基于中医“化瘀止血”治法的茜草炭效应物质基础及作用机制研究

Rubiae radix et rhizome Carbonisatum has been commonly used to eliminate stasis and stanch bleeding in the Chinese traditional clinical treatments. However, the material bases of the actions and mechanisms have not been completely revealed. According to the the treatment of eliminating stasis and stanching bleeding in Traditional Chinese Medicine (TCM) and the blood stasis-eliminating and hemostatic effects of Rubiae radix et rhizome Carbonisatum in the clinical practice, we proposed a hypothesis based on the previous studies about that: the different active site or ingredients (group) of Rubiae radix et rhizome Carbonisatum could affect different links in the blood stasis or bleeding processes through different factors to stanch bleeding and eliminate the blood stasis. This item plans to select several biological indicators which can reflect the blood stasis-eliminating and hemostatic effects of Rubiae radix et rhizome Carbonisatum based on the TCM principles, combined with the application of bio-guided separation technology to screen the active substances and explore the correlation between material basis and the blood stasis-eliminating and hemostatic effects of Rubiae radix et rhizome Carbonisatum. We also use the modern molecular biology and multidimensional chromatography technologies to explore its influences to the blood stasis-eliminating process, hemostatic links and the metabolomics feature indicators in the vitro and vivo activity evaluation models. Then analyze the dose - effect relationship of Rubiae radix et rhizome Carbonisatum, in order to reveal the effective mechanisms in the overall, tissular, cellular and molecular levels. These studies will advisably and scientifically direct the clinical application of Rubiae radix et rhizome Carbonisatum, and provide new ideas and methods to study deeply the mechanisms of drugs with the functions of eliminating stasis and stanching bleeding.

茜草炭为中医临床常用化瘀止血药，但现有研究成果尚未全面解答茜草炭化瘀止血的效应物质基础及其作用机制等科学问题。基于中医化瘀止血治法及茜草炭止血临床实践，结合前期工作基础，提出工作假说：茜草炭不同活性部位/成分（群）可能通过影响血瘀、出血过程不同的关键因子而作用于化瘀止血的多个环节，从而实现活血、止血双向调节，表现出化瘀止血的总体效应。本项目选择符合中医化瘀止血治法的茜草炭止血效应生物学指标，应用生物导向分离技术追踪茜草炭化瘀止血的活性物质，探寻茜草炭化瘀止血物质基础－效应相关性。并采用现代分子生物学及多维色谱联用技术，探索茜草炭对体内外活性评价模型中化瘀止血各环节及代谢组学特征指标等的影响，分析其量-效关系，以期在整体、组织、细胞及分子等层次上揭示茜草炭化瘀止血的作用机制，为茜草炭临床合理应用提供科学依据，并为深入进行其它化瘀止血药物的作用机制研究提供新的思路和方法。

本项目基于中医应用茜草炭化瘀止血的临床实践，复制了符合其辨证用药特点的血瘀模型（急性血瘀大鼠模型）与出血模型（子宫内膜出血大鼠模型），建立茜草炭化瘀止血作用评价体系，采用适应方法与技术开展茜草炭在病理状态下物质基础-效应机制-代谢组学等研究，明确茜草炭化瘀止血效应的物质基础，揭示了茜草炭“化瘀止血”的科学内涵，为茜草炭临床合理应用提供了科学依据，为中医药现代化提供了良好示范，产生了较好的社会效益。.1.通过给予茜草炭、活性部位及成分群，发现上述药物均可通过降低模型大鼠血液黏度，改善血液流变学特性；通过缩短PT、TT和APTT、降低FIB含量，影响Ⅻa因子，启动内源性凝血途径，影响因子Ⅲ的释放而启动外源性凝血途径，从而使得Ⅹ因子被激活进一步促进纤维蛋白原转化为纤维蛋白所形成的共同凝血途径，改善血瘀大鼠与子宫内膜出血大鼠的凝血功能；通过改善PLT、TXA2/PGI2，作用于血小板系统，纠正模型大鼠血小板参数异常；通过回调PAI-1/t-PA，作用于纤溶蛋白，改善纤溶系统。研究了茜草炭活性部位能够保护ox-LDL对人脐静脉内皮细胞的损伤，从而维持内皮功能的稳定，达到保护血管防止出血以及预防血栓形成的目的。.2.利用UPLC-Q-TOF/MS技术及化学成分分离技术，确证了茜草炭活性部位QCT-1+2中主要是大叶茜草素、茜草素、异茜草素、羟基茜草素、甲基异茜草素、1,3,6-三羟基-2-甲基蒽醌和1,3,6-三羟基-2-甲基蒽醌-3-O-α-L-鼠李糖(1→2)-β-D-葡萄糖苷这七种化学成分，并对各成分进行定量研究。.3.采用内皮细胞损伤、大鼠子宫内膜出血模型观察了活性成分、活性部位的代谢组学，表明活性部位及成分均能通过调节花生四烯酸代谢、生物素代谢等相关体内外代谢发挥茜草炭的功效。通过缺活性部位逆向验证，最终证明茜草炭化瘀止血活性部位为QCT-1+2，其主要通过激活内源性凝血途径，纤溶系统，血小板系统，从而改善模型大鼠的凝血系统，达到化瘀止血之功效。 .项目实施期间发表论文10篇，其中SCI收录2篇；获发明专业授权1项；培养研究生5名，其中博士研究生1名。