基于免疫抑制组分谱效关系的东北道地药材类叶牡丹治疗类风湿性关节炎增效机制研究

Caulophyllum robustum Maxim which belongs to the family of Berberidaceae is widely distributed in Shanxi province,Gansu province,Sichuan province,the northeast of china and the far east of Russia. The officinal part of the plant was roots and rhizome which can cultivated. According to “Chinese traditional medicine dictionary”, the herb is a folk medicine that has been utilized widely to cure many diseases such as rheumatoid arthritis, stomachache and bellyache,fracture injuries and menstruate disorder etc. The effective chemical constituents of the plant are alkaloids and triterpenoid saponins..Previous studies have found that these components play a role of immune inhibition synergistic effect. we split effective parts into alkaloids, sapogenins and saponins of different components.Then establish a method for ELSD-HPLC fingerprint determination of the chemical constituents in vivo and vitro ,and a UHPLC-Q-TOF-MS analysis method for Analysis the change of endogenous metabolites in rats. We will evaluate immune synergy effect by metabolomics ,proteomics and genomics.Bsed on components spectrum-structure-activity relationships study synergy immune inhibition mechanism of Caulophyllum robustum Maxim by IL-1,IL-4,IL-10,IL-2,Bcl-2,Bax,FasL etal..We hope to find effective compounds,components,parts and relationships between them. we also hope to find immunosuppression synergy mechanism between components and the relationship between dose and structure-activity relationship. To show anti-rheumatoid arthritis treatment efficacy material base of Caulophyllum robustum Maxim, including the effect of body (a combination of ingredients group)..This study is to explore a new method for traditional Chinese medicine (TCM) in the chemical composition of different components of immunosuppression synergy effect,and to establish a new way of analysis and biological activity evaluation of traditional Chinese medicine efficacy material.At the same time, this study explain class anti-rheumatoid arthritis resistance mechanism of Caulophyllum robustum Maxim in the northeast Chinese. The aim of the study led was to study mechanism of traditional Chinese medicine components, which mixed with alkaloids and saponins for the treatment of RA . Meanwhile we hope to provide scientific reference data for other same research.

类叶牡丹（Caulophyllum robustum Maxim）是一种抗类风湿性关节炎(RA)的东北地区特色中药，皂苷和生物碱为主要药效成分。前期研究发现，各组分间存在着免疫抑制协同作用。本课题在已确定类叶牡丹有效部位的基础上，将其拆分成生物碱、皂苷元及不同结构的皂苷等组分，采用体内外ELSD-HPLC和UPLC-Q-TOF/MS技术，分析大鼠体内的内源性代谢物变化轨迹，筛选类叶牡丹免疫抑制组分协同作用的潜在生物标志物。通过RA模型动物灌胃给药，考察不同组分及其不同剂量配伍，对IL-1、TNFα、IL-4 、Bcl-2及FasL等细胞因子表达的影响。采用多元线性分析等数理统计学方法，对免疫因子信息与配伍组分体内、外HPLC指纹图谱化学信息进行相关性研究，从而寻找出类叶牡丹治疗RA的免疫抑制协同作用机制，揭示含有生物碱与皂苷类治疗RA中药配伍机理，为其他免疫抑制中药作用机制研究提供参考。

类叶牡丹（Caulophyllum robustum Maxim）是一种抗类风湿性关节炎(RA)的东北地区特色中药，性温，味辛、苦。皂苷和生物碱为主要药效成分，然而其抗RA作用机制并不明确。本研究发现类叶牡丹能够调控细胞因子的表达减轻炎症，降低血管翳形成，增加骨保护和促进异常增生的滑膜细胞的凋亡来发挥治疗RA的作用。通过谱效关系研究发现，不同地区、不同采收年限的类叶牡丹药材指纹图谱既有相似也有不同。药材的药效受到纬度和降雨量的影响，其抗炎活性也存在差异，且东北地区的药材抗炎作用较好，谱效结果也证实了类叶牡丹抗RA作用是多种成分协同起效的，这为类叶牡丹中有效物质的辨识奠定了基础。RA模型动物实验结果表明，总皂苷和总生物碱配伍后的作用优于单独使用。61个三萜皂苷化合物被分离得到，包括32个首次从类叶牡丹中分离得到的成分和18个新化合物。药代动力学研究发现，Cauloside C、Cauloside D、Cauloside G、Cauloside H、Leonticin D五种皂苷和Magnoflorine均表现出快吸收慢消除的趋势；小肠的吸收机制为被动转运，十二指肠的渗透系数最高；多分布于肝脏；主要以尿液排泄方式为主。通过代谢组学研究首次发现类叶牡丹对CIA大鼠体内的14个生物标记物（酪氨酸、苯丙氨酸、苯甲酸、马尿酸、假麻黄碱、吲哚酚、吲哚-3-乙醛胺、牛磺酸、酮戊二酸、甘氨酸、鸟氨酸、赖氨酸、蛋氨酸、组氨酸）的含量有显著调节作用，可以明显影响嘧啶、氨基酸和三羧酸循环等代谢网络通路。首次通过基因表达谱分析，发现差异基因表达总数达384条，其中202条为上调，182条为下调，影响了NF-κB、MAPK、TNF、Toll样受体等多种炎性通路，能够抑制类风湿性关节炎、系统性红斑狼疮等自身免疫疾病的通路，对于VEGF、Wnt/Jak-stat骨细胞、细胞凋亡及细胞癌通路等也有调节作用。本研究是由点及网、从宏观到微观、逐层推进的系统性研究，科学阐释了类叶牡丹有效部位抗RA的作用机制，这种作用与多靶点效应和差异性代谢网络是密切相关的，也对其他免疫抑制的抗类风湿性关节炎中药提供了方法学研究参考，为药物的临床应用提供更加可靠的依据。