目的 利用网络药理学结合G蛋白偶联受体和酶活性检测方法探索脑心清片治疗缺血性脑血管病及卒中后抑郁，卒中后失眠障碍的调控机制和药效物质基础。方法 根据前期研究结果，选取脑心清片中13个主要入血成分，利用SwissTargetPrediction、中药系统药理学数据库与分析平台（TCMSP）预测化合物作用靶点，借助STRING数据库与Omicsbean在线分析软件对靶点进行相互作用、生物信息学分析，最后利用Cytoscape软件构建网络药理图。进一步运用胞内钙离子荧光检测和酶抑制剂检测脑心清片及其9个代表性成分对凝血酶（Thrombin）、血栓素A2受体（TP/TBXA2R）、环氧酶-2（COX-2）、核转录因子κB（NF-κB）、单胺氧化酶A（MAO-A）、5-羟色胺受体（5-HT2AR）的抑制活性及对1,1-二苯基-2-苦基肼（DPPH）自由基的清除作用。结果 网络药理学结果表明，13个化合物可通过作用于142个疾病交集靶点干预91条关键信号通路，主要涉及改善血管内皮功能、改善血流动力学、抑制血栓形成、抗氧化应激、抗炎、神经保护、抗抑郁、抗失眠等方面，构建了脑心清片“化合物-靶点-通路-药理作用-功效”网络药理图。对关键靶点的验证实验表明，脑心清片中多个代表性成分（原儿茶酸、原儿茶醛、水杨酸、槲皮素、山柰酚、异鼠李素、白桦脂酸、齐墩果酸、莨菪亭）对Thrombin、TP/TBXA2R、COX-2、NF-κB、MAO-A、5-HT2AR具有较好抑制活性，对DPPH有显著清除作用。结论 脑心清片中代表性成分原儿茶酸、原儿茶醛、槲皮素、山柰酚、异鼠李素、白桦酯酸、齐墩果酸、莨菪亭可能通过作用于Thrombin、TP/TBXA2R、COX-2、NF-κB、MAO-A、5-HT2AR等靶点，调节活血化瘀、通络及抗抑郁、抗失眠相关的多项生物过程，推测上述成分为脑心清片的主要药效物质基础。

Objective To investigate the regulatory mechanisms and pharmacodynamic material basis of Naoxinqing Tablet in treating ischemic cerebrovascular disease (ICVD), post-stroke depression (PSD), and post-stroke sleep disorders (PSSD) using network pharmacology combined with activity assays for G protein-coupled receptors (GPCR) and enzymes. Methods Targets of the 13 components absorbed in plasma in Naoxinqing Tablet were predicted by the Swiss Target Prediction server and TCMSP database. Gene ontology (GO) function enrichment and pathway analysis of the targets were analyzed by Omicsbean analytic system and STRING 10 database. Cytoscape 3.10.4 software was used to construct the network pharmacology map. Further evaluate the effects of Naoxinqing Tablet and nine representative components on thrombin, TP/TBXA2R, COX-2, NF-κB, MAO-A, 5-HT2A, DPPH free radicals by using intracellular calcium ion fluorescence detection and enzyme inhibitor detection technology. Results A total of 13 compounds affected 91 pathways through 142 related targets, of which were associated with improve vascular endothelial function, improve hemodynamics, inhibit thrombosis, anti-oxidative stress, anti-inflammatory, neuroprotection, anti-depression and anti-insomnia. The network of “compound-target-pathway-pharmacological action-efficacy” was also constructed. Representative ingredients (protocatechuic acid, protocatechualdehyde, salicylic acid, quercetin, kaempferol, isorhamnetin, betulinic acid, oleanolic acid, and scopolin) in Naoxinqing Tablet have inhibitory effects on Thrombin, TP/TBXA2R, COX-2, NF-κB, MAO-A, 5-HT2AR, DPPH free radicals. Conclusion The representative components in Naoxinqing Tablet including protocatechuic acid, protocatechualdehyde, quercetin, kaempferol, isorhamnetin, betulinic acid, oleanolic acid and scopoletin may intervene biological processes related to promoting blood circulation for removing blood stasis, dredging collaterals, anti-depression and anti-insomnia by acting on Thrombin, TP/TBXA2R, COX-2, NF-κB, MAO-A, 5-HT2AR, which are presumed to be the pharmacodynamic substances of Naoxinqing Tablet.

R285.5

中国博士后科学基金资助项目（2023M740790）