菌毛蛋白SpaC介导LGG诱发斑马鱼肠道黏膜损伤的分子机制

Probiotics is one of the most perspective substitution of antibiotics applied in fish feeding, intestinal adhesion is necessary for fish feeding probiotics. At early stage, applicant of the project used zebrafish as research model, screened Lactobacillus rhamnosus GG for its highly adhesion of zebrafish intestine, however, zebrafish not showed resistance to infection when fed with different dose of LGG. Further study found that LGG mainly located in zebrafish intestinal mucosa and induced intestinal mucosa injury, which attracted applicant with great research interest on account of SpaC pilin was the decisive factor for LGG adhesion in host intestine. Therefore, applicant plan to establish zebrafish intestinal epithelial cells line model, analyze the molecular mechanism of zebrafish intestinal epithelial cells injury that induced by LGG from the signaling pathways of inflammatory, apoptosis and necroptosis by the technology of histopathology, cell morphology, real-time PCR and western blot. The objective of this study is to provide some valuable references for security application of feeding probiotics, and further put forward a selection criteria of fish feeding probiotics based on the protection function of intestinal mucosa barrier.

益生菌是最有前景的鱼类饲用抗生素替代品之一，肠道粘附是鱼类饲用益生菌的前提条件。申请人前期以斑马鱼为模型，筛选获得鱼类肠道强粘附乳杆菌鼠李糖乳杆菌Lactobacillus rhamnosus GG（LGG），然而LGG梯度饲喂斑马鱼并未获得预期抗感染表型。深究发现LGG主要粘附在斑马鱼肠道黏膜上并诱发黏膜损伤，菌毛蛋白SpaC是LGG肠道粘附核心决定因子，激发申请人极大科研兴趣。因此，申请人拟通过建立斑马鱼肠上皮细胞模型，借助组织病理学、细胞形态学、real-time PCR和western blot等技术，从炎症反应、细胞凋亡和坏死性凋亡信号通路分析菌毛蛋白SpaC介导LGG诱导斑马鱼肠上皮细胞损伤的分子机制，旨在为饲用益生菌的安全应用提供参考，进一步提出基于肠黏膜屏障功能保护的鱼类饲用益生菌优化筛选标准。

鼠李糖乳杆菌GG (Lactobacillus rhamnosus GG, LGG)是一种应用成熟的益生菌，有利于促进哺乳动物肠道黏膜屏障完整性和免疫调控机能。然而，本研究显示，LGG致损斑马鱼肠道黏膜屏障。LGG导致的肠损伤作用可归因于菌毛蛋白亚基SpaC蛋白诱发的肠细胞死亡。SpaC通过直接与TLR4ba受体结合而与肠上皮细胞相互作用，从而介导肠黏膜损伤。此外，我们发现SpaC与TLRs具有物种特异性相互作用，对斑马鱼的TLR4ba具有高的结合力，而不能与人TLR4受体结合，这可能是导致物种特异性肠道损伤的原因。本研究表明，来自不同宿主物种的益生菌可能存在安全性隐患，这有助于我们进一步了解益生菌的宿主特异性有益/有害机制。