基于QOUH理论研究抗UC特色蒙药嘎日迪散修复肠粘膜屏障损伤促进粘膜下组织重建的分子机制

Ulcerative colitis(UC), as one of psychocomatic diseases, belongs among "Bao Ru Diseases" theory of Mongolian Medicine and is regarded as a disease that is very difficult to heal through modern medical treatments. Garidi pulvis is a very effective Mongolian medicine treating UC and without relapse. Based on plenty of previous researches and QOUH theory, from the two core pathological aspects of the UC: the intestinal mucosal barrier injury and differences of submucosal tissue structure and function, the current project is proposed to systematically study cellular immune function imbalance, abnormal cytokine regulatory network and inflammatory changes of the intestinal epithelial mucosa, the relationship of TLRs / NF-kB signal transduction pathway and intestinal mucosal barrier injury. To explore the role of drugs on the regulation of apoptosis of mucosal cells from the mitochondrial pathway and the classical pathway, this reveals the UC recurrent deeply pathological mechanisms. Proposed and verify the hypothesis: UC treatment key is regulating the immune imbalance, correcting proportion of cytokines, restoring the structure of functional status of the submucosal organizational structure . This project will surely provide experimental clues for Garidi pulvis and scientific explanation for the "Bao Ru Diseases" treatment theory "decreasing viscosity and detoxification, transiting Heyi, eliminating necrotic tissues and promoting granulation"of Mongolian medicine.

溃疡性结肠炎（UC）属蒙医"宝如病"范畴，其病机不清，愈后复发，系内科疑难病症。嘎日迪散是治疗UC的特效蒙药，具有愈后不复发的特点。本课题在前期研究工作基础上，基于QOUH理论，从UC发生发展、迁延不愈的两个核心病理环节：肠粘膜屏障损伤和粘膜下组织结构与功能成熟度差异入手，综合研究细胞免疫功能失衡、细胞因子调控网络异常与肠上皮粘膜炎症性变化，TLRs/NF-kB信号转导通路与肠粘膜屏障损伤的关系，并从线粒体途径和经典途径分别探讨药物对粘膜细胞凋亡的调节作用，从而揭示UC反复发作的深层病理机制。提出并验证假说："UC的治疗关键-调节免疫失衡，纠正细胞因子比例，恢复粘膜下组织结构的功能状态"，为确有疗效的民族药嘎日迪散抗UC复发提供实验依据，为蒙医治疗"宝如病" 的"消粘解毒，通行赫依，祛腐生肌"治则提供科学诠释。

本项目为验证“UC治疗的关键—调节细胞免疫失衡，纠正细胞因子比例，恢复黏膜下组织结构的功能状态”这一假说，利用免疫法加TNBS诱导法复制UC大鼠模型，根据治疗期间动物的行为变化，治疗后结肠组织的损伤指数、结肠组织的HE染色、结肠组织的病理学得分以及扫描电镜等结果验证了嘎日迪散能有效修复UC大鼠受损伤的黏膜。通过流式细胞技术检测了UC大鼠外周血和治疗后各组动物外周血中CD4+、CD8+、Th1、Th2和Th17变化，显示嘎日迪散能有效调节UC大鼠CD4+/CD8+、Th1/Th2和Th1/Th17的平衡，证实嘎日迪散能调节UC大鼠细胞免疫失衡。通过ELISA反应检测了各组大鼠外周血和结肠组织中致炎因子IL-1β、IL-6、IL-12、TNF-α、IFN-γ以及抗炎因子IL-4和IL-10变化，结果显示嘎日迪散及阳性药能不同程度的降低各致炎因子在血清和结肠组织中的含量，降低炎症因子对黏膜的进一步的损伤，同时升高抗炎因子IL-4和IL-10含量对抗炎症反应，证实嘎日迪散能纠正失调的细胞因子网络。通过Tunel和凋亡相关因子的检测，证实嘎日迪散能通过Bcl-2/Bax介导的细胞凋亡途径促进黏膜上皮及黏膜固有层炎症细胞的凋亡，而加速上皮细胞的更新和炎症细胞的减少；通过V.G染色，CD105、ICAM-1的免疫荧光染色以及western-blot检测EGF、VEGF、FGF及其受体在结肠组织中的相对表达量，显示嘎日迪散能通过减少ICAM-1在新生黏膜中的表达、减少溃疡愈合处胶原纤维的含量、促进新生组织中毛细血管的成熟及提高EGF、VEGF、bFGF及其受体在新生黏膜中的表达来提高溃疡愈合的组织成熟度和功能成熟度。本项目部分阐释了蒙药治疗包如病的治则：“消粘解毒、通行赫依、袪腐生肌”，的现代科学内涵，揭示了嘎日迪治疗UC愈后不易复发的分子机理