CPT1A Alleviates Senescence and Restores Osteogenic Differentiation of BM-MSC Through SOD2 Succinylation.

Bone marrow mesenchymal stem cells (BM-MSCs) have promising prospects in bone repair and regenerative medicine. However, BM-MSCs gradually lose their original pluripotency and differentiation potential after successive passages. This study aimed to reveal the mechanism underlying the phenomenon. Western blotting, SA-β-gal staining and Alizarin red staining were used to evaluate the senescence phenotype and osteogenic differentiation ability. Mitochondrial ROS levels were detected using flow cytometry. Protein interactions and succinylation modifications were identified by using Co-IP assays. With the increase in passage times, the proliferation and osteogenic differentiation of BM-MSCs were gradually weakened, and the expression level of CPT1A was decreased. BM-MSCs with fewer passages (P1-P5 generations) showed increased mitochondrial ROS production and reduced enzyme activity of superoxide dismutase 2 (SOD2) and the mitochondrial level after the knockdown of CPT1A. In contrast, overexpression of CPT1A in multiple-round-passed BM-MSCs cells (P10-P15 generations) has the opposite effect. Therefore, CPT1A level is associated with the ageing phenotypes and the osteogenic differentiation capacity of BM-MSCs. Knocking down CPT1A significantly reduced the succinylation modification of SOD2, resulting in a decrease in SOD2 enzyme activity and SOD2 levels in mitochondria. Overexpression of CPT1A enhanced the succinylation of SOD2 at the key site K130, thereby reducing cell senescence and promoting osteogenic differentiation. However, this boosting effect was reversed when a mutation occurred at the K130 site of SOD2. CPT1A promotes succinylation modification at the SOD2 (K130) site to induce the accumulation of SOD2 in mitochondria and the enzyme activity, which alleviates BM-MSC senescence and enhances osteogenic differentiation.