| Objective:To explore the mechanism of heshouwuyin regulating DNA-RNA epigenetic crosstalk to delay the senescence of testicular tissue and Sertoli cells in male rats with Kidney essence deficiency.Methods:1 Establishment and identification of aging model of Kidney essence deficiency.1.1 The aging model with kidney essence deficiency rats was established by electro-acupuncture apparatus and single factor electro-stimulation to simulate “Sexual fatigue” and “Panic”.1.2 The aging model was identified by β-galactosidases staining and P16 protein immunohistochemistry.2 Sequencing data analysised by bioinformatics methods.2.1 Me DIP-chip data analysis.Ringo,Limma and MEDM were used to obtain the differentially methylated genes in the testis of the aging group and the heshouwuyin group,respectively,and the young group was used as the control.2.2 RNA-seq data analysis.The differentially expressed genes of young group with aging group and heshouwuyin group were obtained by Limma.2.3 MeRIP-seq data analysis.exome Peak and cluster Profiler were used to obtain the differential peak genes and their GO/KEGG signal pathway distribution and biological function coverage in young group,aging group and heshouwuyin group.3 In vivo experiments were performed to verify the results of sequencing data analysis.3.1 To verify that DNMT1 is regulated by heshouwuyin.RT-qPCR,Western Blot and immunohistochemistry were used to detect the expression of DNMT1 in testis.3.2 Verify the conclusion of Me DIP-chip and RNA-seq data analysis.The expressions of METTL3,FTO and IGF2BP1 in testis tissue were detected by RT-qPCR,Western Blot and immunohistochemistry.Detection of methylation in Mettl3,Fto and Igf2bp1 promoter regions using MSP.3.3 Verify the results of MeRIP-seq data analysis.The methylation of P53 and P16 mRNA was detected by MeRIP-qPCR,and the expression of P53 and P16 mRNA was detected by RT-qPCR,Western Blot and immunohistochemistry.4 Establishment and identification of rat Sertoli cell aging model.4.1 Rat F3 generation Sertoli cells were treated with Fenton’s reagent to established the cell senescence model.Flow cytometry and β-galactosidases staining were used to detect the expression level of β-galactosidases and identify the cell senescence model.4.2 Lentiviral transfection to knock-down Dnmt1 gene in Sertoli cells.The expression of DNMT1 mRNA and protein in Sertoli cells was detected by RT-qPCR and Western Blot to verify the efficiency of Dnmt1 knock-down.5 In vitro experiment and mechanism discussion.5.1 Cell senescence,cell cycle arrest and cell senescence after DNMT1 knockdown and the effect of heshouwuyin were detected by beta-galactosidases staining and Flow cytometry.5.2 Promoter methylation of Mettl3,Fto and Igf2bp1 was detected by MSP,and the effects of heshouwuyin.5.3 mRNA and protein expression of METTL3,FTO and IGF2BP1,and the expression of aging-related genes P53 and P16,were detection by RT-qPCR and Western Blot.5.4 MeRIP-qPCR detection of aging-related genes P53 and P16 mRNA m6A modification.Results:1 Identification of rat aging model.Compared with the young group,the rate of β-Gal positive rate in the aging group was significantly increased(P < 0.01),and the P16 IHC score was significantly increased(P < 0.05).After treat with heshouwuyin,the rate of β-Gal positive cells in the testicular tissue was significantly decreased(P < 0.05),P16 IHC score was decreased(P < 0.05).2 Run a bioinformatics analysis on the sequencing data2.1 Me DIP-chip data analysis showed that METTL3 and IGF2BP1 promoter regions were hypomethylated in the aging group compared with the young group,METTL3 and IGF2BP1 promoter regions showed hypermethylation,but the methylation of FTO promoter regions decreased after treat with heshouwuyin.2.2 RNA-seq data analysis.The expression of METTL3 and IGF2BP1 was up-regulated and the expression of FTO was down-regulated in the testis of the aging group,compared with the young group.2.3 MeRIP-seq data analysis,young group as a control,to obtain the aging model group of differential methylation peak genes and draw the volcano map,the differentially methylated rnas included Tp53,Cdkn2 a,Mtor and Becn1.The differential genes were analyzed by GO/KEGG.The common enrichment pathway in the aging group and heshouwuyin group included cell cycle,mRNA transport,mRNA