目的 基于网络药理学和分子对接技术预测绿原酸治疗脑缺血再灌注损伤（CIRI）的潜在作用靶点和信号通路，并通过大鼠体内实验验证绿原酸是否通过调控表皮生长因子受体（EGFR）/磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（m TOR）信号通路发挥神经保护作用。方法 利用中药系统药理学数据库与分析平台（TCMSP）、GeneCards等数据库筛选绿原酸作用靶点与CIRI疾病靶点的交集，采用STRING构建蛋白质-蛋白质相互作用（PPI）网络，运用DAVID进行基因本体（GO）注释及京都基因与基因组百科全书（KEGG）通路富集分析，使用AutoDock进行分子对接验证。采用线栓法建立大脑中动脉阻塞/再灌注（MCAO/R）模型。通过神经功能评分、2,3,5-氯化三苯基四氮唑（TTC）、病理观察、蛋白免疫印迹法（Western blotting）、免疫组化及TUNEL染色评估绿原酸的神经保护效应及其对EGFR/PI3K/Akt/m TOR通路的影响。结果 网络药理学分析筛选出64个交集靶点，EGFR、胱天蛋白酶3（CASP3）、原癌基因酪氨酸蛋白激酶（SRC）、人表皮生长因子受体2（ERBB2）为核心靶点，主要富集于PI3K/Akt信号通路和细胞凋亡通路。分子对接显示绿原酸与核心靶点结合能良好(-23.4～-33.9 kJ·mol^-1)。动物实验显示，与模型组相比，绿原酸治疗显著改善神经功能缺损，缩小脑梗死体积，改善神经元病理损伤；下调p-EGFR表达，上调p-PI3K、p-Akt、p-mTOR水平；降低促凋亡蛋白Bax和cleaved-Caspase-3表达，升高抗凋亡蛋白B细胞淋巴瘤/白血病-2（Bcl-2）表达，减少神经元凋亡，且呈剂量相关性（P<0.05、0.01）。表皮生长因子（EGF）激动剂可部分逆转绿原酸的神经保护作用（P<0.05、0.01），加重脑损伤，上调p-EGFR并抑制下游PI3K/Akt/mTOR通路。结论 绿原酸通过抑制EGFR过度磷酸化激活，激活PI3K/Akt/mTOR信号通路，抑制神经元凋亡，减轻CIRI，发挥神经保护作用。EGFR激动剂可部分阻断绿原酸的神经保护效应，证实EGFR可能是绿原酸发挥作用的关键上游靶点。

Objective To predict the potential targets and signaling pathways of chlorogenic acid in the treatment of cerebral ischemiareperfusion injury（CIRI） based on network pharmacology and molecular docking, and to validate whether chlorogenic acid exerts neuroprotective effects by regulating the epidermal growth factor receptor（EGFR）/phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt）/mammalian target of rapamycin（mTOR） signaling pathway through in vivo experiments in rats. Methods The intersection of chlorogenic acid action targets and CIRI disease targets was screened using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）, GeneCards and other databases. The protein-protein interaction（PPI） network was constructed using STRING, GO and KEGG enrichment analyses were performed using DAVID, and molecular docking was validated using AutoDock. The middle cerebral artery occlusion/reperfusion（MCAO/R） model was established using the filament method. The neuroprotective effects of chlorogenic acid and its influence on the EGFR/PI3K/Akt/mTOR pathway were evaluated through neurological function scoring, 2,3,5-triphenyltetrazolium chloride（TTC） staining, pathological observation, Western blotting analysis immunohistochemistry, and TUNEL staining. Results Network pharmacology analysis identified 64 intersection targets, with EGFR, CASP3, SRC, and ERBB2 as core targets, which were mainly enriched in the PI3K/Akt signaling pathway and apoptosis pathway. Molecular docking showed good binding affinity between chlorogenic acid and core targets(binding energies ranging from-23.4 to-33.9 kJ·mol^-1). Animal experiments demonstrated that compared with the model group, chlorogenic acid treatment significantly improved neurological dysfunction, reduced cerebral infarction volume, and ameliorated neuronal pathological damage; Downregulated p-EGFR expression while upregulating p-PI3K, p-Akt, and p-mTOR levels; Decreased the expression of pro-apoptotic proteins Bax and Cleaved-Caspase-3, increased anti-apoptotic protein Bcl-2 expression, and reduced neuronal apoptosis in a dosedependent manner（P < 0.05, 0.01）. EGF agonist partially reversed the neuroprotective effects of chlorogenic acid（P < 0.05, 0.01）, exacerbated brain injury, upregulated p-EGFR, and inhibited the downstream PI3K/Akt/mTOR pathway. Conclusion Chlorogenic acid exerts neuroprotective effects against cerebral ischemia-reperfusion injury by downregulating EGFR expression, activating the PI3K/Akt/mTOR signaling pathway, and inhibiting neuronal apoptosis. The EGFR agonist can partially block the neuroprotective effects of chlorogenic acid, confirming that EGFR may be the key upstream target through which chlorogenic acid exerts its effects.

R285.5

四川省医学会科研项目(S22101); 川北医学院附属医院博士科研启动基金(202002); 市校科研合作专项项目(CBY25-ZXB02)