目的 基于GEO芯片联合网络药理学方法探讨黄芎方治疗缺血性脑卒中的作用机制，并构建脑缺血/再灌注（I/R）大鼠模型验证黄芎方药效和核心靶点。方法 利用中药系统药理学数据库与分析平台（TCMSP）数据库及文献检索，获得黄芎方的主要活性成分，通过 Swiss Target Prediction数据库检索其相应的靶点。通过 R软件 limma包对 GEO平台缺血性脑卒中患者数据集 GSE16561 进行差异基因分析。利用 Venny 2.1.0 进行黄芎方成分靶点和缺血性脑卒中差异基因靶点交集靶点分析，采用DAVID数据库对交集靶点进行基因本体（GO）功能富集分析和京都基因与基因组百科全书（KEGG）通路富集分析。采用 Cytoscape 3.9.0 软件构建成分-靶点-通路网络并筛选核心成分，通过 STRING 数据库与 Cytoscape 3.9.0 软件构建蛋白质相互作用（PPI）网络并筛选核心靶点基因。利用PyMOL软件对度值排名靠前的成分和靶点基因进行分子对接。通过single sample Gene Set Enrichment Analysis（ssGSEA）进行免疫浸润分析。采用线栓法制备 I/R 大鼠模型，除假手术组外（n＝21），将69只大鼠随机分为模型组、黄芎方组（3.6 g·kg^-1）组、阿司匹林（9 mg·kg^-1）组。黄芎方组和阿司匹林组每日ig给药1次，假手术组和模型组ig给予等量生理盐水，持续7 d。采用Longa分级评分法对大鼠进行神经行为评分，TTC染色测定大鼠脑梗死体积，免疫组织化学染色法检测离子钙接头蛋白分子1（Iba1）阳性表达，ELISA法检测脑组织中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、白细胞介素-6（IL-6）的表达，实时荧光定量PCR（qRT-PCR）检测大鼠脑组织Toll样受体4（TLR4）、信号转导和转录激活因子3（STAT3）、缺氧诱导因子1A（HIF1A）、髓过氧化物酶（MPO）、基质金属蛋白酶9（MMP9）的mRNA表达。结果 通过TCMSP数据库和文献检索共筛选得到黄芎方的有效活性成分35个，成分靶点754个，缺血性脑卒中基因芯片差异表达基因677个，交集靶点56个。GO富集分析表明，黄芎方治疗缺血性脑卒中与细胞对炎症反应、脂多糖反应和环氧化酶途径等生物学过程相关。KEGG 通路分析显示，主要涉及包括代谢途径、中性粒细胞胞外诱捕网形成、HIF-1 信号传导途径等通路。筛选出 5 个核心成分 α -细辛醚、3，4，5-三甲氧基肉桂酸、β-细辛醚、异阿魏酸、阿魏酸，5个核心靶点TLR4、STAT3、HIF1A、MPO、MMP9。分子对接结果显示，5个核心成分与5个核心靶点具有稳定的结合能力。免疫浸润分析表明，核心靶点基因与中性粒细胞、激活B细胞、自然杀伤细胞、肥大细胞等关系密切。体内实验结果显示，黄芎方显著降低I/R大鼠的神经行为学评分、脑梗死体积、脑组织病理学损伤、脑组织炎症因子的表达，在脑组织中验证了黄芎方对5个核心靶点基因mRNA表达的调节作用。结论 黄芎方可能通过多成分、多靶点、多通路治疗缺血性脑卒中，其可能通过TLR4、STAT3、HIF1A、MPO和MMP9核心靶点发挥治疗作用。

Objective To explore the mechanism of Huangxiong Formula (HXF) in treatment of ischemic stroke (IS) based on GEO chip combined with network pharmacology, and to verify its pharmacodynamics and core targets by using the rat ischemiareperfusion (I/R) model.Methods The active ingredients of HXF were searched by using the TCMSP database and the literature, and their corresponding targets were searched by using the Swiss Target Prediction database. GEO platform IS dataset GSE16561 was used for variance analysis by R software limma package. The Venny 2.1.0 was used to perform intersection target analysis, and the DAVID database GO and KEGG pathway enrichment analysis on intersection target. Cytoscape 3.9.0 software was used to construct the components-target-pathway network and screen the core ingredients, and the STRING database and Cytoscape 3.9.0 software was used to construct the protein-protein interaction (PPI) network and screen the core target genes. The top ranked components and target genes based on the degree values were molecularly docked using PyMOL software. Immuno-infiltration analysis was performed by single sample Gene Set Enrichment Analysis (ssGSEA). A rat model of cerebral I/R was prepared by the wire bolus method. Totally 69 rats were randomly divided into the model group, the Huanxiong Formula (3.6 g·kg^-1) group and the aspirin (9 mg·kg^-1) group, except for the sham-operated group (n=21). Huanxiong Formula and Aspirin groups were given ig once a day, and equal amount of saline was given to the sham-operated and model groups ig for 7 d. The rats were scored for neurobehaviour using the Longa grading scale. TTC staining was used to determine the volume of brain infarction in the rats. Immunohistochemical staining was used to detect positive expression of ion calcium junction protein molecule 1 (Iba1). The levels of tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) in brain tissue was measured by ELISA. The expression of Toll-like receptor 4 (TLR4), signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor 1A (HIF1A), myeloperoxidase (MPO) and matrix metalloproteinase 9 (MMP9) mRNA in rat brain tissue was measured by real-time fluorescence quantitative PCR (qRT-PCR).Results A total of 35 active ingredients, 754 component targets, 677 differentially expressed genes on IS gene chips and 56 intersecting targets of the HXF was screened. GO enrichment analysis indicated that HXF treatment of IS was associated with biological processes such as inflammatory response, response to lipopolysaccharide and cyclooxygenase pathway. KEGG pathway analysis showed that the main signaling pathways were involved in Metabolic pathways, Neutrophil extracellular trap formation and HIF-1 signaling pathway, etc. The top 5 core components such alpha-asarone, 3, 4, 5-trimethoxycinnamic acid, betaasarone, isoferulic acid and ferulic acid were screened, and top 5 targets TLR4, STAT3, HIF1A, MPO, and MMP9. The molecular docking results showed that the core components have stable binding ability to the core targets. Immune infiltration indicated that core target genes were closely related to neutrophil, activated B cell, natural killer cell and mast cell, etc. In vivo experiments showed that Huanxiong Formula significantly reduced the neurobehavioural scores, brain infarct volume, brain histopathological damage and expression of inflammatory factors in brain tissue of I/R rats, and its regulatory effects on mRNA expression of five core target genes were verified in brain tissue.Conclusion Huangxiong Formula may be a multi-component, multi-target and multi-pathway treatment for ischemic stroke, which may exert therapeutic effects through the core targets of TLR4, STAT3, HIF1A, MPO and MMP9.

R285.5

安徽省高校优秀科研创新团队（2022AH010034）；安徽省高等学校科学研究项目（2022AH050478）；安徽中医药大学自然重点项目（2021zrzd10）；安徽中医药大学人才支持计划项目（2020rcyb005）