基于PI3K-Akt-mTOR信号转导通路探讨青风藤配伍白芍治疗类风湿性关节炎"相使"为用的机理

Chinese medicine practitioners often prescribe a number of medicinal herbs together for the treatment of diseases. The composition of TCM prescriptions is basically guided by the traditional Seven Compatibility Laws. Even though TCM prescriptions have demonstrated usefulness in treating patients, the Seven Compatibility Laws, unfortunately, lacks the empirical interpretation of modern language. Therefore, the proof-of-principle studies of the Seven Compatibility Laws on the basis of bioactive constituents from component herbs in TCM prescriptions will have scientific significance for enhancing the modern interpretation of the Laws. QFGJS is a Chinese herbal formula composed of five herbs; it has shown efficacy in patients with rheumatoid arthritis (RA). In QFGJS, Caulis Sinomenii is assisted by Radix Paeoniae Alba to exert the anti-arthritic effect on RA. Studies have revealed that sinomenine and paeoniflorin are the principal bioactive chemicals in Caulis Sinomenii and Radix Paeoniae Alba, respectively. Our previous studies demonstrated that the pharmaceutical preparation of QFGJS and its bioactive constituents, sinomenine and paeoniflorin, possess anti-inflammatory, antinociceptive, and anti-arthritic effects. Fibroblast-like synoviocytes (FLS) is demonstrated to be a key player in RA, which not only drive inflammation and autoimmunity, but also be responsible for cartilage and bone destruction. FLS behave similar to tumor cells, which lead researchers pay attention to the PI3K-Akt-mTOR signaling. Several line of evidence shows that the PI3K-Akt-mTOR signaling mediates the proliferation and apoptosis of RA-FLS. Both sinomenine and paeoniflorin have been reported to inhibit the proliferation of FLS. All of these data lead to the hypothesis that the combination of sinomenine and paeoniflorin guided by the Mutual Assistance Law exerts synergistic effects on RA via the PI3K-Akt-mTOR signaling. To prove our hypothesis, we will utilize integrated pharmacodynamic, cellular and molecular methodologies to investigate the optimal dosage of sinomenine and paeoniflorin for the combinative use, and their synergistic effects on inflammation and pain associated with synovitis and on RA. Furthermore, we will isolate and culture FLS from the synovial tissues of RA patients. The effects of the use of sinomenine and paeoniflorin alone or together on the PI3K-Akt-mTOR signaling in RA-FLS will be investigated. Our study aims to interpret pharmacodynamic foundation and shed light on the molecular mechanisms underlying the synergistic interaction of sinomenine and paeoniflorin when prescribed together in clinic. The data obtained from our research will provide empirical evidence to support the clinical practice of TCM prescriptions for the treatment of RA. It also will lay a substantial foundation for a serial investigation of the Seven Compatibility Laws of TCM prescriptions.

从药物有效成分层次研究方药"七情和合"配伍规律，对促进方剂配伍理论的现代科学诠释具重要意义。前期研究表明，治疗RA临床验方"青附关节舒"复方制剂及其有效成分青藤碱、芍药苷均具抗炎、镇痛、抗RA作用。FLS是介导RA滑膜炎症和关节破坏的主要效应细胞，新近研究表明PI3K-Akt-mTOR信号通路介导RA-FLS增殖及凋亡，而青藤碱和芍药苷能显著抑制其增殖。因此，我们提出"青藤碱配伍芍药苷治疗RA为"相使"为用，两药合用具协同作用，其机理通过作用RA-FLS细胞PI3K-Akt-mTOR信号通路实现"的假说。本项目运用现代多学科技术手段，研究青藤碱配伍芍药苷抗炎、镇痛、抗RA的协同增效作用、最佳配比及对RA-FLS细胞PI3K-Akt-mTOR信号通路的影响，阐释两药"相使"为用治疗RA的药效学基础及分子机理，为中医配伍用药治疗RA提供科学依据，为方药配伍理论之"七情和合"的系列研究奠定基础。

方剂组方配伍理论的研究，就是要探询方剂的药效物质基础和作用机制，揭示方剂配伍的内在规律。从药物有效成分层次研究方药“七情和合”配伍规律，对促进方剂配伍理论的现代科学诠释具重要意义。“青附关节舒”方是治疗类风湿关节炎(RA)的临床验方，方中青风藤与白芍“相使”为用。前期研究表明该方制剂及其中有效成分青藤碱、芍药苷均具有抗炎、镇痛、抗RA及免疫抑制作用等。成纤维样滑膜细胞(FLS)是介导RA滑膜炎症和关节破坏的主要效应细胞，近年研究表明PI3K-Akt-mTOR信号通路介导RA-FLS增殖及凋亡，而青藤碱和芍药苷能显著抑制RA-FLS增殖。基于以上研究基础，我们提出科学假说：青藤碱配伍芍药苷治疗RA “相使”为用，两药合用具协同增效作用，其机理通过作用RA-FLS细胞PI3K-Akt-mTOR信号通路实现。.我们采用急性炎症和镇痛大鼠模型，筛选出青藤碱与芍药苷最佳配伍剂量分别为50mg/kg和120mg/kg，1:1配比具有最佳协同抗炎、镇痛作用。通过优选大鼠品系、II型胶原制备、免疫部位与方案，我们采用不完全弗氏佐剂(IFA)制备牛II型胶原乳剂(CII/IFA)，雌性Wistar大鼠尾根部皮内注射及7天再次加强免疫的方法，成功建立发病率高、病理程度差异性小且病理程度可控的CIA大鼠模型。在此基础上，我们研究显示最佳配比的青藤碱(50 mg/kg)与芍药苷(120 mg/kg)具有协同增效作用抗CIA作用（Q值=0.75）。我们优选建立了组织块培养法分离培养RA-FLS细胞，筛选出青藤碱与芍药苷抑制RA-FLS增殖的最佳配伍药物浓度分别为13μM和22 μM，1:1配比具有最佳协同作用，机制研究发现青藤碱配伍芍药苷针对PI3K-Akt-mTOR分子信号通路不同环节，青藤碱抑制上游Akt表达，而芍药苷主要针对mTOR下游效应分子，二者配伍具有协同作用。.本项目在有效成分配伍层次上，明确了青风藤配伍白芍治疗RA“相使”为用的药效学基础，并基于PI3K-Akt-mTOR信号通路阐明了其分子机理，为中医配伍用药治疗RA提供科学依据，试图为中医经典名方配伍机理的系列探讨搭建药效学-细胞-分子生物学相结合的研究平台。