The Role And Mechanism Of MafG Promoting Liver Fibrosis By Inhibiting Ferroptosis In Hepatic Stellate Cells

Background and objective:Activation of hepatic stellate cells and secretion of extracellular matrix are central to the development of liver fibrosis.In addition to inhibiting hepatic stellate cell activation,promoting the death of activated hepatic stellate cells is also an effective strategy for the treatment of liver fibrosis.Ferroptosis is a novel form of cell death characterized by iron overload and lipid peroxidation.Inducing the ferroptosis of activated hepatic stellate cells has potential applications for the treatment of liver fibrosis.Transcription factors play an important role in the regulation of ferroptosis.Previous studies have shown that Mafb ZIP transcription factor G(MafG)is closely related to ferroptosis,and it promotes the progression of hepatocellular carcinoma and bladder cancer by inhibiting ferroptosis.Meanwhile,MafG is highly expressed in cholestatic liver injury that is a pre-induction factor of liver fibrosis and hepatocellular carcinoma,a malignant outcome of liver fibrosis,suggesting that MafG plays a significant role in the development of liver fibrosis.However,the role of MafG in liver fibrosis and whether it is involved in the ferroptosis of hepatic stellate cells are still unclear.Therefore,the role and mechanism of MafG in liver fibrosis and ferroptosis of activated hepatic stellate cells need to be explored in order to provide new ideas and theoretical basis for the treatment of liver fibrosis.Methods:1.Immunohistochemistry and immunoblotting assays were used to detect the expression of MafG in human and murine liver fibrosis tissues.After stable overexpression or knockdown of MafG expression in human and rat hepatic stellate cells,the effects of overexpression or knockdown of MafG on the expression of liver fibrosis and ferroptosis markers in hepatic stellate cells were detected by real-time fluorescence quantitative PCR,immunoblotting assays and biochemical kits.2.The role and molecular mechanism of MafG in the transcriptional regulation of LCN2 were investigated by dual luciferase reporter gene assay,chromatin immunoprecipitation and electrophoretic mobility shift assay.3.The interaction between myosin heavy chain 9(MYH9)and MafG and its effect on the transcriptional regulation of LCN2 was identified by mass spectrometry,bioinformatics analysis,protein immunoprecipitation and chromatin immunoprecipitation assays.4.By establishing bile duct ligation and carbon tetrachloride murine liver fibrosis model,using adeno-associated virus AAV6 carrying sh RNA to stably knockdown protein in vivo and isolating murine primary hepatic stellate cells to verify the role of MafG in liver fibrosis and ferroptosis as well as the relationship between MafG and LCN2 in vivo.Results :1.MafG was highly expressed in human and mouse liver fibrotic tissues(P < 0.001)and resulted in increased m RNA and protein expression levels of α-SMA(P < 0.01),a marker of hepatic stellate cell activation,and COL1A1(P < 0.01),an extracellular matrix component.2.MafG silencing promoted ferroptosis inducer erastin-mediated inhibition of activated hepatic stellate cells,iron overload(P < 0.001),accumulation of lipid reactive oxygen species,and lipid peroxide production(P < 0.01).In contrast,MafG overexpression blocked erastin-mediated inhibition of activated hepatic stellate cells and ferroptotic events.3.MafG bind directly to the TCAGCA sequence in the LCN2 promoter region and transcriptionally activated LCN2 expression,where GC is the key site for MafG binding to the LCN2 promoter.4.Exogenous expression of LCN2 in hepatic stellate cells blocked the inhibition of activated hepatic stellate cells and promotion of ferroptosis in hepatic stellate cells mediated by MafG knockdown.5.MYH9 and MafG interacted with each other to form a complex.Knockdown of MYH9 inhibited the binding of MafG to the LCN2 promoter,reducing the m RNA(P < 0.01)and protein expression levels of LCN2.6.Hepatic stellate cells-specific knockdown of MafG in vivo reduced the m RNA(P < 0.001)and protein expression levels of LCN2 and promoted the alleviation of liver fibrosis,iron accumulation(P < 0.001)and lipid peroxide production(P < 0.001)mediated by ferroptosis inducer IKE.In addition,MafG knockdown reduced the m RNA levels of LCN2(P < 0.001),α-SMA(P < 0.01)and COL1A1(P < 0.001)in isolated primary hepatic stellate cells.Conclusions:1.MafG is highly expressed in liver fibrosis.MafG promotes the activation of hepatic stellate cells and the secretion of extracellular matrix,which promotes the development of liver fibrosis.2.MafG regulates the metabolism of iron in hepatic stellate cells through transcriptional activating LCN2,which resulted in resistance of ferroptosis.3.MYH9 forms a complex with MafG to modulate MafG mediated the transcriptional activation of LCN2.MafG/MYH9-LCN2 signaling pathway has an important role in ferroptosis of hepatic stellate cells.4.Silencing MafG can promote ferroptosis inducer-mediated antifibrotic effects,indicating MafG can be a potential target for promoting ferroptois of hepatic stellate cells in the treatment of liver fibrosis.17 figures,25 tables,303 references...