干酪乳杆菌通过VIP/MCs调控微生物-脑-肠轴对断奶仔猪肠道屏障损伤的修复机制

Intestinal barrier function impaired by early weaned stress is usually easy to cause illness. Probiotics can improve host intestinal health, but the specific mechanism is unclear. Intestinal flora reconstruction can affect the nerve-endocrine-immune signaling pathways mediated microbiota-brain-gut axis. Vasoactive intestinal peptide (VIP) is an important neurotransmitter mediated brain-gut axis. Mast cells (MCs) is a key effector cells mediated brain-gut axis. Probiotics can inhibit the activation of MCs. Our previous research indicate that lactobacillus casei (L.casei 393) can effectively antagonism pathogenic bacteria ETEC K88 protect porcine intestinal epithelial barrier function, and ameliorate the increase in human intestinal epithelial permeability after activation of mast cells. Moreover, VIP can effectively regulate intestinal mucosal immunity, and maintain the intestinal microecological balance in early weaned piglets. The project will be conducted to investigate the repairing effect of L.casei 393 on the intestinal barrier function injured by early weaned stress or infection and its relationship with the VIP/MCs mediated microbiota-brain-gut axis from the level of molecule-cell-tissue-animal, and through in vitro intestinal tissue perfusion system experiment, especially establishment of L.casei 393/VIP/MCs/intestinal epithelial cell co-culture model, VIP receptor antagonist/MCs blockade model and ETEC K88 induced-intestinal infection model. The research will provides new ideas, and explore new ways for prevention and control of intestinal disease caused by weaned stress or infection.

早期断奶应激导致肠道屏障功能受损而易诱发疾病。益生菌能改善宿主肠道健康，但具体机制尚不清楚。肠道菌群重建会影响微生物-脑-肠轴的神经-内分泌-免疫信号通路，血管活性肠肽VIP是介导脑-肠轴的重要神经递质，肥大细胞MCs是脑-肠轴上关键效应细胞。申请人前期发现，干酪乳杆菌（L.casei393）能拮抗ETEC K88保护猪肠上皮细胞屏障功能，缓解MCs活化所致肠上皮细胞通透性增加；VIP能调节断奶仔猪肠黏膜免疫，维护肠道微生态平衡。本项目拟通过离体肠道组织尤斯灌流系统实验，建立L.casei393/VIP/MCs/肠上皮细胞共培养、断奶仔猪VIP受体拮抗、MCs阻断和ETEC K88肠道感染模型，从分子-细胞-组织-动物水平，探究L.casei393对断奶仔猪肠道屏障损伤修复作用及其与VIP/MCs介导微生物-脑-肠轴的关系，旨在为防治断奶应激或感染所致肠道疾病的发生提供新思路、开拓新途径。

益生菌可以改善肠道微生态，维护肠道健康。肠道菌群重建会影响微生物-脑-肠轴的神经-内分泌-免疫信号通路。血管活性肠肽是介导脑-肠轴的重要神经递质，肥大细胞是脑-肠轴上的关键效应细胞。本项目通过体内外实验探究了干酪乳酸杆菌（L.casei ATCC 393）对肠道屏障功能损伤修复的作用及其与VIP/MCs的分子关联。取得的主要研究结果如下：（1）L.casei ATCC 393可通过模式识别受体PRRs介导的信号通路有效缓解产毒素大肠杆菌ETEC K88感染所致猪肠上皮细胞IPEC-J2屏障功能损伤以及ETEC K88感染所致的C57BL/6小鼠肠道屏障功能损伤；（2）L.casei ATCC 393显著抑制了ETEC K88感染所致的猪肠黏膜肥大细胞的活化及肥大细胞介质β-hex、 histamine、tryptase、 IL-6、IL-8、GM-CSF、IFN-γ和TNF-α的释放，缓解了感染所致的IPEC-J2细胞TEER值的降低以及FITC-Detran跨细胞通透率的增加；L.casei ATCC 393具有MCs稳定剂的作用，其可通过TLRs介导的MCs通路缓解ETEC K88感染所致的肠道屏障功能损伤；（3）VIP前处理显著抑制了ETEC K88和LPS感染所致的MCs的活化以及MCs介质的释放；VIP能够与L.casei ATCC 393协同作用抑制ETEC K88感染所致的IPEC-J2细胞通透性的增加；VIP受体拮抗剂处理显著抑制了L.casei ATCC 393对小鼠肠道屏障功能的保护作用，提示VIP可能通过MCs介导L.casei ATCC 393对肠道屏障功能损伤的保护作用；（4）L.casei ATCC 393及其代谢物能有效拮抗ETEC K88对IPEC-J2细胞的毒性作用；L.casei ATCC 393代谢物能够通过NLRP3介导的信号通路缓解DSS诱导C57BL/6小鼠结肠溃疡性炎症反应；（5）本项目成功构建了L.casei ATCC 393分泌蛋白p40的大肠杆菌重组表达载体，但是其诱导表达及分离纯化技术方法还在探究建立中。该项目研究结果将为应激或感染所致的早期断奶仔猪肠道疾病高效防治措施的建立提供新思路、开拓新途径，将为安全、高效微生态饲料添加剂的研制与应用奠定重要理论基础。