Regulatory Roles Of PGE₂ In LPS-induced Tissue Damage In Bovine Endometrial Explants

Bovine endometritis is the most common uterine disease following parturition.Although PGE2 accumulation is observed in multiple inflammatory diseases,such as endometritis,the association with bovine endometrial damage is unclear.To clarify the role of PGE2 accumulation in lipopolysaccharide(LPS)-induced endometritis in cultured bovine endometrial explants,In this study,CAY 10404 and NS398 as Cyclooxygenase-2(COX-2)inhibitors were used to inhibit the secretion and synthesis of PGE2,and 100073 and SW033291 as 15-hydroxyprostaglandin dehydrogenase(15-PGDH)inhibitors were used to inhibit the degration of PGE2 in order to promote the accumulation of PGE2.From two aspects of inhibition and promotion to verify relationship between of PGE2 accumulation and LPS induced by bovine endometrial secretion and synthesis or release of inflammatory cytokines,damage associated molecular patterns(damage related molecular patterns,DAMPs)and signaling pathways related factors,the relationship between the expression of confirming accumulation of PGE2 may have the effect of inflammatory medium,thereby promoting inflammation in present study.The study may provide an essential foundation for the rational application of non-steroidal anti-inflammatory drugs for treating bovine endometritis and related diseases.The specific research contents of are as follows:1.LPS could damage the bovine endometrial explants.The effects of LPS were detected through the factors for the secretion and synthesis of PGE2 and related rate-limiting enzymes such as cyclooxygenase-2(COX-2),membrane bound prostaglandin E synthetase(mPGES-1),the expression of pro inflammatory cytokines such as interleukin 6(IL-6),tumor necrosis factor a(TNF-?),and inducible nitric oxide synthase/nitric oxide(iNOS/NO)),and damage-associated molecular patterns such as hyaluronan binding protein 1(HABP1)1 and high mobility group box-1(HMGB1)in endometrial explants by using RT-qPCR?Western Blot?Elisa and immunofluorescence(IF).The results showed that LPS could damage the bovine endometrial explants,and the PGE2,COX-2,mPGES-1,pro-inflammatory cytokines(IL-6,TNF-?,and iNOS/NO),DAMPs(HABP1 and HMGB1)expression in bovine endometrial explants were clearly up-regulated after LPS treatment.2.COX-2 inhibitors CAY 10404 and NS-398 affected LPS-induced bovine endometrial explants damage.Both CAY 10404 and NS398 pretreatment attenuated PGE2 secretion,decreased expression levels of COX-2,pro-inflammatory cytokines(IL-6,TNF-a,and iNOS/NO),DAMPs(HABP1 and HMGB1)in bovine endometrial explants after LPS treatment by using RT-qPCR?Western Blot?Elisa and IF.These results suggested that COX-2 expression plays a critical role in PGE2 secretion in bovine endometrial explants after LPS stimulation.And the factors mentioned above are closely associated with PGE2 accumulation.Accumulation PGE2 acted as an inflammatory mediator to promote the occurrence of tissue damage 3.Signaling mechanisms of accumulation PGE2 promoted bovine endometrial explants damage.In this experiment,15-hydroxyprostaglandin dehydrogenase,(15-PGDH)was used to inhibit the decomposition of PGE2,leading to the pathological accumulation of PGE2,and at the same time promoting the high expression of COX-2,mPGES-1,pro-inflammatory cytokines(IL-6,TNF-?,and iNOS/NO),which confirmed the role of pathological accumulation of PGE2 as an inflammatory mediator by using RT-qPCR?Western Blot?Elisa and IF.At the same time,prostaglandin E2 receptor 4(EP4),toll-like receptor 4(TLR4)and myeloid differentiation factor 88(MyD88)are over-expressed,which supports that accumulation PGE2 is involved in the LPS-induced bovine endometrial damage through the PGE2-EP4 affecting TLR4-MyD88 pathway.The results also showed that accumulating PGE2 can activate the phosphorylation of protein kinase A(PKA),mitogen-activated protein kinase(MAPKs)and nuclear transcription factor-?B(NF-?B)related signal transduction molecules,which in turn activate the downstream related signaling pathways and ultimately lead to inflammation.Conclusions:1.LPS could damage the bovine endometrial explants through pathological section,and induce the synthesis and secretion of PGE2 through COX-2/mPGES-1.LPS promoted the over-expression of proinflammatory factors and DAMPs.2.The accumulation of PGE2 in the bovine endometrial explants induced by LPS is mainly regulated by COX-2 as a key PGs synthase.COX-2 inhibitor could reduce the expression of inflammatory cytokines and DAMPs in the bovine endometrial explants,while COX-2 inhibitor can promote the expression of repair factors in the bovine endometrial explants.3.The 15-PGDH inhibitor stimulated the bovine endometrial explants with LPS treated,promoted accumulation of PGE2,high expression of COX-2,inflammatory cytokines and DAMPs.LPS-induced bovine endometrial explants damage through the PGE2-EP4 affecting TLR4-MyD88 pathway,and activated PKA,MAPKs and NF-?B pathway activation,which can promote the inflammatory process.