猪源罗伊氏乳杆菌对受损肠上皮细胞紧密连接的保护作用及其分子机制

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in piglets. Tight junction and cytoskeleton play a vital role in intestinal paracellular permeability by forming physical intercellular barriers in epithelial cells. However, the impact of ETEC on tight junctions and cytoskeleton has not been fully investigated. And, prevent and repair mechanism of Lactobacillus reuteri (L. reuteri), a probiotic bacterium, in tight junction and cytoskeleton of differentiated porcine intestinal epithelial cells exposed to ETEC was also not investigated. In this study, we used porcine-derived L. reuteri as tried strain, enterotoxigenic Escherichia coli to infect IPEC-1, transmission electron microscope to observe ultrastructure of tight junction structure, laser scanning confocal microscope to examine the distribution and structure of tight junction and cytoskeleton proteins, transcriptome profiling to reveal altered multiple pathways, western blot and antibody blocking technique to analyze the key singal targets, in order to reveal the protective role and molecular mechanism of L. reuteri in regulating tight junction IPEC-1 response to ETEC. In addition, may provide a further theoretical basis for the mechanism of L. reuteri eliminating infection of ETEC and relieving disease; and play an important practical significance for developing effective probiotic and promoting the health of piglets.

肠毒性大肠杆菌（ETEC）是引起仔猪腹泻的重要原因之一。紧密连接和细胞骨架对肠道上皮细胞的通透性具有重要作用。但ETEC感染后紧密连接结构损伤如何，以及罗伊氏乳杆菌（L. reuteri）对损伤细胞的紧密连接和细胞骨架的预防和修复机制也尚不清楚。因此本项目拟以猪源L. reuteri作为试验菌株，通过ETEC损伤IPEC-1；采用透射电镜观察上皮细胞间紧密连接的超微结构，激光共聚焦显微镜观察紧密连接及细胞骨架相关蛋白的分布表达，基因芯片转录谱技术揭示细菌与宿主细胞相互作用的代谢途径，结合western blot和抗体阻断技术解析相关通路及关键信号靶点，来揭示L. reuteri对受损的IPEC-1紧密连接的保护作用及相关分子机制，以期为L. reuteri能消除肠道ETEC感染和缓解疾病的机制提供进一步的理论依据，对开发高效的益生菌及促进仔猪健康均具有重要的现实意义。

肠毒性大肠杆菌（ETEC）是引起仔猪腹泻的重要原因之一。紧密连接和细胞骨架对肠道上皮细胞的通透性至关重要，而乳酸杆菌在维护肠道屏障功能完整性方面发挥重要作用。仔猪感染ETEC后肠道紧密连接结构如何损伤，以及罗伊氏乳杆菌（L. reuteri）对损伤细胞的紧密连接和细胞骨架的预防和修复机制也尚不清楚。本项目通过构建ETEC损伤IPEC-1细胞模型，采用激光共聚焦显微镜、透射电镜、荧光定量PCR、信号阻断技术以及Western blotting等技术手段研究ETEC对IPEC-1细胞紧密连接蛋白和细胞骨架蛋白的影响、L. reuteri对ETEC损伤的IPEC-1细胞的保护作用以及L. reuteri与IPEC-1相互作用的信号途径。研究结果发现：1）ETEC能破坏IPEC-1细胞形态完整性，降低IPEC-1细胞活力，破坏细胞之间紧密连接蛋白和骨架蛋白完整性；2）IPEC-1细胞感染ETEC后，紧密连接蛋白claudin-1、occludin和ZO-1以及细胞骨架蛋白F-actin基因转录水平明显下降，相应蛋白表达水平也明显降低；3）L.reuteri菌株可维护ETEC感染的IPEC-1细胞完整性，提高细胞活力，维持紧密连接蛋白和骨架蛋白完整性；4）L.reuteri菌株可提高claudin-1、occludin和ZO-1以及F-actin基因表达水平，提高紧密连接蛋白和细胞骨架蛋白表达水平；5）L.reuteri还可通过竞争性占位机制抑制ETEC对肠上皮细胞的粘附，从而减少ETEC对肠上皮屏障功能的破坏；6）通过对L.reuteri与IPEC-1相互作用关键基因和蛋白表达进行分析，发现L.reuteri菌株可能通过ILK途径与MLCK信号途径与IPEC-1细胞相互作用，从而维护肠道屏障功能完整性。研究结果表明，L. reuteri可消除肠道ETEC感染，并作为有前景的益生菌在养猪生产中使用。