Effect Of DNA Methylation On Expression Of IL-6 And IL-8 Induced By LPS Inbovine Endometrial Cells

Endometritis in cattle is an obstetric disease caused by bacterial infection inpostpartum,which negatively impacts on the dairy economic industry.Previous research focused on the changes rather than mechanism of gene expression.The aim of this study is to evaluate the effects of LPS on the expression of IL-6 and IL-8.1?g /m L LPS was used to stimulate endometrial cells 24 h.Based on this,expression of DNA methylation transfer related enzymes(Me CP2,DNMT1,DNMT3 A and DNMT3B)and effect of DNA methyltransferase inhibitors on IL-6 and IL-8 expression were evaluated.Finally we analyzed the effects of LPS on IL-6 and IL-8 methylation status in the promoter region using BSP.The results are as follows:1?The co-culture system of bovine endometrial stromal cells and epithelial cellsis obtained frombovine endometrial tissue explant,and cellswere challenged with1?g /m L LPSfor 24 h.ELISA showed that LPS can significantly promote IL-6 and IL-8 protein expression(p<0.01).q RT-PCR assay showed that LPS can significantly improve IL-6 and IL-8m RNA expression(p<0.01).2?The mRNA expression of MeCP2,DNMT1,DNMT3 A and DNMT3 Bin LPS treatment cells24 hwas studied.Results indicated that LPS can significantly promote Me CP2,DNMT1,DNMT3 Aand DNMT3B m RNA expression(p<0.01).Then weinvestigated the effects of different concentrations(1 mol/L,2 mol/L,5 mol/L and 10 mol/L)5Aza-dc treated cells fordifferent time(24h,48 h and 72h)on IL-6 and IL-8 m RNA expression in cows endometrial cells of LPS induced.The results indicated that 5Aza-dc can increase IL-6 and IL-8 m RNA expression(p< 0.01).3?The effect of LPS on the IL-6 and IL-8 promoter methylation status was studied by BSP.The results showed that LPS could reduce the promoter methylation rate of IL-6 and IL-8.Compared with the controlgroup,the methylation rate of IL-6 promoter region was decreased by 2%,and the IL-8 decreased by10.4%.The methylation level at methylation site of IL-6 in-660 and IL-8 in-120,-48 were lower than that in the control group.In this study,1?g /m L LPS was used to stimulate cowsendometrial cells 24 h to analyze the expression mechanism of IL-6 and IL-8,showing that DNA methylation is involved in the expression of IL-6 and IL-8 in cows endometrial cells of LPS induced by reducing the methylation level of IL-6 and IL-8 promoter regions.