Li Wanzhi, Lv Ruyue, Zou Tangbin, Chen Ming
Department of Nutrition, School of Public Health, Guangdong Medical University, No.1 Xincheng Avenue, Songshan Lake Science & Technologic Industry Park, Dongguan, China.
Department of Nutrition, School of Public Health, Guangdong Medical University, No.1 Xincheng Avenue, Songshan Lake Science & Technologic Industry Park, Dongguan, China; Dongguan Key Laboratory of Chronic Inflammatory Diseases, The First Dongguan Affiliated Hospital, Guangdong Medical University, No. 42 Jiaoping Road, Tangxia Town, Dongguan, China.
Life Sci. 2024 Aug 15;351:122798. doi: 10.1016/j.lfs.2024.122798. Epub 2024 Jun 7.
The study aims to investigate the role and underlying mechanisms of tricetin in regulating hepatic stellate cells (HSCs) activation.
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.
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.
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.
本研究旨在探讨曲克芦丁在调节肝星状细胞（HSCs）激活中的作用及潜在机制。
我们用曲克芦丁、药理学抑制剂和小干扰RNA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（mHSCs），采用蛋白质免疫印迹法、免疫荧光法、定量聚合酶链反应来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）信号通路。
在此，我们证明曲克芦丁能强烈抑制LX-2细胞的增殖、迁移、脂滴（LDs）丢失以及纤维化标志物I型α1胶原蛋白（Col 1a1）和α平滑肌肌动蛋白（α-SMA）的表达。此外，曲克芦丁能在LX-2细胞中呈时间和剂量依赖性地诱导自噬形成。通过药理学干预或基因敲低自噬相关5（ATG5）抑制自噬，促进了曲克芦丁诱导的LX-2细胞中促纤维化标志物的下调。此外，曲克芦丁处理减少了活性氧（ROS）的积累，促进了LX-2细胞中的Nrf2信号通路，用ROS清除剂N-乙酰半胱氨酸（NAC）预处理可部分逆转曲克芦丁诱导的自噬，并增强曲克芦丁介导的肝星状细胞失活。Nrf2沉默可部分逆转曲克芦丁介导的α-SMA表达抑制。最后，利用原代小鼠肝星状细胞（mHSCs），我们证明曲克芦丁还能诱导自噬激活，抑制转化生长因子-β1（TGF-β1）诱导的脂滴丢失和纤维化标志物表达，用氯喹（CQ）预处理可进一步增强这些作用。
我们的研究表明，曲克芦丁的作用可能是治疗肝纤维化的一种有效策略，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时。
