芪参方药效组分抑制血栓形成关键G蛋白偶联受体激活的多靶点协同机制研究

Acute myocardial infarction (AMI), caused by coronary thrombosis, is one of the most common causes of death. Platelet activation and aggregation play a pivotal role in the generation of occlusive thrombus at the site of coronary arterial plaque rupture and G protein-coupled receptors (GPCR) such as thromboxane A2 receptor (TxA2R), ADP receptor (P2Y1 and P2Y12) and thrombin receptor (PAR-1) are key players in the process. Although Aspirin and other anti-platelet treatment are available, new drugs and combination anti thrombotic therapies are in active development due to side effects and resistance of current drugs. Chinese herbal medicine has been a rich source of cardiovascular drug development and a latest multi-center,large-scale, random,and double blind clinical trial indicated that a Chinese medicinal formula Qishenyiqi (QSYQ), composed of extracts of astragalus, red sage root, notoginseng, dalbergia, was equally effective and safer compared to aspirin in secondary MI prevention. However, the active components and mechanisms of action of QSYQ remain unknown. This application seeks to reveal the molecular basis of QSYQ in MI prevention. We will take a reverse pharmacology approach by first validating the anti-thrombosis activity of the whole (QSYQ) formula in a mouse model and in cell-based activity assays for platelet GPCR (TxA2R, PY1/12, PAR-1) inhibition. High-content screens will be performed in platelets,DAMI cells and a b-arrestin2-GFP expressing U2OS cell line on chemical fractions and the compound library of Qishenyiqi.The double screening combining molecular targets and cell phenotype will identify ingredients and molecules in QSYQ and determine their efficacy on different types of arterial thrombosis. In addition to analyze the Cox-1 /thromboxane A2/thromboxane A2 receptor pathway, we will explore other novel mechanisms and signal transduction pathways for QSYQ role in thrombosis prevention. In particular, we hypothesize that synergistic action of QSYQ component could be mediated in part by influencing the hetero-dimer formation of different platelet GPCRs. The proposed integration approach of drug combination study and network pharmacology will uniquely benefit the understanding of Chinese medicine's multi component and multi targeting characteristics and will open up new avenues for investigation of synergistic effect of complex Chinese medicine.

由冠状动脉血栓形成引起的急性心肌梗死是人类最常见的死亡原因之一。血栓素A2（TXA2）受体、凝血酶受体和ADP受体等G蛋白偶联受体(GPCR)介导血小板活化和聚集，对血栓形成起关键作用。现有抗血小板药物耐药性和副作用局限，加速了新型受体拮抗剂的开发和联合用药抗栓治疗。益气活血类方药具有抗栓功能, 最新临床试验结果和前期动物实验分别表明芪参益气滴丸（芪参方）对心肌梗死二级预防与阿司匹林等效且安全性更好。然而其作用机理是否通过COX-1/ TXA2受体激活途径尚不明确。本课题拟整合分子靶标和细胞表型双重筛选技术，以抑制血小板关键GPCR激活为指标，系统鉴别芪参方中黄芪、丹参、三七、降香组分抑制血栓形成的活性物质基础、信号传导途径和益气活血组分配伍规律，提出并验证"芪参方活性成分通过影响血小板GPCR异聚体相互作用防治血栓形成"的假说,为基于整体药效活性的多组分复方中药协同效应研究开辟新途径。

由冠状动脉血栓形成引起的急性心肌梗死是人类最常见的死亡原因之一。血栓素 A2(TXA2)受体、凝血酶受体和 ADP 受体等 G 蛋白偶联受体(GPCR)介导血小板活化和聚集,对血栓形成起关键作用。现有抗血小板药物耐药性和副作用局限,加速了新型受体拮抗剂的开发和联合用药抗栓治疗。益气活血类方药具有抗栓功能, 最新临床试验结果和前期动物实验分别表明芪参益气滴丸(芪参方)对心肌梗死二级预防与阿司匹林等效且安全性更好。然而其作用机理是否通过 COX-1/ TXA2 受体激活途径尚不明确。本课题整合分子靶标和细胞表型双重筛选技术,以抑制血小板关键 GPCR 激活为指标,系统鉴别芪参方中黄芪、丹参、三七、降香组分抑制血栓形成的活性物质基础、信号传导途径和益气/活血组分配伍规律,鉴定了芪参方主要入血成分，对提出的"芪参方活性成分通过影响血小板 GPCR 异聚体相互作用防治血栓形成"的假说进行了验证和修正, 完成了课题既定的3项目标：1.通过针对TXA2受体，ADP受体和凝血酶受体等介导血小板活化的关键GPCR的药效物质基础和作用机理研究，揭示了芪参方抑制GPCR激活对防止血栓形成的作用机理； 2.通过以反向药理学理念整合分子靶标和细胞表型双重筛选技术，建立了一个适合于复方中药的组分药物研究体系和平台； 3.通过研究不同芪参方活性组分对不同血小板GPCR的药效差异和组合配伍优化，揭示组分中药多靶点协同效应的作用规律; 并发现芪参方主要活性物质对冠状动脉血栓形成前期内皮功能异常和血管损伤的调节和修复作用，初步构建了益气、活血中药芪参方抗血栓形成、逆转内皮功能异常、促进血管新生多靶点作用网络和相互作用药理学评价方法,为基于整体药效活性的多组分复方中药协同效应研究开辟了新途径。