基于PI-IBS肠机械屏障重塑探讨白术/防风药对止泻的机制

Post infectious irritable bowel syndrome（PI-IBS） is the common type of IBS. Intestinal mechanical barrier injury is the key pathological point of PI-IBS diarrhea. Tong-xie-yao-fang prescription is the most commonly used and effective prescription for the treatment of PI-IBS. Theoretical and Experimental Researches have showed that couplet medicines of Rhizoma Atractylodis macrocephalae and Saposhnikovia divaricata which had the effect of restoring deficiency and dispersing evil could synergistically improve the diarrhea and intestinal barrier permeability. It is regarded as the key compatibility of medicines in Tong-xie-yao-fang prescription. Rhizoma Atractylodis macrocephalae can repair the intestinal epithelial cells and the tight juntction, while Saposhnikovia divaricata has the effect of anti-inflammatory and inhibiting the expression of intestinal mucosal PAR-2 and MLCK. Therefore, we assume that the effect of Rhizoma Atractylodis macrocephalae and Saposhnikovia divaricata is through repairing the intestinal mechanical barrier, eliminating the factors which make intestinal barrier damage and regulating the key signaling pathways of intestinal barrier dysfunction, which shows a synergistic effect of multiple targets and multiple ways .Accordingly, the effects of Rhizoma Atractylodis macrocephalae and Saposhnikovia divaricata on intestinal mechanical barrier function, structure and intestinal barrier damage factors in PI-IBS animals will be investigated by fluorescence imaging technique. Moreover, the effects of Rhizoma Atractylodis macrocephalae and Saposhnikovia divaricata on PAR-2 /CaM/MLCK signaling pathway in Caco-2 cell will be studyed by gene silencing technology . These will clarify the effect and molecular mechanism of Rhizoma Atractylodis macrocephalae and Saposhnikovia divaricata on remodeling intestinal mechanical barrier . This project will reveal the scientific connotation of restoring deficiency and dispersing evil and discover new target and new idea for the treatment of this disease in TCM. In addition, it has important academic significance on illustrating the theory of properties of chinese medicinal herbs.

感染后肠易激综合征(PI-IBS)是IBS常见类型，肠机械屏障损伤是导致PI-IBS腹泻的关键病理环节。传统名方痛泻要方为治疗该病最常用有效方剂。理论及拆方实验研究发现，“补散”药对白术/防风能协同止泻、改善肠屏障通透性，为痛泻要方起效的关键配伍，而其中白术可修复肠上皮细胞与紧密连接，防风能抗炎、抑制肠粘膜PAR-2、MLCK表达，故我们认为：白术/防风协同止泻是通过修复肠机械屏障、消除肠屏障损伤因子、调控肠屏障损伤关键信号通路，多靶点、多途径起效的。据此，本项目拟应用活体荧光成像、基因沉默等技术观察白术/防风对PI-IBS动物肠机械屏障功能和结构，肠屏障损伤因子及Caco-2细胞PAR-2 /CaM/MLCK信号通路的调控效应，阐明其重塑肠机械屏障的作用及其分子机制。旨在揭示白术/防风补散配伍的科学内涵，为中医药治疗本病的研究提供新靶点新思路，对阐释中药药性理论具有重要的学术意义。

本项目从整体动物水平观察白术/防风药对对PI-IBS大鼠腹泻、机械屏障功能和结构、肠道屏障损伤因子的作用，发现白术防风药对具有显著的止泻止痛作用，可显著降低D-乳糖含量，修复肠黏膜机械屏障，恢复肠上皮细胞间紧密连接结构，增加结肠粘液蛋白 Muc 1 和 Muc 2的表达，改善PI-IBS大鼠肠机械屏障超微结构，下调肠粘膜屏障Occludin、Claudin-1、ZO-1紧密连接蛋白表达，调节肠道菌群（抑制大肠杆菌和肠球菌，扶植双歧杆菌和乳酸菌），降低炎症因子IL-6、IL-8、IFN-γ、IL-18含量及总蛋白酶活性，阐明了其通过重塑 PI-IBS 肠机械屏障的达到止泻的作用机制；从细胞水平研究白术/防风药对对肠屏障功能和肠机械屏障重塑PAR-2/CaM/MLCK信号通路的调控作用，研究发现白术/防风药对可提高细胞跨膜电阻（TEER）和降低荧光黄透过率（PFP），降低LDH活性，抑制PAR-2基因表达和MLCK活性，下调CAM蛋白表达，揭示白术/防风药对重塑肠机械屏障的分子机制。通过动物和细胞实验，揭示了白术/防风药对“补散兼施、固肠止泻”的作用机制和科学内涵，对阐释中药药性理论具有重要的学术意义。伴随项目的完成和目标的实现，目前已发表SCI论文1篇，中文科技论文2篇，尚有2篇论文待发表。参加学术会议4次，会议交流论文1篇，培养研究生2名。