Study Of Methylation Modification Of PSTPIP2 Regulates Hepatic Macrophage Polarization In CCL4-induced Hepatic Fibrosis And Inflammation

Macrophages play a crucial role in the progression of hepatic fibrosis(HF).Recently,epigenetic mechanisms are increasingly being recognized as crucial controllers of their phenotype,and its regulatory effect on the DNA level of macrophages has attracted extensive attention from researchers.However,the functions of macrophage DNA methylation in experimental models of hepatic fibrosis have not been fully addressed.In this study,CCL4-induced HF was generated by injection of C57BL/6J mice with CCL4(10%CCL4,0.001ml/g,intraperitoneal injection 4 weeks).Then,we isolated hepatic macrophages from CCL4-induced mice and control mice and sorted using Mo Flo High-Performance Cell Sorter to get purify live macrophages.Here,we analyzed isolated liver macrophages DNA methylation level from CCL4-induced mice and control mice using RRBS.RRBS analysis showed that hepatic macrophages in CCL4-induced mice had more higher DNA methylation along the Cp G island(CGI)compared with the vehicle group mice.And 26 genes specifically marking liver fibrosis were differentially expressed in CCL4-induced HF mice.Of these,the significant hypermethylation of PSTPIP2(chr18: 77,843,840–77,843,968)in the 5'-UTR region was particularly interesting.To investgate the protein and m RNA expression of PSTPIP2 in CCl4-induced mice and the vehicle mice,we examined the expression of PSTPIP2 by western blot and q-PCR.The results showed that the expression of PSTPIP2 was significantly downregulated in CCl4-induced mice and reduced in macrophages isolated from the liver.Furthermore,double immunofluorescent(IF)analysis showed that representative colocalization of PSTPIP2 with macrophage marker CD86 immunoreactivity in liver tissues was obviously reduced.To identify the function of PSTPIP2 overexpression in CCL4-induced HF mice,we first confirmed that r AAV9–PSTPIP2 was effectively delivered to CCL4-induced HF mice.Western blot analysis demonstrated that the expression of ?-SMA and COL1 a was elevated in r AAV9-empty treated mice,but declined in r AAV9–PSTPIP2-treated HF mice.In addition,the ELISA data showed that the serum levels of proinflammatory cytokines,including IL-1?,TNF-?,IL-12,and MCP-1,were decreased,and the levels of anti-inflammatory cytokines,such as TGF-? and IL-13,were slightly increased in the r AAV9–PSTPIP2-treated group.The current data provided evidence for a novel role of PSTPIP2 in ameliorating the degree of liver fibrosis and hepatic inflammation in CCL4-induced HF.To examine the mechanism of PSTPIP2 in macrophages polarization in vitro,RAW264.7 cells were treated with LPS(1 ?g/m L)and IFN-?(10 ng/ml)for 24 h to polarize M1 macrophage phenotype,or treated with IL-4(15 ng/m L)for 24 h to induce M2 macrophage phenotype.Western blot and q-PCR analysis showed that DNMT3 a and DNMT3 b participate in the process of PSTPIP2 methylation,and PSTPIP2 inhibits STAT1 phosphorylation but promotes STAT6 phosphorylation in M1 and M2 macrophages,which may provide a possible explanation for CCl4-induced liver fibrosis and M1/M2 macrophage polarization in vitro.Above data indicates that methylation of PSTPIP2 may aggravate hepatic fibrosis,and we first found PSTPIP2 connects DNA methylation to macrophage polarization.It may become a balance point of regulating liver injury and inflammation which regulating the occurrence and development of liver fibrosis.