目的:本研究旨在探讨三酸在调节肝星状细胞(HSCs)活化中的作用及其机制。
方法:我们用三酸处理人肝星状细胞系LX-2和新鲜分离的原代小鼠肝星状细胞(mHSC),药理学抑制剂和siRNA。蛋白质印迹,免疫荧光,定量PCR用于评估纤维化标志物的表达,自噬水平和Nrf2(核因子E2相关因子2)信号。
结果:这里,我们证明了三酸能强烈减弱增殖,迁移,LX-2细胞中脂滴(LDs)丢失和纤维化标志物Col1a1(I型α1胶原)和α-SMA(α-平滑肌肌动蛋白)的表达。此外,在LX-2细胞中,三酸在时间和剂量依赖性地引起自噬形成。通过药理学干预或遗传ATG5(自噬相关5)沉默抑制自噬促进了三酸诱导的LX-2细胞中促纤维化标志物的下调。此外,三酯处理减少活性氧(ROS)的积累,在LX-2细胞中促进Nrf2信号传导和用ROS清除剂NAC预处理部分逆转了三酸诱导的自噬并增强了三酸介导的HSCs失活。Nrf2沉默部分逆转了三酸苷介导的α-SMA表达抑制。最后,利用原代小鼠肝星状细胞(mHSC),我们证明了三酸还能诱导自噬激活,抑制TGF-β1诱导的LDs丢失和纤维化标志物表达,用CQ预处理进一步使这些作用敏感。
结论:我们的研究表明,三尖素的作用可能是治疗肝纤维化的有效策略,并有助于确定新的治疗靶点。特别是与自噬抑制剂联合使用。
OBJECTIVE: The study aims to investigate the role and underlying mechanisms of
tricetin in regulating hepatic stellate cells (HSCs) activation.
METHODS: We treated human hepatic stellate cells line LX-2 and freshly isolated primary mouse hepatic stellate cells (mHSCs) with tricetin, pharmacological inhibitors and siRNAs, western blot, immunofluorescence, quantitative PCR were used to evaluate the expression of fibrotic markers, autophagy levels and Nrf2 (nuclear factor E2-related factor 2) signaling.
RESULTS: Herein, we demonstrated that tricetin strongly attenuated the proliferation, migration, lipid droplets (LDs) loss and fibrotic markers Col 1a1 (type I α 1 collagen) and α-SMA (α-smooth muscle actin) expression in LX-2 cells. Moreover,
tricetin time- and dose-dependently provoked autophagic formation in LX-2 cells. Autophagy inhibition by pharmacological intervention or genetic ATG5 (autophagy related 5) silencing facilitated tricetin-induced downregulation of profibrotic markers in LX-2 cells. Additionally, tricetin treatment reduced reactive oxygen species (ROS) accumulation, promoted Nrf2 signaling in LX-2 cells and pretreatment with ROS scavenger NAC partially reversed
tricetin-induced autophagy and enhanced
tricetin-mediated HSCs inactivation. Nrf2 silencing partially reversed
tricetin-mediated inhibition of α-SMA expression. Finally, utilizing primary mouse hepatic stellate cells (mHSCs), we demonstrated that tricetin also induced autophagy activation, repressed TGF-β1-induced LDs loss and fibrotic marker expression and pretreatment with CQ further sensitized these effects.
CONCLUSIONS: Our study indicates that tricetin\'s actions may represent an effective strategy to treat liver fibrosis and help identify novel therapeutic targets, especially in combination with autophagy inhibitors.