The Effect And Mechanism Of USP25 Regulating STAT3 Pathway In Acute Pancreatitis

Part Ⅰ.Correlation between USP25 and disease severity of patients with acute pancreatitisObjective: To investigate the changes of USP25 expression levels in patients with acute pancreatitis（AP）and the correlation with disease severity and multiple organ injury in patients with acute pancreatitis.Methods: Serum and clinical information were collected from patients with AP and healthy controls on admission: gender,age,and results of blood tests,including serum AMY,LIPA,ALT,AST,Cr,BUN,and CRP.According to the severity of the disease,patients with AP were classified into mild acute pancreatitis（MAP）,moderate severe acute pancreatitis（MSAP）,and severe acute pancreatitis（SAP）,respectively.By using enzyme-linked immunosorbent assay,the serum levels of USP25 protein and the levels of inflammatory factors IL-6,TNF-α and IL-1β were measured;meanwhile,some patients discharged from the hospital after treatment were followed up and their serum data was collected to check USP25 protein levels.The serum USP25 protein levels of patients with acute pancreatitis and healthy controls were compared by using statistical methods;the differences in serum USP25 protein levels of patients with MAP,MSAP and SAP were compared;correlation analysis was employed to analyze the correlation between patients’ serum AMY,LIPA,ALT,AST,Cr,BUN,CRP,inflammatory factors IL-6,and USP25,respectively;finally,the differences of serum USP25 protein levels between patients on admission and after discharge from the hospital were compared.Results: Compared to healthy controls,serum AMY,LIPA,ALT,AST,Cr,BUN,CRP and inflammatory factors IL-6,TNF-α and IL-1β levels of patients with acute pancreatitis were significantly higher and the differences demonstrated statistically significant（p<0.05）.Serum USP25 levels were significantly higher in patients with acute pancreatitis than in healthy controls（p<0.05）.In addition,patients in the SAP group showed the highest serum USP25 levels,while MSAP and MAP group exhibited the lowest serum USP25 levels,and the differences between these three groups showed statistically significant（p<0.05）.Correlation analysis revealed that serum AMY,LIPA,ALT,AST,Cr,BUN,CRP and inflammatory factors IL-6,TNF-αand IL-1β were positively correlated with USP25 levels in patients with acute pancreatitis（p<0.05）.On the other hand,serum USP25 levels of patients discharged from the hospital after treatment were lower than those of patients at the onset of acute pancreatitis（p<0.05）.Conclusion: The expression of USP25 level increased in patients with acute pancreatitis,and the expression level of USP25 is correlated with the severity of disease and the degree of multiple organ injury in patients with acute pancreatitis.Therefore,USP25 may play a significant role in the occurrence and progress of acute pancreatitis and multiple organ injury.Part Ⅱ The study on effect of USP25 in acute pancreatitisObjective: As one of the important roles of the deubiquitination family,USP25 gets involved in a variety of modulations such as immunity,inflammation,neurological diseases and tumors,while studies on the role of USP25 in the development of AP have not been reported yet.In this part,we promoted further investigation on the role of USP25 in AP by carrying out in vivo experiments.Methods: By employing the method of multiple intraperitoneal injections of cerulein combined with lipopolysaccharide induced AP model in mice,Usp25 WT and Usp25^-/-mice were divided into control group and AP group,respectively.The control group was intraperitoneal injected with normal saline,and the AP group was intraperitoneal injected with cerulein and lipopolysaccharide.The serum of mice was collected,the tissues of pancreas,lung,liver,kidney,and intestine were taken to calculate the relative weight of mouse pancreas,and the mice pancreases,lungs,livers,kidneys,and intestines were examined using H&E staining and the histopathological scoring was performed.The inflammation marker CD45 was detected in pancreatic,lung,liver,kidney and intestinal tissues by employing the immunohistochemistry method;the expression of USP25 in mouse pancreatic tissues was detected by the Western Blot and immunohistochemistry methods;the expression of tight junction protein ZO-1 in mouse pancreas was detected by the Western Blot and immunofluorescence methods.In addition,serum AMY,LIPA,ALT,AST,Cr and BUN were detected by a fully automatic chemistry analyzer,and serum levels of inflammatory factors IL-6,TNF-α and IL-1β were measured using an enzyme-linked immunosorbent assay.Results: AP was successfully induced in mice by employing intraperitoneal injection of cerulein combined with lipopolysaccharide.In wild-type mice,the relative weight of the pancreas was significantly higher in the AP group compared with the control group（p<0.05）;significant inflammation-induced pathological changes were