Overexpressed Estrogen Receptor β Suppresses Human Hepatic Stellate Cells Functions And Enhances The Anti-fibrosis Effect Of Calycosin By Inhibiting STAT3 Activation

Background: Liver fibrosis is a sex-related disease which is more likely to be male.Previous studies of our group have shown that phytoestrogens calycosin is effective in anti-hepatic fibrosis in vivo which is related to Janus kinase2-signal transducer and activator of transcription3（JAK2-STAT3）signaling pathway and estrogen receptor β（ERβ）.In vitro,it can inhibit the function of hepatic stellate cells（HSCs）,which is the key cell of hepatic fibrosis,and this effect is also closely related to ER β.Objective: We aim to further investigate the role of ERβ in the anti-fibrosis effect induced by calycosin and its relationship with JAK2-STAT3 signaling pathway in human hepatic stellate cells（LX-2 cells）overexpressed by ERβ.Methods: ERβ overexpression lentivirus was transfected into LX-2 cells,to determine the transfection efficiency of lentivirus,the green fluorescence was detected by fluorescence microscope,the expression of ERβ m RNA and protein level were detected by quantitative real-time PCR and Western blotting.The safe concentration of calycosin（25,50,100,200,300,600μM）was determined by CCK-8 assay.The effects of ERβ and calycosin on proliferation and migration of TGF-β₁ induced LX-2 cells were detected by CCK-8 and Transwell chamber experiments respectively.The expression of α-SMA protein was detected by immunofluorescence and Western blotting.The protein level of COL-I,MMP1,TIMP1,JAK2,p-JAK2,STAT3 and p-STAT3 in LX-2 cells were detected by Western blotting.Furthermore,Western blotting was used to detect the protein level of JAK2,p-JAK2,STAT3 and p-STAT3 in LX-2 cells after treating with estrogen receptor antagonist ICI182,780.Results: 1.ERβ overexpression lentivirus was successfully transfected into LX-2 cells with high transfection efficiency.2.The safe concentration of calycosin on LX-2 cells were 25-200μM and had no obvious effect on the proliferation of LX-2 cells not induced by TGF-β₁.3.The proliferation,migration and α-SMA protein expression of TGF-β₁ induced LX-2 cells were significantly inhibited by ERβ overexpression and calycosin（200μM）（p <0.05）,and the effects of calycosin（200μM）on ERβ overexpressed LX-2 cells was stronger than that of LX-2 cells without overexpression（p <0.05）.4.Overexpression of ERβ and calycosin（200μM）significantly inhibited the expression of COL-Ⅰ and TIMP-1 proteins and increased the protein level of MMP1 in LX-2 cells induced by TGF-β₁（p <0.05）.The effects of calycosin on COL-Ⅰ,TIMP1 and MMP-1 proteins in ERβ overexpressed LX-2 cells were stronger than that in non-overexpressed LX-2 cells（p <0.05）.5.Calycosin had no significant effect on JAK2,p-JAK2 and STAT3 proteins in LX-2 cells with or without overexpression of ERβ.It is worth noting that compared with the model group,ERβ overexpression and calycosin（200 μM）significantly reduce the expression of p-STAT3 protein,thus reducing the ratio of p-STAT3/STAT3.Compared with the non-overexpressed LX-2 cell group,calycosin showed a significant decrease in p-STAT3/ STAT3 ratio in overexpressed LX-2 cells（p <0.01）,while the decrease of p-STAT3 show no significant difference.In addition,the inhibitory effect of calycosin on p-STAT3 protein was reversed after treating with ICI182,780.Conclusion: 1.ERβ participates in the inhibition of cell proliferation,migration,activation and collagen secretion in LX-2 cells,which may be related to the inhibition of intracellular STAT3 protein activation.2.ERβ significantly mediates the inhibitory effect of calycosin on the function of LX-2 cells,which is an important target for calycosin to exert its anti-fibrosis activity.3.The inhibitory effect of calycosin on the function of LX-2 cells is related to the inhibition of intracellular STAT3 protein activation.