GPX4-overexpressing mesenchymal stem cell-derived exosomes ameliorate secondary brain injury after intracerebral hemorrhage by inhibiting neuronal ferroptosis

Abstract

Closely linked to intracerebral hemorrhage (ICH)-related secondary brain injury, ferroptosis lacks effective treatments, whereas exosomes offer novel neuroprotective potential. This study aimed to investigate the neuroprotective effects and mechanisms of exosomes derived from bone marrow mesenchymal stem cells overexpressing glutathione peroxidase 4 (exo-GPX4) in ICH. In in vitro experiments, exo-GPX4 were constructed and exosomes were extracted using ultracentrifugation, followed by characterization through transmission electron microscopy, nanoparticle tracking analysis, and Western blot. In a hemin-induced HT22 cell injury model, cell viability was detected by CCK-8 assay, apoptosis was analyzed by flow cytometry, and Fe²⁺, MDA, and GSH levels were measured by colorimetric assay, and the expression levels of GPX4, ACSL4, and SLC7A11 proteins were detected by Western blot. In vivo experiments, an ICH rat model was established, and histopathological changes were assessed through neurological function scores, H&E staining, Prussian blue staining, and TUNEL assay. Serum ferroptosis-related indicators were detected using kits, while Western blot and immunofluorescence were employed to examine the expression levels of GPX4, ACSL4, and SLC7A11 proteins and the localization of GPX4, respectively. The results demonstrated that in vitro, exo-GPX4 significantly enhanced cell viability, reduced apoptosis, decreased Fe²⁺ and MDA levels, and increased GSH content. In vivo, exo-GPX4 treatment markedly improved neurological function scores, alleviated cerebral iron deposition and neuronal apoptosis, and modulated serum ferroptosis-related indicators. Mechanistic studies revealed that exo-GPX4 upregulates GPX4 and SLC7A11 protein expression while downregulating ACSL4 expression. In conclusion, exo-GPX4 mitigates secondary brain injury after ICH by inhibiting ferroptosis, revealing a novel therapeutic strategy.

Keywords: Exosomes; Ferroptosis; GPX4; Intracerebral hemorrhage; Mesenchymal stem cells.