Overexpression of estrogen receptor β inhibits cellular functions of human hepatic stellate cells and promotes the anti-fibrosis effect of calycosin via inhibiting STAT3 phosphorylation

BACKGROUND: Estrogen receptor β (ERβ) is the major ER subtype in hepatic stellate cells (HSCs). Previously we reported phytoestrogen calycosin suppressed liver fibrosis progression and inhibited HSC-T6 cell functions, suggesting the effects may be related to ERβ. Here, we explore the effect of overexpressed ERβ on human HSCs and the role of ERβ in pharmacological action of calycosin. METHODS: LX-2 cells were transfected with lentivirus to overexpress ERβ. In the presence or absence of overexpressed ERβ, the effects of ERβ and calycosin on proliferation, migration, activation, collagen production and degradation of TGF-β1-induced LX-2 cells and the role of ERβ in the inhibition effect of calycosin were investigated. LX-2 cells overexpressed with ERβ or treated with ER non-selective antagonist ICI182,780 were used to investigate the regulation of ERβ on JAK2/STAT3 signaling pathway. CCK-8 method was used to screen effective doses of calycosin and investigate cell proliferation. The cell migration was detected by transwell chamber assay. The expression of α-SMA was detected by immunofluorescence and western blot. The protein expressions of Col-I, MMP1, TIMP1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by western blot. RESULTS: ERβ overexpressed lentivirus was successfully transfected into LX-2 cells with high efficiency. Overexpressed ERβ or calycosin alone inhibited the TGF-β1-induced LX-2 cell proliferation and migration, downregulated the protein expressions of α-SMA, Col-I, TIMP-1, p-STAT3 and upregulated MMP-1. Both overexpressed ERβ and calycosin had no significant effect on JAK2, p-JAK2 and STAT3 expressions. ERβ overexpression further enhanced the above effects of calycosin. However, after the cells were treated with ICI182,780, downregulation of STAT3 phosphorylation induced by calycosin was reversed. CONCLUSIONS: ERβ mediated the inhibition of major functions of LX-2 cell possibly by inhibiting the phosphorylation of STAT3, and was an important pathway through which calycosin exerted anti-liver fibrosis effect. GRAPHICAL ABSTRACT: [Image: see text]