基于网络药理学探究防衰益寿丸抗衰老作用及实验验证＊

目的 使用网络药理学方法来探究防衰益寿丸中君药抗衰老作用并验证。方法 采用中药系统药理学数据库和分析平台等数据库筛选防衰益寿丸君药的活性成分及活性成分作用靶点;在GeneCards、DigSee和OMIM疾病数据库中检索衰老相关靶点;借助Uniprot数据库查询其活性成分对应靶点基因名称;采用Cytoscape 3.7.2软件构建成分、疾病、两者交集靶点网络图;借助STRING数据库和Cytoscape 3.7.2软件构建蛋白互作网络;通过R 3.6.1软件对防衰益寿丸君药抗衰老靶基因进行GO和KEGG富集分析;将核心靶点与对应的化合物进行分子对接验证,并通过动物实验验证网络药理学预测结果。结果 研究共筛选出31个活性化合物,预测得到152个作用靶点,共收集2257个衰老靶点。通过靶点映射表明药物活性成分作用于AKT1、TNF、MAPK8、MPAK1、CASP3等97个共同表达靶基因,发现靶基因主要参与被动跨膜转运蛋白活性、铵离子结合、血红素结合、脂多糖的反应等生物过程。通过调节糖尿病并发症中AGE-RAGE信号通路、TNF信号通路、MAPK信号通路等发挥抗衰老作用。分子对接验证结果表明核心靶点AKT1、MAPK1、TNF及HMOX-1靶点可与活性成分原阿片碱、花生四烯酸及山奈酚小分子化合物稳定结合。动物实验结果表明防衰益寿丸能够调控多条通路改善小鼠的学习记忆能力,改善大脑胆碱能神经通路功能,增加脑内乙酰胆碱的含量,改善老龄小鼠血脂水平。结论 研究初步探究并验证了防衰益寿丸君药抗衰老作用的核心活性成分（人参皂苷类成分、山奈酚、花生四烯酸、原阿片碱等）及关键靶基因（如AKT1、TNF、CASP3、MAPK1、HMOX1等）,网络关系构建及通路富集（如糖尿病并发症AGE-RAGE通路、TNF信号通路、MAPK信号通路等）,揭示了药物抗衰老的潜在通路和机制,体现了中药的多成分-多靶点-多途径的药理作用,为进一步深入阐释其抗衰老机制奠定了基础。

Study on the Anti-aging Mechanism of Fangshuai Yishou Pills Based on Network Pharmacology and Experimental Verification

Objective To explore and validate the anti-aging mechanism of Jun medicine in Fangshuai Yishou Pills by network pharmacology and molecular docking techniques.Methods The active ingredients and active ingredient action targets of Fangshuai Yishou Pills Jun medicine were screened using the TCMSP and BATMAN-TCM. The aging related targets were retrieved in GeneCards, DigSee and OMIM disease database with the aid of UniProt Database were queried for their active ingredients corresponding target gene names. The network diagram of ingredients, diseases, and the intersection between the two targets was constructed by using Cytoscape 3.7.2 software. The PPI network was constructed using string database and Cytoscape 3.7.2 software. GO functional analysis and KEGG pathway enrichment were performed by R3.6.1 software on the anti-aging target genes of Jun medicine in Fangshuai Yishou Pills. The core targets were subjected to molecular docking validation with corresponding compounds, and the network pharmacology prediction results were validated by animal experiments.Results In this study, 31 active compounds were screened and 152 action targets were predicted, and a total of 2257 senescence targets were collected. Through target mapping, we showed that the active ingredients acted on 97 co expressed target genes including AKT1, TNF, MAPK8, mpak1 and CASP3, and found that the target genes were mainly involved in passive transmembrane transporter activity, ammonium ion binding, heme binding, lipopolysaccharide response and other biological processes. Anti-aging effects was exerted by regulating AGE-RAGE signaling, TNF signaling, MAPK signaling, and so on in diabetic complications. Molecular docking validation results indicated that the core targets AKT1, MAPK1, TNF and HMOX-1 could be stably bound to the active ingredients protoopioid, arachidonic acid and small compounds including kaempferol. The results of animal experiments showed that Fangshuai Yishou Pills can regulate multiple pathways to improve the learning and memory ability of mice, improve the function of cholinergic neural pathways in the brain, increase the content of acetylcholine in the brain, and improve blood lipid levels in aged mice.Conclusion In this study, we initially explores the core active components (e.g. ginsenosides, kaempferol, arachidonic acid, protoopioid, and etc.) and key target genes (e.g. AKT1, TNF, CASP3, and etc.), network relationship construction and pathway enrichment (e.g. diabetes complications AGE-RAGE pathway, TNF signaling pathway, MAPK signaling pathway, and etc.) for anti-aging effect of Fangshuai Yishou Pills, which indicated that multi-component, multi-target, and multi-pathway were involved in anti-aging effect of Fangshuai Yishou Pills.