基于网络药理学及实验验证研究地黄饮子防治阿尔茨海默病作用机制

Network Pharmacology-Based and Experimental Validation Approach to Investigate the Mechanism of Dihuang Yinzi in Treating Alzheimer's Disease

目的:采用网络药理学结合实验验证,探讨地黄饮子防治阿尔茨海默病(AD)的作用机制。方法:利用中药系统药理学数据库与分析平台(TCMSP)、HERB数据库获取地黄饮子化学成分,基于药代动力学信息与文献检索筛选符合条件的有效成分,综合TCMSP、SwissTargetPrediction、STITCH数据库获取对应靶点。利用Therapeutic Target Database、GeneCards、DisGeNET数据库收集AD靶点。利用Cytoscape 3.7.1软件构建“中药-成分-靶点”网络。利用Metascape在线工具对靶点进行GO和KEGG通路富集分析。利用东莨菪碱构建AD小鼠模型,采用Y迷宫、新物体识别、Morris水迷宫评价小鼠学习记忆能力,酶联免疫吸附法(ELISA)检测海马组织乙酰胆碱酯酶(AChE)、肿瘤坏死因子-α (TNF-α)、白细胞介素-6 (IL-6)的水平,Western Blot检测海马组织核因子κB (NF-κB)和磷酸化核因子κB (p-NF-κB)蛋白表达。结果:通过中心网络拓扑分析筛选出地黄饮子中61个关键有效成分,作用于AD的关键靶点106个。GO富集分析发现关键靶点参与肽结合(淀粉样蛋白)、胆碱能结合、神经递质受体的活动,炎症反应的调节、行为活动调控等。KEGG通路富集分析发现AChE、CHRNA7、RELA、TNF等关键靶点富集于胆碱能突触、NF-κB信号通路。体内实验表明地黄饮子预处理能够改善AD模型小鼠的认知功能障碍;抑制小鼠AChE、IL-6、TNF-α水平;抑制p-NF-κB蛋白表达。结论:地黄饮子可能通过抑制小鼠海马AChE的水平,减轻胆碱能损伤,抑制由NF-κB介导的神经炎症,恢复“胆碱能抗炎途径”,改善小鼠认知功能障碍,起到防治AD的作用。

Objective:To explore the mechanisms of prevention and treatment of Alzheimer's Disease(AD) by Dihuang Yinzi using network pharmacology combined with experimental verification.Methods:All chemical components of Dihuang Yinzi were obtained by TCMSP and HERB databases,and the eligible active components were screened based on the pharmacokinetic information of chemical components and literature search,and the corresponding predicted targets were further obtained by TCMSP,SwissTargetPrediction and STITCH databases.AD-related targets were collected using the Therapeutic Target Database,GeneCards and DisGeNET database.Cytoscape 3.7.1 software was used to construct the "herb-component-target" network.The Metascape web tool was used to analyze the targets for GO and KEGG pathway enrichment.The mouse model of AD was constructed by scopolamine.Y-maze,new object recognition and Morris water maze were used to evaluate the learning and memory ability of the mice.The levels of acetylcholinesterase(AChE),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6) in the hippocampus were detected by ELISA.The expressions of nuclear factor(NF-κB) and phospho-NF-κB(p-NF-κB) in the hippocampus were detected by Western Blot.Results:A total of 61 key active components in Dihuang Yinzi were screened by central network topology analysis,and 106 key targets were identified in AD.GO enrichment analysis revealed that key targets were involved in peptide binding(amyloid),acetylcholine binding,neurotransmitter receptor activity,regulation of inflammatory response,behavior,etc.KEGG pathway enrichment analysis revealed that AChE,CHRNA7,RELA,and TNF and other key targets were enriched in cholinergic synapses,NF-κB signaling pathway.In vivo experiments showed that Dihuang Yinzi pretreatment improved the cognitive function of AD mice,inhibited the levels of AChE,IL-6 and TNF-α,and inhibited the expression levels of p-NF-κB proteins.Conclusion:Dihuang Yinzi may reduce cholinergic damage by inhibiting the level of AChE in the mouse hippocampus,further inhibiting NF-κB-mediated neuroinflammation and restoring the "cholinergic anti-inflammatory pathway",thus improve the cognitive dysfunction in mice and preventing AD.