基于靶标网络-蛋白质组学的三黄泻心汤治疗热毒证配伍机制研究

Investigating the compatibility of traditional Chinese medicine (TCM) is one of the vital parts of the formula research. San-Huang-Xie-Xin-Tang (SHXXT) has been applied in the treatment of heat toxin syndrome (HST) and it is firstly documented in “Jin-Gui-Yao-Lve”. Our study group and other academician have already covered the research of SHXXT’s chemistry and pharmacology as well as the compatibility mechanism of dahuang-huanglian drug pair. It is unexpectedly unclear about the whole formula’s compatibility mechanism. We set “SHXXT function as controlling effective constituents-heat toxin syndrome-biological effects-targeting network ” as hypothesis under the guidance of “one center with four elements”, thus study SHXXT’s biological effects centered by the whole formula and drug-pair, effective constituents, body state of disease and targeting network. We employ HST rat model to analyze the effective constituents from different compatibility by UPLC-Q/TOF-MS method. Then proteomics and network pharmacology method is employed to predict advantage compatibility, followed by the validation of animal whole pharmacodynamics, identification of differential protein as well as the key targets in specific inflammatory signaling pathway by biological methods like Western-blot in-vivo and in-vitro. We aim at interpreting the protein network integration compatibility regulation mechanism of SHXXT under the guidance of bioinformatics integration analysis so as to establish formula network equilibrium strategy integration study on the basis of “SHXXT compatibility- effective constituents- heat toxin syndrome- targeting network”.

方剂配伍是中医药研究领域关键科学问题之一。三黄泻心汤(简称三黄方)源自《金匮要略》，为治疗热毒证之要方。本课题组及国内同行对三黄方化学、药理学，以及该方大黄-黄芩药对等配伍进行了部分研究，但全方配伍机制研究尚不系统。拟立足于“一个中心、四个要素”，即围绕生物学效应为中心，探讨三黄方及药对配伍、功效成分、机体疾病状态、靶标网络“四要素”关系。提出“三黄方配伍效应是其功效成分-热毒证-生物学效应-靶标网络密切相关的综合结果”工作假说。用热毒证模型大鼠，以UPLC/Q-TOF-MS技术分析该方不同配伍的功效成分，用网络药理学靶点预测筛选优势配伍，并对模型动物及不同配伍干预后的差异蛋白进行鉴定，对特异性炎症信号通路关键靶位进行Western blot等生物学验证。以生物信息学分析，揭示三黄方配伍与功效成分-热毒证-靶标网络-生物学效应的内在联系，探索方剂配伍机制的"一个中心、四个要素"研究新模式。

本项目针对中药及其复方多成分-多靶点-多途径的作用特点，结合UPLC/Q-TOF-MS、网络药理学、蛋白质组学、生物信息学及分子生物学等技术，分别探讨了三黄泻心汤治疗热毒证的优势配伍成分、作用靶点网络、差异蛋白表达，分析阐明了它们之间的内在关系。UPLC/Q-TOF-MS检测结果表明大黄-黄芩配伍和黄芩-黄连配伍的主要入血成分有大黄酸、黄芩苷、小檗碱、汉黄芩苷等12个成分，确定了其体内效应物质基础。结合网络药理学对上述主要成分进行靶点和通路预测，富集分析获得34条具有显著差异的通路，构建了与内毒素血症相关作用靶点的“药物-化合物-靶点-作用通路”网络图。对以上主要成分及其配伍联用进行了抗炎作用评价，发现大黄酸和汉黄芩苷发挥主要的抗炎作用，且二者与黄芩苷配伍后可发挥良好的协同抗炎效果。在此基础上课题组进行了泻心汤主要成分及其成分配伍联用抗内毒素血症的蛋白组-磷酸化蛋白组-生物学验证-生物信息学整合分析，结果表明代表成分大黄酸、黄芩苷和小檗碱抗炎靶点和通路各有侧重。蛋白组方面，三种成分可同时调节细胞代谢、细胞增殖/分化从而抗内毒素血症，大黄酸侧重于调控炎症和免疫相关信号通路，黄芩苷着重调节代谢，而小檗碱重点调节细胞转录；磷酸化修饰方面，大黄酸还可通过调节细胞周期以发挥其抗炎效应；黄芩苷侧重于细胞自噬和凋亡，而小檗碱着重于调控基因转录和物质转运。而三者联用可同时调节免疫应答、基因表达、信号传导、物质代谢/转运及生物合成等多个途径，且对物质代谢和信号传导等过程蛋白表达的调节强度较单用时有所增强。由此说明三者对炎症过程的调控不是作用于单个信号通路，而是多靶点、多途径，相互协同和全面调节的结果，这也提示多成分合用后对于炎症中各生物学过程的调控不只是简单的加和。以上研究构建了三黄泻心汤的“药材-化合物-靶点-通路”的网络研究体系，阐明了三黄泻心汤组方治疗内毒素血症的蛋白网络整合调控协同起效的配伍机制。