The Role Of DsbA-L In Acute Pancreatitis And Mechanism Study

Acute pancreatitis（AP）is a common acute abdominal disease in general surgery,mainly caused by gallstones,alcohol,obesity,hyperlipidemia,and so on.Although research on acute pancreatitis has been ongoing for decades,there is still no clear explanation for its pathological mechanism.At present,there is a general consensus that abnormal calcium signaling in pancreatic acinar cells leads to mitochondrial dysfunction,leading to cell death and the release of inflammatory factors.Then the infiltrated immune cells in the pancreas increased and worsens the systemic condition.It can be seen that maintaining the homeostasis of mitochondria may have positive influence on inhibiting the development of acute pancreatitis.Disulfide bond A oxidoreductase like protein（DsbA-L）was initially discovered in mitochondria of rat hepatocyte.Researchers have shown that DsbA-L plays an important role in protecting mitochondrial function against endogenous or exogenous oxidative stress.DsbA-L has been shown to be highly expressed in hepatocytes,renal tubular epithelial cells,and adipocytes.However,there is currently no report on the expression of DsbA-L in the pancreas and relationship with acute pancreatitis.Therefore,we investigate the role of DsbA-L in pancreas during acute pancreatitis and mechanisms on inflammation,mitochondrial function,and pathological damage,providing potential targets for the treatment of acute pancreatitis..Aim:To explore the expression of DsbA-L in pancreatic tissue of acute pancreatitis and its effects and mechanisms on inflammation,mitochondrial function,and pathological damage through bioinformatics analysis,animal and cell models of acute pancreatitis,in order to provide potential targets for the treatment of acute pancreatitis.Method:Firstly,we searched for differentially expressed genes related to mitochondria in acute pancreatitis through bioinformatics analysis to target the DsbA-L protein.Then we established the wild type C57BL/6 mice acute pancreatitis model.The expression of DsbA-L was detected at different time points,and the inflammatory factors,mitochondrial function,pathological changes and inflammatory cell infiltration were detected by western blot,reverse transcription-polymerase chain reaction（RT-q PCR）,enzyme-linked immunosorbent assay（ELISA）,Hematoxylin-Eosin（HE）staining and flow cytometry.Then,an in vitro acute pancreatitis cell model was constructed using the rat acinar cell carcinoma cell line AR42J.The expression levels of DsbA-L were detected at different time points,and inflammation,mitochondrial function,and cell apoptosis were detected using WB and RT-q PCR.Finally,the acute pancreatitis model of DsbA-L knockout(DsbA-L^-/-)mice was constructed.The level of myeloperoxidase（MPO）in pancreatic tissue was detected by immunohistochemistry,inflammatory factors and mitochondrial function related genes were detected by RT-q PCR,and NF-κB pathway related proteins was detected by WB,verifying whether DsbA-L affects inflammation and injury in acute pancreatitis through NF-κB pathway.Results:We searched GEO database and selected appropriate AP related dataset,then we perform GO enrichment analysis to obtain 57differentially expressed genes（DEGs）related to mitochondria and selected DsbA-L from them.After analyzing the original data,it was found that the expression of DsbA-L in acute pancreatitis group was significantly reduced.The successful AP model of wild type C57BL/6mice was confirmed by the increase of serum amylase,lipase,TNF-αand IL-6 levels,pathological scores,and immune cell infiltration.DsbA-L expression was significantly downregulated in both protein and m RNA levels,which had significantly positive correlation with mitochondrial function related genes,while had negative correlation with inflammation related genes.In the AP cell model in vitro,the necessity of glucocorticoid to reconstruct the endoplasmic reticulum of AR42J cells and to transform the phenotype of acinar cells was firstly clarified.Subsequently,significant downregulation of DsbA-L expression,significant upregulation of inflammatory factor expression,and significant downregulation of mitochondrial functional genes were detected at both protein and m RNA levels in the AP group,consistent with the results of wild-type mice AP model.Compared with wild type mice AP model,DsbA-L^-/-mice AP model had a significantly increased levels of serum amylase lipase and inflammatory factors,significantly decreased pathological scores and neutrophil infiltration;The expression of mitochondrial function related genes was significantly downregulated,while the expression of inflammatory factors was significantly upregulated;significant increase in P65 phosphorylation and activation of the NF-κB pathway.Conclusion:DsbA-L is significantly downregulated in acute pancreatitis and is significantly correlated with mitochondrial functional homeostasis in pancreatic cells.The absence of DsbA-L leads to a decrease in mitochondrial stability in pancreatic cells,making them more sensitive to the stimulation of cerulein.The possible mechanism is excessive activation of NF-κB,promoting P65 phosphorylation and increasing transcription of inflammatory factors,exacerbating inflammation and pathological damage in AP.