Abstract: Objective: To explore the inhibitory effect and mechanism of urolithin A (UA) on ferroptosis in adipose-derived mesenchymal stem cells (ADMSCs) under a high-glucose high-palmitic acid environment. Methods: The diabetic environment was simulated using a combination of 25 mmol/L glucose and 0.25 mmol/L palmitic acid to induce damage to ADMSCs, and cell viability was detected by the CCK-8 method after UA intervention. The ferroptosis markers malondialdehyde (MDA), glutathione, and ferrous ion were detected. Targets related to UA, type 2 diabetes mellitus and ferroptosis were obtained from online databases and the intersection targets were imported into the STRING database and the Cytoscape 3.9.1 software to construct a protein-protein interaction (PPI) network. The DAVID 6.8 data platform was used to perform signaling pathway enrichment analysis on the intersection targets, and AutoDock Tools 1.5.7 and PyMOL were employed for molecular docking to predict the binding energies between UA and core targets. Western blot (WB) was used to detect the expression of proteins associated with ferroptosis and the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Results: The isolated ADMSCs expressed CD29 and CD90 but not CD11b or CD45 on their surface and had multilineage differentiation capacity. In vitro, ADMSCs were injured under the simulated diabetic condition. The viability was significantly enhanced after UA intervention. In addition, the content of glutathione was significantly increased, while the content of MDA and ferrous ion was reduced. A total of 54 intersection targets were selected by network pharmacology, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that UA might regulate ferroptosis in ADMSCs through the PI3K/AKT signaling pathway. Molecular docking showed that UA could stably bind to key proteins. The results of WB showed that UA intervention significantly increased the expression levels of glutathione peroxidase 4 (GPX4), phospho-protein kinase B (p-AKT) and phospho-phosphoinositide 3-kinase (p-PI3K) in ADMSCs damaged by high glucose and high palmitic acid but decreased the expression level of long-chain acyl-CoA synthetase 4 (ACSL4). Conclusion: UA can reverse ferroptosis induced by high glucose and high palmitic acid in ADMSCs, and the mechanism may be related to the regulation of the downstream GPX4/ACSL4 signaling pathway of ferroptosis by activating the PI3K/AKT signaling pathway.

Key words: urolithin A; high glucose and high palmitic acid; adipose-derived mesenchymal stem cells; ferroptosis; phosphoinositide 3-kinase/protein kinase B