Objective
This study aimed to investigate whether exosomes derived from bone marrow mesenchymal stem cells (BMSCs) under low oxidative stress (H-Exos) exhibit superior anti-aging effects on nucleus pulposus cells (NPCs) and intervertebral disc degeneration (IVDD) compared to exosomes from normal BMSCs (N-Exos), and to elucidate the underlying mechanisms.
Methods
The optimal H2O2 concentration for inducing BMSC oxidative stress was determined using CCK-8. H-Exos and N-Exos were characterized via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and cellular uptake assays. Their effects on TNF-α-induced NPC senescence were assessed by Western blot, SA-β-Gal staining, immunofluorescence, and flow cytometry. A rat tail needle-puncture IVDD model was established, and radiographic/histochemical analyses evaluated the therapeutic effects of H-Exos and N-Exos. RNA-Seq identified BMF as a key target of H-Exos. BMF-knockout (KO) mice and a mouse tail puncture model were used to validate the role of BMF in IVDD. Co-immunoprecipitation (Co-IP) and mass spectrometry revealed that BMF binds to MFN1, inhibiting mitophagy in NPCs. miRNA-Seq and bioinformatics analysis identified miR-29a-3p in H-Exos as a suppressor of BMF. Combined H-Exos and miR-29a-3p inhibitor interventions confirmed the critical role of miR-29-3p.
Results
Both H-Exos and N-Exos mitigated TNF-α-induced NPC senescence and IVDD in rats, with H-Exos showing superior efficacy. RNA-Seq demonstrated that H-Exos significantly downregulated BMF compared to N-Exos. BMF knockdown in NPCs alleviated TNF-α-induced senescence, and BMF-KO mice exhibited reduced IVDD severity post-puncture. Co-IP and mass spectrometry revealed BMF-MFN1 binding, which suppressed MFN1 degradation and impaired mitophagy. Overexpression of BMF and MFN1 exacerbated NPC senescence. miR-29a-3p in H-Exos was found to directly inhibit BMF expression, and miR-29a-3p inhibition attenuated the protective effects of H-Exos.
Conclusion
H-Exos exert stronger protective effects against NPC senescence and IVDD than N-Exos, likely via miR-29a-3p-mediated BMF suppression, which enhances mitophagy.