基于Sirt1多通路协同改善非酒精性脂肪肝的青钱柳药效物质基础和作用机制研究

There is still no therapeutic drug available for the treatment of alcoholic fatty liver disease (NAFLD) in clinical. The research of Traditional Chinese Medicine shows that the antipyretic and detoxifying Chinese medicine can effectively improve the NAFLD, indicating the good curative effect and unique advantage of TCM. The pathogenesis of NAFLD is complex, the "two hit" theory is widely accepted. A large number of studies have confirmed that the activation of Sirt1 can improve lipid metabolism and insulin resistance, reduce oxidative stress and mitochondrial damage, and regulate the NAFLD process in a multiple pathway. Thus, Sirt1 has become a new research target. Moreover, searching for Sirt1 agonists also becomes an effective way for the development of therapeutic drugs. Preliminary studies confirmed that Cyclocarya paliurus show a good effect for the treatment of NAFLD based on Sirt1 target, the triterpenoids are the main active substance. Furthermore, their activities significantly vary from their chemical structures. These triterpenoids could also improve the hepatic lipid accumulation through inhibiting the expression of Srebp-1c, and repair the damage of mitochondria function by activation of Nrf2. But how they coordinate the molecular mechanism and what key targets play the important role in the treatment for NAFLD need to be further clarified. Therefore, the project will carry out in-depth research, clarify the active components of Cyclocarya paliurus to enhance the Sirt1 expression, and elucidate the structure-activity relationship. Furthermore, its molecular mechanism through multiple signal pathways by Sirt1 regulation will be explored. These findings will provide new methods for the research of antipyretic and detoxifying TCM to treat NAFLD.

目前，临床还没有治疗非酒精性脂肪肝病(NAFLD)的药物。中医理论研究表明清热解毒中药可有效改善NAFLD，发挥良好疗效和中药的独特优势。NAFLD发病机制复杂，“二次打击”学说被普遍接受，大量研究证实激活Sirt1能改善脂代谢和胰岛素抵抗，减缓氧化应激和线粒体损伤，多步骤调控NAFLD的疾病进程，成为研究新靶点，寻找Sirt1激动剂也成为开发治疗药物的有效途径。前期研究表明，清热解毒的青钱柳作用于Sirt1显示良好的治疗NAFLD效果，三萜是主要药效物质，结构对其效果有显著影响；该类成分通过抑制Srebp-1c表达改善肝脏脂质积累，激活Nrf2修复线粒体功能损伤，但如何协同治疗的分子机制和关键作用靶点亟待阐明。故本项目拟继续开展深入研究，明确青钱柳作用于Sirt1的活性成分并探明构效关系，阐明其通过Sirt1多通路发挥药效的分子机制，为清热解毒类中药防治NAFLD的研究提供新思路。

本项目依据非酒精性脂肪肝(NAFLD)的“二次打击”学说发病机制，对清热解毒的青钱柳开展Sirt1激动活性物质发现和治疗NAFLD机制研究。从青钱柳分离鉴定155个化合物，包括45个新化合物。在FFA诱导小鼠原代肝细胞、HepG2脂质蓄积模型、HFD诱导的NAFLD小鼠模型上，发现主要活性成分阿江榄仁酸(AA)对细胞和肝内Sirt1表达无影响，但上调NAMPT表达，增加NAD+含量，间接激活Sirt1去乙酰化活性，抑制FATP2和CD36的表达，减少脂肪酸摄取；下调SREBP-1c、ACC和FAS的表达，抑制脂肪酸从头合成；增加PPARα和CPT1α表达，促进脂肪酸的β-氧化；上调ATGL的表达，增加TG水解；下调DGAT的表达，减少TG合成。同时AA上调小鼠肝内LC3Ⅱ/ LC3Ⅰ的表达，减少P62的聚集，诱导自噬，说明AA通过间接激活Sirt1/AMPK调控的脂代谢和诱导自噬发挥降肝脂活性。进一步基于“多重打击”学说全面评估AA对NAFLD的疗效和潜在机制。采用C57BL/6J小鼠HFD饲养16周和12周建立预防和治疗的NAFLD模型，考察不同给药模式下AA对HFD诱导的肝内“打击”和肝外“打击”，即多重“打击”的影响。结果显示，预防给药下，AA可显著缓解HFD诱导的肥胖、血脂紊乱、肝损伤、外周和肝脏胰岛素抵抗及肝内“打击”包括肝脂肪变性、氧化应激和炎症。同时肝外“打击”如脂肪组织炎症(脂肪组织内巨噬细胞浸润)和肠屏障功能紊乱(小肠和结肠黏液分泌减少、紧密连接蛋白表达下调、肠结构损伤和内毒素转位)也被显著改善。机制研究表明，AA间接激活Sirt1/AMPK调控的脂代谢和诱导自噬，共同介导其肝内降脂活性。治疗给药下，AA对Sirt1/AMPK通路和自噬的激活作用消失，减轻体重、降血脂和肝脂、改善胰岛素抵抗及缓解脂肪炎症的活性明显减弱，但AA对肠屏障功能、肝内氧化应激和炎症的改善作用与预防给药效应相当。.本项目研究不仅丰富青钱柳化学物质类型，还发现主要效应分子AA可通过间接激活Sirt1/AMPK通路和诱导自噬减轻肝内“打击”，并抑制脂肪-肝轴“打击”、肠-肝轴“打击”，有效抵御NAFLD进展中的“多重打击”，揭示了青钱柳防治NAFLD的效应分子和作用机制，为NAFLD药物发现提供了新的研究思路和先导分子，培养博士研究生1名，硕士研究生7名，发表SCI论文11篇。