USP15-modified ADMSCs-Exo alleviates chondrocyte damage and effectively relieved osteoarthritis by inducing M2 polarization of macrophages through deubiquitinating FOXC1.

Abstract

The damage to chondrocytes and inflammatory responses are considered the key factors in the pathogenesis of osteoarthritis (OA). Ubiquitin-specific protease 15 (USP15) has been shown to be involved in OA. This study aimed to explore the mechanism of USP15-modified adipose-derived mesenchymal stem cells (ADMSCs) exosome (Exo) in alleviating OA. ADMSC-Exo with USP15 overexpression was isolated by magnetic beads method, and the Exo marker proteins were identified by western blot assay. M1 and M2 phenotypic markers of THP1-M0 cells were analyzed by flow cytometry. ELISA was used to detect the expression of inflammatory factors in cells. CCK-8, EdU, Transwell, and flow cytometry were used to detect the cell activity, proliferation, apoptosis and migration ability. The interaction between forkhead box C1 (FOXC1) and USP15 was verified by Glutathione-S-transferase (GST) pull-down and Co-immunoprecipitation (Co-IP) experiments. The stability of FOXC1 was measured by cycloheximide (CHX), and its ubiquitination level was analyzed by exogenous ubiquitination assay. The Exos from ADMSCs overexpressing USP15 (oe-USP15/Exos) were successfully isolated. It was confirmed that oe-USP15/Exo inhibited the M1 polarization of THP1-M0 cells caused by lipopolysaccharide (LPS) but induced the M2 polarization and the release of inflammatory inhibitory factors. Meanwhile, the damage of chondrocytes caused by LPS was also prevented by oe-USP15/Exo. Besides, USP15 was validated to exert a deubiquitination effect by binding to FOXC1 and positively regulate FOXC1 expression. And the effects of oe-USP15/Exo were abolished after FOXC1 silencing. USP15-modified ADMSC-derived Exos facilitated M2 polarization of macrophages and improved chondrocyte injury by deubiquitination of FOXC1.