Abstract: Objective: To explore the effect of urolithin A (UA), a bioactive compound from pomegranate, on mitophagy and aging in bone marrow mesenchymal stem cells (BMSC) in a simulated diabetic environment and to elucidate the underlying signaling mechanism. Methods: BMSC were isolated, cultured and identified from C57 mice. Cellular senescence was induced by exposing BMSC to 30 mmol/L glucose and 0.5 mmol/L palmitic acid. The senescence cells were then treated with UA at different mass concentrations. Cell viability was detected by the Counting Kit-8 (CCK-8) assay, senescent cells were detected by β-galactosidase staining, reactive oxygen species (ROS) levels were detected using the fluorescent probes 2’,7’-dichlorofluorescin-diacetate (DCFH-DA) and Mito SOX, and the expression levels of proteins related to aging and mitochondrial autophagy were detected by Western Blot. Results: BMSCs showed positive expression of Sca-1 and CD29, and negative expression of CD45 and CD11b. The optimal conditions for inducing cellular senescence were 48 h exposure to 30 mmol/L glucose plus 0.5 mmol/L palmitic acid. Following UA intervention, cell viability increased; cell apoptosis, the rate of galactosidase positive cells, and fluorescence intensities for cellular and mitochondrial ROS decreased. In addition, PTEN induced putative kinase 1 (PINK1) and P62 expression significantly decreased, whereas the expression of Parkin E3 ubiquitin protein ligase (PARKIN) and microtubule-associated protein light chain 3 (LC3) significantly increased. The protein expression of nuclear factor erythroid 2-related factor 2 (NRF2) in the nucleus significantly dropped, while the protein expression of sirtuin 1 (SIRT1) and tumor protein 53 (P53) significantly increased. Meanwhile, the opposite results were observed in the cytoplasm. All the above effects were dose-dependent. Conclusion: UA could inhibit diabetes-induced mitophagy disorder and reduce senescence in BMSC. The underlying mechanisms may involve activation of the PINK1/Parkin mitophagy pathway and regulation of the NRF2/SIRT1/P53 senescence signaling axis, potentially restoring stem cell quality.

Key words: urolithin A; high glucose and palmitic acid; bone marrow mesenchymal stem cells; aging; autophagy