Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis

Background: Chronic inflammatory pain significantly reduces the quality of life and lacks effective interventions. In recent years, human umbilical cord mesenchymal stem cells (huc-MSCs)-derived exosomes have been used to relieve neuropathic pain and other inflammatory diseases as a promising cell-free therapeutic strategy. However, the therapeutic value of huc-MSCs-derived exosomes in complete Freund's Adjuvant (CFA)-induced inflammatory pain remains to be confirmed. In this study, we investigated the therapeutic effect and related mechanisms of huc-MSCs-derived exosomes in a chronic inflammatory pain model.

Methods: C57BL/6J male mice were used to establish a CFA-induced inflammatory pain model, and huc-MSCs-derived exosomes were intrathecally injected for 4 consecutive days. BV2 microglia cells were stimulated with lipopolysaccharide (LPS) plus adenosine triphosphate (ATP) to investigate the effect of huc-MSCs-derived exosomes on Pyroptosis and Autophagy. Bioinformatic analysis and rescue experiments were used to demonstrate the role of miR-146a-5p/ TRAF6 in regulating Pyroptosis and Autophagy. Western blotting, RT-qPCR, small interfering RNA and Yo-Pro-1 dye staining were performed to investigate the related mechanisms.

Results: Huc-MSCs-derived exosomes alleviated mechanical allodynia and thermal hyperalgesia in CFA-induced inflammatory pain. Furthermore, huc-MSCs-derived exosomes attenuated neuroinflammation by increasing the expression of autophagy-related proteins (LC3-II and Beclin1) and inhibiting the activation of NLRP3 inflammasomes in the spinal cord dorsal horn. In vitro, NLRP3 inflammasome components (NLRP3, caspase1-p20, ASC) and gasdermin D (GSDMD-F, GSDMD-N) were inhibited in BV2 cells pretreated with huc-MSCs-derived exosomes. Western blot and Yo-Pro-1 dye staining demonstrated that 3-MA, an Autophagy Inhibitor, weakened the protective effect of huc-MSCs-derived exosomes on BV2 cell Pyroptosis. Importantly, huc-MSCs-derived exosomes transfected with miR-146a-5p mimic promoted Autophagy and inhibited BV2 cell Pyroptosis. TRAF6, as a target gene of miR-146a-5p, was knocked down via small-interfering RNA, which increased Pyroptosis and inhibited Autophagy.

Conclusion: Huc-MSCs-derived exosomes attenuated inflammatory pain via miR-146a-5p/TRAF6, which increased the level of Autophagy and inhibited Pyroptosis.