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.
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.
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.
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.
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.
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.
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.
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.