The Role Of Necroptosis In Pancreatic Injury In Hyperlipidemic Rats With Acute Pancreatitis

Part 1 The establishment and evaluation of acute pancreatitis model in hyperlipidemic ratsObjective:To explore and evaluate the method of establishing acute pancreatitis model in hyperlipidemic rats.Methods: 72 SPF male SD rats were randomly divided into the normal control group(N group),P-407 0.12g/kg(H1 group),P-407 0.25g/kg(H2 group)and P-407 0.50g/kg(H3 group)according to the random number table.Each group was further divided into three subgroups W1 group,W2 group and W4 group,with 6 rats in each group.The hyperlipidemia model was established by intraperitoneal injection of P-407 at the corresponding dose and time,and the acute pancreatitis model was established by intraperitoneal injection of 20% L-arginine after tail vein blood collection.Rats in each group were sacrificed 24 hours after modeling.Automatic biochemical analyzer was used to test the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine(Cr)and urea nitrogen(BUN)in the blood of the rat tail vein,and serum levels of triglyceride(TG),total cholesterol(TC)and amylase(AMY),lipase(LIP).And the histopathological changes of the pancreas were observed under an optical microscope and scored.Results: Compared with the N group,the serum TG and TC levels of the H group were significantly increased,and the difference was statistically significant(P<0.05).In addition,the serum lipid level of rats in H group increased gradually with the dose and time of intraperitoneal injection of P-407.After continuous intraperitoneal injection of 0.25g/kg P-407 for 2 weeks,TG > 1000mg/dl;There was no significant difference in serum ALT,AST,Cr and BUN levels between the H group and the N group(P > 0.05).After the modeling of acute pancreatitis,serum AMY and LIP were significantly increased,and the degree of pathological injury of pancreas under light microscope was gradually aggravated with the dose and time of intraperitoneal injection of P-407.Conclusion: 1.Intraperitoneal injection of 0.25g/kg 10% P-407 for 2 weeks and intraperitoneal injection of 2.5g/kg 20% L-arginine(p H 7.0)at an interval of 1 hour for 2 times can successfully induce the acute pancreatitis model in hyperlipemia rats;2.The serum lipid level of rats gradually increased with the dose and time of intraperitoneal injection of P-407;3.The condition of acute pancreatitis in hyperlipidemic rats was gradually aggravated with the increase of serum lipids levels.Part 2 Pancreatic injury in hyperlipidemic rats with acute pancreatitis and expression of RIP3 in the pancreasObjective : To compare the pancreatic injury of acute pancreatitis in hyperlipidemic rats with that of non-hyperlipidemic rats,and to explore the possible mechanism.Methods: 48 SPF male SD rats were randomly divided into the normal group(N group)and the hyperlipidemia group(H group)according to the random number table.Each group was further divided into the Control group,AP12 h group,AP24 h group and the AP48 h group with 6 rats in each group.The hyperlipidemia group was intraperitoneally injected with 0.25g/kg P-407 daily for 2 weeks.Acute pancreatitis model was established by intraperitoneal injection of 20% L-arginine.Rats in each group were sacrificed at the corresponding time after modeling.The changes of serum amylase(AMY),lipase(LIP),liver function(ALT,AST)and renal function(Cr,BUN)were detected by automatic biochemical analysis.Serum levels of inflammatory mediators TNF-α and IL-6 were detected by ELISA.Histopathological changes of pancreas were observed under light microscope.The expression of MPO in pancreatic tissue was detected by immunofluorescence assay.Ultrastructural changes of pancreatic acinar cells were observed under transmission electron microscope.The RIP3 expression in pancreatic tissue were assessed by immunohistochemical analysis.Results: Compared with the Control group,except the NAP12 h group,the activity levels of serum AMY and LIP,liver and kidney functions,serum inflammatory mediators and pancreatic pathological scores of rats in the AP group at each time point were all increased,and all of them were gradually increased with the extension of time.Compared with NAP group at the same time point,the serum levels of AMY and LIP in HAP group were slightly decreased,while the levels of ALT,AST,Cr,BUN,TNF-α,IL-6 and pancreatic histopathological score and the number of MPO positive cells were significantly increased(P<0.05).In addition,significant pathological injuries were occurred in the pancreas of HAP12 h group,while no significant pathological damage had been found in NAP12 h group.The results of electron microscopy showed that apoptosis of pancreatic acinar cells was dominant in NAP group,and necrosis was dominant in HAP group.Immunohistochemical results showed that RIP3 level in the pancreatic tissues of HAP group were gradually increased with time,and were higher than those of NAP group at the corresponding time point,and the difference was statistically significant(P<0.05).Conclusion: 1.The pancreatic injury of hyperlipidemic rats was earlier and more severe than that of non-hyperlipidemic rats;2.Pancreatic injury in hyperlipidemic rats with acute pancreatitis may be related to necroptosis.Part 3 The role of necroptosis in pancreatic injury in hyperlipidemic rats with acute pancreatitisObjective : To determine the role of necroptosis in pancreatic injury in hyperlipidemic rats with acute pancreatitis.Methods: 24 SPF male SD rats were randomly divided into hyperlipidemic group(H group),hyperlipidemic acute pancreatitis group(HAP group),solvent control group(Vehicle group),and Nec-1 intervention group(N=6)according to the random number table.All rats received intraperitoneal injection of 0.25g/kg P-407 daily for 2 weeks.Acute pancreatitis model was established by intraperitoneal injection of 20% L-arginine.Rats in the Nec-1 group were given 1.65mg/kg Nec-1 intraperitoneal injection 1h before the model formation of acute pancreatitis,and the Vehicle group was given the same dose of solvent,and then the acute pancreatitis model was established.Rats in each group were sacrificed 24 h after modeling.The changes of serum amylase(AMY)and lipase(LIP)in rats were detected by automatic biochemical analysis.Serum levels of inflammatory mediators TNF-α and IL-6 were detected by ELISA.Serum lactate dehydrogenase(LDH)level was detected with the kit.Histopathological changes of pancreas were observed under light microscope.Ultrastructural changes of pancreatic acinar cells were observed under transmission electron microscope.The expression of MPO and IL-6 in pancreatic tissue was detected by immunofluorescence assay.Immunohistochemistry and Western Blot were used to detect the expression levels of RIP1,RIP3 and MLKL in pancreatic tissue.Results: Compared with the H group,the activities of AMY and LIP in the serum of HAP rats were significantly increased,the pancreas showed obvious pathological damage,and the pathological score was increased.In addition,the number of MPO positive cells in the pancreas was increased,and the contents of inflammatory factors TNF-α and IL-6 in the pancreas and serum were also significantly increased.After treatment with Nec-1,the activity levels of AMY and LIP in serum of rats were significantly decreased,the pathological damage of pancreatic tissue was improved,the pathological score was also decreased,the number of MPO positive cells in the pancreatic tissue was also significantly reduced,and the contents of TNF-α and IL-6 in pancreas and serum were significantly inhibited(P<0.05).Further,Nec-1 pretreatment significantly improved acinar cell necrosis,decreased serum LDH levels in rats,and inhibited expression of RIP1,RIP3,and MLKL,the key molecules of programmed necroptosis,in pancreatic tissue.Conclusion: 1.Necroptosis is involved in the process of pancreatic injury in hyperlipidemic rats with acute pancreatitis;2.Nec-1 can reduce pancreatic injury and inflammation by inhibiting necroptosis,and play a protective role in the pancreas.