| Objection:To investigate the methylation mechanism of Heshouwuyin and its monomer compounds on key genes related with senescence in Leydig cells.Method:1.We use the differential adherence method to isolate and culture Leydig cells and identify them by 3?-HSD cytochemical staining.The purity of Leydig cells was more than90%.2.Using free-radical oxidative damage method established cell aging model.The expression levels of?-galactosidase were used to determine whether the aging model was successful.According to the different dosage and time of H2O2 and FeSO4,the positive expression levels of?-galactosidase could determine the optimal conditions for the establishment of cell senescence model.3.According to the purpose of the experiment,Leydig cells are mainly divided into the normal group,aging group,Heshouwuyin group,Icariin group,Echinacoside group,Oleanolic acid group,and Heshouwu group.The optimal concentration of icariin,echinacoside,oleanolic acid,and stilbene glycoside on Leydig cells were selected by MTT method.4.Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of key epigenetic genes:Ttc29,AXL,wnt2b,MAPK10 and C-myb in each group.5.Methylation-specific PCR?MSP?was used to detect methylation levels of key epigenetic genes:Ttc29,AXL,wnt2b,MAPK10 and C-myb in each group.6.Chromatin immunoprecipitation?Ch IP?technique was used to detect the enrichment of H3K27me3,H3K9me3,and H3K9me2 which are located in promoter regions of Ttc29,AXL,wnt2b,MAPK10 and C-myb genes in each group.7.Double-factor and one-way variance analysis were used to statistically analyze the MTT results,and using Person method to analyze the responsibility,others were analyzed by one-way variance analysis.Results:1.According to the positive expression level of?-galactosidase under different conditions,the final conditions for the cell aging model were as follows:the final concentration of H2O2 and FeSO4 were 50?mol L·L-1 and 100?mol L·L-1 respectively,the volume was 2?L and the time was 8 hours.2.MTT assay confirmed that the concentration of drugs in Leydig cells for 72 h were as follows:Icariin 40?M,Echinacoside 20?M,Oleanolic acid 20?M,and Heshouwu 50?M.3.Regulation of DNA and histone methylation by Hehsouwuyin and its monomer compounds on Ttc293.1 mRNA levels:Compared with the normal group,the expression level of the aging group was significantly increased?P<0.05?;The expression level of Heshouwuyin group,Oleanolic acid group and Heshouwu group were much lower than the aging group?P<0.05?.There was no significant difference between Icariin group,Echinacea group and the aging group?P>0.05?;Among them,Heshouwuyin group had the best effect?P<0.05?.3.2 DNA methylation levels:Compared with the normal group,the methylation level of the aging group was significantly increased?P<0.05?;The percentage of methylation level of Heshouwuyin group,Icariin group,Echinacoside group,Oleanolic acid group and Heshouwu group were significantly lower than the aging group?P<0.05?.3.3 histone methylation:The enrichment of H3K27me3,H3K9me3,and H3K9me2 in the promoter regions of Ttc29 were extremely low and even undetectable.4.Regulation of DNA and histone methylation by Hehsouwuyin and its monomer compounds on AXL4.1 mRNA levels:Compared with the normal group,the expression level of the aging group was significantly increased?P<0.05?;The expression levels of Heshouwuyin group,Icariin group and Echinacoside group were much lower than the aging group?P<0.05?;There was no significant difference between Oleanolic acid group and the aging group?P>0.05?;Among them,the Heshouwuyin group had the best effect?P<0.05?.4.2 DNA methylation levels:Compared with the normal group,the methylation level of the aging group was significantly decreased?P<0.05?;The percentage of the methylation level of the Heshouwuyin group,Oleanolic acid group and Heshouwu group were significantly increased than the aging group?P<0.05?;There was no significant difference between Icariin group,Echinacoside group and the aging group?P>0.05?.Among them,the Heshouwuyin group had the best effect?P<0.05?.4.3 histone methylation:The enrichment of H3K27me3,H3K9me3,and H3K9me2 in the promoter regions of AXL were extremely low and even undetectable.5.Regulation of DNA and histone methylation by Hehsouwuyin and its monomer compounds on wnt2b5.1 mRNA levels:Compared with the normal group,the expression level of the aging group was significantly increased?P<0.05?;The expression level of Heshouwuyin group and Heshouwu group were much lower than the aging group?P<0.05?;There was no significant difference between Icariin group,Echinacoside group,Oleanolic acid group and the aging group?P>0.05?;Among them,Heshouwuyin group had the best effect?P<0.05?.5.2 DNA methylation levels:Compared with the normal group,the methylation level of the aging group was significantly lower?P<0.05?;The percentage of the methylation level of Heshouwuyin group,Icariin group,Echinacoside group,and Heshouwu group were significantly increased?P<0.05?;There