基于药物代谢组学和蛋白质组学的防己治疗类风湿关节炎体内药效物质和作用机制研究

Stephania tetrandrae radix (STA) can get rid of rheumatism and relieve pain, its therapeutic benefits towards Rheumatoid arthritis (RA) have been well-defined for centuries. The preliminary study of the project displayed that the major components in rat's plasma were tetrandrine, fanchinoline, corypalmine and its glucuronidated conjugates, after orally administering STA. In addition, STA may benefit RA through MAPK, chemokine signaling pathways and so on. However, the efficacious material basis and the mechanism of STA have not been well revealed. In this project, the STA-derived chemicalome, and the migration courses of endogenous substances and proteome in normal and collagen-induced arthritis (CIA) rats were firstly characterized by combining pharmaco-metabonomics and proteomics. The endogenous metabolome and proteome in combination with classical pharmacological indicators are definined as "efficacy". Consequently, the dynamic spectrum-activity correlation analysis between the "efficacy" and the pharmacokinetic of STA was constructed to screen out the potential medicinal ingredients and targets, which were supplemented by screening the new protein modifications after STA-intervention through noncoding amino acids analysis. The potential medicinal ingredients were obtained by in vitro incubation, LC-MS guided separation, and so on. Their "efficacy" to CIA rats were also evaluated. The anti-rheumatism components and the mechanism of STA were clarified by analyzing the differences in the "efficacy" and regulatory pathways of STA and the potential active ingredients. The project provides an efficient approach to elucidate the material basis and mechanism for traditional Chinese medicine.

防己可以祛风湿止痛，对类风湿关节炎（RA）具有明确的疗效。本项目初步研究表明防己在大鼠血浆中的主要成分为Tetrandrine、Fanchinoline、Corypalmine及其葡萄糖醛酸结合产物；此外，防己可能通过MAPK, Chemokine等信号通路改善RA。然而，防己治疗RA的药效物质和作用机制仍未明确。因此，本项目以CIA大鼠为病理体系，结合药物代谢组学和蛋白质组学技术全面表征体内物质组信息。将内源性小分子代谢轮廓、蛋白表达谱与传统药效指标作为多维“效”，与防己体内过程规律构建“动态组效关系”；同时结合“非编码氨基酸技术”筛选潜在的药效成分和作用靶点。通过购买、体外孵育等手段获得潜在药效成分；再结合CIA大鼠和多维药效指标验证其活性。最后分析防已和各单体成分对CIA大鼠的药效、调控通路等差异，探讨防己治疗RA的药效物质和作用靶点，为中药药效物质和作用机制研究提供新思路。

防己可以祛风湿止痛，多年的临床使用证实防己对类风湿关节炎（RA）具有明确的疗效。针对防己药效机制不明，本项目首先采用LC-TriTOF-MS技术，结合基于苄基异喹啉成分的诊断离子和中性丢失逐级分类解析的策略，快速绘制了防己体内外化学图谱。防己醇提液中主要以双苄基四氢异喹啉和四氢原小檗碱成分为主；而通过灌胃进入大鼠体内主要为粉防己碱（tetrandrine），防己诺林碱（fanchinoline）和千金藤素（cepharanthine）以及紫堇达明碱（corypalmine）及其II相代谢物为主。结合LPS诱导的大鼠腹腔巨噬细胞炎症模型证实cepharanthine和corypalmine抑制炎症因子IL-1β, TNF-α和IL-6表达和分泌的能力和tetrandrine，fanchinoline一致，说明这四个成分是防己治疗RA的主要药效物质。进一步以collagen-induced arthritis (CIA)大鼠为病理体系，开展代谢组学、蛋白组学和泛蛋白修饰组学工作。以传统药效指标和内源性小分子代谢物轮廓为多维药效指标，显示防己给药1.35 g/Kg对大鼠RA的治疗效果最佳。进一步采用ANOVA分析多组学数据，结果表明防己治疗RA的作用途径和双氯酚酸钠显著不同；其中蛋白组显示防己主要调控转录和炎症反应两个生物学过程。泛蛋白修饰组则显示给药防己后，大鼠机体主要反馈为对有机环状化合物和cAMP的细胞反应过程。代谢组表明防己主要调控色氨酸降解和代谢两通路。最后整合多组学工作阐明糖代谢通路关键蛋白的异常修饰，包括：上调的ALDOA@166/Glu->Pro substitution和下调的ENOA@419S/HexNeuAcPhos and hexacosatetraenoyl可能参与调控防己给药后大鼠体内色氨酸代谢过程；而防己通过PI3K-AKT通路的关键蛋白AKT和RAP1分别调控NFkB和MEK/ERK通路发挥抗炎作用；进而治疗类风湿性关节炎症。本项目结合多组学系统阐明防己治疗RA的药效物质和作用靶点，为中药药效物质和作用机制研究提供新思路。此外，本研究还建立了基于修饰反应树的质量偏移系统注释的泛修饰组学并在此基础上发现了新型修饰-同型精氨酸修饰，为新型修饰的挖掘、功能验证提供系统的方法。