目的 基于网络药理学、分子对接和实验验证探究天麻钩藤饮改善高血压血管内皮功能的潜在活性成分、作用靶点及分子机制，明确其是否通过磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）/内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）信号通路发挥作用。方法 通过HERB、SwissTargetPrediction数据库筛选天麻钩藤饮的活性成分及靶点，结合OMIM、GeneCards数据库获取高血压相关靶点，构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络并进行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；选取核心成分与PI3K/Akt/eNOS通路关键靶点进行分子对接验证。以血管紧张素-II（angiotensin-Ⅱ，Ang-Ⅱ）诱导的人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）损伤为模型，通过CCK-8、qRT-PCR、Western blotting、活性氧（reactive oxygen species，ROS）、一氧化氮（nitric oxide，NO）检测等实验验证天麻钩藤饮对细胞活力、氧化应激、炎症反应及PI3K/Akt/eNOS通路的影响。结果 网络药理学筛选出天麻钩藤饮228个活性成分及969个高血压相关交集靶点，核心靶点包括丝氨酸/苏氨酸蛋白激酶1（serine/threonine kinase 1，AKT1）、PI3K家族成员[磷脂酰肌醇-4,5-二磷酸3-激酶催化亚基α（phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha，PIK3CA）、磷脂酰肌醇-4,5-二磷酸3-激酶催化亚基β（phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta，PIK3CB）等]，KEGG富集分析显示PI3K/Akt信号通路为关键通路；分子对接证实山柰酚、芹菜素等核心成分与PI3K/Akt通路靶点结合亲和力强。细胞实验结果显示，25～100 mg/mL天麻钩藤饮对正常HUVECs无细胞毒性，可剂量相关性改善Ang-Ⅱ诱导的细胞活力降低（P＜0.001）；显著抑制血管紧张素-II 1型受体（angiotensin-Ⅱ type 1 receptor，AT1R）、烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NADPH oxidase 4，NOX4）表达及ROS生成（P＜0.001），上调超氧化物歧化酶1（superoxide dismutase 1，SOD1）、过氧化氢酶（catalase，CAT）、谷胱甘肽过氧化物酶1（glutathione peroxidase 1，GPX1）等抗氧化酶水平（P＜0.05、0.01、0.001），下调白细胞介素-6（interleukin-6，IL-6）、IL-1β、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、细胞间黏附分子1（intercellular adhesion molecule 1，ICAM1）、血管细胞黏附分子1（vascular cell adhesion molecule 1，VCAM1）等炎性因子表达（P＜0.01、0.001），增加NO生成并抑制内皮素-1（endothelin-1，ET-1）表达（P＜0.01、0.001），上调p-PI3K、p-Akt、p-eNOS蛋白表达（P＜0.001）；给予PI3K特异性抑制剂Wortmannin后，可显著削弱天麻钩藤饮对PI3K/Akt通路的激活效应，明确其内皮保护作用依赖于PI3K/Akt/eNOS信号通路。结论 天麻钩藤饮可能通过山柰酚、芹菜素等核心成分，多靶点协同激活PI3K/Akt通路，改善氧化应激与炎症反应，调节NO与ET-1的平衡，从而保护高血压状态下的血管内皮功能。

Objective To explore the potential active components, targets and molecular mechanism of Tianma Gouteng Yin (天麻钩藤饮) in improving vascular endothelial function in hypertension based on network pharmacology, molecular docking and experimental verification, and to clarify whether it exerts effects via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signaling pathway. Methods Active components and targets of Tianma Gouteng Yin were screened through HERB and SwissTargetPrediction databases. Hypertension-related targets were obtained from OMIM and GeneCards databases. A protein-protein interaction (PPI) network was constructed, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was performed. Molecular docking was used to verify the binding affinity between core components and key targets in PI3K/Akt/eNOS pathway. Human umbilical vein endothelial cells (HUVECs) injured by angiotensin Ⅱ (Ang-Ⅱ) were used as the cell model. CCK-8, qRT-PCR, Western blotting, reactive oxygen species (ROS) and nitric oxide (NO) assays were applied to evaluate the effects of Tianma Gouteng Yin on cell viability, oxidative stress, inflammatory response and PI3K/Akt/eNOS pathway. Results Network pharmacology identified 228 active components and 969 overlapping hypertension-related targets of Tianma Gouteng Yin. Core targets included serine/threonine kinase 1 (AKT1) and PI3K family members [phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), etc.]. KEGG enrichment indicated that PI3K/Akt pathway was the key pathway. Molecular docking confirmed that core components such as kaempferol and apigenin showed strong binding affinity to PI3K/Akt pathway targets. The results of cell experiment showed that 25-100 mg/mL Tianma Gouteng Yin had no cytotoxicity in normal HUVECs, and dose-dependently attenuated Ang-Ⅱ-induced reduction in cell viability (P < 0.001), significantly inhibited the expressions of angiotensin-Ⅱ type 1 receptor (AT1R) and NADPH oxidase 4 (NOX4), as well as ROS production (P < 0.001), upregulated the levels of antioxidant enzymes including superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase 1 (GPX1) (P < 0.05, 0.01, 0.001), reduced the expressions of inflammatory factors including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) (P < 0.01, 0.001), increased NO production and suppressed endothelin-1 (ET-1) expression (P < 0.01, 0.001), upregulated the protein expressions of p-PI3K, p-Akt and p-eNOS (P < 0.001). After administering the PI3K specific inhibitor Wortmannin, the activation effect of Tianma Gouteng Yin on PI3K/Akt pathway was significantly weakened, indicating that its endothelial protective effect depends on PI3K/Akt/eNOS signaling pathway. Conclusion Tianma Gouteng Yin may activate PI3K/Akt pathway through multi-target synergistic effects of core components such as kaempferol and apigenin, improve oxidative stress and inflammatory response, regulate the balance of NO and ET-1, and protect endothelial function in hypertension.

R285.5

浙江省自然科学基金资助项目（ZCLTGY24H2901）；浙江省自然科学基金资助项目（LY24H280005）