基于体内过程和整体观研究毛冬青抗血栓的药效物质基础及协同作用机制

Ilex pubescens is an effective traditional Chinese medicine in clinic to treat thromboembolic diseases. In our previous study, it was found that the anti-thrombotic activity of Ilex pubescens was closely associated with its ADME process, and we also found that multiple components of Ilex pubescens act synergistically. However, the mechanisms underlying these effects remain unclear. This study will explore the mechanism from a new perspective: integration of physiological disposition and holistic approach. This project will conduct as follows: (1) investigate the disposition of the components of Ilex pubescens in vivo to get the pharmacokinetic profiles of its prototype and metabolites, and then analyze the relationship of the in vitro and in vivo chemomics to confirm its potential active constituents; (2) prepare the Ilex pubescens extract samples based on potential active constituents knock-out/knock-in and compare the differences of their pharmacokinetics, pharmacodynamics and changes of endogenous metabolites in blood-stasis rat model; then correlate and analyze the data to reveal the characteristics of the synergistic anti-thrombotic activity of Ilex pubescens from the perspective of pharmacokinetics, pharmacodynamics and metabonomics; (3) comprehensively analyze the above data and compare the differences of the integrated effects before and after knocking out targeted components to figure out the exact contribution degree of each effective constituents, and elucidate the intrinsic relation and underlying mechanism by metabolic network analysis. The implementation of this project will provide a new insight for the innovation of discovering bioactive constituents and studying integrative mechanism.

毛冬青临床主治血栓性疾病，疗效确切。前期研究发现其抗血栓活性与体内过程密切相关且具有多组分、多途径协同作用，但具体机制尚不清楚。本项目采用体内过程和整体观相结合的新思路，拟先对毛冬青化学物质组进行体内过程研究，获取其进入体内的原型、代谢物及药动学特征，经体内外物质组关联网络分析确定其潜在体内药效组分；继之针对潜在药效组分选择性敲除/敲入组分，建立血瘀证大鼠模型，考察组分缺失前后药动学和药效学差异，并用代谢组学技术分析其干预内源性代谢组应答特征的改变，将获取数据与组分进行关联分析，从药动学、药效学和代谢组学多角度揭示毛冬青多组分抗血栓的协同整合特征和规律；最后综合解读上述数据，通过对比目标成分缺失前后整体活性差异来确定各有效成分对整体药效的贡献度，解码能够表征毛冬青抗血栓的药效成分群，通过代谢网络分析发现毛冬青潜在的抗血栓作用途径和机制。本研究有望为中药药效物质基础及作用机制研究提供新思路。

血栓性疾病严重威胁人类的生命健康，其发病率在我国逐年增加。中药防治血栓性疾病具有多途径、多靶点发挥作用且作用温和的独特优势。因而，从中医药中寻找抗血栓药物并阐明其作用机制，对于心脑血管疾病的防治具有重要的理论意义和应用价值。毛冬青在临床上主要用于冠心病、血栓闭塞性脉管炎等心血管疾病的治疗，疗效确切。前期研究发现其抗血栓活性与体内过程密切相关，且具有多组分、多途径协同作用，但具体机制尚不清楚。本项目采用整体观和体内过程相结合的研究思路，探究了毛冬青抗血栓的药效物质和作用机制。建立并优化了毛冬青复杂化学组分的体内外定性和定量分析方法，建立了能表征毛冬青化学物质组的特征图谱；体内外化学物质组的关联分析，发现三萜及其苷类成分是毛冬青发挥抗血栓作用的潜在药效组分；通过组分的体内过程研究，揭示了毛冬青三萜及其苷类成分在体内的代谢规律及药动学特征，新发现毛冬青三萜类成分在吸收转运过程中，彼此间存在相互作用；药效学研究发现三萜及其苷类成分是毛冬青抗血栓的主要药效物质，非三萜类成分未显示直接的药效作用，但其在参与抗血栓整体药效过程中可能发挥了协同作用；基于血瘀证大鼠模型的UPLC-Q-TOF-MS组学分析方法，描绘出毛冬青组分在机体内干预血瘀证的代谢轮廓，发现了以甘油磷酸胆碱、鞘磷脂等为主的潜在差异性生物标志物；综合解读药动学、药效学和代谢组学研究数据，发现干预鞘脂代谢途径及PI3K信号通路是毛冬青抗血栓作用的潜在途径和机制。项目研究结果不仅有助于阐释毛冬青抗血栓的物质基础和作用机制，还为其质量控制及临床防治血栓性疾病提供了科学依据。本项目以毛冬青为研究范例，建立了基于整体观和体内过程的中药药效物质及作用机制研究方法，可为其他中药多组分、多途径协同机制的研究提供可借鉴的思路与方法。