Background: Inadequate nerve regeneration and an inhibitory local microenvironment are major obstacles to the repair of spinal cord injury (SCI). The activation and differentiation fate regulation of endogenous neural stem cells (NSCs) represent one of the most promising repair approaches. Metformin has been extensively studied for its antioxidative, anti-inflammatory, anti-aging, and autophagy-regulating properties in central nervous system diseases. However, the effects of metformin on endogenous NSCs remains to be elucidated. Methods: The proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot. Results: Metformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK). Conclusion: Metformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.

M2 macrophages-derived exosomes (M2φ-Exos) have been demonstrated to effectively alleviate osteoarthritis (OA) in animal models. Hypoxic preconditioning is commonly used to enhance the biological effects of stem cell-derived exosomes, but its impact on M2φ-Exos remains unclear. This study aims to investigate whether hypoxic preconditioning could enhance the biological effects of M2φ-Exos in OA treatment and to explore the underlying molecular mechanisms, with the goal of providing new insights for the development of safe and effective OA therapeutic strategies. Exosomes were extracted from the supernatants of M2 macrophages cultured under normoxic or hypoxic conditions using low-temperature differential ultracentrifugation and were designated as Nor-Exos and Hypo-Exos. The exosomes were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. To evaluate the impact of hypoxic preconditioning on the biological effects of M2φ-Exos, the therapeutic effects of Nor-Exos and Hypo-Exos were assessed in an IL-1β-induced chondrocyte inflammation model and a rat knee OA model established by surgical intervention. Exosomal miRNAs with differential expression between Nor-Exos and Hypo-Exos were identified through exosomal miRNA sequencing. The miRNA with the highest upregulation in Hypo-Exos was selected for further functional validation. To investigate the role of this miRNA, miRNA inhibitors were used to knock down its expression in Hypo-Exos, and the subsequent impact of this change on Hypo-Exos activity was evaluated. Bioinformatic tools and dual-luciferase reporter assays were used to predict and verify the downstream target genes of the miRNA. Target gene expression was knocked down using small interfering RNA, and the effect of downregulating target gene expression on the inhibitory effect of low miRNA expression on Hypo-Exos was observed at the cellular level. Compared to Nor-Exos, Hypo-Exos exhibited more effective therapeutic effects in both inflammatory chondrocytes and OA rats. miR-124-3p was identified as the most upregulated miRNA in Hypo-Exos, and the suppression of miR-124-3p expression significantly inhibited the biological effects of Hypo-Exos. STAT3 was determined to be a downstream target gene of miR-124-3p. Further cellular experiments revealed that downregulation of STAT3 expression in chondrocytes successfully alleviated the inhibitory effect of low miR-124-3p expression on the biological effects of Hypo-Exos. Hypoxic preconditioning enhances the biological effects of M2φ-Exos in the treatment of OA. The underlying molecular mechanism is associated with increased delivery of miR-124-3p to chondrocytes, which subsequently inhibits the post-transcriptional expression of STAT3. This provides a promising therapeutic strategy for the clinical intervention of OA.