甲基阿魏酸抑制Nox4/ROS-p38MAPK通路抗肝纤维化作用及机制研究

Oxidative stress injury is one of the important pathogenesis of hepatic fibrosis (HF)，When NADPH oxidase 4-derived reactive oxygen species ( ROS ) in the liver is increased , which promotes p38 MAPK phosphorylation in the HSCs, aggravates the apoptosis of hepatic cells and activates HSCs,eventually leads to HF. Therefore the regulation to Nox4/ROS-p38 MAPK pathway is of great significance to the inhibition and reversion for HF。MFA is a monomer isolated from Securidaca vine,which is a Guangxi specialty herb. We found that the MFA can strongly inhibit Nox4 expression and P38 MAPK phosphorylation in the the HSCs, MFA can restore SOD, GSH-Px activity and reduce the serum level of the four diagnostic indicators of hepatic fibrosis in the animal models caused by carbon tetrachloride. According to the reported results and our previous study, we suggest that MFA can act on Nox4/ROS-p38 MAPK to produce anti-hepatic fibrosis effect. In this study, we will utilise RNA interference and overexpress technology in the hepatic fibrosis SD rats model induced by carbon tetrachloride and cell model HSCs LX-2 and SK-hep1 to explain how MFA works on the pathway of Nox4/ROS-p38 MAPK and its anti-hepatic fibrosis effect and its mechanism. This study has great meaning in making clear the role of Nox4/ROS-p38 MAPK in the development of hepatic fibrosis, the mechanism of anti-hepatic fibrosis effect and the acting target of MFA.

氧化应激损伤是肝纤维化(HF)的重要发病机制，肝脏内NADPH氧化酶4(Nox4)是活性氧(ROS)的主要来源,ROS持续升高促进p38MAPK的磷酸化，加剧肝细胞的凋亡和HSCs的激活导致HF。因此调控 Nox4/ROS-p38MAPK通路对抑制与逆转HF意义重大。甲基阿魏酸(MFA)是从蝉翼藤中分离得到的单体，我们发现MFA具有明显抑制HSCs Nox4表达和P38MAPK磷酸化的作用；对动物模型有恢复SOD、GSH-Px酶活性和减轻纤维化血清学指标的作用。结合文献我们推测MFA具有抑制Nox4/ROS-p38MAPK通路抗HF的作用。本研究以CCl4诱导HF大鼠模型和SK-hep1、HSCs LX-2细胞模型，结合RNAi干扰及质粒过表达技术，阐明MFA抑制Nox4/ROS -p38MAPK通路的作用及分子机制。该研究对阐明HF发病机制，明确MFA抗HF作用机制及靶点具有重要意义。

肝纤维化是各种原因导致的肝脏损伤后修复的一种病理状态，主要病理改变是细胞外基质成分的过度合成与异常沉积。肝细胞的持续损害和大量凋亡导致肝内重建失败，肝星状细胞激活分泌大量胶原使得细胞外基质过度沉积是导致HF发生的主要原因。通过观察MFA对乙醇诱导的L02细胞凋亡和TGF-β1诱导的LX-2细胞活化模型的影响以及MFA对CCl4诱导的大鼠急性肝损伤模型、酒精诱导的小鼠急性肝损伤模型、CCl4复合诱导的大鼠肝纤维化动物模型及酒精与CCl4复合诱导的大鼠肝纤维化动物模型的影响，结合细胞RNAi干扰NOX4、p22phox和质粒过表达NOX4、p22phox基因技术，揭示MFA对肝纤维化、肝损伤有保护作用，其机制与MFA调控NOX4/ROS- MAPK通路是有关。目前该研究取得以下成果：.（1）发表SCI论文3篇，中文核心论文5篇；正在投稿论文3篇；（2）专利一项：李勇文 ，李丽，李勇军，容明智，庞勇军，黎荣，程钰，庞文箫，甲基阿魏酸在制备预防和治疗肝纤维化药物中的应用，2013.2.27，中国，CN201210489904.6（3）培养研究生4名。.该研究通过体外培养L02细胞和LX-2 细胞，结合细胞RNAi 干扰和质粒过表达NOX4、p22phox 基因技术以及通过动物试验研究肝细胞凋亡、肝星状细胞活化与NOX4、 p22phox、ROS、MAPK 变化的关系以及甲基阿魏酸对其影响，阐明NOX4/ROS- MAPK 通路与HF 发生、发展的密切相关，加深我们对HF 的发生机制的认识。明确NOX4/ROS- MAPK 通路是抗HF 的关键调控环节，甲基阿魏酸抗HF 的作用靶点及作用机制。该研究为HF 研究提出新思路，为HF 的治疗及预防提供新措施，对HF的防治有重要意义。.