民族药老瓜头抗类风湿性关节炎关键药效成分及其分子机制研究

The incidence of rheumatoid arthritis (RA) is high and it is difficult to cure by now. Its mechanism is related to inflammatory pathways such as JAK/STAT pathway. Previous research has found that the nationalities drugs Cynanchum komarovii can obviously alleviate the inflammation symptom of the CIA model rats and can significantly reduce the expression of IL-6 in the synovial tissue. This means that its anti-RA activity may be related to the JAK/STAT signaling pathway. However, the effective components of anti-RA and its mechanisms are urgent to be solved. This project is based on the previous to select the active site of anti RA from Cynanchum komarovii through the rat acute inflammation model induced by the carrageenan and the CIA rat model. Using a key active substance screening strategy which is a combination of step-by-step activity screening and UPLC-QTOF-MS oriented non-targeted metabolomic analysis methods to characterize the chemical composition of its active moiety and screen its main potent substances. At the same time, the molecular mechanism of the anti-RA effect of Cynanchum komarovii will be by examining its effect on inflammatory cytokines in rats' drug-containing serum, articular cartilage and synovial tissue, and exploring its effect on core pharmacodynamic components on JAK/STAT signaling pathway to investigate the effect of its core pharmacodynamic components on JAK/STAT signaling pathway and its phosphorylation. Implementation of this project will help improve the medicinal value of Cynanchum komarovii and promote its basic research on active ingredents and its comprehensive exploitation.

类风湿性关节炎（RA）发病率高且难以治愈，发病机制与JAK/STAT等炎症通路有关。课题前期发现，回药老瓜头能明显缓解CIA模型大鼠的炎症症状，显著降低滑膜组织中IL-6等细胞因子的表达，提示其抗RA活性可能与JAK/STAT信号通路有关，但其抗RA的关键药效物质及其作用机制尚不明确。故本项目结合角叉菜胶诱导的大鼠急性炎症模型及CIA大鼠模型，以逐步活性筛选方法，寻找老瓜头抗RA活性部位；以基于UPLC-QTOF-MS技术的非靶向代谢组学分析方法，筛选并确定关键活性成分，表征有效部位化学组成并确定主要药效物质；最后，通过考察其对大鼠含药血清、关节软骨及滑膜组织中炎症细胞因子的影响，并探讨核心药效成分对JAK/STAT信号通路STAT1等分子及其磷酸化的调控作用，阐明老瓜头抗RA作用的分子机制，项目实施有助于提升老瓜头药用价值，促进其药效物质基础研究和综合开发利用。

民族药老瓜头多年用于类风湿性关节炎（RA）的治疗。为阐明其抗RA活性物质及作用机制，本项目开展了以下研究工作：通过网络药理学预测老瓜头治疗RA的活性成分、靶点及通路；另外，项目采用Ⅱ型胶原诱导的关节炎（CIA）模型，评价老瓜头提取物及各萃取部位的抗RA药效，获取活性部位；利用各种色谱学技术，分离并阐明其活性部位的化学特征；并在此基础上，探讨其质量控制方法，为其今后更好的临床应用奠定基础。项目取得如下研究成果：.1.通过网络药理学研究，预测得到老瓜头治疗RA的7个关键活性成分和45个关键靶点，主要调控TNF信号通路发挥其对RA的治疗作用。.2.老瓜头含药血清对HFLS-RA细胞的增殖活性有显著的抑制作用，对其迁移和侵袭能力也有显著抑制作用；可促进HFLS-RA细胞的凋亡；有效降低HFLS-RA细胞中凋亡、抑制蛋白Bcl-2的表达，促进了促凋亡蛋白Bax和Cleaved Caspase 3的表达。.3.老瓜头乙醇提取物未引起明显的肝肾病理变化，可明显缓解CIA大鼠关节肿胀程度，显著改善滑膜组织增生和骨破坏，降低炎症因子TNF-α、IL-1β及IL-6的表达，升高IL-10的表达。.4.老瓜头正丁醇、乙酸乙酯萃取部位均能够显著改善CIA大鼠的足肿胀和关节炎评分；有效降低CIA大鼠血清中的IL-1β、TNF-α、IL-18、IL-6等促炎因子的表达水平，提高抑炎因子IL-10在血清中的表达水平；且能够显著改善胸腺指数。.5. 从老瓜头活性部位中共分离得到30个化合物，包括11个苯丙素类、7个挥发油类、6个生物碱类、3个黄酮类、2个甾体类及2个萜类，其中19个化合物为首次从该植物中分离得到。.6. 为了更好的控制药材的质量，本研究对老瓜头药材开展了常规检测，生药学鉴别，主要活性物质的定量分析等研究，为初步建立老瓜头质量标准奠定了工作基础。.7. 本项目共培养硕士研究生3名，培养青年教师1名。本研究共发表论文3篇，申请专利1项。.综上，通过本项目研究，初步阐明了老瓜头的活性部位，明确了其活性部位的化学组成，其抗RA的分子机制可能与TNF信号通路有关，所构建的质量评价方法为其后期的质控和应用提供了参考。