摘要
目的:基于网络药理学方法预测二仙汤治疗焦虑障碍的潜在分子机制,并借助母婴分离结合束缚应激动物模型进行疗效及机制验证。方法:利用中医药系统药理学数据库及分析平台(TCMSP)和成分靶点预测数据库(SwissTargetPrediction)获取二仙汤的活性成分及作用靶点;通过基因数据库(GeneCards)、疗效药靶数据库(TTD)、在线人类孟德尔遗传病数据库(OMIM)和药物数据库(DrugBank)等获取焦虑障碍相关靶点,与药物靶点取交集获得药物-疾病交集靶点;利用STRING数据库构建蛋白质-蛋白质相互作用(PPI)网络图,并基于拓扑参数分析筛选核心靶点;通过Metascape平台对交集靶点进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。采用母婴分离结合束缚应激建立焦虑小鼠模型,在第21天(PD21)离乳至第97天(PD97)束缚完成期间给予二仙汤药混饲料干预。通过开放旷场实验、高架O迷宫实验评估小鼠焦虑状态。酶联免疫吸附测定法(ELISA)检测小鼠血浆皮质酮(CORT)含量;蛋白免疫印迹法(Western blot)、实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠海马蛋白激酶B(Akt1)、哺乳动物雷帕霉素靶蛋白(mTOR)、脑源性神经营养因子(BDNF)、突触后致密物-95(PSD95)及突触素(synaptophysin)的表达水平。结果:共获得二仙汤活性成分97种,作用靶点227个,焦虑障碍相关靶点3863个,药物-疾病交集靶点161个,其中Akt1、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子(TNF)、mTOR等核心靶点可能与焦虑障碍密切相关。KEGG通路分析结果显示二仙汤治疗焦虑障碍主要涉及磷脂酰肌醇3-激酶(PI3K)/Akt、丝裂原活化蛋白激酶(MAPK)及神经活性配体-受体相互作用等信号通路。动物实验结果显示,与模型组比较,二仙汤可显著增加小鼠中央区活动时间及穿越次数、开放臂停留时间及穿越次数、明显上调p-Akt1、p-mTOR、BDNF、PSD95、Syp的表达水平(P<0.05,P<0.01)。结论:二仙汤调治焦虑障碍具有多靶点-多通路的作用特点,其机制可能与二仙汤通过影响Akt1、mTOR、IL-1β、IL-6、TNF等核心靶点及调控PI3K/Akt、MAPK及神经活性配体-受体相互作用等信号通路,改善神经炎症与突触可塑性有关。
Objective:To predict the potential molecular mechanism of Erxian decoction in the treatment of anxiety disorder based on network pharmacology,and to verify the efficacy and mechanism using the animal model of maternal separation combined with restraint stress.Method:Active components and related targets of Erxian decoction were obtained by traditional Chinese medicine system pharmacology database and analysis platform(TCMSP)and SwissTargetPrediction.The targets related to anxiety disorder were screened out through GeneCards,therapeutic target database(TTD),online mendelian inheritance in man database(OMIM),and DrugBank,and the drug-disease intersection targets were obtained by taking intersections with the drug targets.The protein-protein interaction(PPI)network was constructed by the STRING database,and the core targets were screened out based on topological parameter analysis.Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were carried out for the intersection targets through the Metascape platform.Maternal separation combined with restraint stress was used to induce the mouse model of anxiety disorder.From the end of lactation on the 21st postnatal day(PD21)to the completion of restraint stress on the 97th postnatal day(PD97),the mice were fed with Erxian decoction mixed with diet.The anxiety state of mice was evaluated by open field test and elevated O-maze test.The content of plasma corticosterone(CORT)in mice was detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of protein kinase B(Akt1),mammalian target of rapamycin(mTOR),brainderived neurotrophic factor(BDNF),postsynaptic density-95(PSD95),and synaptophysin in the hippocampus of mice were detected by Western blot and real-time quantitative polymerase chain reaction(Real-time PCR).Result:Ninty-seven active components and 227 action targets of Erxian decoction were obtained.There were 3863 targets related to anxiety disorder,with 161 drug-disease intersection targets.Among these intersection targets,core targets such as Akt1,interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor(TNF),and mTOR were presumedly closely related to anxiety disorder.The results of KEGG pathway analysis showed that Erxian decoction mainly treated anxiety disorder through phosphatidylinositol 3-kinase(PI3K)/Akt,mitogen-activated protein kinase(MAPK),and neuroactive ligand-receptor interaction signaling pathways.The results of animal experiments showed that compared with the model group,the Erxian decoction group significantly increased the time of mice spent in the central zone and central crossing times and time spent in the opened arm and opened arm crossing times,with significantly increased expression levels of p-Akt1,p-mTOR,BDNF,PSD95,and synaptophysin(Syp).Conclusion:Erxian decoction has the multi-target and multipathway characteristics in the treatment of anxiety disorder,and its mechanism may be related to the improvement of synaptic plasticity and neuroinflammation by affecting Akt1,IL-1β,IL-6,TNF,mTOR,and other core targets and modulating PI3K/Akt,MAPK,as well as neuroactive ligand-receptor interaction signal pathways.
作者
佘楷杰
杨婧雯
孟丹华
梁文青
巩子汉
岳广欣
SHE Kaijie;YANG Jingwen;MENG Danhua;LIANG Wenqing;GONG Zihan;YUE Guangxin(Institute of Basic Theory for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;School of Basic Medicine,Henan University of Chinese Medicine,Zhengzhou 450000,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第19期185-193,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金面上项目(82174251,81573846)
中国中医科学院科技创新工程项目(CI2021A00607)
中央级公益性科研院所基本科研业务费专项(YZ-202107,YZ-202153)。
关键词
二仙汤
网络药理学
焦虑障碍
动物实验
分子机制
Erxian decoction
network pharmacology
anxiety disorder
animal experiments
molecular mechanism
