M2 Polarization of RAW264.7-Derived Exosomes Inhibits Osteoclast Differentiation and Inflammation via PKM2/HIF-1α Axis

Background: Periodontitis (PD) is a chronic inflammatory disease characterized by alveolar bone loss, primarily driven by excessive osteoclast differentiation and activation. Exosomes derived from M2 macrophages have been implicated in the pathological progression of various diseases, including PD. Pyruvate Kinase (PK)M2, a key metabolic enzyme, promotes inflammation in periodontal disease through enhanced production of pro-inflammatory cytokines. This study investigated whether M2 macrophage-derived exosomes regulate osteoclast differentiation and contribute to PD progression.

Methods: Exosomes were isolated from M2-polarized RAW264.7 cells. Osteoclast differentiation was induced in M2-polarized RAW264.7 cells through treatment with receptor activator of nuclear factor kappa-B ligand (RANKL). Reverse transcription quantitative PCR (RT-qPCR) was used to analyze mRNA levels of osteoclast differentiation-related genes. Enzyme-linked immunosorbent assay (ELISA) quantified inflammatory cytokine production. Co-immunoprecipitation assays were performed to detect the interaction between PKM2 and hypoxia-inducible factor-1α (HIF-1α). To inhibit or activate PKM2 activity, RAW 264.7 cells were treated with Shikonin or TEPP46.

Results: Exosomes were successfully extracted from RAW264.7 cells. M2-derived exosomes significantly suppressed RANKL-induced osteoclast differentiation and inflammatory responses. Furthermore, these exosomes modulated the PKM2/HIF-1α signaling axis. Shikonin treatment inhibited RANKL-induced osteoclastogenesis and inflammation, whereas TEPP46 treatment enhanced these processes.

Conclusion: The M2 polarization of RAW264.7-derived exosomes inhibits osteoclast differentiation and inflammation through the PKM2/HIF-1α pathway, providing potential insights into the molecular mechanisms underlying PD pathogenesis.

Exosome; HIF1-α; M2 polarization; PKM2; inflammation; osteoclast differentiation.