摘要
目的:基于网络药理学、分子对接及动物实验验证,探讨开心散治疗阿尔茨海默病(AD)的作用机制.方法:①采用中药系统药理数据分析平台(TCMSP)和中医药百科全书(ETCM)数据库获取开心散的活性成分及靶点;利用GeneCards、在线人类孟德尔遗传数据库(OMIM)、TTD、PharmGKB和DrugBank数据库获取AD的相关靶点;对开心散的活性成分作用靶点与AD相关靶点取交集(共同靶点);STRING数据库对共同靶点进行网络互作分析,构筑蛋白质-蛋白质相互作用(PPI)网络;应用Cytoscape内的CytoNCA插件筛选出核心靶点;Metascape平台执行基因本体(GO)功能富集分析及京都基因与基因组百科全书(KEGG)通路富集分析;借助Cytoscape 3.8.2建立"药物-活性成分-靶点"互作网络;AutoDock Vina进行分子对接;②利用东莨菪碱(SCOP)造模,腹腔注射,每日1次.将32只雄性C57/BL6小鼠随机分为空白(CON)组(0.9％氯化钠,n=8)、模型(SCOP)组(3 mg·kg-1·d-1,n=8)、盐酸多奈哌齐组(SCOP 3 mg·kg1·d-1+盐酸多奈哌齐3 mg·kg-1·d-1,n=8)、开心散组(SCOP 3 mg·kg-1·d-1+开心散6.5 g·kg-1·d-1,n=8).各组小鼠0.9％氯化钠、开心散或盐酸多奈哌齐灌胃给药,2次/d,连续14 d.采用Morris水迷宫实验观察小鼠学习记忆能力;苏木素-伊红(HE)染色法观察小鼠海马CA1区病理变化;酶联免疫吸附测定法(ELISA)测定小鼠血清乙酰胆碱(ACh)与乙酰胆碱酯酶(AChE)含量;蛋白免疫印迹法(Western blot)检测小鼠海马中信号转导和转录激活因子3(STAT3)、核转录因子(NF)-κB p65蛋白表达水平.结果:①共得到开心散73种活性成分,筛选出开心散对治疗AD的潜在作用靶点(共同靶点)578个;关键活性成分:山柰酚、吉九里香碱等;潜在核心靶点:STAT3、NF-κB p65等;GO功能富集分析得到3 124条生物功能、254条细胞构件、461条分子功能;KEGG通路富集分析获得248条通路,主要涉及癌症相关通路、TRP通路、环磷酸腺苷(cAMP)通路、NF-κB通路等;分子对接显示,关键活性成分与目标靶点结合力较稳定.②Morris水迷宫实验表示开心散可以改善SCOP诱导的小鼠学习记忆能力;HE染色及ELISA检测结果表明开心散对小鼠中枢神经损伤有改善;Western blot检测表明开心散对SCOP模型小鼠海马组织中NF-κB p65磷酸化与STAT3磷酸化水平有下调作用.结论:开心散可以改善SCOP模型小鼠的认知障碍功能,并且可能通过调节NF-κB p65、STAT3等靶点来参与NF-κB信号通路等,减少海马神经元损伤从而发挥治疗AD的作用.
Abstract
Objective:To investigate the mechanism of action of Kaixinsan in the treatment of Alzheimer's disease(AD)based on network pharmacology,molecular docking,and animal experimental validation.Methods:The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and the Encyclopedia of Traditional Chinese Medicine(ETCM)databases were used to obtain the active ingredients and targets of Kaixinsan.GeneCards,Online Mendelian Inheritance in Man(OMIM),TTD,PharmGKB,and DrugBank databases were used to obtain the relevant targets of AD.The intersection(common targets)of the active ingredient targets of Kaixinsan and the relevant targets of AD was taken,and the network interaction analysis of the common targets was carried out in the STRING database to construct a protein-protein interaction(PPI)network.The CytoNCA plugin within Cytoscape was used to screen out the core targets,and the Metascape platform was used to perform gene ontology(GO)functional enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.The"drug-active ingredient-target"interaction network was constructed with the help of Cytoscape 3.8.2,and AutoDock Vina was used for molecular docking.Scopolamine(SCOP)was utilized for modeling and injected intraperitoneally once daily.Thirty-two male C57/BL6 mice were randomly divided into blank control(CON)group(0.9％NaCl,n=8),model(SCOP)group(3 mg·kg-1·d-1,n=8),positive control group(3 mg·kg-1·d-1 ofSCOP+3 mg·kg-1·d-1 of Donepezil,n=8),and Kaixinsan group(3 mg·kg-1·d-1 of SCOP+6.5 g·kg-1·d-1 of Kaixinsan,n=8).Mice in each group were administered with 0.9％NaCl,Kaixinsan,or Donepezil by gavage twice a day for 14 days.Morris water maze experiment was used to observe the learning memory ability of mice.Hematoxylin-eosin(HE)staining method was used to observe the pathological changes in the CAI area of the mouse hippocampus.Enzyme linked immunosorbent assay(ELISA)was used to determine the serum acetylcholine(ACh)and acetylcholinesterase(AChE)contents of mice.Western blot method was used to detect the protein expression levels of signal transducer and activator of transcription 3(STAT3)and nuclear transcription factor(NF)-κB p65 in the hippocampus of mice.Results:A total of 73 active ingredients of Kaixinsan were obtained,and 578 potential targets(common targets)of Kaixinsan for the treatment of AD were screened out.Key active ingredients included kaempferol,gijugliflozin,etc..Potential core targets were STAT3,NF-κB p65,et al.GO functional enrichment analysis obtained 3 124 biological functions,254 cellular building blocks,and 461 molecular functions.KEGG pathway enrichment obtained 248 pathways,mainly involving cancer-related pathways,TRP pathway,cyclic adenosine monophosphate(cAMP)pathway,and NF-κB pathway.Molecular docking showed that the binding of the key active ingredients to the target targets was more stable.Morris water maze experiment indicated that Kaixinsan could improve the learning memory ability of SCOP-induced mice.HE staining and ELISA results showed that Kaixinsan had an ameliorating effect on central nerve injury in mice.Western blot test indicated that Kaixinsan had a down-regulating effect on the levels of NF-κB p65 phosphorylation and STAT3 phosphorylation in the hippocampal tissue of mice in the SCOP model.Conclusion:Kaixinsan can improve the cognitive impairment function in SCOP model mice and may reduce hippocampal neuronal damage and thus play a therapeutic role in the treatment of AD by regulating NF-κB p65,STAT3,and other targets involved in the NF-κB signaling pathway.
国家科技期刊平台
NSTL
维普
万方数据