基于谱效关系-代谢组学-网络分析平台探索半枝莲-白花蛇舌草治疗卵巢癌的配伍规律及其作用机制研究

The couplet of traditional Chinese medicine is a summary of medical practice by ancient and modern experts. It plays an important role on discussing the compatibility principle and its mechanism, which will guide the practice and increase the clinical efficacy. Preliminary results showed that Scutellaria barbata and Hedyotis diffusa possesed the effect of anti-ovarian cancer, and the combination effect is remarkable, their combination is often found in both classical and clinical prescriptions. To enclose the objective law and scientific connotation of the couplet’s compatibility, and guide the medication in clinical effectively, the correlation among the ratio of couplet medicines, the chemical constituents, the metabolites and the efficacy was established through the method of multivariate nonlinear regression and the concept of dimension reduction, the potential pharmacophore substance was presumed, and its validation was verified using the knockout technique of chemical components in this study. The integrated network of active components-target protein-related protein-endogenous metabolites was built by molecular docking technology, the interaction between proteins, network analysis technology, and metabolomics and so on, the specific mechanism of Scutellaria barbata-hedyotis diffusa for treatment of ovarian cancer was explained comprehensively. The prediction model of compatibility principle of Scutellaria barbata-hedyotis diffusa towards ovarian cancer was also established by various methods of data mining, and verified using multi samples. This project will provide ideas for the research of the compatibility principle and mechanism of couplet medicines, lay a theoretical foundation for guiding the rational use and deep development of Scutellaria barbata and Hedyotis diffusa.

中药药对是古今医家长期医疗实践的总结，深入探讨药对的配伍规律及其作用机制对于指导实践，提高临床疗效具有重要意义。前期研究结果表明半枝莲、白花蛇舌草具有抗卵巢癌作用，两者合用效果显著，常常配对出现在经典名方和临床处方中。为揭示药对配伍的客观规律与科学内涵，有效指导临床遣方用药。本研究拟采用多元非线性回归的方法和降维的观念，建立药对配比-化学成分-代谢产物-药效之间的关联，推测潜在的药效成分群，并采用化学成分敲除技术进行验证；利用分子对接技术，蛋白间相互作用，网络分析技术和代谢组学等方法，搭建药效成分-靶点蛋白-相关蛋白-内源性代谢产物的综合网络，阐释药对药效物质群治疗卵巢癌的作用机制；采用多种数学挖掘手段，构建药对半枝莲-白花蛇舌草治疗卵巢癌配伍规律的预测模型，并进行多样本验证。本项目为中药药对配伍规律及作用机制研究提供思路，为指导药对半枝莲-白花蛇舌草的合理配伍使用与深入开发奠定理论基础。

卵巢癌是临床上最致命的妇科恶性肿瘤，五年死亡率高达60%。中药药对是古今医家长期医疗实践的总结，深入探讨药对的配伍规律及其作用机制对于指导实践，提高临床疗效具有重要意义。课题首先通过中医传承辅助平台对128首抗癌方剂进行分析确定半枝莲-白花蛇舌草药对为中医临床中抗癌的常用药对，然后针对含有半枝莲-白花蛇舌草的抗癌方剂进行用药规律研究。采用多种色谱学技术从半枝莲-白花蛇舌草中分离得到28个化合物；通过液质联用技术对半枝莲-白花蛇舌草进行化学成分进行分析，鉴定了76种化学成分；通过网络药理学预测该药对的主要药效成分为槲皮素、芹菜素、木犀草素、穗花杉双黄酮和汉黄芩素等，分子对接进一步验证，推测主要涉及的通路为MAPK通路等，并采用WB实验进行体外验证。在体外实验中，半枝莲-白花蛇舌草可以抑制卵巢癌细胞A2780的生长增殖，增加卵巢癌细胞内ROS水平，诱导其凋亡。蛋白组学和转录组学结果显示半枝莲-白花蛇舌草抑制卵巢癌细胞机制可能与铁死亡密切相关，经WB和Q-PCR验证确定，半枝莲-白花蛇舌草可通过抑癌基因p53上调，影响HO-1的表达升高，分解血红素使其代谢产物Fe2+增多。同时，PCBP1运载Fe2+蛋白，与Fe2+结合的FTH1减少，Fe2+转化为Fe3+减少。另外自溶酶体LC3、ATG5、NCOA4蛋白上调，进一步加剧了FTH1减少，导致Fe2+转化Fe3+减少。Fe2+增多，导致Fenton反应发生，ROS增加，线粒体破坏，从而诱导卵巢癌细胞铁死亡。体内斑马鱼实验显示，半枝莲-白花蛇舌草可抑制肿瘤的生长增殖和转移；裸鼠皮下移植瘤实验显示半枝莲-白花蛇舌草可以抑制皮下瘤增长，并且可调节肠道中Muribaculaceae，UCG-014Clostridia_UCG_014，Proteobacteria，α-Burkholderiales，Clostridium_sp_Culture_27等菌群；瘤体的转录组学分析显示，半枝莲-白花蛇舌草治疗卵巢癌与氧化磷酸化通路和蛋白聚糖通路有关。本项目为中药药对配伍规律及作用机制研究提供思路，为指导药对半枝莲-白花蛇舌草的合理配伍使用与深入开发奠定理论基础。