Dl-3-n-Butylphthalide Protects Human Brain Microvascular Endothelial Cells Against Ischemic Injury Through Dual Modulation of HIF1α/VEGF-Mediated Angiogenesis and COX2-Mediated Inflammation.

摘要

BACKGROUND: Stroke remains a leading cause of death and disability worldwide. Endothelial dysfunction plays a central role in both acute ischemic injury and subsequent recovery. In this study, we investigated the protective effects and underlying mechanisms of Dl-3-n-butylphthalide (NBP) on human brain microvascular endothelial cells (HBMECs) subjected to oxygen-glucose deprivation (OGD), an in vitro model of ischemic stroke. METHODS: HBMECs were divided into three groups: control (normoxia), OGD (10 h hypoxia, followed by reoxygenation for different durations (0-24 h), and then a subsequent 24 h incubation), and NBP-treated OGD (10 μmol/L NBP during reoxygenation). Cell viability and apoptosis were assessed by Cell Counting Kit 8 (CCK8), lactate dehydrogenase (LDH) release, and flow cytometry. Mitochondrial function was evaluated using MitoTracker fluorescence. Molecular mechanisms were examined using Western blot, quantitative real-time PCR (qRT-PCR), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA), focusing on the hypoxia-inducible factor-1α (HIF1α)/vascular endothelial growth factor (VEGF) angiogenic pathway and cyclooxygenase-2 (COX2)-mediated inflammatory response. RESULTS: NBP at 10 μmol/L significantly improved HBMEC viability (37.5% increase, p < 0.01), reduced apoptosis (p < 0.01), and restored mitochondrial membrane potential (p < 0.05) following OGD injury. Mechanistically, NBP demonstrated dual pathway modulation by: (1) promoting angiogenesis-related gene expression through HIF1α upregulation and subsequent increase in vascular endothelial growth factor receptor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) expression (p < 0.05); and (2) suppressing inflammation via COX2 downregulation with concurrent reduction of downstream mediators including inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and Thromboxane B2 (TXB2, p < 0.05). This coordinated regulation created a favorable microenvironment balancing pro-angiogenic signals with anti-inflammatory effects. CONCLUSION: NBP exerts multi-targeted protection on HBMECs after ischemic injury through associated with the activation of HIF1α/VEGF-mediated angiogenesis and suppression of COX2-driven inflammation. This dual modulation strategy, targeting both vascular repair and inflammatory control, provides preliminary in vitro mechanistic insights that warrant further validation in vivo and in clinical settings.