observed in the pancreas,lung,liver,kidney and intestine of the modeling group,and the histological score was significantly higher than that of the control group（p<0.05）;in addition,the inflammation markers CD45 in the pancreas,lung,liver,kidney and intestine of the AP group,as well as serum AMY,LIPA,ALT,AST,Cr,BUN,and inflammatory factors IL-6,TNF-α and IL-1β were significantly higher in the AP group than that of the control group（p<0.05）;and the expression of tight junction protein ZO-1 was significantly lower in the AP group（p<0.05）.Moreover,USP25 expression was upregulated in the pancreatic tissue of mice with AP（p<0.05）.Compared with Usp25 WT AP mice,the relative weight of pancreas was reduced in the Usp25^-/-AP group（p<0.05）,histopathological damage of pancreas,lung,liver,kidney and intestine was reduced（p<0.05）,and serum AMY,LIPA,ALT,AST,Cr,BUN,and inflammatory factors IL-6,TNF-α and IL-1β levels were significantly reduced（p<0.05）,while the expression of tight junction protein ZO-1 was significantly higher in the pancreatic tissues of mice in the Usp25^-/-AP group than that of the mice in the Usp25 WTAP group（p<0.05）.Conclusion: AP and multiple organ injury were successfully induced in mice using cerulein combined with lipopolysaccharide;USP25 expression was upregulated in pancreatic tissues of mice with AP;knockdown of Usp25 attenuated AP and multiple organ injury in mice;in addition,USP25 aggravated AP and multiple organ injury by exacerbating damage to the tight junctions of the pancreas.Part Ⅲ The study on the mechanisms of USP25 exacerbating acute pancreatitis and multiple organ injuryObjective: USP25 can aggravate AP and AP related multiple organ injury,however,the mechanism of how USP25 aggravates AP is still unclear.In this part,we carried out further studying of the mechanisms of USP25 exacerbating AP and multiple organ injury through in vivo and in vitro experiments.Method: To further investigate the mechanism of how USP25 exacerbates AP,Flag-USP25 was overexpressed in 293 T cells,and the proteins interacting with Flag-USP25 were detected by using immunoprecipitation-mass spectrometry（IP-MS）method.Subsequently,both Flag-USP25 and HA-STAT3 were overexpressed in 293 T cells,and protein interaction between USP25 and STAT3 was verified by the immunoprecipitation（CO-IP）method.Mouse pancreatic acinar cells（MPAC）were isolated and extracted from Usp25 WT and Usp25^-/-mice,respectively.Usp25 was lowly expressed in rat pancreatic alveolar cells（AR42J）by using Usp25 Si RNA,and inflammation was induced in AR42 J cells,MPAC cells,Usp25 WT and Usp25^-/-mice by cerulein（or combined lipopolysaccharide）.The expression levels of USP25,STAT3,p-STAT3^Y705 and ZO-1 protein were detected by Western Blot,and the expression of p-STAT3^Y705 in MPAC cells was examined using the immunofluorescence method.STAT3 phosphorylation was suppressed using Stattic in AR42 J cells,MPAC cells,Usp25 WT and Usp25^-/-mice,and STAT3 was activated using Colivelin in AR42 J and MPAC cells.The serum of mice was collected,the tissues of pancreas,lung,liver,kidney,and intestine were taken to calculate the relative weight of mouse pancreas;and the mice pancreas,lung,liver,kidney,and intestine were examined using H&E and the histopathological scoring was performed.Serum AMY and LIPA were detected using a fully automatic chemistry analyzer,and serum levels of inflammatory factors IL-6,TNF-α and IL-1β were detected using an enzyme-linked immunosorbent assay.STAT3,p-STAT3^Y705 and ZO-1 expression in mouse pancreatic tissues and AR42 J and MPAC cells was detected by the Western Blot;and the expression of tight junction protein ZO-1 in mouse pancreas was detected by immunofluorescence.Results: IP-MS and CO-IP results revealed the existence of protein interactions between USP25 and STAT3.In AP models,p-STAT3^Y705 protein levels was decreased in low or no USP25 expression cells or in Usp25^-/-mice（p<0.05）,while tight junction protein ZO-1 expression was upregulated（p<0.05）.Suppression of STAT3 phosphorylation with Stattic led to a significant reduction in the relative weight of the pancreas in mice with AP（p<0.05）;reduced histopathological damage of the pancreas,lung,liver,kidney and intestine in mice（p<0.05）;significantly lower levels of serum AMY,LIPA and inflammatory factors IL-6,TNF-α,IL-1β（p<0.05）;p-STAT3^Y705 protein expression levels was decreased in pancreatic tissue,AR42 J and MPAC cells（p<0.05）,while the expression of tight junction protein ZO-1 was upregulated （p<0.05）.After using STAT3 agonist Colivelin in AR42 J,MPAC cells,the expression level of p-STAT3^Y705 in cells was significantly increased（p<0.05）,while the expression of tight junction protein ZO-1 was significantly decreased（p<0.05）.Conclusion: USP25 protein can interact with STAT3 protein.In AP,USP25 exacerbates tight junction injury in the pancreas by activating the STAT3 pathway,therefore exacerbating AP and AP related multiple organ injury.