MiR-125a-5p in extracellular vesicles of neural stem cells acts as a crosstalk signal modulating neuroinflammatory microenvironment to alleviate cerebral ischemia-reperfusion injury.

Rationale: Ischemic stroke is the second leading cause of death worldwide. Ischemia-reperfusion injury plays a major role in brain function damage and leads to disability. Currently, there are no ideal therapeutic methods for preventing and treating ischemia-reperfusion injury. Extracellular vesicles (EVs) are a promising therapy for cerebral ischemia-reperfusion injury (CIRI). The main purpose of this study was to identify the pivotal miRNAs in EVs that affect functional recovery following CIRI, develop engineered EVs encapsulating key miRNAs, and identify the underlying mechanisms. Methods: Next-generation sequencing was used to identify the crucial differentiating ingredients between EVs from normoxia- and hypoxia-conditioned human neural stem cells (hNSCs). HNSC EVs were electroporated with miR-125a-5p mimics and characterized using nanoparticle tracking analysis and electron microscopy. The role and mechanism by which EV-packaged miR-125a-5p mediates CIRI were investigated in vitro and in vivo. Results: In the present study, miR-125a-5p derived from the EVs of hNSCs was found to signal the crosstalk between different cells, such as microglia and neurons, under ischemic conditions. Furthermore, hNSC-EVs loaded with miR-125a-5p (EVs(miR)) promoted the polarization of anti-inflammatory M2 microglia, resulting in altered inflammatory responses and decreased inflammatory cytokine secretion. Additionally, EVs-miR-125a-5p exerts a significant impact on microglia, subsequently translocating to neurons and inhibiting neuronal death. Moreover, increased miR-125a-5p levels in hNSC-EVs effectively inhibited neuronal apoptosis and improved the axonal ultrastructure and neurological function in vivo. Mechanistically, EVs(miR) regulate the TLR4/NF-κB signaling pathway by targeting IKBKG to alleviate neuroinflammation induced by CIRI. Conclusions: Our findings demonstrate that miR-125a-5p mechanisms contribute to modulating the neuroinflammatory microenvironment and miR-125a-5p-enriched EVs may be a promising therapeutic strategy for CIRI.