川芎嗪通过Sestrin2调控自噬介导的肝细胞程序性坏死防治酒精性肝病的机制研究

Hepatocyte necroptosis is a core event that promotes the occurrence and development of alcoholic liver disease (ALD). Autophagic dysfunction is a critical trigger for cell necroptosis, and Sestrin2/AMPK/mTOR pathway is a central regulator of cell autophagy. Our previous studies showed that ligustrazine exerted a potent curative effect on ALD. However, the molecular mechanisms remain unclear. The preliminary experiments showed that the anti-ALD effect of ligustrazine was correlated with enhancement of Sestrin2-mediated autophagy and inhibition of hepatocyte necroptosis. Hereby, we propose that ligustrazine could inhibit hepatocyte necroptosis in ALD by regulating Sestrin2/AMPK/mTOR-mediated autophagy. To test our hypothesis, this program will first explore whether the anti-ALD effect of ligustrazine is dependent on inhibition of hepatocyte necroptosis at organismal, cellular, and molecular levels. Then, conditional Atg7 knockout mice will be generated for revealing the role of autophagy in the suppression by ligustrazine on alcohol-induced hepatocyte necroptosis. Finally, by using gene overexpression or silence technology, we aim to elucidate the molecular mechanisms of drug action, among which Sestrin2/AMPK/mTOR pathway could play a significant role. This program will provide an innovative insight into the molecular mechanisms underlying the curative effect of ligustrazine on ALD, and develop novel strategies and targets for the medication of ALD.

肝细胞程序性坏死是推动酒精性肝病（ALD）发生发展的核心事件。自噬障碍是细胞程序性坏死的重要诱因，Sestrin2/AMPK/mTOR通路是调控自噬的关键途径。我们前期研究发现川芎嗪具有良好的抗ALD作用，但机制尚不明确。预实验提示川芎嗪能促进Sestrin2介导的自噬并抑制酒精诱导的肝细胞程序性坏死。据此，我们提出假说：川芎嗪可能通过调控Sestrin2/AMPK/mTOR信号介导的自噬抑制肝细胞程序性坏死发挥抗ALD作用。本项目拟先从动物、细胞及分子水平等多层次明确川芎嗪通过抑制肝细胞程序性坏死发挥抗ALD作用；然后利用Atg7条件性敲除小鼠揭示自噬在川芎嗪抑制酒精诱导的肝细胞程序性坏死中的作用；最后运用基因过表达或沉默等技术阐明Sestrin2/AMPK/mTOR信号在川芎嗪作用中扮演的角色。本研究将从新视角阐释川芎嗪抗ALD的作用机制，为药物防治ALD提供新策略与新靶点。

肝细胞程序性坏死是酒精性肝病的核心病理事件。本项目研究发现，川芎嗪减少酒精暴露的肝细胞内受体相互作用蛋白激酶1（RIPK1）/RIPK3程序性坏死小体的形成和混合谱系激酶结构域样蛋白（MLKL）的活化，从而抑制了程序性坏死的发生和损伤相关分子模式的释放。川芎嗪对p-MLKL线粒体转位的抑制有助于损伤线粒体功能的恢复。川芎嗪还通过阻断p-MLKL和RIPK1/RIPK3程序性坏死体之间线粒体活性氧（ROS）依赖的正反馈而打破程序性坏死信号环路。此外，川芎嗪通过恢复PINK1/Parkin介导的线粒体自噬，增加酒精处理的肝细胞中受损线粒体的清除。自噬调控重要因子泛素细胞色素c还原酶核心蛋白2（UQCRC2）在酒精处理的肝细胞中表达降低，经川芎嗪干预后表达恢复。体外敲降肝细胞内UQCRC2表达削弱了川芎嗪的干预作用（包括抑制线粒体ROS积累、减轻线粒体损伤、增强PINK1/parkin介导的线粒体自噬的能力）。类似地，系统性敲除UQCRC2基因抵消了川芎嗪对肝脏自噬信号、程序性坏死信号的调控作用以及对酒精性肝损伤、炎症和ROS过度生成的改善作用。总之，本项目研究表明，川芎嗪恢复了酒精暴露的肝细胞中UQCRC2表达，进而通过促进PINK1/parkin介导的线粒体自噬抑制程序性坏死。这些研究结果揭示了川芎嗪发挥保肝作用的分子基础，并为酒精性肝病治疗提供了优良的候选药物。