Calycosin alleviates blood-brain barrier disruption after cerebral ischemia-reperfusion injury by inhibiting NLRP3-mediated pyroptosis

Ischemic stroke, a severe neurological disorder with a multifactorial pathogenesis, presents significant therapeutic challenges. Calycosin, a natural flavonoid, has diverse biological activities, including antioxidant, anti-inflammatory, and antitumor effects. In this study we investigate the protective effects of calycosin against blood-brain barrier (BBB) damage following cerebral ischemia-reperfusion injury (CIRI) and explore the underlying mechanisms. We employ middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation (OGD) in bEnd.3 brain microvascular endothelial cells to assess neurological function, BBB integrity, the expression of pyroptosis-related proteins, inflammatory mediator release, endothelial barrier permeability, and cell viability. The results reveal that calycosin significantly ameliorates CIRI-induced BBB damage, as evidenced by improved neurological scores, reduced brain water content, and decreased infarct volume. Calycosin suppresses NLRP3-mediated pyroptosis by downregulating HMGB1, NLRP3, caspase 1, GSDMD, N-GSDMD, and IL-18 expression while reducing the secretion of HMGB1, IL-1β, and IL-18. Additionally, calycosin enhances BBB integrity by decreasing MMP9 and AQP-4 expression and upregulating the expression of tight junction proteins (ZO-1, occludin, and claudin-5). In OGD-treated bEnd.3 cells, calycosin inhibits NLRP3-mediated pyroptosis, reduces inflammatory mediator release, and improves cell viability and barrier function. Notably, molecular docking and molecular dynamics simulations demonstrate that calycosin stably binds to NLRP3 with high affinity, supporting its potential as an NLRP3 inhibitor. These findings indicate that calycosin protects against CIRI-induced BBB damage by inhibiting NLRP3-mediated pyroptosis and modulating tight junction protein expression, indicating that calycosin is a potential therapeutic option for ischemic stroke.