| Objective: Based on UPLC-Q-TOF/MS metabolomics and i TRAQ quantitative proteomics,the effects of Yang tonifying formula Jingui Shenqi Pill and Yin tonifying formula Liuwei Dihuang Pill on endogenous metabolites and differential proteins of natural aging mice were observed,and the regulatory effects of Jingui Shenqi Pill and Liuwei Dihuang Pill on natural aging mice were analyzed.Then,combined with network pharmacology and molecular docking technology,through multi database mining,the multi-level network of tonify yin/yang formulas aging target was further constructed.Finally,the multi-dimensional comparative analysis of the effect characteristics of Yang tonifying formula Jingui Shenqi Pill and Yin tonifying formula Liuwei Dihuang Pill on aging was conducted.Methods: Based on the previous research of the research group,3-month-old mice were selected as the low age group,and 20 month old natural aging mice were randomly divided into aging group,Jingui Shenqi Pill group and Liuwei Dihuang Pill group,with 20 mice in each group(10 in female and 10 in male).The dosage of Jingui Shenqi Pill and Liuwei Dihuang Pill was 10.53 g / kg and 9.75 g / kg respectively.The same volume of normal saline was given to the young group and the old group.After 30 days of continuous gavage,plasma,spleen and kidney samples were prepared for UPLCQTOF/MS metabonomics analysis,and liver mitochondria samples were prepared for i TRAQ quantitative proteomics analysis.In network pharmacology research,TCMSP database was used to obtain the corresponding targets of drug-related chemical components of Jingui Shenqi pill and Liuwei Dihuang pill.The aging targets were searched in Gene Cards、OMIM、Pharm GKB、Drug Bank databases respectively,and the aging targets were searched through Cytoscape＿V3.7.0 to construct the multi-level network of tonify yin and yang prescription aging target,explore the correlation between tonify yin and yang prescription and aging,select the common components and common target proteins in the two tonic prescriptions which are more related to the target,and use Auto Dock Vina software for molecular docking.Finally,combined with the analysis of bioinformatics function,the characteristics of action mechanism of Yang tonifying formula Jingui Shenqi Pill and Yin tonifying formula Liuwei Dihuang Pill in regulating aging were discussed.Results: 1 Metabolomics study found that the metabolic substances and their enriched metabolic pathways in various tissue samples were changed during the aging process,and there were differences in the metabolic markers and pathways of aging in different tissue samples.24,14 and 20 metabolic markers were identified in plasma,spleen and kidney tissue samples of female mice,and 22,26 and 34 metabolic markers were identified in plasma,spleen and kidney tissue samples of male mice.For female mice,inosine,9,10-cyclooctadecenoic acid and sphingosine were the same markers in spleen and kidney;D-erythritose-4-phosphate was the same marker in plasma and spleen;l-Dihydrowhey acid was the same marker in plasma and kidney.For male mice,the same markers in spleen and kidney were L-glutamic acid,pantothenic acid,pyroglutamic acid and l-palmitoyl carnitine;the same markers in plasma and spleen were docosahexaenoic acid.There are 8 identical metabolic pathways in spleen and kidney of female mice,4 identical metabolic pathways in plasma and spleen,5 identical metabolic pathways in plasma and kidney,and 3 common metabolic pathways in plasma,spleen and kidney.There are 19 identical metabolic pathways in spleen and kidney of male mice,7 identical metabolic pathways in plasma and spleen,7 identical metabolic pathways in plasma and kidney,and 6 common metabolic pathways in plasma,spleen and kidney.2 Proteomic analysis showed that 163 differential proteins were up-regulated and 97 down regulated in the liver tissues of female mice compared with the aged group(3m)and 165 differential proteins were up-regulated and 91 down regulated in the liver tissues of male mice compared with the aged group(20M).The results showed that there were 155 common differential proteins in both male and female liver samples,and 63 proteins were up-regulated and down regulated.3 Liuwei Dihuang pill and Jingui Shenqi Pill can regulate aging related metabolic markers in different tissue samples.For female mice,Liuwei Dihuang pill had callback effect on 13 aging markers in plasma samples,11 