基于“肠道菌群-肠上皮细胞”途径研究艾灸重建克罗恩病肠道免疫稳态的作用机制

Crohn's disease (CD) is a kind of chronic inflammatory injury of digestive tract which caused by ultra-immune response of ill-natured environmental on people has susceptibility genes. Intestinal flora has been confirmed plays an important role in the development and activation, the changes of intestinal flora may induce or aggravate CD. "Intestinal flora-intestinal epithelial cells" is an important way of immunity homeostasis reconstruction. Currently, adjust the structure of intestinal flora, restrain and reduce the intestinal inflammation, reconstruction of the intestinal immune homeostasis become a new way for the treatment of CD. Research shows that moxibustion can repair the connection between cells, improve the structure and function of epithelial barrier on CD. our study focusing on intestinal flora and intestinal epithelial cells and the interaction of intestinal immune system, stick to two key links of "intestinal flora imbalance" and "imbalances of Th17 / Treg cells " in the process and development on CD, using 16s rDNA gene segment, PCR, RFLP analysis technics, 1H-NMR metabolomics to reveal the regulation mechanism of reconstruction immunity homeostasis of moxibustion on Crohn's disease based on the pathway of "Intestinal flora-intestinal epithelial cells".

克罗恩病(Crohn’s disease，CD) 是具有易感基因的人群对不良的环境因素产生过激的免疫应答，从而导致累及消化道为主的慢性炎症性损伤。肠道菌群已被证实对肠道免疫系统的发育和激活具有重要作用, 肠道菌群变化可能诱发或加重CD，而“肠道菌群-肠上皮细胞” 是重建CD肠道免疫稳态的重要途径。目前，调节肠道菌群结构，抑制和缓解肠道炎症，重建肠道免疫稳态成为治疗CD的一种新方式。研究表明，艾灸能够修复细胞间紧密连接，改善CD结肠上皮屏障结构与功能。本研究以肠道菌群与肠上皮细胞和肠道免疫系统的相互作用为切入点，紧扣CD发生发展过程中的两个关键环节，即“肠道菌群失调”与“Th17/Treg细胞失衡”，运用16S rDNA基因片段PCR、RFLP分析技术、1H-NMR代谢组学技术等多种生物技术手段，基于“肠道菌群-肠上皮细胞”途径进一步揭示艾灸重建克罗恩病肠道免疫稳态的作用机制。

本项目紧扣克罗恩病（CD）发生发展过程中的“肠道菌群失调”与“Th17/Treg 细胞失衡”两个关键环节，采用艾灸疗法，以 CD 大鼠为受试对象，运用 16S rDNA 高通量测序技术、1H-NMR 代谢组学技术、HPLC-ECD、电子显微镜、Western Blotting 等多种生物技术手段，基于“肠道菌群-肠上皮细胞”途径进一步揭示艾灸重建克罗恩病肠道免疫稳态的作用机制。本研究结果显示，①艾灸可改善CD模型大鼠的大便性状、精神状态及活动情况，同时减轻DAI评分与结肠组织的炎症损伤，从而达到治疗或缓解克罗恩病的效果；②艾灸可以调节CD模型大鼠肠道菌群丰富度与多样性，艾灸对肠道菌群的紊乱具有良性的调节作用，其中拟杆菌、肠杆菌、以及乳酸杆菌可能为针灸治疗CD的关键细菌；③艾灸能够改善CD模型大鼠代谢紊乱状态，通过回调亮氨酸、异亮氨酸、赖氨酸、牛磺酸、肌醇、胆碱、肌酸、甜菜碱等代谢物调控肠上皮细胞增殖与凋亡平衡，缓解氧化应激减轻肠道炎症等改善CD症状；④艾灸可抑制CD大鼠结肠组织中IFN-γ、IL-17A的表达，调节黏膜 Th17/Treg 细胞平衡，改善结肠黏膜免疫状态，进而发挥治疗克罗恩病的作用；⑤艾灸可降低CD大鼠血清中TNF-α、IL-1β、IFN-γ的含量，改善结肠上皮黏膜紧密连接状态，重建克罗恩病肠道免疫稳态。以上实验明确艾灸对CD大鼠肠道菌群、Th17/Treg 细胞的调节作用，从机械屏障、免疫屏障等方面揭示艾灸重建克罗恩病肠道免疫稳态的作用机制，为针灸防治CD提供科学的理论基础和实验依据。