Astragaloside IV attenuates hypoxia-reoxygenation-induced endothelial senescence and vascular inflammation by...

Alzheimer's disease (AD) progression involves cerebral microvascular endothelial cell senescence induced by brain hypoperfusion, which contributes to blood-brain barrier (BBB) dysfunction. While Notch1 signaling is known to exacerbate endothelial senescence and neuroinflammation via vascular cell adhesion molecule-1 (VCAM-1), the mechanism by which it mediates hypoxia-induced endothelial aging in AD remains unclear. Furthermore, although Astragaloside IV (AS-IV) has been shown to alleviate cerebral hypoperfusion in AD, whether it acts by directly modulating the Notch1/VCAM-1 axis is unknown. This study aimed to determine whether AS-IV mitigates endothelial senescence and AD pathology by modulating the Notch1/VCAM-1 pathway.

Transcriptomic analysis of AD patient data and network pharmacology identified NOTCH1 as a key target. Molecular docking and 100-ns molecular dynamics simulations (using Desmond) characterized AS-IV-Notch1 interactions. Human brain microvascular endothelial cells (HBMECs) were subjected to hypoxia-reoxygenation (HR) to model AD-associated hypoperfusion. Cells were treated with AS-IV (25, 50, or 100 μM) or N-acetylcysteine (NAC) as a positive antioxidant control. Notch1 signaling was experimentally modulated using a recombinant decoy receptor to simulate signaling blockade, creating a contrast with HR-induced overactivation. Assessments included CCK-8 assays, MDA and ROS detection, qPCR, and Western blotting.

NOTCH1 expression was upregulated in AD patients. Molecular docking and dynamics simulations predicted a stable binding mode between AS-IV and Notch1. HR exposure significantly increased oxidative stress and upregulated senescence markers (p16, p21, and p53), proinflammatory senescence-associated secretory phenotype (SASP) factors (IL-6, IL-1β, and TNF-α), and Notch1/VCAM-1 expression. Conversely, AS-IV treatment improved cell viability, reduced oxidative stress (with efficacy comparable to NAC), and downregulated Notch1/VCAM-1, senescence, and SASP markers. Notably, perturbing Notch signaling, either through HR-induced overactivation or decoy receptor-mediated blockade, exacerbated the senescent phenotype. Importantly, AS-IV co-treatment effectively rescued cellular damage induced by both forms of Notch pathway dysregulation.

AS-IV alleviates HR-induced endothelial senescence by modulating the Notch1/VCAM-1 axis. Our data suggest that AS-IV does not merely inhibit Notch1 but acts by modulating its signaling toward a protective equilibrium, thereby attenuating endothelial senescence and inflammation associated with the restoration of Notch1/VCAM-1 signaling balance. These findings highlight the potential of AS-IV as a therapeutic candidate for AD vascular pathology.