蒙药嘎日迪散纠正肠道菌群失衡调控分子伴侣表达治疗UC的机制研究

Ulcerative colitis (UC) is one of the most intractable diseases at present , and there are no effective therapies currently. New research shows that, the intestinal flora imbalance and chaperone system disorder is the key mechanism of the UC pathogenesis. And the immune and autoimmune regulation change is the intermediate link between their interaction. In this study We plan to research the Mongolian Herbal Medicine GaRiDi pulvis, as it has good curative effect and less recurrence on the UC treatment based on the previous project of the National Natural Science Foundation research. To draw the fingerprint of intestinal flora , the T-RFLP combined with capillary electrophoresis is to be used and through the cluster analysis, GaRiDi pulvis is to be researched about its effect on the intestinal tract flora species; and to investigate the GaRiDi pulvis's effect on the immune and autoimmune regulation of the UC rat model, immunohistochemistry, ELISA, Western-blot and Real-time PCR Technology are to be used; and the double immunofluorescence technique is to be used for observing the GaRiDi pulvis's effect on correcting dysfunctional molecular chaperone system. We would synthetically analysis the relationship among intestinal microflora, immune and autoimmune regulation and molecular chaperone system. And the primarily functional role pathway and effective molecule target of GaRiDi pulvis in the treatment of UC is to be illustrated. The key rule of UC treatment in Mongolian medicine, " Shugan turbidity, decreasing viscosity and detoxification, and transiting Heyi", is to be explained.

溃疡性结肠炎（UC）是现代难治病之一，目前尚无有效根治方法。最新研究表明，肠道菌群失衡和分子伴侣系统失调是UC发病的核心病理机制，而免疫和自免疫调节变化是两者交互作用的中间环节。本课题拟在前期国家自然科学基金项目研究的基础上，以治疗UC疗效好、复发少的特色蒙药嘎日迪散为研究载体，应用T-RFLP结合毛细管电泳法绘制肠道菌群指纹图谱，通过聚类分析，研究嘎日迪散对肠道菌群种属的影响；以IHC、ELISA、Western-blot及Real-time PCR技术探讨嘎日迪散对UC模型大鼠免疫与自免疫调控作用；以双免疫荧光技术观察嘎日迪散对分子伴侣系统失调的纠正作用。综合分析肠道共生菌群、免疫与自免疫调节及分子伴侣系统三者之间的关系，最终阐明嘎日迪散治疗UC的主要作用途径及效应分子靶点，从分子层面阐释蒙医治疗UC的核心治则"疏肝除浊、消粘解毒、通行赫依"的科学内涵。

本项目结合现代医学对UC的最新研究进展和最新研究结果，拟以肠道内环境菌群为靶点，采用高通量测序技术结合生物信息学分析研究DSS诱导的UC模型小鼠肠道内环境菌群失衡特征以及嘎日迪散对失衡菌群的调节作用。本项目从肠道菌群失调与分子伴侣系统障碍是UC的病理核心着手，根据UC发生的两个核心病理环节：肠道共生菌的数量和种类的改变能直接导致肠道共生菌的粘附性及其分泌物的改变，而这些分泌物的改变又影响肠黏膜的屏障作用，引起黏膜固有层先天免疫和获得免疫的改变；分子伴侣系统在机体获得性免疫和先天免疫中发挥着关键的连接交互作用，探讨嘎日迪散对肠道菌群、分子伴侣的调节，最终阐明嘎日迪散治疗UC的部分机制。方法：以肠道细菌16SrRNA为分子靶标，以大鼠结肠内容物中分离的总DNA为模板，采用高通量测序技术结合生物信息学分析探讨肠道菌群变化与UC发病的关系，揭示嘎日迪散对肠道菌群失衡的调节作用。同时通过western-blot和real-timePCR等技术研究与免疫应答相关的信号通路TLR2/4-NF-kB信号通路、与机体自免疫相关的hTM5的变化和分子伴侣热休克在蛋白水平和mRNA水平的变化。通过对结果进行分析得到以下结论：①嘎日迪散可通过直接影响毛螺菌科细菌来发挥对UC的治疗作用。②UC模型小鼠的结肠组织中MUC2的蛋白含量有所下降，其mRNA的水平不变，与疾病状态无关。③TLR2 /4不是NF-kB信号通路的上游调控蛋白。④嘎日迪散能提高hTm5在UC模型小鼠中的表达。⑤嘎日迪散在治疗UC模型小鼠时不是通过分子伴侣发挥其药效的。