基于网络药理学、分子对接和实验验证探讨朱茯苓用于失眠的作用机制

为探讨朱茯苓治疗失眠的可能作用机制,通过TCMSP、BAT-MAN、TCMID和STITCH数据库以及文献挖掘筛选朱茯苓的活性成分及潜在靶点,利用TTD、OMIM、GeneCards和CTD数据库获取失眠类疾病的相关靶点,采用Cytoscape软件和String数据库构建活性成分-靶点网络和靶点蛋白相互作用网络,通过BioGPS数据库进行靶点的器官定位,基于David数据库进行GO功能和KEGG通路的富集分析,利用Autodock_vina软件进行活性成分和核心靶点的分子对接验证,最后通过免疫印迹法(Western blot)验证朱茯苓对γ-氨基丁酸(GABA)的两种受体蛋白α1亚基基因GABRA1和γ2亚基基因GABRG2的影响。最终筛选得到朱茯苓活性成分33种,潜在靶点267个,与失眠的交集靶点36个,多作用于脑、心脏等器官;分子对接结果显示GABRA1、GABRG2两个靶点能够与活性成分自发结合并借助氢键等分子间作用力形成较为稳定的构象;富集分析共获得189个GO条目和24条KEGG通路,主要涉及GABA信号通路、5-羟色胺(5-HT)信号通路等;Western blot实验证明朱茯苓能够增强小鼠脑内GABRA1和GABRG2蛋白的表达。通过网络药理学、分子对接和Western blot实验验证,发现朱茯苓可能通过多成分、多靶点、多途径的协同作用发挥镇静安神的作用,为深入进行朱茯苓治疗失眠的作用机制研究提供新思路和新方法。

Study on the mechanism of Zhufuling in insomnia based on network pharmacology,molecular docking and experimental verification

In order to explore the possible mechanism of Zhufuling in the treatment of insomnia,the active components and potential targets of Zhufuling were screened by TCMSP platform,BAT-MAN database,TCMID database,STITCH database and literature mining;related targets of insomnia diseases were obtained by TTD database,OMIM database,GeneCards database and CTD database,and the active component target network and target protein interaction network were constructed by using Cytoscape software and String database,and the target organs were located by BioGPS database,and GO function and KEGG pathway enrichment analysis were carried out based on David database,and Autodock_Vina software was used to verify the molecular docking of active components and core targets.Western blot was used to verify the effect of Zhufuling on GABA receptorα1 subunit gene(GABRA1)and GABA receptorγ2 subunit gene(GABRG2).Finally,33 kinds of active components,267 kinds of potential targets and 36 kinds of intersecting targets with insomnia were screened,which were mostly used in brain,heart and other organs.The results of molecular docking showed that GABRA1 and GABRG2 could spontaneously combine with the active components and form stable conformations by hydrogen bonding and other intermolecular forces.189 GO entries and 24 KEGG pathways were obtained by enrichment analysis,which mainly involved GABA signaling pathway and 5-hydroxytryptamine(5-HT)signaling pathway.Western blot showed that Zhufuling could enhance the expression of GABRA1 and GABRG2 protein in mouse brain.Through network pharmacology,molecular docking and Western blot experiments,we found that Zhufuling may play a calming and tranquilizing effect through multi-component,multi-target and multi-channel synergistic effect,which provides new ideas and methods for further research on the mechanism of Zhufuling in the treatment of insomnia.