Periplaneta Americana polysaccharides promote and maintain the M2 polarization of macrophages through the O-GlcNAc-HIF-1α/PPARγ pathway

Previous studies have found that the active polysaccharide PAP55-3-1 from Periplaneta americana promotes the expression of HIF-1α in macrophage RAW264.7 cells, induces M2 polarization of macrophages, and maintains the M2 phenotype even under H₂O₂ stimulation, thereby reducing the inflammatory response and promoting wound healing. This study aims to further investigate the molecular mechanisms underlying the anti-inflammatory effects of PAP55-3-1. Untargeted metabolomics revealed that PAP55-3-1 promotes the HBP pathway, thereby enhancing the O-GlcNAcylation of total protein and HIF-1α. Meanwhile, PAP55-3-1 also stabilizes HIF-1α by promoting O-GlcNAcylation. Through mass spectrometry analysis, three specific O-GlcNAc modification sites (T241, S244, and S247) within the PAS domain of HIF-1α were identified for the first time. Site-directed mutagenesis revealed that O-GlcNAcylation at these sites enhances PPARγ transcription, thereby promoting lipid synthesis and M2 polarization of macrophages. Since all three modification sites are located within the PAS domain, it is hypothesized that the maintenance of HIF-1α stability may be mediated by other mechanisms. This study reveals that PAP55-3-1 promotes and sustains the M2 macrophage via the O-GlcNAc-HIF-1α/PPARγ axis. Furthermore, O-GlcNAcylation at T241, S244, and S247 residues of HIF-1α represents key molecular targets through which Periplaneta americana polysaccharides facilitate wound healing.