p38MAPK介导NAFLD大鼠Nrf2/ARE通路对氧化应激相关基因的调控机制及疏肝健脾方药的干预作用

Researches indicate that oxidative stress is one of the central parts to the pathogenesis of nonalcoholic fatty liver disease (NAFLD)；Nrf2/ARE pathway is the most important antioxidant system in vivo；MAPKs play an essential role in regulating Nrf2/ARE pathway. In our previous studies, soothing liver and invigorating spleen recipes have a significantly preventive and curative effect on NAFLD through inhibition of MAPKs activation in hepatocytes and Kupffer cells of rats. However the specific mechanism remains unclear... Objective：To dynamically observe the role regulating effect of Nrf2/ARE pathway mediated by p38MAPK in the regulation of oxidative stress-related genes, such as NQO1,GST,HO-1,CAT, etc. in hepatocytes and Kupffer cells of NAFLD rats in vivo and in vitro, and to investigate the effect of the molecular mechanism of soothing liver and invigorating spleen recipes...Anticipation:.①To observe the mechanism of Nrf2/ARE pathway mediated by p38 MAPK regulating oxidative stress-related genes in pathologic processes of NAFLD..②To observe the effect of recipes of soothing liver and invigorating spleen on Nrf2/ARE pathway mediated by p38 MAPK and oxidative stress-related genes of NAFLD rats..③To find the new drug targets of Chinese medicine..④To enriches the theory of preventing and treating NAFLD with traditional Chinese medicine and modern concepts of the pathogenesis of NAFLD.

研究表明，氧化应激是非酒精性脂肪性肝病（NAFLD）的重要发病机制之一；Nrf2/ARE通路是迄今发现最为重要的体内抗氧化系统；MAPKs对Nrf2/ARE通路具有重要的调控作用。我们前期研究发现，疏肝健脾方药能显著抑制NAFLD大鼠肝细胞、Kupffer细胞MAPKs等作用而发挥良好的防治效果，但具体机制不明。研究目的：体内、体外动态观察p38MAPK介导NAFLD大鼠肝细胞、Kupffer细胞Nrf2/ARE通路对NQO1、GST、HO-1、CAT等氧化应激相关基因的调控作用及疏肝健脾方药干预的分子机制；预期：①明确p38MAPK介导Nrf2/ARE通路在NAFLD病理演变过程中对氧化应激相关基因的调控机制；②揭示疏肝健脾方药对p38MAPK介导NAFLD大鼠Nrf2/ARE通路及氧化应激相关基因的干预作用；③发现中药作用的新靶点；④丰富NAFLD的中医药防治理论和现代发病机制。

1. 项目背景.氧化应激是非酒精性脂肪性肝病（NAFLD）的重要发病机制之一；Nrf2/ARE通路在体内抗氧化系统中有重要作用；p38 MAPK对Nrf2/ARE通路有重要的调控作用。本项目在前期基础上，以p38 MAPK介导的Nrf2/ARE通路对氧化应激的调控作用为切入点，探讨疏肝健脾方药抗NAFLD的作用机制。.2. 主要研究内容.观察疏肝健脾方药对NAFLD大鼠肝组织及肝细胞氧化应激水平和p38MAPK介导的Nrf2/ARE通路的调控机制，探讨NAFLD发病机制及疏肝健脾方药干预作用机理。高脂饲料喂养复制大鼠NAFLD动物模型，同时予柴胡疏肝散（ig, 9.6g/kg/d）、参苓白术散（ig, 30g/kg/d）或二者合方(ig, 39.6g/kg/d)进行16周的干预后，检测血脂等常规生化指标，观察肝组织病理学变化，检测肝组织和肝细胞氧化应激因子含量和p38MAPK/STAT3和Nrf2/ARE通路有关基因和蛋白的表达。.3. 主要结果.病理及生化结果显示，模型组大鼠存在肝细胞脂肪变和脂代谢紊乱，而各药物干预组有不同程度改善，以健脾组和合方组最为明显。与正常组比较，模型组大鼠肝组织、肝细胞SOD、GSH-Px水平明显降低，MDA、NO水平显著升高(P<0.01, P<0.05)；与模型组比较，各药物干预组肝组织、肝细胞SOD、GSH-Px水平升高，MDA、NO水平下降，健脾组和合方组最为显著（P<0.01，P<0.05）。模型组大鼠肝组织、肝细胞p38 MAPK/STAT3和Nrf2/ARE通路有关基因和蛋白表达显著上调（P<0.01），疏肝健脾方药干预能够下调p38 MAPK/STAT3通路和上调Nrf2/ARE通路有关基因和蛋白表达，健脾组和合方组最为显著（P<0.01，P<0.05）。.4. 结论与科学意义.NAFLD大鼠模型肝脏存在氧化-抗氧化失衡，疏肝健脾方药能显著调节肝脏氧化应激水平，可能是疏肝健脾方药抗NAFLD的重要机制之一。NAFLD模型大鼠肝组织和肝细胞存在着p38MAPK/STAT3和Nrf2/ARE通路相关基因及蛋白过度表达，疏肝健脾方药能显著下调肝组织和肝细胞p38MAPK/STAT3和上调Nrf2/ARE通路，p38MAPK/STAT3和Nrf2/ARE通路可能是疏肝健脾方药发挥抗NAFLD作用的有效靶点。