基于网络药理学、分子对接和实验验证探究清金益气颗粒抗疲劳作用机制

Exploring mechanism of action of Qingjin Yiqi Granules against fatigue based on network pharmacology,molecular docking and experimental validation

目的探究清金益气颗粒抗疲劳的作用及潜在作用机制。方法通过中药系统药理学数据库与分析平台(TCMSP)、高通量中药实验和参考指南数据库(HERB)、PubChem、Swiss Target Prediction等数据库获取药物活性成分和作用靶点;通过GeneCards、OMIM数据库收集疲劳的作用靶点;利用VENNY 2.1获取药物与疾病交集靶点,再联合Cytoscape 3.9.1软件构建药物-活性成分-疲劳交集靶点相互关系网络;将交集靶点导入STRING数据库构建蛋白质-蛋白质相互作用(PPI)网络并利用DAVID数据库对交集靶点进行基因本体(GO)和京都基因与基因组百科全书(KEGG)通路富集分析,预测清金益气颗粒抗疲劳的作用机制;选取关键活性成分和核心靶点进行分子对接。通过负重力竭游泳实验观察小鼠负重力竭游泳时间;非负重游泳实验测定小鼠血清乳酸(BLA)、尿素氮(BUN)、乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、肝糖原(LG)、肌糖原(MG)、葡萄糖(GLU)等指标评价清金益气颗粒抗疲劳药效。结果网络药理学和分子对接结果显示清金益气颗粒抗疲劳的210个潜在作用靶点主要富集在磷脂酰肌醇3-激酶-蛋白激酶B(PI3K-AKT)、丝裂原活化蛋白激酶(MAPK)、缺氧诱导因子1(HIF-1)、叉头框蛋白O(FoxO)等信号通路上,其主要活性成分和关键靶点蛋白结合能力较好。动物实验结果表明,清金益气颗粒可通过延长小鼠负重5%力竭游泳时间,降低非负重游泳小鼠血清中BLA、BUN、MDA水平,增加机体LG、MG、GLU水平储存和LDH、SOD、GSH-Px活性发挥抗疲劳作用。结论清金益气颗粒可能通过调节PI3K-AKT、MAPK、HIF-1、FoxO等信号通路影响机体能量代谢和氧化应激发挥抗疲劳的作用。

Objective To investigate the efficacy and potential mechanism of action of Qingjin Yiqi Granules against fatigue.Methods The active ingredients and targets of drug action were obtained through TCM Systematic Pharmacology Database and Analysis Platform(TCMSP),High-Throughput Experimental and Reference Guide Database for Traditional Chinese Medicines(HERB),PubChem,Swiss Target Prediction,and the disease-related targets of fatigue were collected through GeneCards and OMIM databases.VENNY 2.1 was used to obtain drug-disease intersection targets,and then the drug-active ingredient-fatigue-intersection target interrelationship network was constructed in conjunction with Cytoscape 3.9.1 software.The intersecting targets were imported into the STRING database to construct a protein-protein interaction(PPI)network and the intersecting targets were analyzed for gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment using the DAVID database,to predict the anti-fatigue mechanism of Qingjin Yixi Granules.Key active ingredients and core targets were selected for molecular docking.The mice were observed in the weight-bearing exhaustion swimming experiment,and the mice were measured in the non-weight-bearing swimming experiment for serum lactate(BLA),urea nitrogen(BUN),lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),liver glycogen(LG),myoglobulin(MG),glucose(GLU),and so on.The efficacy of Qingjin Yiqi Granules against fatigue was evaluated.Results The results of network pharmacology and molecular docking showed that the 210 potential targets of anti-fatigue of Qingjin Yiqi Granules were mainly enriched in the signaling pathways such as PI3K-AKT,MAPK,HIF-1,FoxO,etc,and the binding ability of its main active ingredients and key target proteins was good.The results of animal experiments showed that Qingjin Yiqi Granules could exert antifatigue effects by prolonging the weight-bearing 5%exhaustion swimming time of mice,decreasing the serum content of BLA,BUN,and MDA in non-weight-bearing swimming mice,and increasing the body's LG,MG,and GLU content storage and the activities of LDH,SOD,and GSH-Px.Conclusions Qingjin Yiqi Granules may exert anti-fatigue effects by regulating the signaling pathways such as PI3K-AKT,MAPK,HIF-1,FoxO and other signaling pathways that affect the body's energy metabolism and oxidative stress.