血府逐瘀汤调控“肠道菌群-TMA-TMAO-血小板”通路的抗血栓作用机制研究

Funded by National Natural Science Foundation of China ‘study of material basis of Xuefuzhuyu Decoction based on process analysis of antiplatelet constituents in vivo’, the study has proved the antithrombotic effect and the compounds with antiplatelet activity of Xuefuzhuyu Decoction. Also, it pointed out that gut flora and the antithrombotic effect of Xuefuzhuyu Decoction had a certain relationship. Aiming at this key question, we pay attention to TMAO and focus on TMAO generation and platelet hyperreactivity enhanced by TMAO by employing Xuefuzhuyu Decoction as model. The project puts forward the scientific hypothesis ‘transitional compounds in intestine of Xuefuzhuyu Decoction modulate gut flora to reduce TMA generation; absorbed compounds into blood of Xuefuzhuyu Decoction inhibit expression and activity of FMOs to ruduce the conversion from TMA to TMAO; therefore, thrombosis induced by platelet hyperreactivity would be alleviated. By employing the technologies and methods of integrated multi-omics, high throughput screening and data mining, the regulation of Xuefuzhuyu Decoction on the network of ‘gut flora-enzyme-metabolite’ related to TMA generation will be clarified. The impact of absorbed compounds into blood of Xuefuzhuyu Decoction on the rate-limiting enzyme (FMOs) related to TMAO generation will be illustrated. Antithrombotic mechanism of Xuefuzhuyu Decoction will be unveiled by regulating ‘gut flora-TMA-TMAO-platelet’ pathway. This project will conduce to the clarification of the scientific connotation of the characteristics of ‘multiple channels’ and ‘multiple targets’ in Xuefuzhuyu Decoction.

在国家自然科学基金“基于抗血小板活性成分体内过程分析的血府逐瘀汤物质基础研究”项目资助下，初步阐明了血府逐瘀汤抗血栓作用及活性成分，并提示血府逐瘀汤抗血栓作用与肠道菌群存在关联。针对此关键问题，继续以血府逐瘀汤为研究对象，以影响血栓形成的肠道菌群关键代谢物TMAO为切入点，聚焦TMAO生成和TMAO增加血小板高反应性两个关键点，提出“血府逐瘀汤‘肠中移行成分’调控肠道菌群减少TMA生成；‘血中移行成分’抑制黄素单加氧酶（FMOs）表达和活性、减少TMA向TMAO转化，抑制血小板激活抗血栓形成”的科学假说，集整合组学、高通量筛选、数据挖掘等技术，分析血府逐瘀汤调控肠道菌群TMA生成相关的“菌-酶-代谢物”网络，阐释“血中移行成分”对TMAO生成限速酶（FMOs）的影响，揭示血府逐瘀汤调控“肠道菌群-TMA-TMAO-血小板”通路的抗血栓作用机制，丰富血府逐瘀汤“多途径”、“多靶点”作用特点。

本研究以理气活血经典方剂“血府逐瘀汤”为研究对象，聚焦影响血栓形成的肠道菌群关键代谢物TMAO和血小板功能系统开展了血府逐瘀汤抗血栓作用评价及其机制研究。围绕心血管危险分子TMAO生成的两个关键因素“肠道中TMA的吸收和肝脏中FMO3对TMA的转化”，采用整体动物实验和体外实验相结合的方法，引入高通量筛选、数据挖掘、超分子传感等多学科交叉技术，研究结果表明：血府逐瘀汤明显抑制肠道中TMA的吸收减少其入血；入血原型成分异甘草素和柴胡皂苷A抑制FMO3的表达，柚皮苷、芍药苷、甘草酸、苦杏仁苷、芍药内酯苷、柴胡皂苷A、β-蜕皮甾酮明显抑制FMO3活性，从而减少TMAO的生成；异甘草素和川陈皮素具有较强的抗血小板聚集作用。该研究阐明了血府逐瘀汤调控“肠道菌群代谢物TMA-TMAO-血小板”通路发挥抗血栓作用，丰富了血府逐瘀汤“多途径”、“多靶点”的作用特点，对口服活血化瘀复方的药效评价和作用机制阐释具有重要参考价值，部分研究成果也成功应用于中成药大品种血府逐瘀胶囊、血府逐瘀丸的化学物质解析、质量标志物辨析、作用机制阐释。