Study On The Effect And Mechanism Of Shouwuwan On Aging From DNA Methylation Angle

Objective:Through literature research,network pharmacology research and DNA methylation bioinformatics analysis,this paper discusses the mechanism of aging,explores the action target and action pathway of Shouwuwan in delaying aging,and analyzes the core action target and main action pathway of Shouwuwan in delaying aging from the perspective of DNA methylation.At the same time,it is verified by animal experiments in order to lay a theoretical foundation for the clinical application of Shouwuwan in delaying aging and preventing senile diseases.Methods:1.Theoretical research:Explore the mechanism of aging by consulting various literatures,and analyze the composition and research status of Shouwuwan.2.Based on network pharmacology and DNA methylation bioinformatics analysis,explore the mechanism of Shouwuwan in delaying aging:TCMSP and ETCM databases were used to obtain the active components of 13 traditional Chinese medicines in Shouwuwan and the corresponding drug targets of the active components of each drug.At the same time,Genecards database was used to screen the targets related to aging.STRING website was used to construct PPI network for the targets of Shouwuwan for delaying aging.R language cluster Profiler package was used to conduct enrichment analysis based on GO database and enrichment analysis based on KEGG database.Use the GEO database to obtain the data set of DNA methylation sequencing and the data set related to aging.Define the age≥60 year old sample as the elderly group,and define the age<45 year old sample as the youth control group.Use Ch AMP to detect the quality of DNA methylation data and find the differential DNA methylation sites between the elderly group and the youth control group.Use the R language cluster Profiler package to conduct enrichment analysis based on the KEGG database for the differential DNA methylation target genes of Shouwuwan in delaying aging.The core action target and main action pathway of Shouwuwan affecting DNA methylation and delaying aging were screened out,and the core action target was linked with the corresponding drug active molecule,and the structure with the lowest binding free energy in the output result was selected.3.Experimental study:The rats were randomly divided into normal group,model group,Shouwuwan group,and vitamin E group.The model group,Shouwuwan group,and vitamin E group were subcutaneously injected with 0.5% D-galactose physiological saline solution into the head and neck to create an aging model.At the same time,the Shouwuwan group was given Shouwuwan suspension by gavage,the vitamin E group was given vitamin E suspension by gavage,and the normal group and model group were given the same amount of physiological saline by gavage for 6 weeks.Six weeks later,liver and kidney tissues were taken.ELISA was used to detect the overall DNA methylation level.MSP was used to detect the change of DNA methylation level in the promoter region of TNF and p21 genes.Real-time fluorescence quantitative PCR was used to detect the expression level of DNMT1,DNMT3 a,DNMT3b,TNF-α,p21,AKT1,p65,p53,Rb1,IL-6 m RNA.WB was used to detect the expression level of DNMT1,DNMT3 a,DNMT3b,TNF-α,p21,AKT1,p-AKT1,p65,p-p65,p53,p-p53,Rb,p-Rb,IL-6 protein.The frozen sections of liver and kidney tissues were β-Galactosidase staining.Results:1.The results of theoretical research:Yin deficiency is an important factor leading to aging and is also the pathological basis of aging.Kidney Yin is the root of whole body’s Yin,liver and kidney seminal blood homologous interaction,liver and kidney yin deficiency is the basic disease mechanism of aging,and invigorating the Yin of the liver and kidney is an important law to delay aging and prolong life.Shouwuwan can tonify liver and kidney,strengthen muscles and bones,and blacken beard and hair.Shouwuwan’s medicinal propertie is gentle,and it is suitable for the elderly with yin deficiency of liver and kidney to take it regularly to delay aging and prolong life span.2.The results of the study on the mechanism of Shouwuwan in delaying aging based on the network pharmacology and DNA methylation bioinformatics analysis:554 drug targets of active ingredients of Shouwuwan were obtained,8305 age-related targets were obtained,and 479 anti-aging targets of Shouwuwan were obtained after the intersection of the two,mainly including TP53,HSP90AA1,AKT1,JUN,RELA,MAPK1,CTNNB1,RXRA,MAPK14,ESR1,TNF,FOS,IL6,EGFR,MAPK8,CYP1A1,NCOA1,MYC,NFKB1,HIF1 A,CYP2E1,PPARA,RB1,CAV1,CYP3A4,CDKN1 A,CCND1,CDK1,NR3C1,AR.The results of KEGG enrichment showed that the anti-aging target of Shouwuwan was significantly related to IL-17 signal