SPP1 exacerbates ischemic stroke by promoting ferroptosis induced brain injury.

Ischemic stroke persists as a leading global cause of mortality and disability. Studying its mechanisms of occurrence and seeking effective treatments hold significant research value. Single-cell sequencing analysis revealed increased Spp1 levels after ischemia-reperfusion (I/R). Recent evidence suggests its involvement in reactive oxygen species (ROS) accumulation during I/R injury. Given the known association between ferroptosis and cerebral I/R injury, we explored the relationship between SPP1 and ferroptosis and downstream pathways. Using oxygen-glucose deprivation/reoxygenation (OGD/R)-treated HT22 cells and middle cerebral artery occlusion/reperfusion (MCAO/R) models, SPP1 expression patterns were analyzed via Western blot. Spp1-knockdown HT22 cells and mice treated with the SPP1-specific inhibitor Compound 11 were employed to assess molecular changes in OGD/R and MCAO/R models, and we found SPP1 inhibition alleviated ferroptosis. Proteomic analysis identified the PI3K/AKT cascade as the primary signaling mechanism. In OGD-treated HT22 cells, SPP1 was shown to regulate neuronal death via the PI3K/AKT pathway. Western blot and immunofluorescence analyses demonstrated that SPP1 knockdown reduced p-PI3K and p-AKT phosphorylation levels. Pharmacological inhibition of PI3K with LY294002 recured SPP1 overexpression-aggravated ferroptosis, concomitant with reduced ACSL4 expression. These findings were corroborated by electron microscopy observations and ROS quantification. Collectively, our results identify SPP1 as a promising therapeutic target for cerebral ischemia through modulation of the PI3K/AKT signaling pathway, suggesting its clinical potential for improving treatment strategies in cerebral ischemia management.