草鱼肠道黏膜屏障的损伤与益生芽孢杆菌的保护机制

Intestinal disease is one of the most important diseases in aquaculture. Nevertheless, the integrity of intestinal mucosal barrier plays an important role in protection of fish to resist the invasion of pathogens and hazardous substances. Thus, maintaining the integrity of intestinal mucosal barrier is a significant means to keep animal healthy. Until now, the damage of intestinal mucosal barrier by pathogens infection and the repairing and protection mechanism of probiotic Bacillus is still unknown in the grass carp(Ctenopharyngodon idellus). In this project, we focuses on the mechanism of damage the intestinal mucosal barrier and protection of probiotic Bacillus in grass carp. Aeromonas hydrophila、TNF-α and probiotic Bacillus or alone was used to treat grass carp and primary cultured intestinal epithelium cells of grass carp, respectively. Then the structure and permeability of intestinal mucosal epithelial cells, and their the expression profile of tight junction related molecules, apoptosis related molecules, and inflammation and oxidation injury related molecules were analyzed by QPCR、western blot、and ELISA. The Aim is to reveal the impact of Aeromonas hydrophila on the intestinal mucosal barrier and its repairing and protection mechanism of probiotic Bacillus in grass carp, which could provide new sight for treating the fish intestinal inflammatory diseases and blocking the invasion pathway via the intestinal mucosa.

肠道疾病是当前水产动物重要病害，肠道黏膜屏障的完整对鱼类抵御病原生物及其他有害物质的入侵有重要作用，对肠道黏膜屏障功能的维持和保护是保持动物体健康的重要手段。鱼类肠道黏膜屏障损伤与益生芽孢杆菌的修复和保护机制，目前并不清楚。本项目围绕草鱼肠道黏膜屏障的损伤和益生芽孢杆菌对黏膜屏障保护的分子基础与作用机制，通过利用病原性嗜水气单胞菌、TNF-α、益生芽孢杆菌分别处理或共处理草鱼及其肠上皮培养细胞，采用免疫荧光、Western blot、ELISA、荧光定量PCR等方法，从肠黏膜上皮细胞的结构、通透性、紧密连接相关分子、细胞凋亡因子和凋亡通路信号因子，以及炎性因子及抗氧化应激因子等方面，揭示草鱼肠道黏膜屏障损伤机理及损伤修复过程中益生芽孢杆菌的作用及其保护机理，研究结果为改善鱼类肠道炎性疾病和阻断病原通过肠道黏膜的入侵途径提供新的思路。

肠道疾病是当前水产动物重要病害，肠道黏膜屏障的完整对鱼类抵御病原生物及其他有害物质的入侵有重要作用，对肠道黏膜屏障功能的维持和保护是保持动物体健康的重要手段，而饲用微生物（益生菌）是改善鱼类肠道健康状态、提高鱼体抗病力的重要手段。本项目通过利用病原性嗜水气单胞菌、LPS、益生芽孢杆菌分别处理或共处理草鱼及其肠上皮培养细胞以及Caco-2细胞，采用组织学、免疫荧光、Western blot、ELISA、荧光定量PCR等方法，分析了益生芽孢杆菌对草鱼肠黏膜上皮细胞的结构、通透性、紧密连接相关分子、细胞凋亡因子和凋亡通路信号因子、炎性因子及抗氧化应激因子、以及益生菌对病原菌毒力等方面的影响。结果显示，益生芽孢杆菌能有效地保护和修复肠黏膜和肠上皮细胞结构、改善肠上皮细胞的紧密连接、降低由嗜水气单胞菌感染引起的通透性升高、减少病原菌对肠上皮细胞的黏附、调节肠上皮细胞的凋亡过程，减轻因嗜水气单胞菌感染造成的损伤；同时，益生芽孢杆菌能有效地提高草鱼的抗氧化能力和免疫能力，使其免受由嗜水气单胞菌感染引起的氧化应激损伤，减轻嗜水气单胞菌感染引起的炎症反应；益生芽孢杆菌还可通过群体感应作用，降低嗜水气单胞菌的毒力基因表达水平和溶血能力，减弱其毒性。研究结果充实了益生芽孢杆菌对草鱼肠道黏膜屏障损伤的修复与保护的分子基础和作用机理等理论知识；对在水产养殖上更好地利用益生菌来修复肠道黏膜损伤、保护鱼类肠道黏膜功能、促进鱼体的健康、防治肠道炎性疾病、阻断病原通过肠道黏膜的侵入途径，具有重要的意义。