基于G蛋白偶联受体相关通路的隐丹参酮抗血小板聚集机制研究

Cryptotanshinone is the representative of the fat-soluble constituents in Salvia Miltiorrhiza Bge. The recent researches have verified its promising application prospect in treatment of cardiovascular and cerebrovascular diseases. However, the mechanism is still unclear. Our previous researches have found that the cryptotanshinone exerted strong activity in resistance to platelet aggregations, especially when the aggregations were induced by AA and ADP. And it was also found that the G protein on the platelet membrane changed dramatically in the measurement with high content screening. Besides, literature reports demonstrated that the altered G protein receptor is relevant to the TXA2/PGI2 and P1Y2/P2Y12 signaling pathway. Therefore, based on high content screening is applied for observation the change of platelet membrane protein and interactions between proteins to screen the distinct targets with cryptotanshinone as subject and platelet as carrier. Moreover, metabolomics can be used to quantitatively analyze the small molecules activating platelet and explore distinct metabolic substances. And the effect of cryptotanshinone on platelet aggregation will be clarified with molecular biotechnology by detecting the phosphorylation level of cytoplasm protein. Finally, the target can be confirmed in the animal model of blood stasis and the mechanism behind the anti-platelet aggregation of cryptotanshinone will be clear, which provides the experimental basis for the research about function of anti-platelet aggregation of Salvia Miltiorrhiza Bge.

隐丹参酮是活血化瘀类中药丹参脂溶性成分中的代表性物质，近年研究已证实其在治疗心脑血管疾病方面具有良好的应用前景，但作用机制不明确。课题组前期研究发现隐丹参酮在抗血小板聚集方面具有较强活性，并且对AA、ADP诱导血小板聚集的抑制作用最强；在高内涵筛选技术评测血小板活化过程中，血小板膜上G蛋白受体发生显著变化，文献显示变化的G蛋白受体与TXA2/PGI2和P1Y2/P2Y12信号通路相关。因此，本课题以隐丹参酮为研究对象，离体血小板为载体，采用高内涵筛选技术观察活化血小板膜蛋白变化及蛋白之间相互作用，筛选可能作用靶标；采用代谢组学方法对活化血小板的小分子代谢物进行定量分析，发现差异代谢物质；结合分子生物学技术，检测活化血小板胞浆蛋白磷酸化水平的变化，阐述隐丹参酮对血小板聚集作用影响；最后在血瘀动物上进行验证，筛选其作用靶点，明晰隐丹参酮抗血小板聚集的生物学机制，为丹参的抗血小聚集机制提供参考。

隐丹参酮是活血化瘀类中药丹参脂溶性成分中的代表性物质，近年研究已证实其在治疗心脑血管疾病方面具有良好的应用前景，但作用机制不明确。课题组已完成隐丹参酮抗血小板聚集的信号通路和代谢相关标志物的筛选，构建了隐丹参酮抗血小板聚集作用的信号网络，隐丹参酮代谢组学研究共筛选出腺嘌呤、5-甲基脱氧胞苷和脯氨酰-苏氨酸等52种差异代谢物，其抗血小板聚集的作用可能是通过影响鞘脂代谢、硫胺素代谢和酪氨酸代谢产生；通过药代动力学-药效学的联合模型，确认体内高活性的抗血小板聚集的活性成分为隐丹参酮的代谢产物，其代谢产物丹参酮2A可能是其体内抗血小板聚集的活性成分，从而进一步阐明了其体内作用机制；另外，本研究发现丹参酮可以通过调节Akt/Nrf2信号通路抑制坏死，从而在体内外发挥心血管保护作用。此外，本研究发现小粒径的丹参饮片的抗血小板聚集的效应成分指数值最高，丹参与郁金、三七和川芎等配伍煎煮后的提取液的抗血小板聚集的效应成分指数值最高。总之，本研究可为抗血小板聚集作用的药物隐丹参酮的进一步开发提供实验依据，也可为其他活血化瘀类中药的抗血小板聚集作用研究和开发提供新思路。课题组完成了预期研究结果：完成筛选发现得到了隐丹参酮抗血小板聚集的主要作用靶点和通路，已发表了论文2篇，其中SCI 论文1篇，已申请了专利1项。