鼠李糖乳杆菌GR-1通过MyD88依赖性和非依赖性信号途径抗大肠杆菌诱导奶牛子宫内膜上皮细胞损伤的分子机制

Endometritis is an important disease of dairy cows that leads to significant economic losses in the dairy cattle industry. Endometrial infection of Escherichia coli is one of the major causes of this disease. Previous research has shown that Lactobacillus rhamnosus GR-1 can inhibite the activation of NF-κB and the release of inflammatory factors induced by Escherichia coli, resulting the reduction of bovine endometrial epithelial cells (BEEC) inflammatory injury. However, the molecular mechanism still remains unclear. Thus, in the present study, BEEC damage model induced by Escherichia coli will be established. Using the MyD88-dependent and independent signaling pathways as an entry point, combined with the RNAi method and specific transduction inhibitors, then the BEEC apoptosis will be detected by flow cytometry, and the expression levels of key signaling molecules in the NF-κB signaling pathway, tight junction proteins, TLR4 and its downstream adaptor molecules will be tested by immunoblot assays and real-time quantitative PCR. Then to explore the effect of Lactobacillus rhamnosus GR-1 on BEEC injury induced by Escherichia coli and explain the molecular mechanism and target of Lactobacillus rhamnosus GR-1 against BEEC inflammatory injury induced by Escherichia coli. Furthermore, this project will contribute significantly to provide a theoretical basis for the clinical application of lactic acid bacteria in the treatment of bovine endometritis.

子宫内膜炎是严重危害奶牛产业经济效益的奶牛常发病，而子宫内膜感染大肠杆菌是本病发生的主要原因之一。前期研究发现，鼠李糖乳杆菌GR-1能够通过抑制NF-κB的激活，抑制大肠杆菌诱导奶牛子宫内膜上皮细胞（BEEC）炎性细胞因子的大量释放，减轻细胞的炎性损伤，但其分子机制尚不明确。在此基础上，本项目拟建立大肠杆菌诱导BEEC损伤模型，以MyD88依赖性和非依赖性信号途径为切入点，结合RNAi方法和特异性信号转导抑制剂，采用流式细胞术、实时荧光定量PCR及免疫印迹等技术，检测细胞凋亡、紧密连接蛋白的表达、TLR4及其下游衔接分子、NF-κB信号通路中关键信号分子的表达情况，探求鼠李糖乳杆菌GR-1抗大肠杆菌诱导BEEC损伤作用，以期阐明鼠李糖乳杆菌GR-1抗大肠杆菌致BEEC炎性损伤的分子机制及其作用靶点，为临床应用乳酸菌治疗奶牛子宫内膜炎提供一定的理论依据。

奶牛子宫内膜炎是奶牛产后常发病，引起本病的主要原因是病原微生物感染，其中以大肠杆菌最为常见，是造成奶牛不孕症的主要原因之一，严重危害奶牛产业的经济效益。目前一般采用抗生素对该病进行治疗，但是长期以来大量不当的使用抗生素治疗本病，引起了严重的交叉耐药，导致了严重的食品安全问题。因此，找到一种能够替代抗生素治疗和预防本病的安全高效、无残留污染的绿色制剂已成为畜牧业的当务之急。益生菌作为潜在的抗生素替代品，可以通过调节机体自身的免疫状态来治疗和预防疾病，极大的减少了生物安全危害和食品安全顾虑。大量研究表明乳酸菌是健康奶牛生殖道内的常住菌群，且围产期奶牛阴道内灌注乳酸菌，可以显著提高机体的免疫反应，降低子宫炎的发病率。但是乳酸菌在治疗和预防奶牛子宫内膜炎的具体机制尚不清楚。本项目通过原代培养的奶牛子宫内膜上皮细胞与大肠杆菌相互作用，建立大肠杆菌诱导奶牛子宫内膜上皮细胞损伤模型，证实了鼠李糖乳杆菌GR-1抗大肠杆菌诱导奶牛子宫内膜上皮细胞损伤作用，发现鼠李糖乳杆菌GR-1可以通过抑制大肠杆菌诱导的奶牛子宫内膜上皮细胞中MyD88和TRIF mRNA的高表达，及信号通路下游关键蛋白p65、IκB-α、JNK、ERK1/2和p38的磷酸化水平，进而抑制IL-1β、IL-6、IL-8和TNF-α mRNA的高表达，阐明了鼠李糖乳杆菌GR-1通过MyD88依赖性和非依赖性信号途径抑制大肠杆菌诱导奶牛子宫内膜上皮细胞损伤的调控机制，为临床应用乳酸菌治疗奶牛子宫内膜炎提供了一定的理论依据。