目的 基于GEO芯片聯(lián)合網(wǎng)絡(luò)藥理學(xué)方法探討黃芎方治療缺血性腦卒中的作用機制，并構(gòu)建腦缺血/再灌注（I/R）大鼠模型驗證黃芎方藥效和核心靶點。方法 利用中藥系統(tǒng)藥理學(xué)數(shù)據(jù)庫與分析平臺（TCMSP）數(shù)據(jù)庫及文獻(xiàn)檢索，獲得黃芎方的主要活性成分，通過 Swiss Target Prediction數(shù)據(jù)庫檢索其相應(yīng)的靶點。通過 R軟件 limma包對 GEO平臺缺血性腦卒中患者數(shù)據(jù)集 GSE16561 進行差異基因分析。利用 Venny 2.1.0 進行黃芎方成分靶點和缺血性腦卒中差異基因靶點交集靶點分析，采用DAVID數(shù)據(jù)庫對交集靶點進行基因本體（GO）功能富集分析和京都基因與基因組百科全書（KEGG）通路富集分析。采用 Cytoscape 3.9.0 軟件構(gòu)建成分-靶點-通路網(wǎng)絡(luò)并篩選核心成分，通過 STRING 數(shù)據(jù)庫與 Cytoscape 3.9.0 軟件構(gòu)建蛋白質(zhì)相互作用（PPI）網(wǎng)絡(luò)并篩選核心靶點基因。利用PyMOL軟件對度值排名靠前的成分和靶點基因進行分子對接。通過single sample Gene Set Enrichment Analysis（ssGSEA）進行免疫浸潤分析。采用線栓法制備 I/R 大鼠模型，除假手術(shù)組外（n＝21），將69只大鼠隨機分為模型組、黃芎方組（3.6 g·kg^-1）組、阿司匹林（9 mg·kg^-1）組。黃芎方組和阿司匹林組每日ig給藥1次，假手術(shù)組和模型組ig給予等量生理鹽水，持續(xù)7 d。采用Longa分級評分法對大鼠進行神經(jīng)行為評分，TTC染色測定大鼠腦梗死體積，免疫組織化學(xué)染色法檢測離子鈣接頭蛋白分子1（Iba1）陽性表達(dá)，ELISA法檢測腦組織中腫瘤壞死因子α（TNF-α）、白細(xì)胞介素1β（IL-1β）、白細(xì)胞介素-6（IL-6）的表達(dá)，實時熒光定量PCR（qRT-PCR）檢測大鼠腦組織Toll樣受體4（TLR4）、信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3（STAT3）、缺氧誘導(dǎo)因子1A（HIF1A）、髓過氧化物酶（MPO）、基質(zhì)金屬蛋白酶9（MMP9）的mRNA表達(dá)。結(jié)果 通過TCMSP數(shù)據(jù)庫和文獻(xiàn)檢索共篩選得到黃芎方的有效活性成分35個，成分靶點754個，缺血性腦卒中基因芯片差異表達(dá)基因677個，交集靶點56個。GO富集分析表明，黃芎方治療缺血性腦卒中與細(xì)胞對炎癥反應(yīng)、脂多糖反應(yīng)和環(huán)氧化酶途徑等生物學(xué)過程相關(guān)。KEGG 通路分析顯示，主要涉及包括代謝途徑、中性粒細(xì)胞胞外誘捕網(wǎng)形成、HIF-1 信號傳導(dǎo)途徑等通路。篩選出 5 個核心成分 α -細(xì)辛醚、3，4，5-三甲氧基肉桂酸、β-細(xì)辛醚、異阿魏酸、阿魏酸，5個核心靶點TLR4、STAT3、HIF1A、MPO、MMP9。分子對接結(jié)果顯示，5個核心成分與5個核心靶點具有穩(wěn)定的結(jié)合能力。免疫浸潤分析表明，核心靶點基因與中性粒細(xì)胞、激活B細(xì)胞、自然殺傷細(xì)胞、肥大細(xì)胞等關(guān)系密切。體內(nèi)實驗結(jié)果顯示，黃芎方顯著降低I/R大鼠的神經(jīng)行為學(xué)評分、腦梗死體積、腦組織病理學(xué)損傷、腦組織炎癥因子的表達(dá)，在腦組織中驗證了黃芎方對5個核心靶點基因mRNA表達(dá)的調(diào)節(jié)作用。結(jié)論 黃芎方可能通過多成分、多靶點、多通路治療缺血性腦卒中，其可能通過TLR4、STAT3、HIF1A、MPO和MMP9核心靶點發(fā)揮治療作用。

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

安徽省高校優(yōu)秀科研創(chuàng)新團隊（2022AH010034）；安徽省高等學(xué)校科學(xué)研究項目（2022AH050478）；安徽中醫(yī)藥大學(xué)自然重點項目（2021zrzd10）；安徽中醫(yī)藥大學(xué)人才支持計劃項目（2020rcyb005）