植物乳杆菌调控仔猪肠细胞先天免疫应答和谷氨酰胺转运功能的研究

Lactic acid bacteria are well-accepted as the probiotics that could provide health benefits to the animal and human gut. However, the molecular mechanism of their regulation on intestinal innate immune system remains unclear. Our previous studies have shown that Lactobacillus plantarum CGMCC 1258 can improve newborn piglet growth potential and regulate the innate immune responses in the intestinal mucosa upon enterotoxigenic Escherichia coli K88 (ETEC) challenge. In this proposal, we will therefore study the molecular mechanism of the regulation of innate immune responses in piglet enterocytes by L.plantarum, and unravel the potential interaction between innate immune responses and the capability of glutamine transport. With the established co-culture systems of different bacteria with piglet enterocytes (IPEC J2 cells), advanced quantitative proteomic approaches and a series of molecular and immunological techniques will be utilized to systematically analyze the cell permeability, cell integrity, major signaling proteins in innate immune responses and the capability of glutamine transport. Together, these studies should lead to fundamental insights into immune control of bacterial infection by L.plantarum on piglet intestinal mucosa, and will provide new perspectives on application of L.plantarum as a safe substitute for antibiotics to preventatively alleviate piglet diarrhea caused by ETEC and improve piglet growth in the swine industry.

乳酸菌是公认的改善动物和人类肠道健康的益生菌，但其对肠道先天免疫应答的作用机理尚不清楚。本课题组前期研究发现，植物乳杆菌可改善新生仔猪生长性能，并调控肠道黏膜应对大肠杆菌ETEC K88感染的先天免疫应答。本课题拟通过建立微生物-肠细胞共培养模型，采用最新的定量蛋白质组学、蛋白质印迹等技术，从细胞和分子水平，系统研究植物乳杆菌和大肠杆菌ETEC K88对肠细胞屏障功能、胞内信号通路关键蛋白、细胞因子等先天免疫应答特征和谷氨酰胺转运功能的影响，以阐明植物乳杆菌调控仔猪肠道上皮细胞应对大肠杆菌ETEC K88感染的先天免疫应答的作用机理，探讨肠道上皮细胞免疫状态与谷氨酰胺转运功能的相互关系。该研究将为益生菌替代抗生素应用于预防仔猪腹泻和改善生长提供理论依据。

产毒性大肠杆菌（Enterotoxigenic Escherichia coli, ETEC）引起的新生和断奶仔猪腹泻是困扰养猪业的重大问题。本课题组前期发现，植物乳杆菌（Lactobacillus plantarum, LP）可显著降低新生仔猪的腹泻率，改善生产性能，可能与改善肠道屏障和免疫功能有关，但其作用机理有待研究证实。本项目以仔猪肠上皮细胞系（IPEC-J2）为研究对象，摸索了LP最佳MOI为75，处理时间为6 h，摸索了ETEC最佳MOI为50，处理时间为3 h，建立了LP-ETEC-IPEC-J2细胞共培养模型，并在该模型下研究了预处理LP对ETEC感染的IPEC-J2细胞屏障功能、先天免疫功能和转运载体表达的影响，最后通过定量蛋白质组学阐释LP的可能作用机理。结果表明，ETEC显著降低了IPEC-J2细胞紧密连接蛋白ZO-1、Occludin和Claudin-1的mRNA表达量以及Claudin-1和Occludin的蛋白表达量，显著降低了细胞TEER值，显著下调葡萄糖转运载体和氨基酸转运载体的表达量，但显著提高了炎性细胞因子的表达量以及氯离子转运载体的表达量；而预处理LP能够抑制IPEC-J2细胞的TEER值和紧密连接蛋白的表达量的下降，降低炎性因子的表达，调节转运载体的表达，缓解ETEC对细胞屏障的损伤，发挥保护肠上皮细胞屏障功能及营养物质转运功能的作用。定量蛋白质组学显示，ETEC组与对照组有90个差异蛋白；LP 与对照组有105个差异蛋白，LP+ETEC组与ETEC组有133个差异蛋白，其中ETEC攻毒后肠上皮细胞差异表达蛋白主要富集在MAPK信号通路，而且氨基酸合成和碳代谢通路显著受到抑制。Western blotting验证显示，LP的保护作用可能与TLRs和MAPK信号通路有关。本研究结果为植物乳杆菌在养猪生产中的应用提供科学依据，可为抗生素替代品的研发提供理论支持，有助于养猪业的健康发展。