天然产物研究与开发 ›› 2022, Vol. 34 ›› Issue (12): 2119-2129.doi: 10.16333/j.1001-6880.2022.12.016

• 数据研究 • 上一篇    下一篇

基于网络药理学和实验验证探讨黄芪散治疗阿尔茨海默病的作用机制

张运辉1,2,杨梦琳1,2*,周小青2,伍大华2,3,刘   霞1,李   祥1   

  1. 1重庆三峡医药高等专科学校,重庆 404120;2湖南中医药大学,长沙410208;3湖南省中医药研究院附属医院,长沙410006
  • 出版日期:2022-12-28 发布日期:2022-12-29
  • 基金资助:
    湖南省研究生创新课题(2020CX10);国家自然科学基金(81874462);重庆市中医药重点学科(中医基础理论)建设项目(渝中医[2021]16号)

Study on the mechanism of Huangqisan in treating Alzheimer′s disease based on network pharmacology and experimental verification

ZHANG Yun-hui 1,2,YANG Meng-lin 1,2*,ZHOU Xiao-qing 2,WU Da-hua2,3,LIU Xia1,LI Xiang1   

  1. 1Chongqing Three Gorges Medical College,Chongqing 404120,China;2Hunan University of Chinese Medicine,Changsha 410208,China;3Affiliated Hospital of Hunan Provincial Academy of Traditional Chinese Medicine,Changsha 410006,China
  • Online:2022-12-28 Published:2022-12-29

摘要:

运用网络药理学方法探讨黄芪散治疗阿尔茨海默病(Alzheimer′s disease,AD)的作用机制。通过 TCMSP数据库检索黄芪散的有效成分及相关靶点;采用DisGeNET和GeneCards数据库搜集AD靶点,通过String在线数据库构建靶蛋白相互作用网络,采用R语言对关键靶点进行GO和KEGG富集分析。采用Aβ25-35诱导PC12细胞损伤作为AD细胞模型,通过MTT法检测细胞存活率,采用显微镜观察细胞形态和突触生长,通过透射电镜观察PC12细胞自噬小体,利用试剂盒检测活性氧(reactive oxygen species,ROS)含量;最后采用ELISA法和Western blot实验对网络药理学主要预测的生物过程与信号通路进行验证。结果共获得黄芪散44个有效成分,对应靶点134个,与AD相关靶点共102个,KEGG相关信号通路前20条,GO分析前20个生物学过程。细胞实验证实了黄芪散能有效增加PC12细胞的存活率和突触长度,有效促进Aβ25-35诱导的PC12细胞自噬小体包裹受损线粒体,降低其炎症因子IL-1β、IL-18、TNF-α的含量,降低ROS水平,升高LC3Ⅱ/Ⅰ比值,上调PINK1、parkin、BDNF蛋白表达,下调p62、NLRP3蛋白表达。黄芪散可能是通过激活PINK1/parkin通路促进线粒体自噬,降低ROS水平进而抑制NLRP3炎症小体的活化和改善突触可塑性而发挥治疗AD作用。

关键词: 黄芪散, 网络药理学, 阿尔茨海默病, 线粒体自噬

Abstract:

To study the mechanism of Huangqisan in treating Alzheimer′s disease(AD) by network pharmacology.To search the active ingredients and corresponding targets of Huangqisan through the TCMSP databases,Then obtained the targets of AD through the DisGeNET and GeneCards database.Constructed protein interaction network map by String online database.At the same time GO and KEGG enrichment analysis of key targets were carried out by using R language.Induction of PC12 cell injury by adopted Aβ25-35 as AD cell model.Detection of cell viability by MTT assay.Observation of cell morphology and synaptic growth by microscope.Observation of Autophagosomes in PC12 cells by transmission electron microscopy.Detection of reactive oxygen species (ROS) content by using Kits.Finally,ELISA and Western blot were used to verify the main biological processes and signaling pathways predicted by network pharmacology.A total of 44 active ingredients of Huangqisan were screened,134 corresponding targets,102 targets related to Alzheimer′s disease.The top 20 KEGG related signaling pathways and GO analysis of the top 20 biological processes.Cell experiments proved that Huangqisan effectively increased the survival rate and synaptic length of PC12 cells,effectively promoted Aβ25-35 induced PC12 cells autophagosome wrapped damaged mitochondria,reduced the content of inflammatory factors IL-1β,IL-18,TNF-α,reduced ROS level,increased LC3 II/I ratio,up-regulated PINK1,parkin,BDNF protein expression,down-regulated p62,NLRP3 protein expression.Huangqisan had a therapeutic effect on AD by activated PINK1/parkin pathway to promoted mitophagy and reduced ROS levels,thereby inhibited the activation of NLRP3 inflammasome and improved synaptic plasticity.

Key words: Huangqisan, network pharmacology, Alzheimer′s disease, mitophagy

中图分类号:  R285.5