Endothelial progenitor cell exosomes combined with miR-222-3p alleviate cognitive impairment after subarachnoid hemorrhage by inhibiting neuronal ferroptosis

Exosomes derived from endothelial progenitor cells (EPCs) attenuate neuronal ferroptosis in an in vitro hemorrhagic stroke model. Building on these findings, we investigated whether EPC-derived exosomes (EPC-EXs) carrying miR-222-3p alleviate neurological deficits and cognitive impairment in a rat subarachnoid hemorrhage (SAH) model, and explored the underlying molecular mechanisms. Candidate miRNAs were identified by differential expression and machine learning analyses of the public cerebrospinal fluid microRNA dataset GSE211002 (SAH patients vs. healthy controls). EPC-EXs overexpressing miR-222-3p were administered intranasally in a rat endovascular perforation SAH model. Behavioral assessments (modified Garcia, beam balance, Morris water maze, and novel object recognition) and histopathology (Fluoro-Jade C, Nissl, and iron staining) were performed. OxyHb-treated HT22 cells were used for in vitro mechanistic studies. miR-222-3p targets were identified by bioinformatic prediction and validated by dual-luciferase reporter assay and CRISPR/Cas9 activation. miR-222-3p was identified as a hub miRNA associated with SAH and ferroptosis, with good diagnostic performance (AUC=0.907, 95% CI: 0.836–0.978, sensitivity: 100%, specificity: 80.70%). Intranasal EPC-EXs miR-222-3p elevated brain miR-222-3p levels, improved neurological scores, reduced brain edema, and preserved hippocampal neurons. EPC-EXs miR−222−3p suppressed ferroptosis markers (iron deposition, lipid peroxidation, and COX2) and restored GPX4 expression in vivo and in vitro. Mechanistically, miR-222-3p directly targeted CYP1B1 (validated by luciferase assay), and CYP1B1 activation abolished the anti-ferroptotic effects of EPC-EXs miR−222−3p . The cAMP/PKA/CREB signaling axis mediated CYP1B1-regulated ferroptosis. Exosomal miR-222-3p derived from EPC-EXs alleviates neurological deficits and cognitive impairment following SAH by modulating CYP1B1-mediated neuronal ferroptosis via the cAMP/PKA/CREB pathway.