was no significant difference between the Oleanolic acid group and the aging group?P>0.05?;Among them,the Heshouwuyin group had the best effect?P<0.05?.5.3 histone methylation:The enrichment of H3K27me3 in the wnt2b promoter region of the aging group was significantly lower than the normal group?P<0.05?,but there was no significant difference in the enrichment of H3K9me3 and H3K9me2?P>0.05?.The enrichment of H3K27me3 in wnt2b promoter region of Heshouwuyin group,Icariin group,Oleanolic acid group,and Heshouwu group was significantly higher than that of aging group?P<0.05?,but there was no significant difference between Echinacetin group and aging group?P>0.05?.There was no significant difference in the enrichment of H3K9me3 and H3K9me2 between the aging group and other groups?P>0.05?.6.Regulation of DNA and histone methylation by Hehsouwuyin and its monomer compounds on C-myb6.1 mRNA levels:Compared with the normal group,the expression level of the aging group was significantly lower?P<0.05?;The expression level of Heshouwuyin group,Icariin group,Echinacoside group,Oleanolic acid group and Heshouwu group were much higher than the aging group?P<0.05?;Among them,Heshouwuyin group had the best effect?P<0.05?.6.2 DNA methylation levels:Compared with the normal group,the methylation level of the aging group was significantly lower?P<0.05?;The percentage of the methylation level of Heshouwuyin group,Icariin group,Echinacoside group,Oleanolic acid group and Heshouwu group were significantly increased than the aging group?P<0.05?;Among them,the Heshouwuyin group had the best effect?P<0.05?.6.3 histone methylation:Compared with the normal group,the enrichment of H3K9me3 in the C-myb promoter region was significantly increased in the aging group?P<0.05?,while the enrichment of H3K27me3 and H3K9me2 had no difference?P>0.05?.Compared with the aging group,the enrichment of H3K9me3 in the C-myb promoter region of Heshouwuyin group,Echinacoside group,Oleanolic acid group and Heshouwu group were significantly decreased?P<0.05?,and there was no significant difference between Icariin group and aging group?P>0.05?;The enrichment of H3K27me3 in the Oleanolic acid group was significantly higher than that in the aging group?P<0.05?,and other groups had no difference?P>0.05?;The enrichment of H3K9me2 between the aging group and other groups had no difference?P>0.05?.7.Regulation of DNA and histone methylation by Hehsouwuyin and its monomer compounds on MAPK107.1 mRNA levels:Compared with the normal group,the expression level of the aging group was significantly increased?P<0.05?;The expression level of Heshouwuyin group,Icariin group,Echinacoside group,Oleanolic acid group,and Heshouwu group were significantly lower than the aging group?P<0.05?;Among them,Heshouwuyin group had the best effect?P<0.05?.7.2 DNA methylation levels:Compared with the normal group,the methylation level of the aging group was significantly lower?P<0.05?;the percentage of the methylation levels of Heshouwuyin group,Icariin group,Echinacoside group,Oleanolic acid group,and Heshouwu group were significantly increased than the aging group?P<0.05?;Among them,the Heshouwuyin group had the best effect?P<0.05?.7.3 histone methylation:The enrichment of H3K27me3,H3K9me3,and H3K9me2 in the promoter regions of MAPK10 were extremely low and even undetectable.8.The expression levels of AXL,wnt2b and MAPK10 were highly negatively correlated with the methylation levels?P<0.05?;the expression level and methylation level of C-myb were highly positively correlated?P<0.05?;There was no correlation between the expression level and the methylation level of Ttc29?P>0.05?.Conclusion:1.Heshouwuyin delayed the aging of Leydig cells by down-regulating the mRNA levels of AXL,wnt2b,MAPK10 and Ttc29 genes,up-regulating the mRNA level of C-myb gene,and the effect is obviously better than other monomer compound groups.2.Heshouwuyin delayed the aging of Leydig cells by up-regulating the methylation levels of AXL,wnt2b,MAPK10,and C-myb genes,down-regulating the methylation level of Ttc29 gene,and the effect is obviously better than other monomer compound groups.3.There was a negative correlation between the methylation levels and mRNA expression levels of AXL,wnt2b,and MAPK10 genes.And there was a positive correlation between the methylation level and the mRNA expression level of C-myb gene.There was no correlation between the methylation level and the mRNA expression level of Ttc29 gene.Heshouwuyin inhibited Leydig cells aging by regulating the methylation and mRNA expression levels of genes.4.The mechanism of Heshouwuyin to inhibit the process of Leydig cells aging indicated that it may inhibit the transcription of wnt2b gene by up-regulating the expression of H3K27me3,and may also promote the transcription of C-myb gene by down-regulating the expression of H3K9me3,and the effect is obviously better than other monomer compound groups. |
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