目的 通过网络药理学与分子对接技术探究钩藤Uncaria rhynchophylla治疗热性惊厥的作用机制，并进行实验验证。方法 通过TCMSP与BATMAN-TCM数据库检索钩藤活性成分及相关作用靶点，用TCMSP、PubChem数据库和SwissADME平台对活性成分进行筛选；通过GeneCards数据库和OMIM数据库提取热性惊厥疾病靶点，经Uniprot数据库进行蛋白-基因symbol转换；药物靶点与疾病靶点取交集绘制Venn图，通过STRING数据库绘制蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络，使用Cytoscape 3.7.2软件构建“中药-活性成分-关键靶点-疾病”网络，采用DAVID v6.8在线分析平台对关键靶点进行基因本体（gene ontology，GO）功能及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析，利用Cytoscape 3.7.2软件将富集分析结果可视化并将分析所得关键化合物及靶点进行分子对接。通过免疫组化、Western blotting及脑片膜片钳技术对钩藤的作用机制进行验证。结果 得到钩藤活性成分29个，活性成分对应靶点297个，活性成分靶点与疾病靶点取交集得到关键靶点81个。KEGG通路富集分析结果显示，钩藤治疗热性惊厥主要作用于神经元配体-受体相互作用、尼古丁成瘾、环磷酸腺苷信号通路。GO功能富集分析结果显示，尼古丁反应、质膜和细胞外配体门控离子通道活性显著性最大。免疫组化及Western blotting结果显示，钩藤显著抑制热性惊厥模型乳鼠脑组织中N-甲基-D-天冬氨酸受体2A（N-methyl-D-aspartic acid receptor 2A，NMDAR2A）、N-甲基-D-天冬氨酸受体2B（N-methyl-D-aspartic acid receptor 2B，NMDAR2B）的表达（P＜0.05、0.01、0.001）。脑片膜片钳实验结果显示，钩藤对海马CA1区锥体神经元NMDA受体诱发兴奋性突触后电流（NMDA receptor evoked excitatory postsynaptic currents，eEPSC_NMDA）具有抑制作用。结论 通过网络药理学及分子对接技术发现了钩藤治疗热性惊厥的活性成分及作用靶点，通过免疫组化、Western blotting及膜片钳技术证实了钩藤对NMDAR的抑制作用，为进一步研究钩藤治疗热性惊厥的作用机制、药物研发等提供理论基础。

Objective To explore the mechanism of Uncaria rhynchophylla (UR) in treatment of febrile seizures by network pharmacology, molecular docking technology and experimental verification and experimental verification. Methods The active ingredients and related targets of UR were searched from TCMSP and BATMAN-TCM database, and the active ingredients were screened by TCMSP database, PubChem database and SwissADME platform; The disease targets of febrile seizures were extracted from GeneCards and OMIM database, the protein-gene symbol conversion was performed through Uniprot database; The Venn diagram was drawn by the intersection of drug targets and disease targets, protein-protein interaction (PPI) network was drawn by STRING database, “traditional Chinese medicine-active ingredient-key target-disease” network was constructed by Cytoscape 3.7.2 software. Gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis on key targets were performed by DAVID v6.8 online analysis platform. The enrichment analysis results were visualized by Cytoscape 3.7.2 software. The key compounds and targets obtained from analysis were performed molecular docking. Immunohistochemistry, Western blotting and brain patch clamp techniques were used to verify the mechanism of UR. Results A total of 29 active ingredients of UR were obtained, with 297 corresponding targets. The intersection of active ingredient targets and disease targets resulted in 81 key targets. The KEGG pathway enrichment analysis results showed that UR mainly acted on neuronal ligand-receptor interaction, nicotine addiction and cyclic adenosine monophosphate signaling pathway in the treatment of febrile seizures. The GO functional enrichment analysis results showed that nicotine response, plasma membrane and extracellular ligand gated ion channel activity were most significant. Immunohistochemical and Western blotting results showed that UR significantly inhibited the expressions of N-methyl-D-aspartate receptor 2A (NMDAR2A) and N-methyl-D-aspartate receptor 2B (NMDAR2B) in brain tissue of neonatal rats with febrile seizures (P < 0.05, 0.01, 0.001). The results of brain patch clamp experiment showed that UR had an inhibitory effect on NMDA receptor induced excitatory postsynaptic currents (eEPSC_NMDA) in hippocampal CA1 pyramidal neurons. Conclusion The active ingredients and targets of UR in the treatment of febrile convulsions were discovered through network pharmacology and molecular docking technology. The inhibitory effect of UR on NMDAR was confirmed through immunohistochemistry, Western blotting and patch clamp technology. This study provides a theoretical basis for further research on mechanism and drug development of UR in the treatment of febrile seizures.

R285.5

河北省科技厅资助项目（H2022406026，H2022329001）；承德医学院高层次人才科研启动基金（202106）；河北省神经损伤与修复重点实验室开放课题（NJKF202103）；河北省大学生创新创业训练计划项目（2023074，2024004，2024014）