RNF2 Mediates Hepatic Stellate Cells Activation By Regulating ERK/p38 Signaling Pathway In Liver Fibrosis

Objective:To explore the effect of RNF2 on the activation and proliferation of hepatic stellate cells and its mechanism,which may provide a new target and theoretical basis for the treatment of liver fibrosis.Methods:Human normal liver tissues and fibrotic liver tissues were collected and the histopathological changes were observed by HE staining and Masson staining.At the same time,the expression of α-SMA and RNF2 were detected by immunohistochemical staining and Western blot.TGF-β1 was used to induce LX-2 cells.According to the expression changes of RNF2,we have selected an appropriate induction time and concentration to establish a liver fibrosis model in vitro.LX-2 cells were transfected or infected with p EGFP-C2-RNF2 plasmid or RNF2-sh RNA lentivirus respectively to increase and decrease the expression level of RNF2.And then LX-2 cells were induced by TGF-β1.The expression of α-SMA,Col1aⅠ,MMP2 and TIMP1 were detected by Western blot and RT-q PCR.The secretion and expression of inflammatory cytokines TNF-α,IL-6 and IL-1β were detected by ELISA and RT-q PCR.MTT method and Ed U staining were used to detect the proliferation of LX-2 cells.Flow cytometry was used to detect the apoptosis of LX-2 cells.Finally,the effect of RNF2 on MAPK signaling pathway activation was detected by Western blot.Results:The results of H&E staining and Masson staining showed that human fibrotic liver tissues have severer steatosis and necrosis compared with normal liver tissues,which formed regenerative nodules and fibrotic septa.The results of immunohistochemistry and Western blot demonstrated that the expression of α-SMA and RNF2 were up-regulated in fibrotic liver tissues.Notably,the expression of RNF2 was obviously upregulated in TGF-β1-induced LX-2 cells by Western blot analysis,and the expression of RNF2 reached the peak after 24 h induced by 20 ng/ml TGF-β1.Therefore,TGF-β1at a concentration of 20 ng/ml was selected to induce LX-2 cells for 24 h as the condition for activation of hepatic stellate cells.LX-2 cells were induced by TGF-β1 on the condition of successful overexpression or silence of RNF2 respectively.Results showed that overexpression of RNF2 promoted the production of α-SMA and Col1aⅠ,upregulated the expression of TIMP1 and downregulated the expression of MMP2.Meanwhile,overexpression of RNF2 promoted the expression and secretion of inflammatory cytokines TNF-α,IL-6 and IL-1β.However,RNF2 silencing could reverse these effects.Notably,overexpression of RNF2 promoted the proliferation of activated LX-2 cells and inhibited the apoptosis of LX-2 cells.On the contrary,RNF2 silencing inhibited the proliferation of LX-2 cells and promoted apoptosis.The results of Western blot indicated that overexpression of RNF2 up-regulated the phosphorylation of ERK and p38 in TGF-β1-induced LX-2 cells,and RNF2 silencing inhibited the phosphorylation of ERK and p38.However,the change of RNF2 expression had no effect on the phosphorylation level of JNK.Conclusions:Based on the above results,RNF2 could promote the activation and proliferation of hepatic stellate cells and inhibit the apoptosis,which promoting the progression of liver fibrosis through ERK/p38 signaling pathway.