基于网络药理-分子对接-SPR技术研究甘麦大枣汤联合百合知母汤治疗抑郁症的分子机制

Study on molecular mechanism of Ganmai Dazao Decoction combined with Baihe Zhimu Decoction in treating depression based on network pharmacology⁃molecular docking⁃SPR technology

目的通过网络药理学探析甘麦大枣汤联合百合知母汤的活性成分及治疗抑郁症的潜在靶点,并通过分子对接、表面等离子共振(SPR)实验、热迁移实验对主要活性成分-关键靶点相互作用进行验证。方法运用中药系统药理学技术平台(TCMSP)、草本成分靶点平台(HIT)、中药分子机制生物信息学分析工具数据库(BATMAN-TCM)、中医百科全书数据库(ETCM)筛选甘麦大枣汤联合百合知母汤的有效成分。运用TCMSP数据库、比较毒理基因组学数据库(CTD)、PHARMAPPER药物靶点数据库获得甘麦大枣汤联合百合知母汤的潜在作用靶点,运用人类基因数据库(GeneCards)和基因表达数据库(GEO)获得抑郁症相关靶点,与上述药物作用靶点取交集后得到潜在治疗抑郁症靶点。运用String数据库构建靶点蛋白-蛋白相互作用(PPI)网络,运用DAVID数据库和京都基因与基因组百科全书(KEGG)数据库进行基因本体(GO)和KEGG富集分析,运用Cytoscape软件构建相应网络,筛选甘麦大枣汤联合百合知母汤抗抑郁的关键靶点和相应的活性成分。运用Discovery studio软件进行分子对接,运用SPR技术检测活性成分与靶点结合活性,运用热迁移实验检测主要活性成分与靶点的结合作用。结果经药物吸收-分配-代谢-排泄-毒性数据库(ADMET)筛选后获得甘麦大枣汤联合百合知母汤的有效成分160个,与抑郁症相关靶点、抑郁患者差异表达基因取交集后获得潜在抗抑郁靶点763个。经PPI网络分析、GO及KEGG富集分析发现,丝裂原活化蛋白激酶(MAPK)信号通路等10条信号转导通路可能发挥重要作用,其中MAPK14处于关键位置。分子对接和SPR实验结果显示,刺芒柄花素(formononetin)、甘草查尔酮A(licochalcone A)和维斯体素(vestitol)为甘麦大枣汤联合百合知母汤的主要活性成分,可与MAPK14蛋白结合,亲和力分别为2.36μmol/L、26.01μmol/L、11.30μmol/L。热迁移实验显示这3个成分与MAPK14结合后可显著提高MAPK14的热稳定性,表明这3个成分与MAPK14能发生相互作用。结论MAPK14是治疗抑郁症的潜在靶点,甘麦大枣汤联合百合知母汤通过其中3种活性成分与MAPK14结合,首次从活性分子直接作用靶点角度解释了甘麦大枣汤联合百合知母汤治疗抑郁的分子机制。

Objective To explore the active ingredients of Ganmai Dazao Decoction(GDD)combined with Baihe Zhimu Decoction(BZD)and the potential targets for the treatment of depression by network pharmacology,and verify the interaction between the main active ingredients and the key targets by molecular docking,surface plasmon resonance(SPR),and thermal shift assay experiments.Methods TCMSP,HIT,BATMAN-TCM and ECTM databases were used to screen the effective compounds in GDD combined with BZD.TCMSP,CTD and PHARMAPPER databases were used to obtain the potential targets of GDD combined with BZD.GeneCards and GEO databases were used to obtain depression-related targets,and the potential therapeutic targets for depression were obtained after intersection with above targets.The target protein-protein interaction(PPI)network was constructed using String database.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed using DAVID and KEGG databases.Cytoscape software was used to construct the corresponding network to obtain the main active ingredients and key targets.Discovery studio software was used for molecular docking.SPR technology was used to detect the binding activity of active ingredients and target.Thermal migration assay was used to detect the binding effect of main active ingredients and target.Results After ADMET screening,160 active components of GDD combined with BZD were obtained,and 763 potential anti-depression targets were obtained after intersection of depression-related targets and differentially expressed genes of depressed patients.PPI network analysis,GO and KEGG enrichment analysis showed that 10 signaling pathways such as mitogen-activated protein kinase(MAPK)signaling pathway may play an important role,of which MAPK14 was the most important.Molecular docking and SPR experiments showed that formononetin,licochalcone A and vestitol were the main active ingredients of GDD combined with BZD,which could bind to MAPK14 protein with the affinity of 2.36μmol/L,26.01μmol/L,11.30μmol/L,respectively.The thermal migration assay showed that these 3 molecules could significantly improve the thermal stability of MAPK14 after binding to MAPK14,indicating that these 3 molecules could interacted with MAPK14.Conclusions MAPK14 is a potential target for the treatment of depression,GDD combined with BZD bind to MAPK14 through 3 active ingredients.The molecular mechanism of GDD combined with BZD in treating depression was explained from the point of view of direct action of active molecules for the first time.