Study On Effect And Mechanism Of PGE₂ In Process Of Inflammatory Factors Expression Of Bovine Endometrial Tissue Induced By Staphylococcus Aureus

Endometritis caused conception delayed,infertility and milk production decreased for dairy cow.Bacterial infection is mainly etiology for bovine endometritis.Antibiotics are often treated for bovine endometritis in clinical.However,the antibiotics treatment methods cannot repair damaged of endometrial tissue,so it is influence for promoting re-pregnancy ratio.In addition,the inflammtory responses in staphylococcus aureus(S.aureus)-infected bovine endometrium,is not fully clarify,it restricts effect of preventing and treating for endometritis.The researches have been showed that prostaglandin E2(PGE2)involoves in the process of multiple inflammatory responses,and PGE2 plays an important role for human endometrial repair process through regulates the expression of IL-8 with effect of vascular regeneration.The effect and mechanism of PGE2 for inflammatory responses in S.aureus-infected bovine endometrial tissue was studied in this research,and the study aimed to clarify the mechanism of molecule of endometritis induced by staphylococci,the specific research contents and results are as follows:1.In order to built research method that culture bovine endometrial in vitro for endometritis caused by bacterium.The test used different concentration of heat killed(HK)-S.aureus to treat the bovine endometrial tissue in vitro,and the expression of pro-inflammatory factors IL-6 and TNF-α mRNA were detected by qPCR.The results showed that IL-6 and TNF-α mRNA expression in HK-S.aureus-treated bovine endometrial tissue was significantly higher than control groups(P<0.05).It is consistent with the result that IL-6 and TNF-α expression for dairy cow with endometritis are significantly higher than healthy dairy cow.2.In order to clarify the signaling pathway of PGE2 secretion in S.aureus-induced endometrial tissue,and the effects of PGE2 for pro-inflammatory factors expression.The test used HK-S.aureus to treat the bovine endometrial tissue in vitro,and following treated with COX-2 inhibitors(CAY-10404,NS-398),prostaglandin-degrading enzymes-15-PGDH inhibitor(SW033291),respectively.The expression of COX-2,IL-6,TNF-α and PGE2 generation were detected by qPCR,western-blot and Elisa.Following treated with EP4 receptor agonist(CAY-10598),EP4 receptor antagonist(AH-23848)and p38 MAPK signaling pathway inhibitor(SB202190)to measure the expression of COX-2 and PGE2 generation.The results showed COX-2,IL-6,and TNF-α expression and PGE2 generation were decreased when treated CAY-10404,NS-398,AH-23848 and SB202190,comparison with HK-S.aureus treated bovine endometrial tissue(P<0.05).The expression of COX-2,IL-6,TNF-α and PGE2 generation were increased when treated CAY-10598 and SW033291,comparison with HK-S.aureus groups(P<0.05),and SB202190 markedly decreased CAY-10598-promoted COX-2 expression and PGE2 generation(P<0.05).3.To clarify the effects of regulation of PGE2 for tissue damage in S.aureus-infected bovine endometrial tissue.The test used HK-S.aureus-treated bovine endometrial tissue,and following treated with COX-2 inhibitors,prostaglandin-degrading enzymes 15-PGDH inhibitor,EP4 receptor agonist,EP4 receptor antagonist,and p38 MAPK signaling pathway inhibitor,respectively.The expression of damage associated molecule factors HMGB-1 and HABP-1 were detected by qPCR,western-blot and immunofluorescence,and different treatment groups were stained by HE methods to observe pathological changes of bovine endometrial tissue.The results showed the expression of HMGB-1 and HABP-1 were decreased when treated CAY-10404,NS-398,AH-23848 and SB202190,comparison with HK-S.aureus treated bovine endometrial tissue(P<0.05),and the degree of damage of endometrial epithelial cells were alleviated.The expression of HMGB-1 and HABP-1 expression was increased when treated CAY-10598 and SW033291 compared with HK-S.aureus groups(P<0.05),and the degree of endometrial epithelial cells disintegration and necrosis were more seriously.4.To clarify the mechanism of inflammatory responses in bovine endometrial tissue caused by S.aureus.The test used live S.aureus(LSA)and heat killed S.aureus(HK-SA)treated bovine endometrial tissue in vitro.The mRNA expression of IL-6,IL-1β and TLR2 were detected by qPCR,western-blot and Elisa.The results showed that IL-6,IL-1β and TLR2 expression in LSA-infected bovine endometrial tissue was significantly lower than HK-S.aureus treated groups(P<0.05),but the mRNA expression of COX-2 in S.aureus-infected bovine endometrial tissue was markedly increased,comparison to HK-S.aureus treated groups.To utilize LSA,HK-SA,E.coli,LSA and E.coli mixture infected endometrial tissue,and IL-6,IL-1β expression were detected by qPCR and Elisa.The results showed that the expression of IL-6 and IL-1βin LSA and E.coli mixture group were significantly higher than E.coli-infected group(P<0.05).It demonstrated that the pro-inflammatory factors expression in LSA-infected endometrial tissue may regulate indirectly.To clarify the effect of PGE2 in LSA-infected groups,COX-2,mPGES-1 mRNA,EP4 receptor expression,PGE2,cAMP generation were detected by qPCR,Elisa and western-blot,and the difference of activation signaling pathway in two infection ways were investigated by western-blot.The results showed the expression IκB-α in HK-SA treated endometrial tissue was significantly higher than LSA-infected groups(P<0.05),but the COX-2,mPGES-1 mRNA,EP4 receptor expression,PGE2,cAMP generation and p38 MAPK,JNK phosphorylate in LSA-infected groups were markedly higher than HK-SA treated groups(P<0.05),and it was no significant difference for ERK1/2 signaling pathway in two infection ways(P>0.05).The research showed:(1)The method of bovine endometrial tissue cultured in vitro suit for research of endometritis induced by bacteria;(2)Staphylococcus aureus induces the secretion of PGE2 through the EP4-p38 MAPK signaling pathway in bovine endometrial tissue;(3)PGE2 regulated IL-6 and TNF-α expression in S.aureus-infected bovine endometrial tissue in vitro;(4)PGE2 regulated tissue damage in S.aureus-infected bovine endometrial tissue via the EP4-p38 MAPK signaling pathway;(5)LSA and HK-SA evoked different inflammatory associated factors in bovine endometrial tissue in vitro;LSA induce secretion of PGE2 via TLR2-MAPK signaling pathway in bovine endometrial tissue in vitro,but HK-SA induced expression IL-6 and IL-1β via TLR2-NF-κB signaling pathway in bovine endometrial tissue in vitro.