基于“PPARα捕集—超滤质谱”平台探讨葛花治疗酒精性脂肪肝的“体内显效形式”及作用机制

Peroxisome proliferater-activated receptor alpha (PPARα) is closely related to alcoholic fatty liver (AFL), thus exploring PPARα agonists is significant to the treatment of AFL. Previous study has shown that isoflavonoids from Pueraria Flos (IFPF) could treat AFL by activating PPARα. And phase II conjugated metabolites were the major forms of IFPF in vivo, some of which showed better PPARα agonistic activity. Therefore, it is reasoned that phase II metabolites exhibit AFL beneficial activity by agonizing PPARa and serve as majority effective forms of IFPF in vivo. However, the effective form and mechanism have not been elucidated. In the present study, in vivo active metabolites of IFPF agitating PPARα will be screened based on biology trapping-ultrafiltration mass spectrometry platform, and such effective forms will be isolated from rat urine and bile after oral administration of IFPF by multiple chromatographic separation technique. Cell model and molecular biology technology will be used for activity determination. Finally, the mechanism will be revealed by three-dimensional quantitative structure-activity relationship established through molecular docking technique and activity analysis. The present study will not only provide an experimental basis for the treatment of AFL by Pueraria flos, but also make beneficial exploration in seeking for the leading compounds for the treatment of AFL from natural resources.

过氧化物酶体增殖物激活受体α（PPARα）与酒精性脂肪肝（AFL）密切相关，探寻PPARα激动剂对AFL治疗具有重要意义。前期研究发现：葛花异黄酮可通过激活PPARα表达治疗AFL；其在体内主要以II相结合型代谢产物形式存在；部分代谢产物显示出更好的PPARα激动活性。由此推测葛花异黄酮主要以II相代谢产物为“体内显效形式”叠加在PPARα靶点产生药效作用，然而具体显效形式和作用机制尚不明确。本课题以葛花异黄酮为研究对象：运用“生物捕集—超滤质谱”平台快速识别药效血清中具有PPARα激动活性的“体内显效形式”；通过多重色谱分离技术从大鼠胆汁和尿液中定向分离目标组分；采用细胞模型和分子生物学技术进行活性评价；基于PPARα激活位点—活性的三维定量构效关系探讨葛花异黄酮“体内显效形式”的作用机制。本研究为葛花治疗AFL提供实验依据，是从天然药物资源中寻找治疗AFL先导化合物的有益探索。

随着人民生活水平的提高，长期大量饮酒所致的酒精性肝病在临床上发病率逐年上升。葛花为作为传统医学中最具代表的解酒药物，应用历史悠久，临床疗效确切。本项目以“阐明葛花解酒保肝药效物质基础及作用机制”为总体思想，采用“体外表征-体内代谢-生信分析-靶向筛选-活性验证”层层递进的研究模式：运用UPLC-QTOFMS表征了粉葛花提取物43个化学成分，测定了主成分含量；建立大鼠酒精肝损伤模型，从整体动物水平明确葛花提取物对酒精肝的保护作用及通过激活PPARα表达发挥药效的分子机制；首次系统鉴定和解析了灌胃给予粉葛花提取物的大鼠血浆、尿液、胆汁和粪便中的25个原型和82个相关代谢产物，采用网络药理学平台构建了葛花治疗酒精肝的“代谢成分-关键靶点-通路”网络图，结合KEGG富集分析及分子对接，挖掘出4个异黄酮类潜在活性成分(Te-7XG，genistein-7G-4'S，tectoridin-4'S 和Te-7XG-4'S)和2个关键靶点（PPARa和MAOA），从整体水平揭示其通过调控糖酵解/糖异生、氨基酸和脂质代谢、炎症和免疫调控相关靶点发挥功效的机理；收集灌胃给药大鼠胆汁，分离制备II相代谢产物，开展4种葛花体外主要成分和3种体内II相代谢产物对2种酒精代谢关键酶超滤实验，结合活力：黄酮苷元＞黄酮苷，原型成分＞体内代谢产物；建立酒精诱导LO2细胞损伤模型，检测细胞活力、AST、ALT、TG和油红O水平以及PPARα的蛋白表达，从体外细胞水平验证了葛花提取物及其活性成分对酒精肝的保护作用及激活PPARa表达的分子机制。综上所述，课题揭示了葛花治疗酒精肝的“显效形式”和作用机制，为保障葛花资源的合理开发和临床安全用药提供实验依据，同时为中药药效物质基础研究提供可借鉴的研究思路。