基于Nrf2信号通路调控枯否细胞极性的雷公藤甲素肝毒性作用机制研究

The imbalance of immune response induces the toxicity of Tripterygium wilfordii Hook F (TWHF). But there are only a few studies about the immune response in liver. Kupffer cells (KCs) are resident macrophages of the liver and play an important role in some drug induced-hepatotoxicity. In this project we investigate the pathomechanism of hepatotoxicity caused by perturbation of KCs polarization after treatment with triptolide, which is the main active and toxic component of TWHF, both in vitro and in vivo. We propose a novel hypothesis to elucidate the mechanism of triptolide-induced hepatotoxicity by perturbation of KCs polarization: triptolide regulate the KCs polarity by inhibition the nuclear factor erythroid-2 related factor 2 (Nrf2) pathway, thus the KCs’ function of phagocytosis and cytokine-release will be changed. The KCs polarization will change the sensitization endotoxin and aggravate the hepatocyte injury. In this project, the number of KCs, polarity and function including phagocytosis and cytokine-release will be assessed by Western bolt, realtime-PCR, molecular probe, cloning technique, cells co-culture, Nrf2 knockout or KCs depletion assay. The possible role of KCs polarity mediated by Nrf2 pathway in triptolide-induced hepatotoxicity will be investigated. The goal of this project is to provide vigorous experimental data for the clinical application of TWHF and provid a potential therapeutic target for hepatotoxicity induced by TWHF.

免疫失衡介导的雷公藤毒性近年引起关注，但对肝脏免疫影响的研究相对薄弱。枯否细胞是肝脏固有免疫重要部分，参与多种药物性肝损伤。本研究以雷公藤活性和毒性主要成分-雷公藤甲素(TP)为研究对象，首次提出以对枯否细胞极性影响为研究主线，采用Western、定量PCR、分子克隆、分子探针、细胞共培养、基因敲除小鼠及枯否细胞耗竭小鼠等技术或模型，从分子、细胞和动物整体水平探索TP对枯否细胞极性、数量、吞噬、分泌功能及对LPS反应性的影响，对核因子E2相关因子2(Nrf2)调控枯否细胞极性的机制进行探讨，考察枯否细胞在TP诱导肝毒性中关键作用，揭示枯否细胞介导TP产生肝毒性机制：TP通过抑制Nrf2及其下游通路调控枯否细胞极化，引起枯否细胞分泌、吞噬及免疫功能改变，增加枯否细胞对内毒素敏感性，参与并加重肝细胞损伤，从而产生肝脏毒性，为雷公藤临床合理应用提供实验依据，为治疗雷公藤导致肝损伤寻找潜在靶点。

免疫失衡介导的雷公藤毒性近年引起关注，但对肝脏免疫影响的研究相对薄弱。肝脏巨噬细胞是肝脏固有免疫重要部分，参与多种药物性肝损伤。本研究以雷公藤活性和毒性主要成分-雷公藤甲素(TP)为研究对象，以对肝脏巨噬细胞极性影响为研究主线，针对肝脏巨噬细胞极性变化的复杂性特点，从整体、器官、细胞、分子水平进行多层次的深入研究，引进药理、毒理及生物化学等学科的新方法与新技术，采用野生型小鼠、Nrf2基因敲除小鼠等模型建立雷公藤甲素诱导的肝毒性模型，以此客观、全面的考察雷公藤甲素对肝脏巨噬细胞极性状态的影响。我们通过考察雷公藤甲素给药后大鼠对低剂量LPS的敏感性变化，以及考察Nrf2基因敲除小鼠中雷公藤甲素对肝脏巨噬细胞极性的影响，证明了雷公藤甲素通过Nrf2信号通路影响肝脏巨噬细胞的M1/M2极化状态，改变了机体应对外界环境刺激的能力进而导致肝损伤。研究表明，Nrf2可能是治疗雷公藤甲素引起的肝脏损伤的潜在靶点。研究揭示了Nrf2信号通路在雷公藤甲素对肝脏巨噬细胞极性的改变过程中的关键作用，从新的角度揭示雷公藤甲素产生肝毒性的分子机制，为临床雷公藤制剂肝毒性的防治提供新思路和新靶点。