NCF1 aggravates cerebral ischemia-reperfusion injury by amplifying NOX2-Dependent oxidative stress and inflammatory responses.

Cerebral ischemia-reperfusion injury (CIRI) is a major contributor to poor outcomes after ischemic stroke, yet the mechanisms linking oxidative stress and inflammation remain incompletely understood. Neutrophil cytosolic factor 1 (NCF1), a key organizer subunit of the NOX2 NADPH oxidase complex, is involved in reactive oxygen species (ROS) generation and immune activation. Here, we investigated the expression pattern, pathological role, and mechanistic significance of NCF1 in CIRI. A public spatial transcriptomic dataset of ischemic mouse brain tissue was reanalyzed to assess the spatial distribution of Ncf1 after stroke. A transient middle cerebral artery occlusion (MCAO) model was established, and NCF1 expression was evaluated by spatial transcriptomics, immunofluorescence staining, RT-qPCR, and Western blotting. Loss-of-function experiments were performed using NCF1 knockdown, followed by neurological deficit scoring, TTC staining, ROS detection, and inflammatory gene analysis. We found that NCF1 was markedly upregulated in the ischemic brain after stroke and was enriched in infiltrating neutrophils. NCF1 knockdown significantly improved neurological outcomes and reduced infarct volume in MCAO mice. Mechanistically, silencing NCF1 suppressed NOX2 expression and ROS accumulation, indicating attenuation of oxidative stress. In addition, NCF1 knockdown decreased the expression of multiple pro-inflammatory mediators, including IL-1β, IL-2, IL6, HMGB1, TNF-α, and iNOS, while increasing the expression of the anti-inflammatory and reparative factors Arg-1 and IGF-1. These findings indicate that NCF1 aggravates cerebral ischemia-reperfusion injury by amplifying NOX2-dependent oxidative stress and inflammatory responses, and suggest that NCF1 may serve as a potential therapeutic target for ischemic stroke.