PI3K/AKT信号通路参与菖蒲参麦方改善急性脑梗死后神经功能缺损的分子机制

PI3K/AKT signaling pathway is involved in the molecular mechanism of Changpu Shenmai Decoction in improving neurological deficits after acute cerebral infarction

关键词:  急性脑梗死  菖蒲参麦方  补气扶正治法  PI3K/AKT信号通路  网络药理学  大脑中动脉梗死模型

Key words:  acute cerebral infarction  Changpu Shenmai Decoction  Buqi Fuzheng therapy  PI3K/AKT signaling pathway  network pharmacology  MCAO model

摘要:

[目的] 基于网络药理学、分子对接技术探讨菖蒲参麦方治疗急性脑梗死（ACI）的潜在分子机制，并通过动物实验初步验证。[方法] 通过中医药系统药理学平台（TCMSP）、中药分子机制生理信息学分析工具（BATMAN-TCM）数据库检索菖蒲参麦方活性成分及成分靶点，通过人类基因数据库（GeneCards）、在线人类孟德尔遗传数据库（OMIM）、遗传药理学与药物基因组学数据库（PharmGkb）、DrugBank数据库获取ACI的潜在靶点，映射后通过Cytoscape 3.9.1软件构建菖蒲参麦方治疗ACI的“活性成分-潜在作用靶点”网络图。利用蛋白互作网络数据库（STRING）构建蛋白互作用网络图（PPI）并筛选得到核心靶点基因。运用R语言软件进行基因本体（GO）富集分析及京都基因与基因组百科全书（KEGG）通路富集分析。最后采用Autodock软件进行分子对接处理，用Pymol软件对其绘图观察核心靶点与核心活性成分氢键的形成情况。构建大鼠大脑中动脉梗死（MCAO）模型，通过改良神经功能评分（mNSS）、抓力实验、转棒实验、2，3，5-三苯基氯化四氮唑（TTC）染色、免疫蛋白印迹（Western Blot）实验初步验证药效、药理学及分子对接结果。[结果] 菖蒲参麦方活性成分靶点与ACI靶点相映射得到270个菖蒲参麦方治疗ACI潜在靶点，对应55个活性成分。PPI网络分析计算后筛选得到ESR1、MAPK14、AKT1、Caspase-3、Caspase-9共5个核心靶点。GO功能富集分析得到3 129个条目，富集后发现其主要参与对氧气的反应、对异物的反应等生物过程。KEGG通路富集分析共筛选得到188条信号通路筛选后提示其机制可能与AGE-RAGE、PI3K-AKT等信号通路相关。分子对接结果显示核心成分与核心靶点对接结果良好。动物实验表明菖蒲参麦方可改善大鼠神经功能，差异具有统计学意义（P<0.05），抓力及在转棒上的运动时间，减少脑梗死面积，差异均具有统计学意义（P<0.001）；可上调p-AKT/AKT的比值、下调Caspase-9的表达，差异均具有统计学意义（P<0.001），其脑保护作用随着PI3K抑制剂LY294002的使用而被削弱。[结论] 菖蒲参麦方治疗ACI的药理作用是通过多成分、多靶点及多通路的调控而实现的，其机制可能与激活PI3K/AKT信号通路相关。

Abstract:

[Objective] To explore the potential molecular mechanism of Changpu Shenmai Decoction in the treatment of acute cerebral infarction(ACI) based on network pharmacology and molecular docking technology,and to conduct a preliminary study through animal experiments. [Methods] The active ingredients and component targets of Acorus calorus ginseng wheat recipe were retrieved from Traditional Chinese Medicine Systems Pharmacology Database(TCMSP) and Batmanic-TCM databases,and the potential targets of acute cerebral infarction were obtained from GeneCards,Online Mendelian Inheritance in Man Database(OMIM),PharmGkb and DrugBank databases. Cytoscape 3.9.1 software was used to construct the network diagram of "active ingredients-potential targets" in the treatment of ACI. The STRING database was used to construct the PPI network and screen the core target genes. R language software was used to perform Gene Ontology(GO) gene function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Finally,Autodock software was used for molecular docking,and Pymol software was used for mapping to observe the hydrogen bond formation between the core target and the core active component. The middle cerebral artery occlusion(MCAO) model was constructed,and the drug efficacy,pharmacology and molecular docking results were preliminarily verified by neurological function score,grip strength test,rotarod test,triphenyltetrazolium chloride(TTC) staining and Western blot. [Results] A total of 270 potential targets for the treatment of acute cerebral infarction,corresponding to 55 active components,were obtained by mapping the targets of active components of Acorus calarinosus and ACI. Five core targets including estrogen receptor 1(ESR1),mitogen-activated protein kinase 14(MAPK14),v-akt murine thymoma viral oncogene homolog 1(AKT1),Caspase-3 and Caspase-9 were screened after Protein-Protein Interaction Network Analysis(PPI) network analysis and calculation. GO functional enrichment analysis obtained 3129 items. After enrichment,it was found that they were mainly involved in biological processes such as the reaction to oxygen and the reaction to foreign bodies. A total of 188 signaling pathways were screened by Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis,suggesting that the mechanism may be related to Advanced glycosylation end product-specific receptor(AGE-RAGE),phosphatidylinositide 3-kinases-protein kinase B(PI3K-AKT) and other signaling pathways. Molecular docking results showed that the core components docked well with the core targets. Animal experiments showed that Changpu Shenmai Decoction could improve the neurological function(P<0.005),grip strength(P<0.001) and movement time on the rod(P<0.001),reduce the area of cerebral infarction(P<0.001),up-regulate the ratio of p-AKT/AKT(P<0.001) and down-regulate the expression of Caspase-9(P<0.001) in rats,and its brain protective effect was significantly weakened by the use of PI3K inhibitor LY294002. [Conclusion] The pharmacological effect of Changpu Shenmai Decoction on acute cerebral infarction is achieved through the regulation of multi-components,multi-targets and multi-pathways,and its mechanism may be related to the activation of PI3K/AKT signaling pathway.