Role And Mechanism Of P53 Acetylation In Acinar Cell Death Response In Cearulein Induced Acute Pancreatitis In Mice

Background and purpose: The way of pancreatic acinar cells death in acute pancreatitis(AP)is an important pathophysiology mechanism that determines disease prognosis.Necrosis of pancreatic acinar cells leads to more severe AP,while apoptosis relieves the severity.However,mechanisms underlying the regulation of pancreatic acinar cell death in response to stimuli remain to be uncovered.p53 is a vital protein in the regulation of apoptosis.And acetylation of p53,a posttranslational modification,guarantees the execution of its normal function.Therefore,this study aimed at deciphering whether acetylation of p53 involves in regulating the way of pancreatic acinar cells death during AP and the mechanisms underway.Contents and methods: Firstly,using the model of cearulein induced AP in C57BL/6 mice,we observed the severity of AP and the time course changes of pancreatic acinar cells underwent necroptosis or apoptosis.Secondly,we investigated the effects of Compound 14,an apoptosis inducing compound,on the apoptosis of pancreatic acinar cells and the severity of AP,and further studies the pathophysiology effects of pancreatic acinar cells apoptosis on AP.Thirdly,we explored the time course of p53 acetylation and its transcriptional activity changes during AP in vitro and in vivo.Furthermore,we enquired the impacts of TSA,a histone deacetylation enzyme inhibitor,on the acetylation level of p53 and its transcriptional activity in the pancreatic acinar cells,as well as its influence in the way of pancreatic acinar cell death in vitro and in vivo.Fourthly,we detected the expression of phosphorylated RIPK3 and MLKL and their membrane translocation,and testified whether the RIPK3/MLKL necroptosis signaling pathway was involved in the pancreatic acinar cell necrosis during AP.Results: The pathology changes,levels of pancreatic acinar cell necrosis and inflammatory reaction of induced AP in C57BL/6 mice were exacerbated in a time course-dependent manner.In addition,positive rate of TUNEL in pancreatic tissues remained low,and the expression of cleaved caspase-3/8/9 was suppressed along this process.However,when we treated AP mice using apoptosis inducing compound(Compound 14),positive rate of TUNEL and the expression of cleaved caspase-3/8/9 in AP tissues were significantly elevated,and levels of pancreatic acinar cell necrosis and inflammatory reaction were attenuated at the same time.When interrogating the mechanisms,we found that cearulein inhibited the acetylation level of main acetylation sites(lys375,lys370 and lys305)of p53,and also down-regulated the protein content of tatol p53 in pancreatic tissues or pancreatic primary acinar cells.In addition,Fas and Puma,target genes that were transcriptional activated by p53,were also significantly down-regulated.Furthermore,the acetylation level of α-tubulin was inhibited.The transcription level of p53 was also inhibited in the primary pancreatic acinar cells when induced by cearulein in vitro.Therefore,we interfered this process using TSA and found that TSA could significantly elevate the protein contents of p53 and enhance its acetylation level in aforementioned acetylation sites,and promote the protein level of its target genes,Puma and Fas,through its enhanced transcriptional activity,at the same time.So,TSA could significantly promote apoptosis of pancreatic acinar cells,and markedly alleviated the damage of pancreatic tissue and inflammatory reaction.However,during AP,the protein content and phosphorylation level of RIPK3 and MLKL were significantly down-regulated in pancreatic tissue in vivo.Cearulein treatment didn’t change the phosphorylation level of RIPK3 and MLKL in primary acinar cell.Meanwhile,cearulein didn’t influence the protein contents of p-MLKL,MLKL(N terminal),MLKL(C terminal)and RIPK3 in the membrane of primary pancreatic acinar cells.The RIPK1 inhibitor Necrostatin-1 didn’t influence the phosphorylation level of RIPK3 and MLKL as well as the ATP content in primary pancreatic acinar cells when treated by cearulein,either.Conclusion: The apoptotic signaling pathway remains repressive in pancreatic acinar cells during AP induced by cearulein.When apoptosis was induced by compounds,the necrosis of pancreatic tissues and inflammatory reaction wound be markedly alleviated.The repressed apoptosis could be attributed to the inhibited acetylation level of p53 and the consequent slience of p53/Puma/Fas apoptosis pathway in pancreatic acinar cells during AP.TSA could restore the acetylation level of p53 and its transcriptional activity,which activates the p53-dependent apoptosis pathway and leads to alleviated necrosis of pancreatic acinar cells and inflammatory reaction during AP.Furthermore,RIPK3 and MLKL may not be involved in the necrosis of pancreatic acinar cells in cearulein induced AP mice mode.