pathway,HIF-1 signal pathway,TNF signal pathway and other pathways.In the aging related DNA methylation sequencing data set GSE40279,there are 395 samples in the elderly group and 60 samples in the youth control group.There are 5149 differential methylation sites between the two groups,with 2659 genes annotated.The anti-aging target of Shouwuwan was intersected with the acquired genes of DNA methylation difference sites related to aging,and a total of 67 targets were obtained.The targets mainly include HTR2 A,CD44,MET,FTH1,ACACA,NQO1,CASP8,PTGS2,GSTP1,GSTM1,SHBG,FXN,CDKN1 A,PRKCA,HK1,COL1A1,MTHFR,CYP2E1,PPARG,SCN1 B,DRD2,TNF,CTSD.The results of KEGG enrichment showed that the target genes of differential DNA methylation were significantly related to C-type lectin receptor signaling pathway,HIF-1 signaling pathway and other pathways.Intersect the top 30 targets of Degree with the top 30 targets of |delta Beta| to obtain CDKN1 A,CYP2E1 and TNF.Among the top 50 pathways in the enrichment results of KEGG pathway of the anti-aging target of Shouwuwan and the anti-aging differential DNA methylation target of Shouwuwan,the pathways with significant correlation with aging research were screened,and three pathways were obtained,namely TNF signaling pathway,PI3K-Akt signaling pathway,NF-kappa B signaling pathway.At the same time,TNF,CDKN1 A,TP53,AKT1,RELA,which are significantly related to the main action pathway and aging,were selected as the core action targets among the top 10 targets of Degree and CDKN1 A,CYP2E1 and TNF.The molecular docking between the core target and the corresponding drug active molecule shows that there are binding hydrogen bonds between the protein and small molecules.3.The results of experimental study:Compared with the model group,the overall DNA methylation rate in liver and kidney tissues of Shouwuwan group was significantly increased(P<0.01).The DNA methylation rate of TNF and p21 gene promoter region in liver and kidney tissue was increased(P<0.05).Liver tissue DNMT1,DNMT3 a,and DNMT3 b m RNA expression levels were significantly increased(P<0.01),p21,AKT1,p65,and Rb1 m RNA expression levels were significantly decreased(P<0.01),and TNF-α,p53,IL-6 m RNA expression levels were decreased(P<0.05).Kidney tissue DNMT1 and DNMT3 a m RNA expression levels were significantly increased(P<0.01),DNMT3 b m RNA expression was increased(P<0.05),TNF-α,p21,AKT1,p65 m RNA expression levels were significantly decreased(P<0.01),and p53,Rb1,IL-6 m RNA expression levels were decreased(P<0.05).In liver tissue,DNMT1,DNMT3 a protein expression levels were significantly increased(P<0.01),DNMT3 b,p-Rb protein expression levels were increased(P<0.05),TNF-α,p21,IL-6 protein expression levels were significantly decreased(P<0.01),p-AKT1,p53,p-p53 protein expression levels were decreased(P<0.05),the ratios of p-AKT1/AKT1,p-p65/p65 and p-p53/p53 were all decreased(P<0.05),and the ratio of p-Rb/Rb was significantly increased(P<0.01).Kidney tissue DNMT3 a protein expression levels were significantly increased(P<0.01),DNMT1,DNMT3 b protein expression levels were increased(P<0.05),TNF-α,p21,IL-6,p-AKT1,p65 protein expression levels were significantly decreased(P<0.01),and p-p65,p-p53,Rb protein expression levels were decreased(P<0.05),the ratios of p-AKT1/AKT1 and p-p53/p53 were significantly decreased(P<0.01),the ratio of p-p65/p65 was decreased(P<0.05),and the ratio of p-Rb/Rb was increased(P<0.05).The ratio of the areas positive for β-gala-ctosidase was significantly lower in the liver and kidney(P<0.01).Conclusions:1.Yin deficiency is an important cause of aging,and liver and kidney yin deficiency is the basic pathogenic mechanism of aging.Shouwuwan’s medicinal propertie is gentle,and it is suitable for elderly people with liver and kidney yin deficiency to delay aging and extend life span.2.TP53,HSP90AA1,AKT1,JUN,RELA,MAPK1,CTNNB1,RXRA,MAPK14,ESR1 are the main targets of Shouwuwan in delaying aging.HTR2 A,CD44,MET,FTH1,ACACA,NQO1,CASP8,PTGS2,GSTP1 and others are the differential DNA methylation targets of Shouwuwan for delaying aging.CDKN1 A,CYP2E1,TNF are the central targets through which Shouwuwan affects DNA methylation to delay aging.TNF signaling pathway,PI3K-Akt signaling pathway,NF-kappa B signaling pathway are the main acting pathways by which Shouwuwan regulate DNA methylation and thus delay aging.3.Shouwuwan may inhibit the expression of TNF,p21 by up regulating DNA methylation levels.By TNF-α/AKT/NF-κB pathway inhibition of NF-κB signaling pathway,thereby inhibiting the expression of p53.Ultimately,it can be used to delay the onset of cellular senescence as well as inhibit the secretion of SASP such as TNF-α and IL-6 by inhibiting the p53/p21/Rb pathway.