The Function And Regulatory Mechanism Of SENP2 In Progression And Reversal Of Mice Liver Fibrosis

Background:Liver fibrosis?LF?,a pathological process of abnormal hyperplasia of intrahepatic connective tissue,often caused by multiple chronic injury pathogenesis factors.Abnormal accumulation of extracellular matrixs?ECMs?with collagen deposition in hepatic tissue is mian characteristic of LF,untimately leading to damage of the liver normal structure and decline of normal function.Without timely intervention in the progression of LF,mild liver fibrosis can become liver cirrhosis and cancer eventually.The deposition of ECMs is highly correlated with the collagen production of hepatic stellate cells?HSCs?,which are often activated in LF.Studies have shown that LF can be reversed when the causative agent is removed.Apoptosis of HSCs,senescence and transformation of quiescent phenotype can be considered as common phenomena in the process of reversion.The study of how LF are reversed is of great significance in the treatment of this disease.SUMOylation and deSUMOylation,a dynamic process,is proved to be involved in various fibrotic diseases.In liver fibrosis,UBC9,the only known E2-conjugating enzyme involved in SUMOylation,plays a role in aggravates fibrosis by promoting the activation and proliferation of HSCs.However,the function and mechanism of SUMO-specific protease?SENP?in liver fibrosis are still unclear.The purpose of this study was to investigate the functions of SENP2,a member of SENP family,during the progression and reversal of hepatic fibrosis,and its regulation of hepatic stellate cell activation,proliferation and apoptosis,as well as its regulation of Wnt/?-catenin pathway.Methods:In vivo,45 healthy male C57BL/6J mice were randomly divided into three groups:normal control group,model group and reversal group?15 mice in each group?.The olive oil solution of 10%CCl₄ was injected into the abdominal cavity of mice twice a week for a total of 4 weeks.Mice in the reversal group were stopped from injecting CCl₄ after 4 weeks and returned to normal feeding for 6 weeks.After the end of modeling,serum,liver tissue and primary hepatic stellate cells of mice were taken for detection.The successful establishment of the mouse liver fibrosis progression and reversal phase model was firstly verified,and the expression changes of SENP1-3 and SENP5-7 in primary HSCs were detected.Then,the localization and expression of SENP2 were detected by western blot,RT-qPCR and immunofluorescence double staining.In vitro,HSC-T6 cells were activated by TGF-?1,cell reversions induced by MDI medium,and SENP2 protein expression and localization were detected by western blot and immunofluorescence single staining.HSC-T6 cells were transfected with pEX3-SENP2 and SENP2-siRNA,followed by stimulation by TGF-?1,respectively.Flow cytometry and western blot were used to detect whether SENP2 would affect the activation,proliferation,apoptosis and reversal of HSC-T6 cells.Result:In vivo,SENP2 was abnormally down-regulated in primary HSCs and liver tissues during the progression of liver fibrosis,and almost returned to normal level in the reversal group.In vitro,SENP2 was significantly down-regulated in HSC-T6 cells activated by TGF-?1,and recovered in the MDI reversal group.Overexpression of SENP2 inhibited the activation of HSC-T6 cells,showing a significant down-regulation of?-sma and col1a1.What's more,the proliferation of HSC-T6 cells was inhibited,which was manifested as decreased cell viability and cycle arrest.High expression of SENP2 also promoted apoptosis of HSC-T6 cells by increasing apoptosis rate and upregulation of apoptosis-related proteins,while silencing SENP2 has the opposite effect.Mechanistically,SENP2 significantly inhibited the expression of key proteins in the Wnt/?-catenin pathway,such as?-catenin,c-myc,and cyclinD1,while silencing SENP2 did the opposite effect.Conclusion:According to our results,SENP2 can inhibit the activation and proliferation of HSCs and promote the apoptosis and reversal of activated HSC,and we also found that SENP2 may play a role in alleviating liver fibrosis by inhibiting the Wnt/?-catenin pathway.