基于肠道微环境探讨脾虚导致痹症（类风湿关节炎）加重的机制研究

Rheumatoid arthritis (RA) is one of Bi syndrome in traditional Chinese medicine, and spleen deficiency is one of the basic pathogenesis of Bi syndrome. Our previous studies showed that the disorders of intestinal mucosal immune system under the condition of spleen deficiency can aggravate the progress of arthromyodynia. It was also reported that intestinal flora disturbance is not only an important pathological factor of spleen deficiency syndrome, but also involved in the pathogenesis of RA. As an important part of intestinal micro environment, intestinal flora and mucosal immunity are closely related to the pathogenesis of RA. Above all, we put forward the new hypothesis that the spleen deficiency could induce the aggravation of Bi syndrome (CIA): In the state of spleen deficiency, the disorder of intestinal flora leads to the disorder of intestinal mucosal immune system, which further aggravates the Bi syndrome. In the present research, the pre-established stable spleen deficiency CIA model of DBA / 1 mice and metagenomics sequencing technologies combined with bioinformatics method will be used to draw the intestinal bacteria group lineages and to elucidate intestinal micro environment characteristic of spleen deficiency with Bi syndrome. Flow cytometry and ELISA will be used to investigate the relationship and the effect of intestinal flora disturbance on intestinal mucosal immune system. Finally, Xiongfu powder will be used as the tool medicine to verify the mechanism of spleen deficiency aggravated Bi syndrome. The results will provide new ideas on the treatment of rheumatism from the spleen.

类风湿关节炎（RA）属中医“痹证”范畴，脾虚是其基本病机之一。课题组前期研究发现，脾虚证小鼠肠道黏膜免疫失调可加重关节炎发病。新近研究表明，肠道菌群紊乱不仅是脾虚证的重要病理变化之一，而且参与RA发病。作为肠道微环境重要组成部分的肠道菌群和黏膜免疫均与RA发病密切相关。基于此，我们认为脾虚加重痹症与肠道微环境密切相关，并提出新的假说：脾虚状态下，肠道微环境的肠道菌群发生了紊乱，这种紊乱导致与之紧密联系的肠道黏膜免疫系统失调，进而加重了痹症。因此，本项目拟基于前期建立的DBA/1小鼠脾虚痹症（CIA）模型，运用宏基因测序结合生物信息学方法，绘制DBA/1小鼠脾虚痹症肠道菌群谱系，初步阐明其肠道微环境特征；采用流式细胞术、ELISA等方法，研究肠道菌群紊乱对肠道黏膜免疫系统的影响并进行性相关性分析。最后以芎附散为工具药，对脾虚加重痹症的机制进行反证。研究结果将为痹症从脾论治提供新的思路和靶标。

类风湿关节炎（Rheumatoid arthritis，RA）属中医学“痹证”范畴，中医理论认为，脾虚是痹证（RA）的基本病机之一。本课题前期基于DBA/1小鼠脾虚（大黄泻下加饥饱失常）叠加痹症（Collagen Induced Arthritis，CIA）模型研究发现，脾虚痹症小鼠肠道黏膜免疫系统存在异常，且脾虚能够加重痹症。另有研究表明，脾虚与肠道菌群紊乱联系紧密，肠道菌群紊乱及肠道黏膜免疫失调与RA疾病程度关系密切。因此，深入探讨脾虚证肠道菌群紊乱导致肠黏膜免疫系统改变对痹症进展的影响，阐明脾虚加重痹症的内在机制，对中医药痹症从脾论治的治疗靶点选择和药物疗效评价研究均具有非常重要的意义。本研究按照任务书的计划进度执行了相关科研任务，并根据课题研究需要，设计完成了任务书外的其他内容，增加了胶原诱导关节炎（CIA）大鼠不同部位肠道菌群特征研究，结果表明CIA大鼠回肠菌群变化与疾病特征更相关，为本研究肠道菌群部位的选择提供了有力支持；对任务书中个别指标进行调整，如考虑到样本量获取的可行性，将实验动物由DBA/1小鼠变更为SD大鼠，变更后不影响实验动物造模成功率。本课题在前期工作基础上，基于SD大鼠脾虚痹症（CIA）模型复制及古方芎附散疗效观察，运用16s测序、宏基因测序、流式细胞、Micro CT、分子生物学等技术，结合组织细胞病理学、酶联免疫组织化学、生物信息学、微生物学等方法，阐明了脾虚痹症（CIA）模型大鼠肠道菌群特征，探究了肠道菌群改变诱发肠道黏膜免疫系统变化导致痹症加重的内在机制，并予以芎附散反证，从肠道微环境层面阐释了脾虚加重痹症机理。本项目表明脾虚状态下肠道菌群紊乱能诱发肠道黏膜免疫失调，从而加重痹症；也证实了脾虚状态下肠道菌群紊乱与肠道黏膜免疫失调存在相关性。