The Effects Of PGF_2?-PTGFR And PGE₂-PTGER2 Pathway On Regulating Proliferation In Bovine Endometrial Epithelial Cells

PGF2a and PGE2 are the most representative compounds of prostaglandins with reproductive physiological activity and inflammatory medium,which are generally resided in organism.Recently some studies demonstrated that PGE2 and PGF2a play an important role in human endometrial repair.Whether in bovine endometrial epithelial cells or in bovine endometrial explants,our laboratory studies have also confirmed that the pathways mediated by PGF2a and PGE2 could promote the expression of growth factors(such as VEGF,CTGF,TGF-?1,IL-8)related to tissue repair.Any damaged tissue or organ must undergo a complex process to return to normal,as well as the inflammatory endometrial tissue,in which the proliferation of bovine endometrial epithelial cells is essential.In the present study,based on the above background,the following questions were raised.What is the mechanism by which pathways mediated by PGF2a and PGE2 regulate the process of proliferation in bovine endometrial epithelial cells.Detailed research contents are as follows:(1)The culture of bovine endometrial epithelial cells and the detection of relative expression of PTGFR and PTGER.In order to obtain the bovine endometrial epithelial cells more quickly,our study further optimized the cell culture methods previously established in the laboratory.The obtained cells were comprehensively evaluated by using cell morphological observation,immunofluorescence staining and cell real-time detection technology(RTCA).The relative expression of PTGFR and PTGER mRNA were detected by Quantitative real-time PCR assays in bovine endometrial epithelial cells.The result showed that the relative expression of PTGFR mRNA was the highest,followed by PTGER2 compared with PTGER1,PTGER3 and PTGER4.(2)Study on effect of PGF2a-PTGFR pathway on regulating proliferation in bovine endometrial epithelial cells.First,RTCA and WST-1 were used to detect the effects of different doses of PGF2? and Fluprostenol on proliferation in bovine endometrial epithelial cells.The result showed that PGF2? and Fluprostenol of 10-6M could significantly promote the proliferation.In order to further confirm that PGF2?and Fluprostenol are promoting cell proliferation through their specific receptor PTGFR,RTCA and WST-1 were used to detect the effect of specific receptor antagonist AL8810 on cell proliferation.The result showed that AL8810 of 10-5M inhibited the proliferation of bovine endometrial epithelial cells.Subsequently,the results of RTCA and WST-1 showed that the effects of PGF2a and Fluprostenol on promoting proliferation were significantly inhibited compared with AL8810 in bovine endometrial epithelial cells.The above results fully indicated that the PGF2+-PTGFR pathway is an important signaling to regulate the proliferation in bovine endometrial epithelial cells.The effect of PGF2?-PTGFR pathway on the expression of PCNA was detected by Quantitative real-time PCR assays and Western blot.The result showed that both PGF2a and Fluprostenol significantly promoted the expression of PCNA.The effect of PGF2a-PTGFR pathway on the proteins expression of the cell cycle network system(Cyclins-CDKs-CKIs)was detected by Quantitative real-time PCR assays and Western blot.The results showed that PGF2?-PTGFR pathway promoted proliferation of bovine endometrial epithelial cells by up-regulating Cyclins(Cyclin A,B1,D1,D2,D3,E2)and Cyclin-dependent kinasesl,2,3,4(CDK1,2,3,4)and down-regulating Cyclin-kinases inhibitor(CKIs)p15,p21.The expression of EGF and PTGFR was detected by Quantitative real-time PCR assays and Western blot.The result showed that the expression of EGF and PTGFR could be significantly up-regulated by PGF2a-PTGFR pathway.Finally,we investigated the effects of COX-1 and COX-2 on the proliferation of bovine endometrial epithelial cells from both positive and negative aspects.The results of Quantitative real-time PCR assays and Western blot showed that PGF2a-PTGFR pathway could significantly promote the expression of COX-1 and COX-2.The results of RTCA and WST-1 showed that COX inhibitors Indomethacin and Aspirin could significantly inhibited the proliferation of bovine endometrial epithelial cells.Thus,endogenous PGs synthesized by COX-1 and COX-2 play an important regulatory role in the proliferation of bovine endometrial epithelial cells.(3)Study on effect of PGE2-PTGER2 pathway on regulating proliferation in bovine endometrial epithelial cells.First,RTCA and WST-1 were used to detect the effects of different doses of PGE2 and Butaprost on proliferation in bovine endometrial epithelial cells.The result showed that PGE2 and Butaprost of 10-6M could significantly promote the proliferation.When the specific receptor antagonist AH6809 of PGE2 and Butaprost existed,the effect of PGE2 and Butaprost on promoting the proliferation was significantly inhibited in bovine endometrial epithelial cells.The above results fully indicated that the PGE2-PTGER2 pathway is another important signaling to regulate the proliferation in bovine endometrial epithelial cells.The results of Quantitative real-time PCR assays and Western blot showed that PGE2-PTGER2 pathway could significantly promote the expression of PCNA,Cyclin A,Cyclin B1,Cyclin D1,Cyclin D2,Cyclin D3,Cyclin E2,CDK1,CDK2,CDK4,CDK6,EGF,COX-1,COX-2 and PTGER2.Conclusions:Both PGF2?-PTGFR and PGE2-PTGER2 pathways can significantly promote the proliferation of bovine endometrial epithelial cells.Both PGF2?-PTGFR and PGE2-PTGER2 pathways can regulate the proliferation process by regulating the expression of Cyclins,CDKs,CKIs,PCNA,EGF,COX-1 and COX-2 in bovine endometrial epithelial cells.The PGF2?-PTGFR and PGE2-PTGER2 pathway can promote the proliferation of bovine endometrial epithelial cells by up-regulating the expression of relative receptor PTGFR and PTGER2 respectively.