新型松香烷型二萜抗RA的作用机制和构效关系研究

n our previous study, it was found that abietane diterpenoids possessing a peroxide bridge showed significantly inhibitory activities against LPS-induced NO production in RAW264.7 macrophages, the possible mechanism of action is to inhibit the overproduction of NO by inhibiting IKK and regulating IKK/NF-κB signaling pathway to exert anti-inflammatory effects. In order to further clarify the mechanism and structure-activity relationship of this class of compounds against RA, the project aims to use multi-directed separation methods combining biology, chromatography, and spectroscopy to conduct further separation, identification, and concentration of this type of compounds from Callicarpa macrophlla Vahl. and Callicarpa kwangtungensis Chun. At the same time, the mechanism of action of monomer compounds with significant activity was studied by using the experimental methods in vitro and in vivo. Based on the results of in vitro and in vivo studies, the anti-RA effect targets and the structure-activity relationship of these compounds was explored by using the “molecular docking” technique, “LC-MS combination” technology, and computer-aided drug design in order to provide lead compounds of new RA treatment drugs.

前期研究发现紫珠属特有成分含过氧桥环松香烷型二萜能显著抑制LPS诱导的RAW264.7巨噬细胞中NO的生成，其可能的作用机制是通过抑制IKK，调控IKK/NF-κB信号传导通路来抑制NO过量生成从而发挥抗炎作用。为进一步明确该类化合物抗RA的作用机制和构效关系，本项目拟采用生物学-色谱学-波谱学相结合的多重导向分离方法进一步对大叶紫珠及广东紫珠中该类化合物进行定向分离、鉴定和富集；同时采用体内、体外相结合的实验方法对活性显著的单体化合物进行作用机制研究；并基于体内外活性研究结果，利用“分子对接”技术、“液质联用”技术和计算机辅助药物设计等探讨该类化合物抗RA作用靶点和构效关系，以期为新型RA治疗药物的发现提供先导化合物。

本项目按原计划完成。本课题研究形成了一套生物学-色谱学-波谱学相结合、多重导向分离活性化合物的方法，从大叶紫珠和广东紫珠95%乙醇提物氯仿层中发现了二萜类成分的有效部位和活性馏分，并通过运用硅胶柱色谱、Sephadex LH-20柱色谱、ODS柱色谱、沃特世DAD-HPLC、岛津半制备型高效液相色谱法等现代分离手段从中分离得到38个二萜类成分，并运用现代波谱技术(NMR、HR-MS、ECD、X-ray单晶衍射法等)、与标准品对照、参考文献数据对照及理化常数测定等鉴定了全部化合物结构。同时建立佐剂性关节炎（AA）大鼠模型，对有效部位和活性显著的含过氧桥环松香烷型二萜抗炎活性进行验证。建立了体内外模型(佐剂性关节炎（AA）大鼠模型和LPS诱导的滑膜细胞模型), 并采用Western Blot、RT-PCR、ELISA和免疫组化等分子生物学技术，研究活性显著的含过氧桥环松香烷型二萜抑制IKK和调控IKK/NF-κB信号传导通路的作用机理，系统的阐述了含过氧桥环松香烷型二萜抗RA 的作用靶点和构效关系。已发表相关SCI论文6篇，申请国家发明专利1项。