| SAP(Severe acute pancreatitis) accounts for 10%-25% of AP(acute pancreatitis ) .SAP result in 20%-30% fatality rate and is often complicated with MODS( Multiple-organ dysfunction syndrome).The incidence of acute renal failure is about 14%-43% and next to acute lung failure.The mechanism of renal injuries induced by AP is still unknown completedly.It may have relationship with the following factors :the disturbances of hemodynamics and dysfunction of microcirculation in kidney ,translocation of bacteria and endotoxin from intestine,the releasing of inflammatory mediators and cytokines, oxygen -free radicals and so on.Phosphatidylinositol 3-kinase is a second messenger that is related to intracellular signal transduction. Protein kinase B is a downstream effector of PI3K. PI3K∕PKB signal pathway is a important signal system ,which cause celluar response through mediating extracelluar signaling. It plays important roles in the following fields: cancer progression,activation and chemotaxis of inflammatory factors, ischemia-reperfusion,interstitial fibrosis and so on.Recently ,the relationship between PI3K∕PKB signal pathway and pancreatitis obtain more and more attention. PI3K∕PKB signal pathway increases Ca2+ release from the pool of calcium,and this process participates in the formation of intracellular Ca2+ overload,then trypsinogen is activated into the digestive enzyme trypsin.The digestive enzyme trypsin can cause pancreas injury . Simultaneously,it was found out this signal pathway can promote neutrophlic recruitment and produce excessive inflammatory factors such as TNF-α,IL-1βand so on,and these effects can worsen pancreas injury.Normally,phosphorylated p-PKBcan measure the activation of PI3K∕PKB signal pathway. In our test,the expressional levels of p-PKB and PKB in kidney were measured, which is used to study the relationship between PI3K∕PKB signal pathway and kidney injuries induced by AP in rat. Our study use the specific inhibitor of PI3K wortmannin to interfere in renal injuries induced by AP and investigate its possible mechanism.Objective: Studying the pretreatment of AP rat by wortmannin which is a specific inhibitor of PI3K,investigate the role of wortmannin in renal injuries induced by severe acute pancreatitis in rats and its mechanism.Methods :Fifty-four healthy male SD rats were randomly divided into 3 groups : SO group(sham operation group), AP group (acute pancreatitis group), WAP group ( acute pancreatitis plus wortmannin). (n=18 per group).Each group is divided into 3 subgroups at 3h, 6h, 12h, and there are 6 rats in each subgroup. The rats were anaesthetized with an intraperitoneal injecton of 10% chloral hydrate,then perform a operation into abdomen through median upper abdominal incision.After these, occlude pancreatic duct adjoining to hepatic portal with a noninvasive artery clip. AP modle was induced by retrograde injection of 50g/L sodium taurocholate into the biliopancreatic duct of rats with dosage of 1 ml/kg and rate of 0.2 ml/min. Next, pinch the injection site with the left thumb and the duration is 3 minutes. The site is the joint of pancreatic duct and duodenum. 3 minutes later, remove the clip.wortmannin was administered to WAP group introperitoneally with the dosage of 0.35mg/kg 4 hours before the modle was made. SO group and AP group were injected introperitoneally the equivalence DMSO (dimethylsulfoxide) 4 hours before the modle was made. DMSO is a solvent of wortmannin. All the animals were sampled blood from inferior vena cava and taken kidney and pancreas at 3h, 6h, 12h under sterile circumstances, then sacrificed .The serum levels of tumor necrosis factor-alpha ( TNF-α), amylase, creatinine and urea nitrogen were measured. Changes of gross anatomy and pathology in kidney and pancreas were observed. Myeloperoxidase (MPO) activity in kidney was minitored. p-PKBcan measure the activation of PI3K∕PKB signal pathway, the expressional levels of PKB and p-PKB were measured by western blot. By way of this, study the relationship between PI3K∕PKB signal pathway and renal injuries induced by AP in rat. After the pretreatment of AP rat by wortmannin, monitor changes of the foregoing measurements.RESULTS: The measurements of SO group at all trial times have no significant difference .The serum levels of tumor necrosis factor-alpha ( TNF-α), amylase, creatinine and urea nitrogen and myeloperoxidase (MPO) activity in kidney of AP group at all trial times were significantly elevated ( P < 0. 05) compared with SO group. The pathological changes in kidney and pancreas in AP group were significantly aggravated than SO group at all trial times;all indices of all trial times in WAP group were significantly elevated ( P < 0. 05) compared than SO group and significantly decreased ( P < 0. 05) compared than AP group.In our study, MPO activitiy represents neutrophilic infiltration. The MPO activity in SO group rats at 3h,6h and 12h are (1.04±0.17)U/g,(1.02±0.17)U/g,(1.05±0.19)respectively;the MPO activity in AP group rats at 3h,6h and 12h are (2.12±0.26)U/g,(7.45±0.41)U/g,(9.97±0.23)U/g respectively;the MPO activity in WAP group rats at 3h,6h and 12h are (1.59±0. 20)U/g,(4.48±0.23)U/g,(6.14±0.20)U/g. The MPO activity of SO group rats at all trial times has no significant difference. Compared with so group, the MPO activity of AP group and WAP group is significantly increased at all trial times ( P < 0. 05 ). In comparison with AP group, the MPO activity of WAP group is significantly decreased at all trial times ( P < 0. 05 ). The serum levels of TNF–αof all trial groups are as following: (78.2±17.62)ng/L,(75.86±16.58) ng/L,(67.13±12.55) ng/L respectively of SO group rats at 3h,6h and 12h; (274.57±22.37) ng/L,(500.39±30.96) ng/L,(764.83±41.61) ng/L respectively of AP group rats at 3h,6h and 12h;(170.60±25.99)ng/L,(312.31±35.66)ng/L,(397.21±33.45)ng/L respectively of WAP group rats at 3h,6h and 12h. The serum levels of TNF–αof SO group rats at all trial times has no significant difference. Compared with so group, the serum TNF–αlevels of AP group and WAP group is significantly increased at all trial times ( P < 0. 05 ). In comparison with AP group, the serum TNF–αlevels of WAP group is significantly decreased at all trial times ( P < 0. 05 ) .There is no significant difference of PKB expression among the 3 groups. The p-PKB expression in AP group and WAP group was significantly elevated ( P < 0. 05) compared with SO group, and the p-PKB expression in WAP group wassignificantly decreased ( P < 0. 05) compared with AP group . Conclusions: After the AP modle was made ,pathology damages took place in pancreas .the infiltration of inflammatory cells and degree of necrosis were worsened as time prolonging .All these prove the modle was successful. The p-PKB expression in AP group was significantly elevated compared with SO group, which prove that PI3K∕PKB signal transduction pathway is involved in the pathogenesis of renal injuries induced by acute pancreatitis in rats.After the pretreatment with wortmannin, the expressional level of p-PKB in WAP group is significantly decreased than AP group.These show wortmannin can inhibit the PI3K∕PKB signal transduction pathway in reanl injuries induced by AP.The changes of gross anatomy and pathology in kidney of WAP group is significantly alleviated than SO group,the same as the serum levels of creatinine, urea nitrogen and TNF-α. Meanwhile, the aggregation of neutrophil in kidney of WAP group is obviously lessened than AP group. These give evidence wortmannin can relieve the renal injuries induced by acute pancreatitis. The mechanism may be related to the inhibition of release inflammatory factors and neutrophil aggregation. The inflammatory factors include TNF-αand so on. To sum up the above arguments, wortmannin has a preventive effect in renal injuries induced by AP.
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