Effect Of Progesterone On Inflammatory Response Of Bovine Endometrial Epithelial Cells Induced By Escherichia Coli

Endometritis is a common postpartum disease in dairy cows.This disease often leads to an increase in the number of insemination,repeated infertility,and other reproductive problems,which increase the elimination rate and cause great economic losses to the farm.Escherichia coli(E,coli)is a common pathogen of bovine endometritis.E.coli is a gram-negative bacterium,which mainly exerts pathogenic effects through lipopolysaccharides(LPS).However,the pathogenesis of E.coli-induced bovine endometritis and the role of LPS in pathogenesis remain to be explored.Clinical studies have shown that changes in progesterone levels during perinatal period have a certain impact on the incidence of cow endometritis,but the mechanism is still unclear.In the current experiment,by construction of the LPS-and E.coli-induced bovine endometrial epithelial cell(BEEC)inflammation model,we investigated the effect of progesterone on the inflammation-related cytokines and pathways of BEECs,providing theoretical foundation and experimental data in the prevention and treatment of bovine endometritis.The cells used in this study were primary cells cultured in vitro.We combined mechanical and enzymatic digestion methods to culture BEECs.The cell viability was measured by CCK8 assay.The protein levels of MAPK and NF-κB signaling pathways were detected by Western blot.The result showed that progesterone of various concentrations had no effect on viability of the cells.There was no significant change in the protein level of MAPK and NF-κB signaling pathways when treated with progesterone of various concentration at 0.5 h.To explore the effect of progesterone on E.coli-induced BEECs inflammatory response,the cells were treated with 105 CFU/mL E.coli as an inflammation model group.In test groups,the cells were treated with progesterone(1,3 and 5 ng/mL)plus E.coli.The gene expressions of the inflammatory cytokines,including TNF-α,IL-1β,IL-6 and IL-8 were measured by qPCR at 2,4 and 6 h.The key protein expressions of MAPK and NF-κB signal pathways were detected by Western blot at 0.5 h.The results showed that,compared with the blank group,the expressions of inflammatory cytokines in the model group increased at 2,4 and 6 h(p<0.01).The phosphorylation levels of p38,JNK,ERK,p65 and IκBa were higher than those in blank group(p<0.01).Compared with model group,the RNA expressions of inflammatory cytokines were lower(p<0.01)at 2,4 and 6 h.The protein phosphorylation levels of the cells in test groups were lower(p<0.01)compared with the model group.This part of the experiment aims to explore the effect of progesterone on the LPS-induced inflammatory response in BEECs.The LPS concentration was determined by CCK8 assay,based on its influence on the cell viability,to establish the LPS inflammation model.Cells were co-treated with different concentrations(1,3,5 ng/mL)of progesterone and 1 μg/mL LPS.The gene expressions of inflammatory cytokines were detected at 3,12 and 18 h.The expressions of key proteins in MAPK and NF-κB signaling pathways were detected at 0.5 h.As a result,compared with the blank group,the phosphorylation levels of inflammatory cytokines and signal pathway proteins in the model group increased,which was consistent with the E.coli test.Compared with the model group,the cells in progesterone(1,3.5 ng/mL)and LPS co-treated groups showed decreased gene expression of inflammatory cytokines at 12 h and 18 h(p<0.01).The phosphorylations of the key proteins of MAPK and NF-κB pathways decreased in co-treatment groups of LPS and progesterone(3 ng and 5 ng/mL),compared with the model group(p<0.01).In conclusion,progesterone could exert anti-inflammatory effects in E.coli-and LPS-induced BEECs by inhibiting the activation of MAPK and NF-κB signaling pathways,and reduction of the gene expression of inflammatory cytokines.