雌激素受体 β 的过表达抑制人肝星状细胞的细胞功能，并通过抑制 STAT3 磷酸化促进毛蕊异黄酮的抗纤维化作用。
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.
机构信息
Department of Pharmacology, School of Basic Medical Sciences of Anhui Medical University, NO.81 Meishan Road, Hefei, 230032, Anhui Province, China.
Department of Gastroenterology, the Second Hospital of Anhui Medical University, NO.678 Furong Road, Hefei, 230601, Anhui Province, China.
出版信息
BMC Pharmacol Toxicol. 2022 Oct 7;23(1):77. doi: 10.1186/s40360-022-00617-y.
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.
背景
雌激素受体β（ERβ）是肝星状细胞（HSCs）中主要的 ER 亚型。我们之前报道过植物雌激素毛蕊异黄酮苷能抑制肝纤维化进展，并抑制 HSC-T6 细胞功能，这表明其作用可能与 ERβ有关。在这里，我们探讨了过表达 ERβ对人 HSCs 的影响，以及 ERβ在毛蕊异黄酮苷药理学作用中的作用。
方法
用慢病毒转染 LX-2 细胞以过表达 ERβ。在存在或不存在过表达 ERβ的情况下，研究 ERβ和毛蕊异黄酮苷对 TGF-β1 诱导的 LX-2 细胞增殖、迁移、激活、胶原产生和降解的影响，以及 ERβ在毛蕊异黄酮苷抑制作用中的作用。用 ER 非选择性拮抗剂 ICI182，780 处理过表达 ERβ或用 ER 非选择性拮抗剂 ICI182，780 处理过表达 ERβ的 LX-2 细胞，以研究 ERβ对 JAK2/STAT3 信号通路的调节作用。用 CCK-8 法筛选毛蕊异黄酮苷的有效剂量并研究细胞增殖。用 Transwell 室法检测细胞迁移。用免疫荧光和 Western blot 检测 α-SMA 的表达。用 Western blot 检测 Col-I、MMP1、TIMP1、JAK2、p-JAK2、STAT3 和 p-STAT3 的蛋白表达。
结果
高效地将 ERβ过表达慢病毒转染到 LX-2 细胞中。过表达 ERβ或毛蕊异黄酮苷单独作用均可抑制 TGF-β1 诱导的 LX-2 细胞增殖和迁移，下调 α-SMA、Col-I、TIMP-1、p-STAT3 的蛋白表达，上调 MMP-1 的蛋白表达。过表达 ERβ和毛蕊异黄酮苷对 JAK2、p-JAK2 和 STAT3 的表达均无明显影响。过表达 ERβ进一步增强了毛蕊异黄酮苷的上述作用。然而，在用 ICI182，780 处理细胞后，毛蕊异黄酮苷诱导的 STAT3 磷酸化下调作用被逆转。
结论
ERβ可能通过抑制 STAT3 的磷酸化来介导 LX-2 细胞主要功能的抑制，这是毛蕊异黄酮苷发挥抗肝纤维化作用的重要途径。
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