Mechanical Role Of Wortmannin(PI3K/AKT Pathway Inhibitor) In Severe Acute Pancreatitis Associated Thyroid Injury In Rats

Objective:To investigate the changes of thyroid tissue morphology and to study the changes of PI3K/AKT pathway in the course of the evolution of thyroid tissue in SAP rats.To investigate the dose effect relationship of PI3K/AKT inhibitor Wortmannin on SAP induced thyroid injury in rats.To observe the pathological effects of PI3K/AKT pathway on thyroid tissue in SAP rats, and to detect the activation of NF-kB in thyroid tissue, and to investigate the mechanism of PI3K/AKT inhibitor Wortmannin on the protective effect of SAP on thyroid injury.Method:Part One:The 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), we incision in the middle of abdom and reversed pancreas and duodenum then 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 (LIP) level of the experimental rats were detected.Histology in pancreas and thyroid 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+Wortmannin group(Wortmannin 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 Wortmannin 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 and LIP level observed the pancreatic tissue and pathological changes, then we could determine the optimal dose of Wortmannin.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), Wortmannin preconditioning group (SAP+Wortmannin group, n=30) in experimental group. There were 3 time point (3h,6h,12h) in the SAP group and the Wortmannin preconditioning group.5%DMSO (2ml/kg) was impact injected at 30 min before operation in SAP group and CON group, different doses of Wortmannin 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-?B p65 was detected by immunohistochemistry in the thyroid tissue, and the pathological changes of pancreas and thyroid in rats were observed by HE staining, and the levels of serum amylase AMY and LIP were observed. The survival rate group (n=30) were randomly divided to 3 group (CON group, SAP group, Wortmannin group, n=10).The treatment of each group was the same as that of the research experiment one. The effect of PI3K/AKT inhibitor Wortmannin on the survival rate of SAP rats in 24h was observed.Results:Part One With the progress of the disease, the serum AMY and LIP 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 thyroid 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, and 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 Wi group, higher than those in W2 and W3 group in SAP group.Ascites volume, serum amylase, serum amylase, serum lipase and survival rate of Wi 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, serum AMY and LIP level 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,Wortmannin——PI3K/AKT pathway inhibitor——can significantly reduce the AMY, LIP and the pathological score of the pancreas in severe acute pancreatitis rats, reduce the pathological damage of thyroid tissue, improve the survival rate of SAP rats.NF-kB p65 and p-AKT protein was increased in thyroid tissue after SAP model induced (p<0.05), and AKT was decreased (p<0.05).Wortmannin blocking PI3K/AKT pathway,whichcan inhibit the expression of NF-kB 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 thyroid of SAP rats pathological damage of progressive increase, phosphorylate level of AKT in thyroid tissue activation was 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 thyroid injury of SAP.2. NF-kB activation was enhanced in the thyroid injury of SAP rats, and the pathological injury of thyroid tissue was obviously.The intervention of Wortmannin (0.5mg/kg) by intraperitoneal shock therapy can improve the SAP condition. Perhaps it is the optimal dosage of safety and efficiency. That Wortmannin inhibited PI3K/AKT pathway can reduce the activation of p-AKT, inhibit the activation of NF-kappaB pathway in the thyroid acinus, reduce the thyroid necrosis of SAP rats, and reduce the thyroid inflammatory injury, which had effected to protect SAP related thyroid injuryed.