Mocecular Mechanism Underlying Cell Apoptosis Promoted By APD For SAP Treatment

Objective:Severe acute pancreatitis(SAP)is a common clinical acute abdominal disease,often accompanied by systemic inflammatory response and even important organ failure and other complications.So far,SAP's death rate is still as high as 20-30%.For a long time,the strategy of early treatment of acute pancreatitis has been to support the treatment of the main,the temporary lack of specific treatment.Our previous studies have shown that early peritoneal puncture and drainage(APD)can effectively control the degree of inflammatory response during SAP by elicitation of pancreatic inflammatory ascites(PAAF)and reducing the concentration of pro-inflammatory factors in blood and ascites,thus achieving the purpose of treating SAP.However,the potential mechanism of treating SAP through APD has not been further explored.It has been demonstrated that apoptosis in pancreatic tissue is a key pathological change in the pathogenesis of SAP and is considered to be an important protective mechanism.Therefore,this study aimed to investigate the apoptosis of pancreatic tissue cells during the treatment of SAP with APD and the underlying molecular mechanism.Methods:Male adult SD rats aged about 200g at 8 weeks were randomly divided into SO group(sham operation group),SAP group and APD group,with 18 rats in each group.The three groups of rats were respectively preparedby the following methods.In the severe acute pancreatitis group,severe acute pancreatitis was induced in rats by retrograde injection of 5% sodium sulphurate into the pancreatic bile duct.In the APD group,a drainage tube was placed into the abdominal cavity immediately after the model of severe acute pancreatitis was prepared according to the above methods for subsequent drainage of pancreatic ascites.The drainage tube was fixed in the right lower abdomen of the rats.In the sham operation group,after opening the abdominal cavity,only the pancreas was gently turned over,and the abdomen was closed without any surgical operation.Aortic blood and pancreatic tissue were collected from each group at 6h,12h and 24h after operation.HE staining was used to make pathological scores on pancreatic tissues of rats in each group,and cell necrosis rate of pancreatic tissues was calculated to evaluate the effect of APD on the degree of pancreatic tissue damage during SAP treatment.Serum amylase,inflammatory factors(TNF-?,il-1,il-6)and endotoxin concentrations in rats of each group were detected by blood biochemistry to determine the effect of APD on the degree of systemic inflammation during SAP treatment.The apoptosis of pancreatic cells in rats in each group was detected by TUNEL staining.Western blot and immunohistochemical staining were used to detect the expression of apoptosis-related proteins in pancreatic tissues.The expression of PI3K/AKT signaling pathway was detected by western blot.Results:(1)In the three groups of animal models,the degree of pancreatic tissue damage and systemic inflammatory response in the SAP group and APD group were significantly worse than that in the SO group at each time point;The degree of pancreatic tissue injury in APD group was significantly reduced than that in SAP group.Meanwhile,compared with the SAP group,the serum levels of amylase,endotoxin,TNF-,il-1,and il-6 in the APD group were also significantly decreased.(2)Compared with the SAP group,the number of apoptosis increased in the pancreatic tissue of the APD group,while the degree of necrosis of pancreatic tissue cells decreased.However,apoptosis and necrosis of pancreatic tissue cells were not observed in SO group.In addition,Western blot and histochemical staining showed that pro-apoptotic proteins(Cleaved caspase-3 and bax)were significantly increased in APD group compared with SAP group,while anti-apoptotic proteins(bcl-2)were significantly decrease.(3)Through western blot,we found that after APD treatment,phosphorylation activation of PI3K/AKT/ nf-kb signaling pathway was significantly decreased compared with SAP group.(4)In addition,we performed intraperitoneal injection of PAAF and PAAF+PI3K inhibitors wortmannie into the mild pancreatitis model,It has been confirmed that PAAF can inhibit the apoptosis of pancreatic tissue by promoting the phosphorylation activation of PI3K/AKT/ nf-kb signaling pathway,thus aggravating the pancreatic injury and inflammatory response of acute pancreatitis.The experimental results were verified in reverse that the drainage of PAAF by APDcould indeed regulate the apoptosis of pancreatic tissue cells through the PI3K/AKT/ nf-kb signaling pathway to reduce the severity of SAP.Conclusion:The above results showed that during SAP treatment,APD could inhibit the phosphorylation and activation of PI3K/AKT signaling pathway in pancreatic tissue by draining toxic substances from PAAF,thus enhancing the apoptosis of pancreatic tissue and reducing the degree of pancreatic tissue damage and systemic inflammation during SAP treatment.This study provides new insights into the mechanism of APD in the treatment of SAP patients.