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.

Tianma Gouteng Yin  /  hypertension  /  vascular endothelial function  /  oxidative stress  /  inflammation  /  PI3K/Akt/eNOS signaling pathway  /  network pharmacology  /  kaempferol  /  2-apigenin