补阳还五汤治疗阿尔茨海默病的网络药理学研究及实验验证

Network pharmacology research and experimental verification of Buyang Huanwu decoction for Alzheimer’s disease

目的 基于网络药理学和动物实验,探讨补阳还五汤(BYHWD)治疗阿尔茨海默病(AD)的作用机制。方法 通过TCMSP、HERB数据库和文献检索筛选BYHWD的活性成分和作用靶点;运用UniProt校正作用靶点基因名,SwissADME和SwissTargetPrediction平台预测地龙作用靶点;利用OMIM、TTD、DisGeNET、HPO数据库获取AD的相关靶点,借助Venny平台选取BYHWD与AD的交集靶点;采用STRING 11.5在线数据库构建PPI网络;使用Cytoscape 3.7.2软件构建“组方药材-活性成分-疾病靶点”网络图;通过Metascape数据库对交集靶点进行GO注释和KEGG通路富集分析,借助微生信平台将其可视化。动物实验通过新物体识别、避暗实验检测APP/PS1小鼠学习记忆能力;苏木精-伊红染色观察海马神经元细胞形态;免疫组化法检测海马中RAGE、LRP1、ApoE和VCAM-1蛋白表达。结果 筛选出163个BYHWD抗AD的潜在靶点,如ApoE、VCAM-1,主要富集在癌症、AD、胆固醇代谢、脂质和动脉粥样硬化、AGE-RAGE等信号通路;行为学实验显示BYHWD给药后APP/PS1小鼠的新物体识别指数有所改善、避暗潜伏期增长,错误次数减少;HE染色显示BYHWD可以改善海马形态结构;IHC结果显示,给药后海马LRP1、ApoE表达水平升高,RAGE、VCAM-1表达水平降低。结论 本文结合动物实验对网络药理学预测结果进行验证,发现BYHWD可能通过调控RAGE/LRP1转运体改善APP/PS1小鼠的学习记忆障碍。

Objective To determine the mechanism of action of Buyang Huanwu decoction (BYHWD) against Alzheimer’s disease (AD) based on network pharmacology and animal experiments.Methods The active components and targets of BYHWD were screened by TCMSP,HERB database and literature search.The target gene names were corrected by UniProt,and the targets of earthworm were supplemented by the SwissADME and SwissTargetPrediction platforms.The OMIM,TTD,DisGeNET,and HPO databases were used to obtain the AD-related targets,and the Venny platform was used to select the intersection targets of BYHWD and AD.The PPI network was constructed by using the STRING online database.The network of “recipe herbs-active ingredients-disease targets” was constructed by Cytoscape 3.7.2 software.GO annotation and KEGG pathway enrichment analysis were performed on the intersection targets through the Metascape database,and were visualized with the help of the WeChat.In animal experiments,the learning and memory ability of APP/PS1 mice was detected by novel object recognition and dark avoidance experiments.Hematoxylin-eosin (HE) staining was used to observe the morphology of hippocampal neurons.Immunohistochemical (IHC) method was used to detect hippocampal RAGE,LRP1,ApoE and VCAM-1 protein expression.Results BYHWD has 118 active components,of which 163 intersected with AD,which were mainly enriched in cancer,Alzheimer’s disease,cholesterol metabolism,lipid and atherosclerosis,AGE-RAGE signaling pathways,etc.The behavioral experiments showed that the novel object recognition index of APP/PS1 mice was improved after the BYHWD administration,the dark avoidance latency increased,and the number of mistakes decreased.HE staining showed that BYHWD improved the morphological structure of the hippocampus.IHC showed that the expression levels of LRP1 and ApoE in the hippocampus were increased.While the expression levels of RAGE and VCAM-1 were decreased after the administration.Conclusion Network pharmacology combined with animal experiments has been verified.BYHWD may improve the learning and memory impairment of APP/PS1 mice by regulating the RAGE/LRP1 transporter.