The Protective Effect And Molecular Mechanism Of Lactobacillus Rhamnosus GR-1 Against Inflammatory Injury Of Bovine Endometrial Epithelial Cells Induced By E.coli

Endometritis is a prone disease for dairy cows after childbirth,which seriously affects the economic benefits of dairy cow breeding.At present,the use of antibiotics,and the resulting resistance problems are becoming more serious,so there is efficient and no side-effect therapy has a huge demand.Lactic acid bacteria(LAB)is a key member of the probiotic family.It can use its antagonist pathogenic bacteria and secrete beneficial biomass and other functions to promote body growth and prevent and control related diseases.In this test,take healthy cow uterus tissue,using improved tissue mass digestion and adhesion,primary bovine endometrial epithelial cells(BEECs),of origin identification of BEECs purity using keratin-18 immunofluorescence,determination of cell activity by MTT method,successful cultivation of BEECs.The experimental team has successfully established a cell inflammatory injury model in the early stage.The inflammatory concentration and action time of Escherichia coli(E.coli),the protection concentration and action time of Lactobacillus rhamnosus GR-1(L.rhamnosus GR-1)were determined.The experiment was divided into four groups:blank control group(CONT),E.coli group(ECOL),L.rhamnosus GR-1 alone treatment group(LRGR),L.rhamnosus GR-1 pretreatment 3 h plus E.coli group(LRGR+ECOL),E.coli action concentration was 5×10~5 CFU/mL,L.rhamnosus GR-1 action concentration is 5×10~6 CFU/mL,common role 6 h.Apoptosis was then detected using an apoptosis kit;Using Real-time PCR techniques to detect TLR4,MyD88,TNF-α,IL-1β,IL-6 and IL-8 mRNA expression;The phosphorylation level of IκBα,p65,ERK1/2,JNK1,p38 protein was detected by Western Blot technique.Results:Apoptosis detection revealed a significant increase in apoptosis of cells and death in the ECOL group compared with the CONT group(P<0.01),LRGR group had no significant change;The number of apoptosis and death in LRGR+ECOL group was significantly lower than that in ECOL group(P<0.05).Using Real-time PCR techniques,the expression of TLR4,MyD88,TNF-α,IL-1β,IL-6 and IL-8 mRNA in ECOL group was significantly higher than that in CONT group(P<0.05).There was no significant change between LRGR group and CONT group.The expression of TLR4,MyD88,TNF-α,IL-1βand IL-6 mRNA was significantly down-regulated in LRGR+ECOL group and ECOL group(P<0.05).And by Western Blot means,Comparedto CONT,the p-p65/p65 and p-IκBα/β-actin ratios of ECOL groups were significantly higher(P<0.01),there was no significant difference in p-p65/p65 ratio between LRGR groups,p-IκBα/β-actin ratio decreased significantly(P<0.05);Compared to ECOL,the p-p65/p65 and p-IκBα/β-actin ratios of LRGR+ECOL groups weresignificantly decreased(P<0.01);The ratio p-JNK1/GAPDH,p-ERK1/2/GAPDH,p-p38/GAPDH higher than CONT(P<0.05),LRGR group had no significant difference;Compared to ECOL group,the p-JNK1/GAPDH,p-ERK1/2/GAPDH,p-p38/GAPDH ratio decreased significantly(P<0.05).Conclusion:L.rhamnosus GR-1 reduces the apoptosis of BEECs induced by E.coli;E.coli can induce BEECs to activate NF-κB and MAPKs signaling pathway and promote the expression of TNF-α,IL-1β IL-6 and IL-8 mRNA;L.rhamnosus GR-1 can reduce inflammatory damage induced by NF-κB and MAPKs signaling pathway and reduces the expression of TNF-α,IL-1β,IL-6 mRNA.