Regulation Of DNA Methylation On Expression Of TNF-α Induced By LPS In Bovine Endometrial Cells

The mastitis is a major disease that affects the development of dairy in the whole world,and causes the serious economic losses in our country.In order to find out whether DNA methylation is involved in the mastitis induced by lipopolysaccharide(LPS),this study investigated the effect of DNA methylation on the tumor necrosis factor(TNF-α)expression induced by LPS in mammary epithelial cells.The bovine mammary epithelial cells were treated by LPS and inflammatory modle is successful established.The TNF-α level of cells treated with DNA methyltransferase inhibitors and the expression of DNA methylation transferase(DNMT)were detected.The methylation level of TNF-α was detected by methylation-specific PCR(MSP)and bisulfite sequencing PCR(BSP).The results are as follow: 1.The mammary epithelial cells were treated with LPS at 0μg/ml,1μg/ml,10μg/ml and 100μg/ml for 6h,12 h and 24 h,and detected by qRT-PCR.The IL-6,BNBD-5 and TNF-α mRNA level is increased significantly compared with the control group,and the level of TNF-α protein detected by ELISA is increased.2.The mammary epithelial cells were treated with the 5-Aza-2’-deoxycytidine(5-AZA)at 1.0umol/L,2.5umol/L and 5.0umol/L for 24 h,48h and 72 h.The TNF-α mRNA level was detected by qRT-PCR.The expression of TNF-α in 5-AZA treatment group was significantly increased compared with that of LPS treatment group.The expression of DNMT(DNMT-1,DNMT-2,DNMT-3A and DNMT-3B)was significantly decreased at 3h,6h,12 h after treating with 1ug/ml LPS.3.The mammary epithelial cells were treated with 1ug/ml LPS for 6h.MSP and BSP were used to detect the degree of methylation in TNF-α promoter.The results showed that LPS treatment group for TNF-α promoter methylation was significantly lower than the control group.The promoter methylation at-323,-270 and-245 sites showed the most obvious change in the different group.This study investigated the role of DNA methylation in the bovine mammary inflammatory response.The results showed that LPS promoted the expression of TNF-α by inhibiting DNA methylation in gene promoter region.