基于NF-κB和MAPK通路的舒筋草抗类风湿关节炎药效物质及作用机制探索

Lycopodiastrum casuarinoides is a folk medicine commonly used against rheumatoid arthritis(RA) in Southern China. However, its anti-rheumatic chemical components and mechanism of actions remain unknown. L. casuarinoides, like most species of the families Lycopodiaceae and Huperziaceae, produces two major classes of secondary metabolites, namely Lycododium alkaloids and serratane-type triterpenoids. The Lycopodium alkaloids are a series of structurally related, yet diverse piperidine alkaloids. Some of them, such as huperzines A and B, exhibit potent acetylcholinesterase inhibitory activities, attracting great interests from synthetic, biogenetic, and biological points of view. The serratane-type triterpenoids are unusual. Unlike most other triterpenoids reported so far, the serratane-type usually possesses a seven-membered C-ring. The biological activity of this type of triterpenoids has not been thoroughly investigated and only limited reports on their possible cytotoxic activities. Recently, as a continuation of our investigation on anti-rheumatic chemicals from traditional Chinese medicines, the total alkaloid and triterpenoid extracts of L. casuarinoides were subjected to in vivo model of adjuvant arthritis rats to evaluate their possible therapeutic effects against RA. Fortunately, both of the two extracts, especially the alkaloid extract, exhibited promising therapeutic effects against RA-like symptoms on rats. Therefore, the purpose of the present project is to 1) confirm the best anti-RA effective extract of L. casuarinoides 2) isolate and elucidate the structures of natural products from the best effective extract 3) reveal the mechanism of actions of L. casuarinoides against RA. To confirm the best anti-RA effective extract, the total alkaloid and triterpenoid extracts were analyzed for their effects on adjuvant arthritis rats. To isolate chemicals from best effective extract, some integrated technologies already established in our laboratory, such as HPLC-ELSD, prepHPLC-ELSD, are adopted. NMR, MS and X-ray techniques were applied to elucidate the molecular structures. Finally, to reveal the possible mechanism of actions, the best effective extract as well as the single compounds is analyzed in vivo and in vitro experiments to evaluate their effects on important inflammatory mediators, pro-inflammatory cytokines(IL-1, IL-6, TNF-α), MAPK and NF-κB closely relating to RA pathology.

以舒筋草为代表的石松和石杉科药用植物在民间用于类风湿性关节炎（RA）的治疗，然而至今药效物质和作用机制不明。本项目先期的研究发现，舒筋草主要所含的生物碱和三萜两大类成分（尤其是生物碱），对大鼠佐剂关节炎病变显示了显著的缓解作用，很可能通过调控与炎症紧密相关的细胞因子及信号通路（NF-κB和MAPK）而发挥药效。本项目拟通过对这两类成分在动物模型中的药效学评价，筛选出舒筋草抗RA的最佳有效部位。采用高效液相-多级质谱联用等新技术，在体外活性指导下，对最佳有效部位进行系统化学分离，以相关活性实验确证舒筋草抗RA的有效成分。采用分子生物学技术，观察舒筋草有效部位和有效成分对NF-κB和MAPK信号通路的影响，探讨作用机制。研究结果将有助于揭开具有类似化学成分的石松和石杉科众多传统中药抗RA的神秘面纱，为开发和合理使用具有我国自主知识产权的抗RA新药提供坚实的理论基础

石松类药用植物具有祛风除湿的功效，多用于治疗关节风湿痛。这类植物主要含有石杉型三萜和石松生物碱成分。自从具有极显著抑制乙酰胆碱酯酶活性的石杉碱甲发现以来，石松类药用植物活性成分的筛选几乎全部指向了针对阿尔茨海默病的治疗。这也意味着，石松类药用植物治疗关节炎的物质基础尚不明确。针对以上现状，本项目以舒筋草的总生物碱和总三萜为对象在动物RA实验模型上的作用效果进行评价，确定最佳有效部位和可能的作用机制。进一步对最佳活性部位的有效成分进行系统的化学分离和结构鉴定，并试图通过体外筛选获得活性化合物。我们发现舒筋草的总生物碱对动物RA模型具有更好的防治效果，其在动物体内通过调节NF-κB通路改善关节炎症状。利用色谱学方法，特别是制备液相色谱法对生物碱部位进行了系统的化学分离，利用现代波谱技术测定化合物的结构，包括平面结构、相对构型和绝对构型。总共分离并鉴定了59个单体生物碱成分，均为石松定碱型生物碱，其中新化合物54个。然而，对单体化合物的体外抗炎活性筛选中，单体化合物并未显示出活性。将舒筋草的主要生物碱单体huperzinine直接用于动物炎症模型（二甲苯诱导的小鼠耳肿胀和完全弗氏佐剂诱导的大鼠关节炎模型），发现其具有较强的抑制炎症作用。这些结果表明舒筋草的单体生物碱通过尚未阐明的抗炎通路发挥效果。其中一种可能性是通过胆碱能抗炎通路，因为舒筋草所含的主要生物碱成分具有较显著的中枢乙酰胆碱酯酶抑制活性。这些单体生物碱在体内发挥抗炎效果的确切靶点仍需进一步探索。以上研究结果，目前共发表基金第一标注的SCI论文3篇，会议论文1篇，申请发明专利1项，其余结果正在总结和撰写论文中。本项目的研究结果证明石松生物碱成分在体内具有确切的抗炎效果。大量单体生物碱的获得为今后深入研究抗炎作用确切靶点提供了充足的化合物保障，丰富了天然产物的结构类型，从而为开发和合理使用具有我国自主知识产权的抗RA新药提供理论基础。