α-酮戊二酸调控TNF-α/MLCK通路保护鲤肠道紧密连接的机制

There are a large number of pathogenic bacteria in the aquatic environment, some bacteria cause diseases by destroying the intestinal barrier function. The integrity of the intestinal barrier to prevent the invasion of the bacteria, protect the body health, tight junction (TJ) is an important component of the intestinal barrier. Previous studies have shown that α-ketoglutarate (AKG) can improve the expression of intestinal protein synthesis gene and reduce intestinal permeability of fish, but its mechanism of regulating intestinal barrier is not clear. Therefore, this project is through the normal hysiological state, Aeromonas hydrophila stimulation, combined with intestinal epithelial cells in vitro, using transmission electron microscope, RT-PCR, Western Blotting and immunofluorescence technique, to investigate the effects AKG on intestinal ultrastructure, the expression of TJ protein, and the regulation pathway (TNF-α / NF-κB / MLCK) of TJ protein, and to explore the mechanism of regulating intestinal TJ. The results provide scientific basis and guidance for protecting the intestinal barrier through nutrition regulation and improving the intestinal disease resistance.

养殖水环境中存在大量的致病菌，部分致病菌通过破坏肠道屏障功能导致疾病发生。完整的肠道屏障能够阻止致病菌的侵入，保障机体健康，紧密连接（TJ）是肠道屏障的重要组成部分。本课题组前期研究表明，α-酮戊二酸（AKG）能够提高鱼类肠道蛋白合成基因的表达量、降低肠道通透性，但其调控肠道屏障的机理尚不明确。为此，本项目拟通过正常生理状态、嗜水气单胞菌感染，同时结合体外肠道上皮细胞培养，利用透射电镜、RT-PCR、Western Blotting、免疫荧光等技术，考察AKG对鲤鱼肠道超微结构、TJ蛋白表达量的影响，挖掘AKG对肠道TJ蛋白调节通路（TNF-α/NF-κB/MLCK）的作用，以探明AKG调控肠道TJ的机制。其成果为通过营养调控保护肠道屏障、提高肠道抗病力提供科学依据和指导。

养殖水环境中存在大量的致病菌，部分致病菌通过破坏肠道屏障功能导致疾病发生。完整的肠道屏障能够阻止致病菌的侵入，保障机体健康，紧密连接（TJ）是肠道屏障的重要组成部分。试验旨在研究饲料中添加α-酮戊二酸（AKG）对松浦镜鲤生长性能、肠道健康的影响，结果发现，在饲料中添加0.4%AKG有利于提高松浦镜鲤的生长性能、促进肠道健康。感染嗜水气单胞菌后，饲料中添加1%的α-酮戊二酸能够显著提高松浦镜鲤的存活率。同时，α-酮戊二酸还可显著提高肠道免疫消化酶（溶菌酶、碱性磷酸酶）、补体C4浓度和总抗氧化能力。此外，α-酮戊二酸还可通过TLR4/MyD88/NF-KB信号通路显著下调TNF-α、IL-1β、IL-6和IL-8等促炎细胞因子的基因表达水平；并通过MLCK信号通路上调紧密连接蛋白（claudin-1、claudin-3、claudin-7等）的基因表达水平，并从细胞层面阐明了AKG调控紧密连接蛋白信号通路的机制。