Methylation Mechanisms Of Heshouwuyin In Regulating Leydig Cells Senescence In Rats

Objection:To observe the effect of Heshouwuyin on DNMTs activity of rat Leydig cells,to investigate the methylation mechanism of Heshouwuyin on aging of rat Leydig cells.Method:1.Leydig cell(LC)was isolated and cultured by differential adherence method.The purity of Leydig cell was identified by 3 β-HSD specific staining.2.The aging model was established by oxidative damage method.The expression level ofβ-galactosidase,cell cycle distribution and the expression level of P16 protein were detected to determine whether the aging model was successfully constructed.3.Lentivirus was transfected into Leydig cells and Lentiviral transfection was used to knock down DNMT1 in Leydig cell.4.MTT assay was used to detect the viability of Leydig cell,and Real-time fluorescence quantification PCR was used to detect the expression level of DNMT1 mRNA.The optimal concentration and time of DNA methyltransferase inhibitors were screened out.5.According to the purpose of the experiment,the cell groups were: normal group,aging group,Heshouwuyin group,Methyltransferase inhibitors group,Methyltransferase inhibitors and Heshouwuyin co-incubation group,DNMT1 knockdown group,DNMT1 knockdown and Heshouwuyin co-incubation group,Negative control transfection group.6.Methylation-specific PCR(MSP)was used to detect promoter methylation levels of epigenetic genes Wnt2 B and C-myb in each group.Results:1.Identification of Leydig cells of rat testis aging model: After persistent oxidative damage,the expression level of β-galactosidase increased significantly,The proportion of G1 phase in cell cycle increased while that of S phase decreased significantly.,and the expression of P16 protein increased significantly,which was significantly different from that of normal group(P < 0.05).It suggested that the aging model was successfully established.2.The effect of Heshouwuyin on the expression of β-galactosidase in Leydig cells of rat testis: Heshouwuyin can significantly reduce the positive rate of β-galactosidase,which is significantly different from aging group(P < 0.05).Effect of Heshouwuyin on the Aging Leydig cells of rat testis Cell Cycle : After treatment with Heshouwuyin,the proportion of G1 phase in Leydig cells decreased,and the proportion of S phase increased significantly.(P < 0.05).Effect of Heshouwuyin on the Aging Leydig cells of rat testis P16 protein: After treatment with Heshouwuyin,the expression of P16 protein decreased which is significantly different from aging group(P < 0.05).3.DNMT1 knockdown can induce Leydig cell senescence: When DNMT1 was knocked down,the expression level of β-galactosidase increased significantly.The proportion of G1 phase in cell cycle increased while that of S phase decreased significantly.The expression levels of P16 protein increased significantly compared with the normal group(P < 0.05).These results suggest that DNMT1 knockdown can induce the aging of Leydig cell.4.The effect of Heshouwuyin on p16 expression: The expression of P16 protein in aging group,DNMT1 knockdown group and DNA methyltransferase inhibitor group increased significantly,and Heshouwuyin could partly reverse this phenomenon.5.Effect of Heshouwuyin on methylation levels of Wnt2 b and C-myb:5.1 wnt2b:Compared with the normal group,the methylation level of aging group and DNMT1 knockdown group increased significantly(P < 0.05).Compared with aging group,methylation level of the DNMT1 knockdown and co-incubation of Heshouwuyin group and Heshouwuyin group decreased(P > 0.05).The methylation level of DNMT1 knockdown and Heshouwuyin co-incubation group was significantly lower than that of the DNMT1 knockdown group(P < 0.05).The methylation level of methyltransferase inhibitor group was lower than that of normal group(P > 0.05).The methylation level of methyltransferase inhibitors and Heshouwuyin co-incubation group was significantly higher than that of methyltransferase inhibitors group(P < 0.05).5.2 C-myb:Compared with the normal group,the methylation level of aging group,methyltransferase inhibitor group and DNMT1 knockdown group decreased significantly(P< 0.05).Compared with aging group,Methylation levels of Heshouwuyin group,methyltransferase inhibitors and Heshouwuyin co-incubation group and DNMT1 knockdown and Heshouwuyin co-incubation group increased significantly.Methyltransferase inhibitors and Heshouwuyin co-incubation group and aging group had statistical difference(P < 0.05),the other two groups had no statistical difference(P > 0.05).The methylation level of DNMT1 knockdown and Heshouwuyin co-incubation group was significantly higher than that of DNMT1 knockdown group(P < 0.05).Compared with methyltransferase inhibitor group,the methylation level of DNMT1 knockdown group decreased significantly(P < 0.05).Conclusion:1.Knocking down DNMT1 can induce senescence of Leydig cells.2.Heshouwuyin inhibits the expression of p16 by up-regulating the activity of DNMT1.3.Heshouwuyin can delay the senescence of Leydig cells of testis by improving theactivity of DNMTs and regulating the methylation level of genes.