基于网络药理学结合分子动力学模拟技术探讨愈梗通瘀汤治疗心绞痛的作用机制

Mechanism of Yugeng Tongyu Decoction for Treating Angina Pectoris Based on Network Pharmacology Combined with Molecular Dynamics Simulation Technique

目的:通过网络药理学、分子对接和分子动力学模拟技术,探讨愈梗通瘀汤治疗心绞痛的作用机制,通过体外实验进行验证。方法:通过查询TCMSP和TCMIP中药成分数据库,筛选愈梗通瘀汤的化学成分及相应的蛋白靶点,进一步通过UniProt数据库查询基因名称。根据数据库查询结果,构建“中药-有效成分-靶点”相互作用网络。同时,通过GeneCards和OMIM疾病靶点数据库筛选与心绞痛的疾病相关靶点,将愈梗通瘀汤的作用靶点与心绞痛疾病相关靶点取交集,获得两者的共同靶点。随后利用Cytoscape与STRING数据库分别构建了“活性成分-潜在作用靶点”及“药物-靶点-基因本体(GO)/京都基因与基因组百科全书(KEGG)”网络关系图。使用AutoDock Vina进行分子对接验证,利用OpenMM软件对分子对接后的蛋白-配体体系进行模拟验证,最后通过体外细胞实验进行了验证。结果:共筛选获得了265种活性成分及671个潜在作用靶点;进一步筛选出了182条信号通路;GO富集分析表明,主要涉及炎症反应、组织修复、氧化应激反应等;KEGG富集分析表明,主要涉及动脉粥样硬化、糖尿病并发症以及非酒精性脂肪性肝病等相关通路;分子对接结果显示,10对关键活性成分和核心靶点最小结合能均<-5.0 kJ/mol,同时小于靶点原配体的结合能,提示对接效果良好;分子动力学模拟中展示人参皂苷Rh2与磷脂酰肌醇-3-激酶催化亚单位α(PIK3CA)具有良好的结合稳定性和活性;体外细胞实验表明,人参皂苷Rh2具有明显的心肌保护作用,同时激活磷脂酰肌醇3-激酶(PI3K)-蛋白激酶B(AKT)通路。结论:通过网络药理学预测愈梗通瘀汤可能通过槲皮素、木犀草素、山柰酚、异黄酮、黄芩素、隐丹参酮、芦荟大黄素、人参皂苷Rh2、金黄紫堇碱、延胡索单酚碱等关键活性成分,调控低氧诱导因子-1(HIF-1)、肿瘤坏死因子(TNF)、PIK3CA、雌激素受体1(ESR1)等潜在通路靶点,这些通路与代谢性疾病中的炎症及氧化应激关系最为密切;且通过分子对接及分子动力学模拟和细胞实验表明人参皂苷Rh2的药理作用与激活PIK3-AKT通路关系密切;预测结果提示抗炎作用可能是愈梗通瘀汤治疗心绞痛的潜在作用机制之一。

Objective:Mechanism of Yugeng Tongyu decoction for treating angina pectoris was studied through network pharmacology,molecular docking and molecular dynamics simulation techniques.Methods:By searching the traditional Chinese medicine(TCM)system pharmacology database(TCMSP)and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine(TCMIP)traditional Chinese medicine component databases,the chemical components and corresponding protein targets of Yugeng Tongyu decoction were screened,and the gene names were further queried through UniProt database.According to the query results of the database,the interaction network of"TCM-active ingredient-target"was constructed.At the same time,the disease-related targets of angina pectoris were screened through GeneCards and Online Mendelian Inheritance in Man(OMIM)disease target databases,and the target of Yugeng Tongyu decoction were intersected with the target of angina pectoris disease to obtain the common target.The"active ingredient-potential target"and"drug-target-gene ontology(GO)/Kyoto Encyclopedia of Genes and Genomes(KEGG)"network diagrams were constructed by Cytoscape and STRING databases respectively.AutoDock Vina was used for molecular docking verification.OpenMM software were used to simulate and verify the protein-ligand system after molecular docking.Finally,it was verified by cell experiments in vitro.Results:A total of 265 active ingredients and 671 potential targets were obtained.Further,182 signal pathways were screened.GO enrichment analysis showed that it mainly involves in inflammatory reactions,tissue repair,oxidative stress reactions.KEGG enrichment analysis showed that it mainly involved atherosclerosis,diabetes complications and non-alcoholic fatty liver disease related pathways.The results of molecular docking showed that the minimum binding energy of 10 pairs of key active ingredients and core target was less than-5.0 kJ/mol,which was smaller than the binding energy of the primary ligand of the target,indicating better docking effect.Molecular dynamics simulation showed that ginsenoside Rh2 and phosphatidylinositol-3-kinase catalytic subunitα(PIK3CA)showed better binding stability and activity.In vitro cell experiments showed that ginsenoside Rh2 showed obvious myocardial protective effect and activated the phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT)pathway.Conclusion:Through network pharmacology,it was predicted that Yugeng Tongyu decoction might be composed of quercetin,luteolin,kaempferol,isoflavone,baicalein,cryptotanshinone,aloe emodin,ginsenoside Rh2,corydaline,fumarine monophenol,and other key active ingredients.The regalation of hypoxia inducible factor-1(HIF-1),tumor necrosis factor(TNF),PIK3CA,estrogen receptor 1(ESR1)and other potential pathway targets were most closely related to inflammation and oxidative stress in metabolic diseases.Through molecular docking,molecular dynamics simulation and cell experiments,the pharmacological effects of ginsenoside Rh2 were closely related to the activation of PIK3-AKT pathway.The results suggest that the anti-inflammatory effect may be one of the potential mechanisms of Yugeng Tongyu decoction for the treatment of angina pectoris.