槐果碱激活AMPK信号通路治疗非酒精性脂肪性肝炎的机制研究

Non-alcoholic steatohepatitis (NASH) is one of the most important liver diseases in the world, and there are insufficient outstanding concerns on treatment for NASH. Metabolism disturbances and inflammation are the most important pathogenesis for NASH. AMPK is an important regulatory protein for cellular energy balance and is considered as a master switch of glucose and lipid metabolism. Sophocarpine, a tetracyclic quinolizidine alkaloid derived from Sophora alopecuroides L, has been documented that it can regulate adipocytokines in alleviating non-alcoholic steatohepatitis (NASH) in vivo. Moreover, leptin promotes AMPKα2 phosphorylation at Ser-491 and deactivates Thr-172. We guess that sophocarpine may be a novel target for NASH by activating AMPK signaling pathways. In this study, the relationships of AMPK、ACC、HNF-4α、Srebp-1 and NASH will be detected in human beings. Then, steatosis and AMPK signaling pathways’ proteins will be investigated in sophocarpine intervented NASH models in vitro and in vivo. In addition, AMPK will be blocked by compound C or adenovirus carrying miR-200b to confirm the importance of AMPK in sophocarpine’s anti-NASH process. Moreover, the activation and subsequence of Ser-491 and Thr-172 will be detected. Furthermore, the model of hepatocyte-specific AMPK knock-out mice will be made and get steatosis with high fat food or 30% fructose water, the anti-NASH effect of sophocarpine in these mouse may be lightened or disappeared. This result will certify the importance of activating AMPK signaling pathway in sophocarpine’s anti-NASH process. Taken together, sophocarpine is one of new therapeutic targets for NASH, and activating AMPK signaling pathway could be one of the probable mechanisms in sophocarpine’s anti-NASH process.

非酒精性脂肪性肝炎（NASH）发病涉及脂代谢紊乱和炎症等因素，纠正脂代谢紊乱是治疗的基础。AMPK是肝细胞的能量控制点，Thr-172活化调节脂代谢。我们前期发现槐果碱能够体内抗NASH并下调肝脏脂质因子leptin表达，而leptin已被证实可以通过促进AMPKα2 Ser-491磷酸化抑制Thr-172活化,提示槐果碱可能通过激活AMPK信号通路发挥抗NASH作用。本课题拟于临床标本验证AMPK信号通路活性与NASH发病的相关性；构建体内外大鼠NASH模型并检测槐果碱的治疗作用及对AMPK信号通路的激活；采用共振荧光能量转移法检测AMPKα2 Ser-491和Thr-172活性的变化和发生顺序；使用靶向阻断剂Compound C、miR-200b或肝细胞特异性基因敲除小鼠阻断AMPK后观察槐果碱治疗作用的减弱或消失。本课题旨在阐明槐果碱抗NASH作用的分子机制，为临床应用提供理论依据。

非酒精性脂肪性肝炎（NASH）是危害人类健康的重要肝病，发病率逐年上升，防治手段缺乏特异性，关于NASH的发病机制涉及脂代谢紊乱和炎症等多方面因素，有效的调控肝细胞脂肪代谢成为近期NASH治疗研究的核心内容，其中AMP蛋白激酶（AMPK）是肝脏脂代谢调控的重要靶点，AMPK信号通路的活性可以反应肝细胞脂肪代谢功能情况。槐果碱是从中药苦参和苦豆子中提取的一种生物碱，本课题前期研究发现槐果碱能够体内抗NASH并下调肝脏脂质因子leptin表达，而leptin已被证实可以促进AMPKα2活化,提示槐果碱可能通过激活AMPK信号通路发挥抗NASH作用。本次课题研究内容和研究目标按照计划书执行。在前期研究基础上，首先筛选临床NASH病例并检测到人体NASH发病后肝组织内AMPK信号通路活性受到抑制，同时发现肝组织中miR-200b高表达；进一步通过体外研究证实槐果碱可以抑制油酸诱导的大鼠原代肝细胞脂肪变性，该过程中存在AMPK信号通路的激活；构建腺病毒载体Ad-miR-200b，双荧光素酶法证实Ad-miR-200b可以靶向阻断AMPK蛋白的表达，将Ad-miR-200b导入脂肪变性的肝细胞后，槐果碱的抗NASH作用减弱；构建高脂饮食所致大鼠NASH模型，实验证实槐果碱具有明显的抗NASH作用，该过程中存在AMPK信号通路的激活；AMPK基因敲除后，槐果碱对高果糖所致小鼠NASH发生的治疗作用明显减弱。本课题通过人体标本检测和体内外实验证实了槐果碱具有体内外抗NASH作用，激活AMPK信号通路是其发挥作用的重要途径。本项目是前期槐果碱治疗慢性肝病研究工作的深入，旨在阐明槐果碱抗NASH作用的分子机制。槐果碱具有良好的药物临床应用前景，为NASH治疗提供新的策略。项目负责人目前已发表槐果碱抗NASH治疗相关研究SCI论文三篇，本次课题开展过程中完善了实验室建设并培养博士后1名。