Nrf2/ARE信号通路参与小柴胡汤抗大鼠肝纤维化的机制研究

Oxidative stress is an important factor of the activated hepatic stellate cell(HSC) which is the center component of hepatic fibrosis. While, the anti-oxidation of Nrf2/ARE signaling pathway play a key point effect on the creation and development of hepatic fibrosis. XIAO CHAIHU TANG has the effect of hepatic fibrosis treatment, but molecule mechanisms are indefinite. Elementary finding was that XIAO CHAIHU TANG has interference effect on the expression of Nrf2 mRNA. On the foundation, we plant to study the anti liver fibrosis effects of XIAO CHAIHU TANG through in vivo and in vitro experiments further more. We will investigate the intervention effect of the activation of hepatic stellate cell when liver fibrosis induced by carbon tetrachloride and the mechanisms involved the signal pathway of ERK, PI3K and JAK/STAT. The intervention effect of Nrf2 signal pathway and its downstream factor ARE, HO-1, Noq-1 and GCLM expression of XIAO CHAIHU TANG were also our investigation. Prior study was revealing the regulation action of Nrf2 signaling pathway on the activation of hepatic stellate cell via the related mRNA and protein expression detection of hepatic stellate cell activation and Nrf2 signaling pathways. The finding will identify the molecular mechanisms of XIAO CHAIHU TANG in the treatment of hepatic fibrosis and provide new research ideas and theoretical basis for traditional Chinese medicine in the treatment of liver fibrosis.

肝星状细胞活化是肝纤维化的中心环节，氧化应激是肝星状细胞活化的重要因素，Nrf2/ARE信号通路的抗氧化作用在肝纤维化发生发展中起到重要作用。小柴胡汤具有治疗肝纤维化的作用，但其分子机制尚不明确。初步研究证实小柴胡汤能很好的改善四氯化碳引起大鼠肝纤维化肝功能改变和肝脏的病理学变化，并且对Nrf2基因表达有干预作用。本项目拟在此基础上，通过在体和离体实验探讨小柴胡汤对四氯化碳引起肝纤维化时肝星状细胞活化的干预作用及主要信号通路ERK、PI3K及JAK/STAT途径的机制；探讨小柴胡汤对Nrf2信号通路的干预作用及对其下游因子ARE、HO-1、Noq-1及GCLM表达的影响；揭示Nrf2信号通路对肝星状细胞活化的调节作用。进一步明确小柴胡汤治疗肝纤维化的分子机制，为中药治疗肝纤维化机理研究提供新的思路。

活性氧 (reactive oxidative species，ROS)在肝纤维化的发生发展过程起到重要的促进作用，同时氧化应激能够增加肝纤维化等级。核因子相关因子-2 (Nuclear factor erythroid 2-related factor 2，Nrf2)在细胞抗氧化应激系统中发挥重要作用。经典传统中药复方小柴胡汤 (Xiaochaihutang，XCHT)广泛应用于抗炎、保肝、解热、镇痛等治疗，是一种潜在的治疗肝纤维化的药物，但其作用的分子机制尚不明确。本研究旨在探明小柴胡汤的抗肝纤维化作用和可能的作用机制。我们通过血清生化学、组织形态学分析、Real time RT-PCR、Western blot、基因沉默等技术手段，采用四氯化碳诱导的大鼠肝纤维化模型和HSC-T6细胞系，观察XCHT治疗大鼠肝纤维化的作用和对HSC-T6细胞增殖和活化的抑制作用，并着重从Nrf2信号通路和氧化应激方面分析了XCHT抗肝纤维化的作用机制。结果发现XCHT能够显著改善四氯化碳诱导的大鼠肝纤维化，其作用机制涉及以下方面：①激活Nrf2/ARE信号通路，增强对抗氧化应激的能力，进一步抑制HSC的增殖和活化；②下调ERK2、PI3K、JAK2和STAT3的mRNA表达，可能抑制ERK2、PI3K和JAK2/ STAT3信号通路，干预HSC的活化；③降低肝组织CYP2E1蛋白及mRNA的表达；④调节MMP-2和TIMP-2的表达，减少细胞外基质 (extracellular matrix, ECM)的过度沉积；⑤下调TGF-β1的 mRNA表达，抑制Smad2/3的磷酸化，上调Smad7的蛋白表达有关。研究结果与预期基本一致。本项目的系列发现为XCHT在临床上治疗肝纤维化的应用提供了基础药理学依据。