markers in spleen samples,17 markers in kidney samples;for male mice,Liuwei Dihuang pill had callback effect on 17 aging markers in plasma samples,27 markers in spleen samples;for male mice,Liuwei Dihuang pill had callback effect on 17 aging markers in kidney samples In the sample,there are 18 markers of Liuwei Dihuang pill.Jingui Shenqi Pill can recall 10 aging markers in plasma samples,12 in spleen samples and 15 in kidney samples of female mice;for male mice,Jingui Shenqi Pill can recall 20 aging markers in plasma samples,25 in spleen samples and 32 in kidney samples.4 Liuwei Dihuang pill and Jingui Shenqi Pill can regulate aging related differential proteins.In female liver samples,Liuwei Dihuang Pill recalled 117 aging related differential proteins,and in male liver samples,Liuwei Dihuang Pill recalled 46 aging related differential proteins;Jingui Shenqi Pill recalled 115 aging related differential proteins in female mice and 58 aging related differential proteins in male mice.For female mice,Liuwei Dihuang pill and Jingui Shenqi Pill increased 44 senescence related proteins and decreased 66 senescence related proteins;for male mice,Liuwei Dihuang pill and Jingui Shenqi Pill increased 3 senescence related proteins.There are 14 common pathways for Liuwei Dihuang pill and Jingui Shenqi Pill to regulate natural aging female mice and 4 common pathways to regulate aging related protein enrichment in male mice.5 Network pharmacology research found that 46 major components in Liuwei Dihuang Pill correspond to 190 target genes,of which 159 are related to aging;48 major components in Jingui Shenqi Pill correspond to 194 target genes,of which 162 are related to aging.Both Liuwei Dihuang pill and Jingui Shenqi Pill can regulate the aging process by participating in the biological processes such as cell response to hypoxia,response to lipopolysaccharide,negative regulation of apoptosis,aging,positive regulation of angiogenesis,cell aging,positive regulation of vascular endothelial growth factor production,protein phosphorylation,and cell response to insulin stimulation.6 Molecular docking studies showed that c-fos(FOS)had the strongest binding activity with quercetin,α-serine / threonine protein kinase(AKT1)with diosgenin,kaempferol,quercetin,and mitogen activated protein kinase 1 MAPK1)with quercetin.Conclusion:1 In the process of aging,there are changes in metabolism and protein,and there are differences in different tissues and genders.2.The results showed that tonify yin(Liuwei Dihuang pill)and tonify yang(Jingui Shenqi Pill)formulas could regulate the metabolism markers and metabolism of aging in different tissue samples.The results showed that the effects of tonify yin(Liuwei Dihuang pill)on metabolic markers in plasma,spleen and kidney of natural aging female mice were more significant than those of tonify yang(Jingui Shenqi Pill);the effects of tonify yang(Jingui Shenqi Pill)on metabolic markers in plasma,spleen and kidney of natural aging male mice were more significant than those of tonify yin(Liuwei Dihuang pill).3 Both of Liuwei Dihuang pill and Jingui Shenqi Pill can regulate aging by intervening aging,cell adhesion regulation,protein phosphorylation,apoptosis,myocardial contraction,lipid metabolism and other biological processes.Combined analysis of metabonomics and proteomics showed that 113 differential proteins regulated by Liuwei Dihuang pill and Jingui Shenqi Pill were bioinformatics related to 72 metabolic markers in common metabolic pathway.4 Network pharmacology research found that the main targets of the anti-aging effects of the Yang tonifying agent Jingui Shenqi Pill and the Yin tonifying agent Liuwei Dihuang pill are closely related to the biological processes such as the response of cells to hypoxia,the negative regulation of apoptosis,inflammatory response,the response to lipopolysaccharide,aging,cell aging,and the positive regulation of vascular endothelial growth factor production.The results of molecular docking showed that the main active components of the two formulas had good affinity with FOS,TP53,mapk1,AKT1,Myc,Jun and m TOR.Compared with the results of proteomics and network pharmacology,the common enrichment pathways of anti-aging target / protein include plaque,PI3 K Akt signaling pathway,HIF-1 signaling pathway,thyroid hormone signaling pathway and Alzheimer’s disease. |
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