Early Diagnosis Of Endometritis Of Dairy Cows And The Mechanism Of Probiotics Against Inflammatory Injury Of Bovine Endometrial Epithelial Cells Induced By Escherichia Coli

Background and Aims:Escherichia coli(E.coli)is one of the major pathogens in dairy cow endometritis,causing serious economic losses to the dairy industry.It is particularly important to explore the diagnostic indexes of early dairy cow endometritis,and to detect and remedy the disease as soon as possible.Probiotics have been taken as a potential alternative to antibiotics,however,the mechanism in the treatment and prevention of dairy cow endometritis is still unclear.Therefore,this study was conducted to evaluate the mechanism of Lactobacillus rhamnosus GR-1(L.rhamnosus GR-1)against bovine endometrial epithelial cells(BEECs)inflammatory injury induced by E.coli,and to provide theoretical basis for its clinical application.Methods:Through the cytological examination of uterine contents and vaginal secretions at 21 d among 200 postpartum Holstein dairy cows,21 cows were divided into three groups:clinical endometritis group(CE,n = 7),subclinical endometritis group(SE,n = 7)and healthy control group(HC,n = 7).Serum was collected at 1,7,14,21 and 30 days postpartum.The changes of serum enzyme activity and ion concentration were measured by automatic biochemical analyzer.The changes of inflammatory cytokines and acute phase proteins were detected by ELISA.The mechanism of L.rhamnosus GR-1 against inflammatory injury induced by E.coli was evaluated by using primary BEECs.A total of four groups were assigned:control group(CONT),Escherichia coli group(ECOL),L.rhamnosus GR-1 group(LRGR)was pretreated with L.rhamnosus GR-1 only for 3 h,and pretreated with L.rhamnosus GR-1 for 3 h and then added to the E.coli group(LRGR + ECOL).The concentration of E.coli was 1 x 105 CFU/mL and L.rhamnosus GR-1 was 1 x 107 CFU/mL.The morphological and ultrastructural changes of BEECs were observed by inverted microscope,scanning electron microscope and transmission electron microscopy,respectively.The flow cytometry was used to detect the apoptosis of BEECs and western blot assay was used to detect the expression of tight junction protein and signal pathway protein.Real-time PCR was applied to detect the mRNA expression levels of cell-associated receptors and inflammatory factors.Results:The level of AST in serum of CE group was significantly higher than that of HC group at 7 days postpartum.The level of CK in serum of CE group was significantly higher than that of HC group at 14,21 and 30 days postpartum,and the level of CK in serum of SE group was significantly higher than that of HC group at 21 days postpartum.The level of TNF-a in serum of CE group was significantly higher than that of HE group and SE group at 21 and 30 days postpartum.The level of Hp in serum CE group was significantly higher than that of HC group at 7,14,21 and 30 days postpartum,and the level of Hp in serum of SE group was significantly higher than that of HC group at 7,14 and 21 days postpartum.L.rhamnosus GR-1 attenuated the E.coli-induced cell structure damage and significantly reduced the rate of apoptosis.L.rhamnosus GR-1 significantly reduced the increased mRNA expression levels of pattern recognition receptors(TLR2,TLR4,NOD1,and NOD2),inflammasome(NLRP3,ASC,and Caspase-1),TLR4 downstream adapter molecules(MyD88 and TICAM2),negative regulatory factors TOLLIP,transcriptional regulatory factors(NF-?B and IRF3),inflammatory cytokines(TNF-?,IL-1?,IL-6,IL-8,IL-10,IL-18,and IFN?),and other immune response molecules caused by E.coli on BEECs.L.rhamnosus GR-1 significantly increased the expression of the decrease of claudin-1/2,Occludin and ZO-1 induced by E.coli,and significantly decreased the expression of NF-?B2 and p65.Conclusion:The levels of TNF-? and Hp in serum of dairy cow can be used as assistant diagnostic indicators for clinical endometritis.Compared with other diagnostic indicators,Hp in dairy cow has higher sensitivity to inflammation as assistant diagnostic indicators for subclinical endometritis.L.rhamnosus GR-1 attenuates MyD88-dependent and non-dependent signaling pathway and enhances the interaction between NLRs and TLRs to inhibit the activation of NF-?B signaling pathway in E.coli-induced bovine endometrial epithelial cells and then reduces the release of inflammatory cytokines,meanwhile alleviating the destruction of tight junctions and reducing the apoptosis rate of the cells.Through those pathways mentioned above,L.rhamnosus GR-1 weakens the inflammatory injury of E.coli-induced bovine endometrial epithelial cells.