Objective To explore proteomic changes of M2 macrophages⁃derived exosomes under different oxygen concentrations and their impact on bone repair. Methods Murine⁃derived macrophage cell line (RAW264.7 cell line) was cultured and induced into M2⁃type macrophages. M2⁃type macrophages were assigned to normoxia group (cultured in an environment of 37 ℃ and 5% O2) or hypoxia group (cultured in an environment of 37 ℃ and 1% O2), and corresponding conditions were given to culture. The differential centrifugation was adopted to obtain M2 macrophage⁃derived exosomes. The transmission electron microscope and Western blot were employed to perform morphological identification and identification of surface markers, respectively. After preparing sequencing samples of exosomal proteins, liquid chromatography⁃mass spectrometry coupling technique was adopted to analyze protein components of exosomes. Differentially expressed proteins of the two groups were screened, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to perform functional and pathway enrichment analyses on differentially expressed proteins, and protein⁃protein interaction (PPI) network chart was plotted. Results Under transmission electron microscopy, M2 macrophages⁃derived exosomes in both groups exhibited the typical cup⁃shaped morphology with clearly visible bilayer membranous vesicular structures. Western blot analysis confirmed the presence of exosomal surface markers Alix and Tsg101 proteins in both groups. A total of 1199 differentially expressed proteins were identified between the normoxia and hypoxia groups, among which 696 were up⁃regulated and 503 were down⁃regulated. GO functional enrichment analysis results revealed that these differentially expressed proteins were primarily involved in biological processes such as ribonucleotide triphosphate biosynthesis and metabolic processes, in cellular components like cytoplasmic ribosomes, and in molecular functions including receptor ligand activity. KEGG pathway enrichment analysis indicated differentially expressed proteins were mainly enriched in signaling pathways such as pentose phosphate pathway and hypoxia inducible factor 1 signaling pathway. PPI network analysis suggested that Rack1, Eef1g, Mrto4, Rps9, and Rps15a might serve as key target proteins. Conclusion Under different oxygen concentrations, the protein expression profiles of M2 macrophage⁃derived exosomes exhibit significant differences. Signaling pathways such as pentose phosphate pathway and hypoxia inducible factor 1 signaling pathway, as well as proteins such as Rack1, Eeflg, Mrto4, Rps9, and Rps15a, may play important regulatory roles in bone repair.

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