Abstract
OBJECTIVE: M2 macrophage-derived exosomes (M2-exos) hold promise for patients with bone cancer pain (BCP). This study aimed to investigate the therapeutic effect and related mechanisms of M2-exos in both in vitro and in vivo models of BCP. METHOD: RAW 264.7 macrophages were treated with IL-4 to generate M2-polarized macrophages. M2-exos were characterized by transmission electron microscope and Western blotting. A mouse model of BCP was established, and BV2 microglia were activated by lipopolysaccharide stimulation. The effects of M2-exos were evaluated in vitro by coculture with reactive BV2 microglia and in vivo by microinjection into the rostral ventromedial medulla (RVM) of BCP mice. CCK-8 assays, ELISAs, flow cytometry and immunofluorescence were used to determine the effects of M2-exos on microglial activation. The therapeutic effects of M2-exos were evaluated via pain behavior experiments. Bioinformatic analysis and rescue experiments were performed to investigate the mechanisms through which M2-exos affect the progression of BCP. RESULTS: In vitro, M2-exos administration repolarized microglial toward the anti-inflammatory M2 phenotype in coculture systems. In vivo analysis indicated that microinjection of M2-exos into the RVM region improved neuroinflammation. Notably, miR-216a expression was significantly increased in M2-exos and could be delivered into BV2 microglia. Blockade of miR-216a abolished the therapeutic effects of M2-exos in vitro and in vivo. Mechanistically, miR-216a negatively regulates high mobility group Box 1 protein (HMGB1) expression, further inhibiting Toll-like receptor 4 (TLR4)/NF-κB and inducing M2 microglial polarization, thereby delaying BCP progression. CONCLUSION: M2 macrophage-derived exosomal miR-216a could delay BCP progression by targeting HMGB1/TLR4/NF-κB-mediated microglial M2 polarization.