基于SIRT1-AMPK信号系统探讨酒精性脂肪肝能量代谢变化与分子调节机制及葛花解酒涤脂汤干预研究

Alcoholic fatty liver (AFLD) is a major risk factor for advanced liver injuries such as steatohepatitis, fibrosis, and even hepatocellular carcinoma (HCC), which no effective treatment measures.SIRT1 - AMPK signal has an important regulatory role in cellular energy and redox status, and the role of both in AFLD has been sure. Liver is thought as a key organ to store energy and adjust metabolism, it’s metabolism and energy balance are closely related with the body life activities. Therefore, based on the above research foundation and theory, we conceive hepatic energy metabolism and its change has significant correlation with the AFLD, but without enough literature support. Our team preliminary experiment found Gehua Jiejue Dizhi Decoction could significantly ameliorates the formation of liver steatosis, which was better than that of resveratrol. This project plans to probe the changes of hepatic energy metabolism and how SIRT1-AMPK signalling system impact on energy metabolism in vitro and in vivo.Further clarify the molecular mechanism to AFLD occur, at the same time, to provide scientific laboratory basis for the unique advantage of traditional Chinese medicine in the treatment of AFLD.

酒精性脂肪肝(AFLD)是酒精性肝病进行性发展的重要起始病理阶段，尚无有效的治疗措施。SIRT1-AMPK信号对细胞能量和细胞氧化还原状态具有重要调节作用，且二者在AFLD中的作用已得到肯定。肝脏作为机体储存能量和调节代谢的重要器官，其代谢与能量平衡同机体生命活动密切相关。因此，基于上述研究基础与理论，我们设想肝脏能量代谢及其变化与AFLD发生具有重要相关性，但缺乏文献支持。课题组前期预实验发现，由经方与自拟方临证加减后形成的葛花解酒涤脂汤能够显著改善AFLD小鼠肝脏脂肪变性，其作用显著优于白藜芦醇。本课题拟通过动物体内和体外细胞实验，探讨能量代谢与AFLD发生的相关性、乙醇暴露后AFLD相关microRNAs对SIRT1- AMPK信号表达与活性调节机制及其对能量代谢的影响，进一步阐明AFLD发生的分子机制，同时为发挥中医药在AFLD治疗中的独特优势提供科学的实验室依据。

目前仍缺乏对酒精性脂肪肝（AFLD）的针对性治疗药物，更多地了解AFLD发生的分子机制与寻找新的治疗药物显得非常迫切。基于课题组前期对自拟方葛花解酒涤脂汤具有显著干预小鼠AFLD的发现及已有的肝脏能量代谢相关研究基础与理论，我们设想肝脏能量代谢及其变化与AFLD发生具有重要相关性，但缺乏文献支持。采用C57BL/6J小鼠酒精性脂肪肝体内模型（NIAAA法）和HepG2酒精性脂肪肝细胞模型，经组织病理学方法检测了模型制备及药物的干预作用。采用RP-HPLC方法对小鼠肝组织ATP、ADP、AMP含量进行了检测和能荷（EC）计算；采用分光光度法及蒽酮法完成了小鼠肝组织Na+-K+-ATP酶、Ca2+-ATP酶、SDH酶活性及肝糖原含量检测；采用qRT-PCR、Western blot等方法对SIRT1-AMPK信号系统关键靶位及上下游指标进行了检测；采用网络药理学方法分析了葛花解酒涤脂汤干预AFLD的作用及其靶点。体内、外实验均表明葛花解酒涤脂汤具有显著的AFLD干预作用；AFLD小鼠肝组织ATP/AMP及EC水平显著降低，经药物干预后均能够显著提升。葛花解酒涤脂汤干预能够显著降低AFLD小鼠肝组织Na+K+-ATP酶活性，但对其Ca2+Mg2+-ATP酶活性无影响；能够显著增加AFLD小鼠肝组织SDH酶活性；AFLD小鼠肝组织糖原含量显著增加，药物干预并未影响其含量。与已有研究结果一致，AFLD小鼠SIRT1表达显著降低，经药物干预后显著上升。AFLD小鼠AMPKβ1表达增加，药物干预后显著降低至正常相似水平；但AMPKα1、AMPKα2表达水平与正常组相似，药物能够显著提升AFLD小鼠肝组织AMPKα2表达水平。采用网络药理学对葛花解酒涤脂汤9味药进行了化合物筛选、对应靶点查找，总结通路与生物学功能。细胞学实验验证发现槲皮素、甘草素对AFLD具有显著的干预作用。与体内实验一致，AFLD模型组SIRT1、AMPK表达显著降低，其中槲皮素能够显著增加其SIRT1表达水平。结果表明，葛花解酒涤脂汤具有显著干预AFLD的作用，能量代谢变化参与了AFLD的发生和葛花解酒涤脂汤的干预作用，槲皮素是方药中具有显著干预作用的化学成分之一。