The Effect Of Epigenetic Genes And The Mechanism Of Heshouwuyin On Testicular Retrogression In Natural Aging Rats

Objection:To investigate the effect of epigenetic genes and the mechanism of Heshouwuyin on testicular retrogression in natural aging rats.Methods:1. Wistar male rats were randomly divided into six groups with 10 rats in each group: the young control group, natural aging group, Heshouwuyin group, Heshouwu group,Roucongrong group and Huainiuxi group. The young control group was sacrificed at the age of 12 months; Heshouwuyin group, Heshouwu group, Roucongrong group, Huainiuxi group were fed to the age of 18 months, and were intragastrically given Heshouwuyin, Heshouwu extract, Roucongrong extract, Huainiuxi extract respectively from the age of 16 months, once a day for 60 days; the natural aging group was given same amount of normal saline at the same time. DNA methylation chip was conducted with appropriate testicular tissue from the young control group, natural aging group and Heshouwuyin group to screen out differentially methylated genes and then bioinformatics analysis was processed.2. MSP to detect the methylation levels of the epigenetic genes: AXL, C-myb, Ttc29,MAPK10, wnt2 b in the testes of young control group, natural aging group, Heshouwuyin group, Heshouwu group, Roucongrong group and Huainiuxi group.3. Real-time quantitative PCR to detect the m RNA levels of the epigenetic genes: AXL,C-myb, Ttc29, MAPK10, wnt2 b in the testes of young control group, natural aging group,Heshouwuyin group, Heshouwu group, Roucongrong group and Huainiuxi group.4. Immunofluorescence to detect the Bromodeoxyuridine(Brd U) expression levels in the testes of young control group, natural aging group, Heshouwuyin group, Heshouwu group,Roucongrong group and Huainiuxi group.Results:1. The screening result of gene chip: DNA methylation gene chip screened out 1728 genes between young control group and natural aging group, among these 1728 genes, 238 genes were directed regulated by Heshouwuyin. Compared with the young control group,methylation levels of 1176 genes were significantly increased in the natural aging group while Heshouwuyin intervention could reduce the methylaltion levels of 135 genes; in the natural aging group, the methylation levels of 552 genes were decreased than the young control group while Heshouwuyin intervention could increase the methylation levels of 103 genes. GO analysis and pathway analysis indicated that these 238 genes were distributed in 23 cell pathways which were related to disease, cell proliferation and apoptosis, cell adhesion and so on.2. Methylation level of AXL, C-myb, Ttc29, MAPK10, wnt2 b were detected by MSP The results of MSP indicated that, compared with the natural aging group, the methylation percentage of AXL, wnt2 b, MAPK10 in young control group, Heshouwuyin group,Heshouwu group, Roucongrong group and Huainiuxi group were increased significantly(P<0.05), the methylation percentage of Roucongrong group and Huainiuxi group were significantly decreased than Heshouwuyin group(P<0.05), there was no significant difference between Heshouwu group and Heshouwuyin group(P>0.05); the methylation percentage of C-myb, Ttc29 were significantly decreased(P<0.05), the percentage of Heshouwu group, Roucongrong group and Huainiuxi group were significantly increased than Heshouwuyin group(P<0.05), there was no significant difference between young control group and Heshouwuyin group(P>0.05).3. m RNA levels of AXL, C-myb, Ttc29, MAPK10, wnt2 b were detected by q RT-PCR The results of q RT-PCR indicated that, compared with the natural aging group, the levels of wnt2 b,MAPK10 in the young control group, Heshouwuyin group, Heshouwu group, Roucongrong group and Huainiuxi group were significantly decreased(P<0.05), the levels of AXL, Ttc29,C-myb were significantly increaed(P<0.05); compared with the Heshouwuyin group, the level of Wnt2 b in Huainiuxi group was significantly increased(P<0.05), the level of AXL,Ttc29 and C-myb were significantly decreased(P<0.05), there was no significantly difference between the young control group and Heshouwuyin group(P>0.05).4. Brd U staining Immunofluorescence staining found that Brd U positive products were red patches or granular and were located in the nucleus. Brd U positive cells were located in spermatogonia close to the basilar membrane of seminiferous tubule. Statistical analysis showed that compared with the natural aging group, the number of positive cells was significantly increased in the young control group, Heshouwuyin group, Heshouwu group,Roucongrong group and Huainiuxi group(P<0.05), the number of Heshouwu group,Roucongrong group and Huainiuxi group were significantly decreased compared with the Heshouwuyin group(P<0.05), and there was no significant difference among these three groups(P>0.05).Conclusion:1. There were 1728 differential methylated genes between the natural aging group and young control group, among these 1728 differential methylated genes, there were 238 differential methylated genes were regulated by Heshouwuyin and they were distributed in 23 signaling pathways related to disease, cell proliferation and apoptosis, cell adhesion and so on.2. Heshouwuyin, Heshouwu extract, Roucongrong extract and Huainiuxi extract could increase the methylation level of promoters in wnt2 b, MAPK10, AXL and down-regulate wnt2 b, MAPK10; and could reduce the methylation level of promoters in Ttc29, C-myb, and up-regulate Ttc29, C-myb, AXL. The effect of Heshouwu extract was stronger than Roucongrong extract and Huainiuxi extract, and the effect of Heshouwuyin was stronger than the other three groups.3. Heshouwuyin, Heshouwu extract, Roucongrong extract and Huainiuxi extract could promote the proliferation of spermatogonia in natural aging rats, and the effect of Heshouwuyin was stronger than the other three groups.