Gingival Mesenchymal Stem Cell-derived Exosomes USP13 Inhibit NF-κB Signaling Pathway and Induce M2 Polarization of Macrophages

doi:10.13701/j.cnki.kqyxyj.2026.05.009

Abstract

Abstract: Objective: To investigate the regulatory effect of ubiquitin-specific protease 13 (USP13) in gingival mesenchymal stem cell (GMSCs)-derived exosomes (GMSCs-Exos) on macrophage polarization. Methods: GMSCs-Exos were isolated and identified. Their morphological structure was observed via transmission electron microscopy, and the expression levels of exosomal markers CD9 and CD63 were determined using Western blotting. THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Immunofluorescence staining verified the uptake of GMSCs-Exos by macrophages. Macrophages were divided into five groups: control group (macrophages cultured alone), model group (macrophages with M2 polarization induced by IL-4 and IL-13), GMSCs-Exos group (macrophages co-cultured with GMSCs-Exos), GMSCs-Exos + USP13 siRNA group (macrophages co-cultured with GMSCs-Exos and USP13 small interfering RNA), and GMSCs-Exos + nuclear factor kappa B (NF-κB) agonist (Compound-32) group (macrophages co-cultured with GMSCs-Exos and Compound-32). The interaction between USP13 and inhibitor of nuclear factor kappa B alpha (IκBα) was analyzed using co-immunoprecipitation (Co-IP). Western blotting was used to determine the expression levels of USP13, IκBα, p65, phosphorylated p65 (p-p65), and M2 markers (CD206 and Arg1) in macrophages. Flow cytometric analysis measured the proportion of CD206-positive cells on the cell surface. Enzyme-linked immunosorbent assay (ELISA) quantified the levels of transforming growth factor-β (TGF-β), interleukin-10 (IL-10), and arginase-1 (Arg1) in the macrophage supernatant. Results: GMSCs-Exos exhibited a typical vesicular morphology and high expression levels of CD9 and CD63. Following internalization of GMSCs-Exos by macrophages, USP13 bound to IκBα, which reduced the ubiquitination level of IκBα. Compared with the control group, the model group and GMSCs-Exos group showed decreased levels of p-p65 and nuclear p65, along with increased cytoplasmic p65 levels. Additionally, these two groups exhibited elevated expression levels of M2 markers (CD206 and Arg1), an increased proportion of CD206-positive cells on the cell surface, and higher levels of TGF-β, IL-10, and Arg1. By contrast, compared with the GMSCs-Exos group, the GMSCs-Exos + Compound-32 group displayed opposing trends in the aforementioned M2 polarization indicators. Conclusion: Following internalization of GMSCs-Exos by macrophages, the interaction between USP13 and IκBα reduces the ubiquitination level of IκBα. This, in turn, inhibits the phosphorylation and nuclear translocation of p65 in macrophages, ultimately promoting macrophage M2 polarization.

Key words: gingival stem cells, exosomes, USP13, IκBα, NF-κB

GAO Jiaming, YUE Jiasheng, ZHANG Naixin. Gingival Mesenchymal Stem Cell-derived Exosomes USP13 Inhibit NF-κB Signaling Pathway and Induce M2 Polarization of Macrophages[J]. Journal of Oral Science Research, 2026, 42(5): 414-420.

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