NADPH exerts neuroprotection in ischemic stroke by reinforcing blood-brain barrier integrity and stimulating angiogenesis.

Ischemic stroke (IS) represents a significant global health burden with increasing incidence, creating an urgent need for novel therapeutic approaches. This study explored the neuroprotective effects of nicotinamide adenine dinucleotide phosphate (NADPH) in preserving blood-brain barrier (BBB) integrity and promoting angiogenesis after IS. Through network pharmacology analysis and molecular docking, five key molecular targets of NADPH in IS were identified: HIF-1α, SRC, NLRP3, CASP3, and AKT1. In vivo, NADPH treatment conferred significant protection against cerebral ischemia in the transient middle cerebral artery occlusion (tMCAO) model. At the optimal dose of 7.5 mg/kg, it substantially reduced infarct volume (∼50%), attenuated cerebral edema (from 81% to 76%), improved neurological function (∼58%), and preserved BBB integrity. Mechanistically, NADPH protected the BBB by upregulating key tight junction (TJ) proteins, including a ∼29% increase in ZO-1 expression, with electron microscopy confirming strengthened TJ structure. NADPH also reduced the protein levels of matrix metalloproteinase-9 (MMP9) and caveolin-1 by ∼23% and 50%, respectively. Furthermore, it suppressed NLRP3 inflammasome activation, decreased the expression of NLRP3 (∼14%), ASC (∼24%), Caspase-1 (∼30%), and interleukin-1β (IL-1β; ∼16%), thereby attenuating inflammation. In vitro, NADPH enhanced endothelial cell proliferation, migration, and tube formation under oxygen-glucose deprivation (OGD) conditions. Additionally, NADPH further elevated the expression of key pro-angiogenic markers, increasing HIF-1α protein by ∼77.1% and vascular endothelial growth factor (VEGF) by ∼44.8% at day 7 post-tMCAO. These findings suggest that NADPH confers neuroprotection in IS by preserving BBB integrity, inhibiting NLRP3 inflammasome-mediated damage, and stimulating angiogenesis through HIF-1α/VEGF signaling. Our results highlight NADPH's dual therapeutic mechanisms and its potential as a promising neuroprotective agent for IS.