Effect Of Endogenous PGE₂ On Escherichia Coli-Induced Bovine Endometrial Damage

Bovine endometritis is the most common bovine uterine disease after parturition,which is one of the major diseases that cause the bovine’s fertility to decline.It is well known that the stability of the mammalian uterus microenvironment is very important for the normal development of the embryo,and the maintenance of this steady state is directly related to the occurrence of endometrial diseases and the therapeutic effects after the disease.It is demonstrated prostaglandin E2（PGE₂）plays a crucial role in the regulation of endometrial inflammation and repair.Therefore,this study explored the effects of PGE₂5 which is both a reproductive physiologically active substance and an inflammatory mediator,on the course of bacterial bovine endometritis and the mechanism of tissue damage regulation,in order to lay a theoretical foundation for the effective treatment of bacterial bovine endometritis.The present study included five aspects as followed:（1）Cultured bovine endometrial tissue and established tissue model of E,coli infection in vitroBased on endometrial explants culture method,were stimulated by endometrial pathogenic E.coli at different concentrations,followed by the detection of endometrial explants damage by HE,the section of IL-1β,IL-6,and PGE₂ by ELISA in the endometrial explants supernatant and the differential expression of IL-1β and IL-6 by RT-qPCR.The results showed that the histological sections of E.coli induced endometrium indicated that the endometrial glands were not clearly visible,and a quantity of endometrial epithelial cells and glandular epithelial cells had disappeared after treatment of E.coli for 12,24 h.In addition,IL-1β,IL-6,and PGE₂ section and IL-1β,IL-6 mRNA expression were significantly up-regulated compared with control group.The results indicated that the cultivation method of E.coli infected bovine endometrial tissue was successfully established in vitro,and IL-1β,IL-6,and PGE₂ may play an important role in the inflammatory response of bovine endometrium infected by E.coli.（2）Study on the effect of E.coli on the synthesis of PGE₂ in bovine endometrium in vitroTo determine how PGE₂ production was regulated in E.coli-infected bovine endometrium,the expression levels of PLA2,COX-2,mPGES-1,AA,PGE₂,and CysLT were measured by RT-qPCR,Western blot,and ELISA in E.coli-infected endometrial tissues following treatment with COX-2 inhibitors（CAY 10404 and NS398）and mPGES-1 inhibitors（MK886 and MF63）.The mRNA and protein expression levels of PLA2,COX-2,and mPGES-1 were remarkably upregulated in E.coli-infected endometrial tissues compared with control group.However,the increased expression levels were significantly inhibited in the E.coli-infected groups treated with COX-2 and mPGES-1 inhibitors.Additionally,15-PGDH inhibitors markedly increased PLA2,AA,PGE₂,and CysLT secretion in E.coli-infected tissues,while treatment with the EP4 inhibitor AH23848 or the PKA inhibitor H89 significantly reduced PLA2,AA,PGE₂,and CysLT accumulation,both as compared to the untreated E.coli-infected group and the E.coli-infected group treated with 15-PGDH inhibitors.（3）Effect of PGE₂-EP4/PKA pathway on the expression of pro-inflammatory cytokines in E.coli-infected endometrial tissuesTo further explore how the PGE₂,EP4,and PKA were associated with the pro-inflammatory cytokines（IL-1β,IL-6,IL-8,TGF-α,NOS2,PAFR,and MMP-2）in the E.coli-infected bovine endometrial tissue,RT-qPCR and ELISA were used to examine the effects of the inhibitors of COX-2,mPGES-1,15-PGDH,EP4,and PKA on the expression of pro-inflammatory cytokines.The results showed that the expression and secretion of these pro-inflammatory cytokines were significantly increased in the E.coli-infected endometrial tissue compared with control group,additionally,treatment with the 15-PGDH inhibitors the increased levels of these pro-inflammatory cytokines were more higher than that in untreated E.coli-infected endometrial tissue.In contrast,treatment with the COX-2,mPGES-1,EP4 and PKA inhibitors significantly reduced these pro-inflammatory cytokines expression,both in the untreated E.coli-infected endometrial tissue and in the E.coli-infected endometrial explants treated with 15-PGDH inhibitors.（4）Effect of PGE₂ synthase inhibitor on the mRNA expression of chemokine in E.coli-infected bovine endometrial tissueIn this study,E.coli-infected bovine endometrial tissue were treated by COX-2 and mPGES-1 inhibitors,followed by the detection of the mRNA expression of chemokines by RT-qPCR.The results showed that CXCL1,CXCL2,CXCL3,and CXCL5 expression were significantly upregulated in E.coli-infected endometrial tissue compared with control group.However,COX-2 and mPGES-1 inhibitors markedly decreased chemokines expression in E.coli-infected endometrial tissue.（5）Effects of PGE₂-EP4/PKA on tissue damage in E.coli-infected bovine endometrial tissueTo evaluate tissue damage resulting from E.coli infection and COX-2,mPGES-1,15-PGDH,EP4?and PKA inhibitors treated with E.coli-infected endometrial tissue,damage-associated molecules（DAMP）HMGB-1 and HABP1 expression levels were measured using RT-qPCR,Western blotting,and immunofluorescence staining.And the morphology of tissue damage was observed by H&E staining.The mRNA and protein expression levels of DAMPs were significantly higher in the E.coli-infected endometrial tissues compared with control group,which resulted the endometrial glands were not clearly visible,and several endometrial epithelial cells and glandular epithelial cells had disappeared.In addition,the expressions of DAMPs were higher in 15-PGDH treated with E.coli-infected endometrial tissue than that in E.coli-infected endometrial tissue.Furthermore,endometrial epithelial cells and endometrial glands had absolutely destroyed.However,the effects of E.coli infection and E.coli infection treatment with 15-PGDH inhibitors on endometrial tissue were significantly reduced by COX-2 inhibitors,mPGES-1 inhibitors,EP4 inhibitor,and PKA inhibitor,which significantly decreased DAMPs expression and endometrial epithelial cells and endometrial glands had partly destroyed in E.coli-infected endometrial explants and that in 15-PGDH inhibitors treated with E.coli-infected endometrial tissue.The above results suggest that E.coli infection can cause the accumulation of PGE₂ in bovine endometrial tissue in vitro,which can regulate the expression of pro-inflammatory cytokines,chemokines,and DAMP,promoting damage process of E.coli infected endometrial tissue via EP4/PKA pathway.