Exosomes miR-369-3p Alleviates Early Brain Injury After Subarachnoid Hemorrhage by Promoting Ferroptosis of M1 Microglia via Inhibiting iNOS/GPX4 Axis.

Background: Ferroptosis in pathophysiological mechanisms in early brain injury after subarachnoid hemorrhage (SAH-EBI) has been demonstrated. MicroRNAs (miRNAs) are involved in various aspects of neurological disorders. A growing number of studies suggest that intense inflammation mediated by M1 microglia after subarachnoid hemorrhage (SAH) may lead to neurological damage. According to our research and related reports, exosomal miR-369-3p is involved in the pathophysiological process of SAH, and miR-369-3p has a potentially central role in regulating inflammatory responses. Therefore, targeted delivery of miR-369-3p across the blood-brain barrier (BBB) into the brain to alleviate SAH-EBI is a promising therapeutic approach.

Methods: In this study, we extracted exosomes from RBCs and then modified RVG peptide onto the exosome surface using the click chemistry principle. Finally, miR-369-3p mimic was loaded into the RVG peptide-modified exosomes to form RVG-Exo/miR-369-3p (RVG-Exo/miR) by electroporation. Tail vein injection of RVG-Exo/miR was used to achieve delivery of miR-369-3p into the brain of SAH mice. The effect of miR-369-3p on SAH-EBI was examined by neurobehavioral scores, brain water content, Fluoro-Jade C (FJC) staining, and Nissl staining. MDA and GSH kits were used to assess the extent of ferroptosis occurrence. Western blotting analysis, immunofluorescence staining, and qRT-PCR were used to detect the levels of each protein, mRNA, and miRNA.

Results: The exosome system (RVG-Exo/miR) successfully delivered miR-369-3p to the mouse central nervous system across the blood-brain barrierBBB. This exosomal system reduced the number of M1 microglia by enhancing their sensitivity to ferroptosis by inhibiting the expression of iNOS and GPX4. In addition, miR-369-3p treatment alleviated neurobehavioral disorders, brain edema, and neuronal damage after SAH-EBI.

Conclusions: RVG-Exo/miR promotes ferroptosis in M1 microglia by inhibiting the iNOS/GPX4 axis, which may be a new and effective therapeutic strategy for treating SAH-EBI.