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The study employed a serum pharmacology approach to investigate the mechanism by which the blood-absorbed components of Dengzhan Shengmai Formula (DZSM) promote angiogenesis in mouse brain microvascular endothelial cells (bEND.3) following oxygen-glucose deprivation/reoxygenation (OGD/R) injury. The blood-absorbed constituents of DZSM were identified using ultra-high performance liquid chromatography coupled with quadrupole electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive-Orbitrap). An OGD/R-injured bEND.3 cell model was established. Cell viability was measured by CCK-8 assay, cellular injury was assessed by lactate dehydrogenase (LDH) levels, reactive oxygen species (ROS) expression was detected by confocal microscopy, and the apoptosis rate was determined by flow cytometry. The pro-angiogenic effect of DZSM-medicated serum was evaluated through wound healing assay, Transwell migration assay, tube formation assay, transepithelial electrical resistance (TEER), and NaF permeability test. Protein expression related to the hypoxia inducible factor-1/vascular endothelial growth factor A (HIF-1α/VEGFA) signaling pathway was detected by Western blot, and molecular docking was performed to screen key active components. The results showed that twelve components, including scutellarin, 4, 5-dicaffeoylquinic acid, and ginsenoside Rb2, were absorbed into the blood. The optimal concentration of DZSM-medicated serum for intervening in OGD/R-injured bEND.3 cells was 20% (v/v). Compared with the control group, the model group exhibited significantly decreased cell viability, migration rate, tube formation ability, and expression of pathway-related proteins, while ROS content and apoptosis rate were markedly increased (P < 0.05). Compared with the model group, the DZSM blood-absorbed components group showed significantly increased cell viability, migration rate, tube formation ability, and expression of tight junction proteins, as well as significantly decreased ROS content and apoptosis rate (P < 0.05). Moreover, the protein expression levels of HIF-1α and VEGFA were notably upregulated. Molecular docking results indicated strong binding affinities between the blood-absorbed components (e.g., scutellarin) and the HIF-1α/VEGFA targets. In conclusion, the blood-absorbed components of DZSM promote angiogenesis in OGD/R-injured bEND.3 cells, and the mechanism is closely related to activation of the HIF-1α/VEGFA signaling pathway and upregulation of its associated proteins. The experimental protocol strictly adhered to the guidelines of the Ethics Committee of Animal Research of Guangdong Pharmaceutical University (approval No.: gdpulacspf2022137).
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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