基于Nrf2 抗氧化信号通路探讨金钗石斛生物碱保肝作用机制

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a battery of cytoprotective genes in the cells, which can upregulate the expression of a series of antioxidant proteins and detoxification enzymes, protecting against toxicants-induced liver injury. A great deal of research has demonstrated that the activation of Nrf2 antioxidant pathway is implicated in the mechanism of many Chinese traditional herbs and natural plant extracts mediated prevention and treatment of cancers and many other diseases. Our recent study found that dendrobium nobile lindl alkaloids (DNLA) can significantly reduce the AST and ALT activities in serum, and ameliorate the pathological injury in liver tissue of mice induced by CCl4. Moreover, DNLA can upregulate the gene expression of Nrf2 in mice liver, suggesting a role of Nrf2 activation in DNLA elicited liver protection. This project aims to utilize Nrf2 knockout mice and primary hepatocytes to further investigate the alteration pattern of the expression of Nrf2 and Nrf2 downstream target genes in response to CCl4 induced liver toxicity. The molecular mechanism of DNLA mediated liver protection will be unraveled through the study of the activation of Nrf2/ARE signaling pathway. The project will also determine whether DNLA can inhibit CCl4 induced mitochondrial dysfunction, through modulating the activation of Nrf2 pathway. This project will provide an experimental basis for treatment and prevention of chemical-induced liver injuries.

转录因子NF-E2相关因子2（Nrf2）是肝细胞防御过氧化应激的重要调节因子，可以上调一系列抗氧化蛋白和解毒酶的基因表达，保护肝细胞免受毒物损害。研究表明,许多中草药和天然植物提取物抗癌和抗疾病的机理与激活Nrf2抗氧化信号通路有关。我们最近研究发现，金钗石斛生物碱能显著降低CCl4诱导的小鼠血清中AST、ALT活性，明显减轻小鼠肝组织病理损伤；研究还发现，金钗石斛生物碱能显著上调小鼠肝组织中Nrf2 基因的表达，提示其保肝作用与Nrf2抗氧化系统激活有关。本项目拟采用Nrf2敲除小鼠和肝原代细胞研究Nrf2和Nrf2下游靶基因的表达在CCl4诱导的肝细胞损伤中的作用。通过研究Nrf2/ARE信号通路激活，探讨金钗石斛生物碱保肝作用的机理。并进一步采用分子生物学手段观察金钗石斛生物碱保肝与Nrf2激活导致的线粒体遗传物质和功能改善的相关性，为金钗石斛生物碱用于保护和治疗肝损伤提供实验依据。

肝脏疾病为临床常见多发病，严重危害人类健康。目前临床上尚缺乏有效的治疗药物。近年来发现Nrf2是肝细胞对抗各种应激损伤，抵御外来有害刺激的重要转录因子，而Nrf2/ARE抗氧化损伤通路的激活是保护肝细胞免受攻击损伤的重要机制。深入研究Nrf2/ARE信号调控的分子机制，并开发基于Nrf2途径的保肝中药材可具有广泛应用前景。本研究组发现金钗石斛总生物碱（DNLA）具有抗肝损伤的作用，但其机制尚未完全阐明。本项目采用CCl4诱导的小鼠急性肝损伤模型，观察金钗石斛总生物碱（DNLA）对CCl4诱导的肝损伤的保护作用，并采用Nrf2基因敲除小鼠及LO-2肝细胞进一步探讨Nrf2/ARE信号通路在DNLA的保肝作用机理。研究发现,DNLA减轻CCl4诱导的肝损伤模型小鼠血清中ALT、AST含量，减轻肝脏病理损伤，提高肝组织抗氧化酶SOD、GSH活性，并降低MDA含量，上调小鼠肝组织中Nrf2、Keap1、Nqo1、Ho-1和Gclc mRNA表达，同时提高Nrf2及其下游蛋白 HO-1、NQO1、GCLC表达水平。DNLA可降低CCl4引起的肝损伤模型小鼠肝脏ROS含量，亦可降低线粒体H2O2、MDA及8-OHdG的含量，提高线粒体Mn-SOD、GSH活性水平，降低线粒体Cytochrome C蛋白表达，抑制肝细胞凋亡率，增加肝脏ATP含量,提高肝脏线粒体呼吸功能。而在Nrf2缺失的小鼠中，DNLA对肝线粒体功能的保护作用减弱，且对Nrf2信号通路基因和蛋白表达未见激活作用，肝细胞凋亡亦无明显抑制作用。DNLA可提高CCl4引起的LO-2肝细胞损伤模型细胞存活率，降低LDH的释放，抑制肝细胞ROS生成，上调LO-2肝细胞Nrf2、Keap1、Nqo1、Ho-1和Gclc基因表达，提高LO-2肝细胞NRF2、HO-1蛋白表达，促进 Nrf2 的入核。综上所述，表明DNLA可通过激活肝脏Nrf2抗氧化信号通路，增加肝脏抗氧化应激性损伤相关蛋白NRF2、GCLC、NQO1和HO-1的表达，减轻肝损伤小鼠线粒体氧化应激损伤，保护线粒体功能，从而发挥抗肝损损伤作用。本研究发现为后续DNLA用于肝损伤的防治提供了药理学实验依据，并可为基于激活Nrf2抗氧化信号通路肝损伤治疗新策略打下基础，具有潜在的临床应用前景。..关键词：金钗石斛；生物碱; 氧化应激；Nrf2；肝损伤