stability,WNT signal pathway and m TOR signal pathway.3 Performed in vivo experiments to verify the results of sequencing data analysis.3.1 The expression of DNMT1 mRNA and protein in the testis of the aging model group was significantly lower than that of the natural aging group(P < 0.001),after treat with heshouwuyin,the expression of DNMT1 mRNA and protein increased(P < 0.05).3.2 Results of Mettl3,Fto and Igf2bp1 MSP in testicular tissue.Compared with young group,the methylation of METTL3 and IGF2BP1 promoter was significantly decreased(P <0.001)and FTO increased(P < 0.001)in aging group.After treat with heshouwuyin METTL3 and IGF2BP promoter methylation increased(P < 0.05)and FTO decreased(P < 0.05).3.3 The results of RT-qPCR and Western Blot of METTL3,FTO and IGF2BP1 in testicular tissues.The mRNA expressions of METTL3 and IGF2BP1 in the aging group were significantly higher than those in the young group(P < 0.001),while the expression of FTO was decreased.After the treat with heshouwuyin,the mRNA expression of METTL3 and IGF2BP1 decreased,while the expression of FTO increased(P < 0.01).3.4 The results of P53,P16 MeRIP-qPCR,RT-qPCR and Western Blot in testicular tissues.Compared with the young group,the mRNA and protein expressions of P16 and P53 in the aging group were significantly increased(P < 0.01).P16 and P53 m6A were decreased(P < 0.05),and their mRNA and protein expressions were decreased.4 Identification of the aging model of Sertoli cells.4.1 Results of β-Gal staining and cell cycle assay.Compared with the control group,the ratio of blue staining positive cells was increased and the cell cycle was arrested in G 1/S phase(P < 0.01).The combination of the two indicates that the establishment of the Sertoli cell senescence model is successful.After treatment with heshouwuyin containing serum,the ratio of blue-stained positive ratio decreased and G 1/S phase arrest relieved(P < 0.05).4.2 Results of lentiviral transfection of Sertoli cells.The green fluorescence intensity of GFP was high at 24 h after transfection,and the expression of DNMT1 was significantly decreased by RT-qPCR and Western Blot(P < 0.05).5 In vitro experiment were performed for mechanism study.5.1 DNMT1 knockdown of testicular Sertoli cells Mettl3,Fto and Igf2bp1 MSP results,compared with the control group.The methylation of METTL3 and IGF2BP1 decreased and FTO increased in the aging group.After treat with heshouwuyin contained serum the methylation of METTL3 and IGF2BP1 increased and the methylation of FTO decreased(P <0.01).Compared with empty load group,METTL3 and IGF2BP1 in shDNMT1 group showed a decrease in methylation and an increase in FTO methylation after DNMT1 knock-down(P <0.05),but there was no significant change in shDNMT1 + heshouwuyin group.5.2 DNMT1 knock-down of Sertoli cells METTL3,FTO and IGF2BP1 by RT-qPCR and Western Blot.Compared to the control group,METTL3,IGF2BP1 mRNA expression in the aging group increased significantly,and the FTO expression decreased.The heshouwuyin group METTL3 and IGF2BP1 mRNA expression decreased,and FTO expression increased(P< 0.05).Compared with the empty load group,the shDNMT1 group METTL3,IGF2BP1 and FTO mRNA and protein expression increased,shDNMT1 + heshouwuyin group METTL3,IGF2BP1,FTO mRNA and protein expression have not changed significantly.5.3 DNMT1 knock-down testicular Sertoli cells P53,P16 MeRIP-qPCR results.Compared with the control group,the degree of P53 and P16 mRNA m6A modification has increased in the aging group,and mRNA expression has increased.The P53 and P16 mRNA m6A modification increased in the heshouwuyin group,and mRNA expression decreased(P <0.05).The empty load group,the shDNMT1 group m6A modification increased,and the mRNA expression increased(P < 0.05).shDNMT1 + heshouwuyin group P53,P16 mRNA m6A decoration and mRNA expression have not changed significantly.Conclusion:1 Heshouwuyin could decrease the promoter methylation of Mettl3 and Igf2bp1 gene,and increase the promoter methylation of Fto gene.2 Heshouwuyin can delay the aging of Sertoli cells by regulating the methylation level of P53 and P16 RNA.3 Heshouwuyin can delay the aging of Sertoli cells by affecting the expression of Dnmt1,Metttl3,Fto and Igf2bp1 and participating in DNA-RNA epigenetic crosstalk. |
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