独活抗类风湿性关节炎体内外药效物质组整合指纹表征新方法研究

Radix Angelicae Pubescentis (RAP) possesses Qufengchushi, Tongbizhitong effect according to traditional Chinese medicine (TCM) theory and is clinically used to treat rheumatic disease, but the material basis and mechanism of action is not clear. In this study, the profiling was selected as a starting point. The novel strategy was proposed to capture reversely the substance and estrogen -mediated mechanisms of action. Compared with the fingerprint of in vivo plasma and that of TCM, the absorbed chemical composition could be obtained. The activity integrated profilings were performed to capture the components used to following research.The estrogenic activity ntegrated profiling was used to determine the active ingredient with phytoestrogens in vitro and in vivo . The phytoestrogens activity (transcriptional activator, receptor binding and cell proliferation) integrated fingerprints were developed to screen the active ingredient and its metabolites of RAP. The effects of immune tolerance of dendritic cells (differentiation, migration , antigen presentation , T cell activation )were used to explore the efficacy of anti-rheumatic traditional Chinese medicines RAP; the scientific hypothes that RAP could induce immune tolerance mechanisms dendritic cells by estrogen -mediated pathways to exert the effect on the anti- rheumatic joints was verified. This program intends to clarify the material basis and immune mechanism of action and provide adequate scientific basis for modern usage of RAP and its preparation.

独活具有祛风除湿、通痹止痛之功效。临床用于类风湿疾病治疗、疗效确切、但物质基础及作用机制不明确。本研究以指纹图谱技术为切入点，提出基于体内外药效物质组活性整合指纹的反向捕获雌激素介导的免疫逃逸活性的药效物质研究策略。通过体外化学成分与体内吸收物质指纹图谱的比较，确定其可能吸收入血的化学成分；建立基于色谱分离技术的植物雌激素（转录激活、受体结合和细胞增殖）活性整合指纹图谱，确定独活及代谢物雌激素活性成分；建立树突细胞免疫耐受机制（分化、迁移、抗原递呈、T细胞激活）的活性整合指纹技术，探讨中药独活抗类风湿性关节炎药效物质基础，验证中药独活通过雌激素介导的途径诱导树突细胞免疫耐受机制发挥抗风湿性关节作用”的科学假说。本研究拟阐明独活抗风湿疾病的效应物质及其免疫作用机制，为独活的现代为主的现代中药制剂提供充分的科学依据。

独活具有祛风除湿、通痹止痛之功效。临床用于类风湿疾病治疗、疗效确切、但物质基础及作用机制不明确。本研究以指纹图谱技术为切入点，提出基于体内外药效物质组活性整合指纹的反向捕获雌激素介导的免疫逃逸活性的药效物质研究策略。本项目优化了独活有效成分提取制备工艺，建立了独活药材中有效成分同时定量的分析方法，建立了独活药材中多种有效成分绿色提取及定量的分析方法，开展了独活化学成分（二氢欧山芹醇-β-D-葡萄糖苷、二氢欧山芹醇当归酸脂、二氢欧山芹醇乙酸酯、没药当归烯醇）体内吸收、代谢研究，阐明了中药独活多种有效成分体内吸收代谢过程特点，明确其可能吸收入血的雌激素活性成分。建立树突细胞免疫耐受机制的活性整合指纹技术，从DC表型、吞噬、趋化、刺激T细胞增殖等环节、筛选了独活中树突细胞具有抑制活性的化合物，并通过ELISA和Western blotting实验从分子水平阐释其发挥免疫抑制活性的机制。在完成原计划基础上增加了独活药材抗炎活性成分指纹图谱的建立与抗炎活性成分筛选；在了解树突细胞抑制活性成分二氢欧山芹醇当归酸脂和欧前胡素基础上，开展了二氢欧山芹醇当归酸脂和欧前胡素免疫抑制作用机制研究；开展类似功效中药蛇床子、桑枝、羌活的化学成分分析方法研究；发表学术研究论文20篇，其中SCI收录的论文14篇。培养硕士生4名。