目的 基于网络药理学、分子对接与细胞实验，探究葛根素治疗脑梗死的神经保护机制。方法 通过SEA、CTD等数据库预测葛根素的靶点，GeneCards、Pharmgkb等数据库收集脑梗死靶点，筛选药物与疾病交集靶点；构建“药物-靶点-疾病”网络及蛋白相互作用（PPI）网络，进行基因本体（GO）功能与京都基因与基因组百科全书（KEGG）通路富集分析；利用分子对接技术验证葛根素与核心靶点的结合活性。体外实验采用SH-SY5Y细胞氧糖剥夺/复氧（OGD/R）模型，设对照组、模型组、葛根素组，以MTT法检测细胞活力，流式细胞术检测活性氧（ROS）水平及细胞凋亡率。RT-qPCR与Western blotting分别检测丝裂原活化蛋白激酶1（MAPK1）、丝裂原活化蛋白激酶3（MAPK3）、Fos原癌基因（FOS）mRNA与磷酸化蛋白（p-MAPK1、p-MAPK3）及总蛋白表达变化。结果 网络药理学筛选获得82个葛根素与脑梗死交集靶点，PPI网络分析确定MAPK3、MAPK1、FOS为核心靶点，GO与KEGG富集分析显示主要涉及凋亡调控、氧化应激反应及MAPK信号通路；分子对接验证葛根素与3个核心靶点均具有良好结合活性。体外实验显示，葛根素可显著提高SH-SY5Y细胞活力（P＜0.001），降低ROS水平及细胞凋亡率（P＜0.01、0.001）；葛根素可下调FOS mRNA及p-MAPK1、p-MAPK3、FOS蛋白表达（P＜0.05）。结论 葛根素可能通过调节MAPK3、MAPK1、FOS靶点，调控MAPK信号通路，发挥抗氧化、抗凋亡的神经保护作用。

Objective To explore the neuroprotective mechanism of puerarin in treatment of cerebral infarction based on network pharmacology, molecular docking, and cellular experiments. Methods Targets of puerarin were predicted using databases such as SEA and CTD, while targets related to cerebral infarction were collected from databases including GeneCards and Pharmgkb. The overlapping targets between the drug and the disease were screened. A “drug-target-disease” network and a PPI network were constructed, followed by GO functional enrichment analysis and KEGG pathway enrichment analysis. Molecular docking technology was used to verify the binding activity of puerarin to core targets. In vitro experiments were performed using the OGD/R model of SH-SY5Y cells, which were divided into control group, model group, and puerarin group. Cell viability was detected by MTT assay, and ROS levels and cell apoptosis rate were measured by flow cytometry. RT-qPCR and Western blotting were used to detect the expression changes of MAPK1, MAPK3, and, FOS mRNA level, as well as the expression of their phosphorylated proteins (p-MAPK1, p-MAPK3) and total proteins. Results A total of 82 overlapping targets between puerarin and cerebral infarction were identified through network pharmacology. PPI network analysis confirmed MAPK3, MAPK1, and FOS as core targets. GO and KEGG enrichment analyses showed that these targets were mainly involved in apoptosis regulation, oxidative stress response, and the MAPK signaling pathway. Molecular docking verified that puerarin had good binding activity with the three core targets. In vitro experiments showed that puerarin significantly increased the viability o SH-SY5Y cells (P< 0.001), reduced ROS levels and cell apoptosis rate (P < 0.01, 0.001). Puerarin downregulated the abnormally increased expression of FOS mRNA, p-MAPK1, p-MAPK3, and FOS protein (P < 0.05). Conclusion Puerarin may exert neuroprotective effects of antioxidation and anti-apoptosis by regulating the MAPK3, MAPK1, and FOS targets and modulating the MAPK signaling pathway.

R285.5;R286.1

上海市嘉定区卫生系统重点专科建设资助项目（ZK2024B02）