point after operation.The expression levels of P-AKT and AKT were detected by Western blot, and the expression level of NF-κB p65 was detected by immunohistochemistry in the intestinal tissue, and the pathological changes of pancreas and intestine in rats were observed by HE staining, and the levels of serum amylase (amylase, AMY) and LPS (lipase) were observed.The survival rate group(n=30) were randomly divided to 3 group (CON group,SAP group,wortmanin group,n=10).The treatment of each group was the same as that of the research experiment one. The effect of PI3K/AKT inhibitor wortmanin on the survival rate of SAP rats in30h was observed.Results:Part One With the progress of the disease, the serum amylase and lipase levels were increased in the rats. The pathological changes of pancreatic tissue, such as edema, hemorrhage and necrosis were gradually increased.The expression level of P-AKT was very low in CON group, The expression of P-AKT in intestinal tissues increased rapidly (P<0.05) at 3h after operation,reached peak at 12h in SAP group.In contrast to AKT,it gradually reduce (P<0.05).Part Two The rats spirit and activities were as usual,No rat died in CON group.Compared with CON group,ascites volume,serum amylase and serum lipase values were significantly higher than those in CON group (P<0.05), the survival rate was lower than those in W1 group, higher than those in W2 and W3 group in SAP group.Ascites volume, serum amylase, serum amylase, serum lipase and survival rate of W1 group were significantly lower than that of SAP group, W2 group and W3 group, and the difference was statistically significant (P<0.05).Serum amylase and serum lipase in W2 group were significantly lower than those in SAP group and W1 group, but the survival rate was significantly lower than that in SAP group, and the amount of ascites was significantly increased and become red and black.Compared with those in W3 group,AMY and LPS increased, the survival rate was higher,the difference was statistically significant (P<0.05). Serum amylase and serum lipase levels in W3 group were significantly lower than that in SAP group, W1 group and W2 group, but the survival rate was significantly lower than that of group, and the difference was statistically significant (P<0.05).Part Three Compared with SAP model group,Wortmanin——PI3K/AKT pathway inhibitor——can significantly reduce the AMY, LPS and the pathological score of the pancreas in severe acute pancreatitis rats, reduce the pathological damage of intestinal tissue, improve the survival rate of SAP rats.NF-κB p65 and P-AKT protein was increased in intestinal tissue after SAP model induced (P<0.05), and AKT was decreased (P<0.05).Wortmanin blocking PI3K/AKT pathway,whichcan inhibit the expression of NF-κB p65 and AKT, and decrease the transformation of P-AKT to (P<0.05), thus improving the survival rate of rats.Conclusion:1.With the progress of the disease, the intestina of SAP rats pathological damage of progressive increase, AKT of intestinal tissue activation time-dependent enhanced. It has the effect of promoting cell necrosis.It is suggested that the PI3K/AKT pathway is involved in the process of intestinal injury of SAP.2. NF-κB activation was enhanced in the intestinal injury of SAP rats, and the pathological injury of intestinal tissue was obviously.The intervention of Wortmanin(0.5mg/kg) by intraperitoneal shock therapy can improve the SAP condition.Perhaps it is the optimal dosage of safety and efficiency. That wortmanin inhibited PI3K/AKT pathway can reduce the activation of P-AKT, inhibit the activation of NF-kappa B pathway in the intestinal tract, reduce the intestinal necrosis of SAP rats, and reduce the intestinal inflammatory injury,which had effected to protect SAP related intestinal injuryed.