Study On The Role And Mechanism Of ZSWIM3 Modified By DNA Methylation In Alcoholic Liver Injury

Alcoholic liver disease(ALD)is a liver-damaging disease caused by long-term drinking.The most typical features are steatosis,hepatitis,and fibrosis/cirrhosis.Alcohol promotes liver inflammation by increasing the sensitivity of macrophages and activating the immune response.In addition,alcohol increases membrane fluidity and the production of intestinal endotoxins(called lipopolysaccharides,LPS),which further promotes the activation of macrophages.Although there are extensive studies describing its pathogenesis,there are few effective treatment strategies to prevent and treat alcoholinduced liver injury(ALI).Effectively inhibiting the inflammatory response regulated by macrophages is one of the effective ways to treat alcoholic liver injury.At present,epigenetic regulation is increasingly closely related to the pathogenesis and progression of diseases,and abnormal macrophage DNA methylation is closely related to inflammation.Our preliminary screening results of macrophage DNA methylation demonstrated the zinc finger SWI2/SNF2 and Mu DR(SWIM)-domain containing 3(ZSWIM3)were hypermethylated in the 5? untranslated region(5?-UTR)region.Baseline,DNA hypermethylation of genes can cause a decrease in gene expression.ZSWIM3,a novel zinc finger-chelate domain of SWIM,is predicted to function in DNAbinding and protein-binding interactions.Although the SWIM domain has been found in a relatively limited variety of domain,the information about ZSWIM3 is relatively limited,and its structure or function has not yet been studied.In order to explore the expression,function and mechanism of ZSWIM3 in alcoholic liver injury,this study is divided into the following five parts.1.The expression of ZSWIM3 in peripheral blood mononuclear cell(PBMC)of patients with alcoholic liver disease,primary liver macrophages,hepatocytes,and hepatic stellate cells in mice with alcoholic liver injury.First,we extracted PBMC from the serum of patients with alcoholic liver disease,and detected the protein level and m RNA level of ZSWIM3.Western blot and real time-PCR data showed protein level and m RNA level of ZSWIM3 were significantly decreased in PBMC from ALD patients.Next,liver macrophages,hepatocytes and hepatic stellate cells were extracted from ALI mice by in situ perfusion respectively,and the expression of ZSWIM3 in different cells was detected.The results showed that ZSWIM3 protein and m RNA levels were significantly reduced in macrophages(MΦ,mouse primary hepatic macrophages)isolated from Et OH-fed mice,but there was no statistical difference in the expression of hepatocytes and hepatic stellate cells.The results of immunofluorescence(IF)double staining showed that ZSWIM3 could co-localize with CD68,further showing that it was expressed on macrophages.These finding indicated the down-regulation of ZSWIM3 in macrophages in chronic-plus-binge ethanol feeding mice.2.The effect of overexpression of ZSWIM3 on alcohol-induced liver injury in vivo.Based on the reduced expression of ZSWIM3 in the alcoholic liver injury model group,ZSWIM3 was over-expressed in mice after the systemic administration of recombinant adeno-associated virus-9(r AAV9)vectors which have shown efficient transduction into the liver via tail vein.r AAV9-ZSWIM3 reversed the reduction of ZSWIM3 in Et OH-fed mice.The results of HE staining,oil red staining,F4/80 immunohistochemistry,serological ALT,AST,TG,T-CHO levels showed that overexpression of ZSWIM3 in the liver alleviated liver damage including fatty degeneration,cell necrosis,reduced macrophage infiltration and decreased ALT,AST,TG,T-CHO levels.Real time-PCR and ELISA results showed that r AAV9-ZSWIM3 treatment can inhibit alcohol-induced TNF-α,IL-6,IL-1β and MCP-1 levels.The above results indicated that overexpression of ZSWIM3 in the liver can protect against alcohol-induced liver injury and inhibit inflammation in vivo.3.The effect of overexpression or silencing of ZSWIM3 on alcohol-induced inflammation in vitro.In alcoholic liver injury,macrophages are activated by alcohol and LPS.In order to better simulate the in vivo environment,100 m M Et OH and 1 μg/m L LPS were used to stimulate RAW264.7 cells to establish an in vitro model.On the one hand,the overexpression of ZSWIM3 by transfecting ZSWIM3 overexpression plasmid can effectively reduce the release and expression of inflammatory cytokines TNF-α,IL-6,IL-1β and chemokines MCP-1 induced by Et OH + LPS.On the other hand,the silence of ZSWIM3 by transfecting ZSWIM3 si RNA further aggravated the inflammatory response stimulated by Et OH + LPS.The above results further confirmed the regulating effect of ZSWIM3 on alcohol-induced inflammation.4.The regulation mechanism of ZSWIM3 on inflammation in alcoholic liver injury.Interact predicted that ZSWIM3 and TNF receptor-associated factor 2(TRAF2)have an interaction relationship.The binding effect of ZSWIM and TRAF2 was verified by molecular docking technology and co-immunoprecipitation experiment.TRAF2 plays an important role in the activation of the nuclear transcription factor kappa B(NF-κB)pathway.In Et OH + LPS-treated RAW 264.7 cells,overexpression of ZSWIM3 reduced TRAF2 and inhibited the activation of NF-κB signaling,including the phosphorylation of P65 and IκBα and the transfer of P65 into the nucleus.These results indicate that ZSWIM3 may regulate inflammation through TRAF2-mediated activation of the NF-κB pathway in ALI.5.The relationship between down-regulation of ZSWIM3 and DNA methylation in alcoholic liver injury models.Bisulfite sequencing(RRBS)identified and analyzed the DNA methylation of ZSWIM3 gene.There are two Cp G islands in the promoter region of the ZSWIM3 structure.Methylation-specific PCR(MSP)and bisulfite sequencing PCR(BSP)experiments verified the methylation of ZSWIM3 in alcoholic liver injury.DNA methyltransferases(DNMTs)are key regulatory enzymes responsible for DNA methylation.In order to study the effect of DNMTs on ZSWIM3,firstly,Decitabine and 5-azad C,the broad-spectrum inhibitors of DNMTs,were used in vivo and in vitro,which can significantly reduce alcohol-induced liver injury and inflammation,and reverse the expression level of ZSWIM3.To further analyze which DNMTs affected ZSWIM3 methylation,DNMT1,DNMT3 a,or DNMT3 b were silenced with si RNA in Et OH+LPS-treated RAW264.7 cells.The results showed that silencing DNMT3 b can significantly restore the expression of ZSWIM3.Ch IP results and MSP results further verified DNMT3 b binds to the ZSWIM3 promoter region to regulate its methylation.Molecular docking technology also further demonstrated the combination of DNMT3 b and ZSWIM3.The results suggest that the aberrant expression of ZSWIM3 was associated with DNA methylation,and in particular,DNMT 3b regulated modification in ALI.In summary,in alcoholic liver injury,alcohol inhibits ZSWIM3 protein and m RNA levels in macrophages.The low expression of ZSWIM3 promotes the inflammatory response of the liver by activating the NF-κB pathway mediated by TRAF2.The down-regulation of ZSWIM3 may be related to the DNA hypermethylation of ZSWIM3 mediated by DNMT3 b.Therefore,this study provides detailed insights into the role and mechanism of DNA methylation-modified ZSWIM3,thereby contributing new substantial research in the elucidation of the pathogenesis of ALI.Aiming at epigenetic regulatory modification and ZSWIM3,it provides a new therapeutic target for ALI.