基于RIP激酶介导坏死状凋亡信号通路的四氯化碳诱导肝损伤新分子机制研究

Necroptosis is a novel cell death pathway different from apoptosis and necrosis. The receptor interacting protein kinase (RIP) 1 and RIP3 are central mediators in this necroptotic pathway. Recent data demonstrated that RIP kinase signaling pathway is responsible or partially responsible for liver injury induced by xenobiotics. CCl4-induced liver injury model is a classic pathological model used to test the hepatoprotective potential of drug candidates. The liver damage of CCl4 needs the metabolic activation by CYP2E1 to a highly reactive trichloromethyl radical intermediate as the initiation step for toxicity. However, the underlying toxic mechanisms of this radical intermediate and the potential role of necroptotic RIP kinase signaling pathway in CCl4-induced liver injury remain unclear. Our preliminary studies provided clear evidence that inhibition of RIP1 or knockout of RIP3 ameliorated CCl4-induced acute liver necrosis in mice. Thus, the aim of the current project is to determine if necroptotic RIP kinase signaling pathway is involved in CCl4-induced acute liver necrosis and chronic liver fibrosis and to investigate the protective mechanisms of three hepatoprotective natural products (silymarin, salvianolic acid B, and glycyrrhizic acid) on CCl4-induced liver injury by regulation of necroptotic RIP kinase signaling, using multiple techniques, such as metabolomics, molecular biology, and gene knockout animals. This research will provide scientific basis for the further development of the hepatoprotective drugs and the discovery of new target for liver protectants.

受体相互作用蛋白（RIP）激酶调控的坏死状凋亡是区别于坏死和凋亡的新型细胞死亡途径，介导或参与外源物引起的肝损伤。CCl4致肝损伤模型是经典的病理模型，广泛用于保肝药物筛选。CYP2E1代谢活化生成高反应性的三氯甲基自由基被认为是导致CCl4肝损伤的主要因素，但该中间体的致毒机制有待深入研究，特别是坏死状凋亡在其所致肝损伤过程中的作用尚未阐明。前期实验结果揭示抑制RIP1活性或基因敲除RIP3均可显著改善CCl4所致小鼠急性肝坏死。本项目拟在文献和前期工作的基础上，综合运用代谢组学、分子生物学、基因敲除小鼠等技术和手段，从基因-蛋白-细胞-整体动物多个层面，研究CCl4经CYP2E1代谢活化作用激活RIP激酶介导坏死状凋亡信号通路而导致急性肝坏死和慢性肝纤维化的分子机制，评价保肝中药成分丹酚酸B对该通路的影响，为现有保肝药物的进一步开发和保肝药物新靶点的发现提供科学依据。

受体相互作用蛋白（RIP）激酶介导的坏死状凋亡信号通路是当前学术领域的研究热点之一，但在肝脏疾病中的作用最近才引起关注。有研究揭示RIP激酶介导的坏死状凋亡和对乙酰氨基酚及乙醇引起的肝损伤间存在密切联系，RIP激酶可能是肝脏疾病治疗的潜在靶点。CCl4诱导的肝损伤模型在保肝中药成分筛选和药效评价中最为常用，对其肝损伤机制的研究有助于更好的理解和评价保肝药物活性。本项目针对CCl4诱导肝损伤动物模型机制研究存在的不足，综合应用药物代谢、细胞与分子生物学和基因敲除小鼠等方法和技术体系，从基因-蛋白-细胞-组织-整体动物多个层面，阐明了CCl4经CYP2E1代谢生成三氯甲基自由基，引起ROS，导致少部分肝组织损伤，进而级联激活RIP1和RIP3激酶介导坏死状凋亡信号通路导致急性肝坏死，但是该通路不影响CCl4诱导的慢性肝纤维化。保肝中药活性成分丹酚酸B具有良好的抗氧化应激的功能，可以有效清除CCl4活性代谢产物三氯甲基自由基诱导的ROS，从而抑制RIP1和RIP3激酶的活化，改善CCl4所致急性肝损伤。本研究结果为现有保肝药物的进一步开发和保肝药物新作用靶点的发现提供新的理论基础和科学性依据。