Study On The Mechanisms Of Yiqi Huoxue Recipe For Preventing Vascular Aging By Network Pharmacology Approach

As a major risk factor for various cardiovascular diseases?CVDs?,vascular aging contributed to the increasing morbidity and mortality from CVDs.Therefore,it is of great significance to take effective measures to prevent vascular aging.Renshen Sanqi Chuanxiong?RSC?,a Chinese herbal formula used to supplement Qi and activate blood circulation,has been proven effective for the prevention of vascular aging.However,the action mechanisms of RSC for preventing vascular aging have not been completely elucidated.This thesis is divided into two sections to discuss the mechanism of RSC delaying senescence.Section 1.Dissection of the Molecular Mechanisms of Renshen Sanqi Chuanxiong for Preventing Vascular Aging Using a Network Pharmacology ApproachObjective:In this study,an integrative network pharmacology approach based on bioinformatics,chemical,pharmacokinetic,and pharmacological data was constructed to evaluate the underlying molecular mechanisms of RSC in the delay of vascular aging.Method:First,all chemical components in RSC were collected from the Traditional Chinese medicine systems pharmacology platform?TCMSP?,and the compounds of which the drug-like?DL?value was lower than 0.18 were further excluded.Second,the candidate compounds identified the corresponding targets by mapping known drug-target interactions?DTIs?and predicted new targets by balanced substructure-drug-target-network-based reasoning?bSDTNBI?methods.Then,the human vascular senescence-related genes which were constructed by NCBI gene database,AgeFactDB database and HAGR database,were matched with the targets of the above compounds to further obtain targets related to vascular senescence in RSC.Finally,the compound-target network and target-function network were constructed by Cytoscape software,the ClueGo analysis and integrated“vascular aging”pathway analysis were performed.Result:One hundred twenty-two potential active components of RSC were identified through drug-likeness screening and their corresponding 692 direct targets were retrieved via target prediction and identification.Forty-nine vascular aging-associated targets were identified for RSC by overlapping 692 potential targets with 146 human vascular aging-associated genes.The results from the compound-target network indicated that most components acted on common targets and displayed synergistic action,which showed that the magnifying effects of RSC were based on these common targets.The target-function network revealed that each target was involved in multiple function modules,suggesting that RSC was multi-functional for the treatment of vascular aging.The results of the ClueGo analysis indicated that most of the targets were associated with the HIF-1 signaling pathway,thyroid hormone signaling pathway,and FOXO signaling pathway.In addition,the results from the pathway analysis indicated that an integrative vascular aging-related pathway mainly included the angiogenesis regulation module,cell survival module,oxidative stress resistance module,and DNA repair module for further exploration of the underlying therapeutic effects of the formula.Conclusion:This study suggested that the integrated network pharmacology approach provided new perspectives to scientifically clarify the molecular mechanisms of RSC for delaying vascular aging.This may promote the use of traditional Chinese medicine in modern medicine and provide new options for modern medicine to fight these complex diseases.Section 2.The intervention effect and action mechanism of ginsenoside Rg1 on delaying vascular endothelial cell senescenceObjective:The aim of present study was to investigate the effect of microRNA-34a/SIRT1/p53 signal pathway on ginsenoside Rg1 delaying H₂O₂-induced vascular endothelial cell senescence.Method:In this study,human umbilical vein endothelial cells?HUVECs?were selected as the research object,senescence induced by hydrogen peroxide?H₂O₂?was established as the aging model and resveratrol was used as the positive drug.HUVECs were randomly divided into four groups,which were youth group,senescence model group,ginsenoside Rg1 group and resveratrol group.The ginsenoside Rg1group and resveratrol group were stimulated with 100?moL·L^-1 H₂O₂ for4 h after 24 h treatment with ginsenoside Rg1(20?moL·L^-1)and resveratrol(10?moL·L^-1)respectively.In the end,each group was replaced with complete medium to cultivate cells for 24 h.The degree of cellular senescence detected with senescence-associated?-galactosidase?SA-?-gal?staining kit,the cell viability detected with cell counting kit-8,the cell cycle distribution analyzed by flow cytometry and the cellular SOD activity detected by WST-1 method was performed to each group respectively.The expression of SIRT1?p53?p21 and p16 proteins in HUVECs were detected by western blot.In addition,the expression level of microRNA-34a?SIRT1 mRNA and p53 mRNA in HUVECs were assayed by real-time PCR.Result:Ginsenoside Rg1 significantly reduced the positive staining rates of senescent cells,enhanced cell's proliferative capacity and intracellular SOD activity,decreased the proportion of cells in G₀/G₁phase and increased the percentage of cells in G₂/M phase simultaneously compared with the senescence model group.Moreover,ginsenoside Rg1decreased the level of microRNA-34a?p53 mRNA and the expression of p53?p21 and p16 proteins.At the same time,ginsenoside Rg1 increased the protein and mRNA expression of SIRT1.The differences in these results between the senescence model group and ginsenoside Rg1 group were all statistically significant?P<0.05?.But there were statistically insignificant?P>0.05?in these results between the ginsenoside Rg1 group and resveratrol group.Conclusion:Senescence of endothelial cells which was induced by H₂O₂ can be used as a model for aging research,the anti-aging effect of ginsenoside Rg1 on vascular endothelial cells was obvious in this study.The mechanism is possibly that ginsenoside Rg1 can delay the process of vascular endothelial cell senescence induced by H₂O₂ through regulating microRNA-34a/SIRT1/p53 signal pathway.