Kinsenoside Regulates Ferroptosis and Enhances Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells through the AMPK/SIRT1 Pathway.

OBJECTIVE

Differentiation imbalance of bone marrow mesenchymal stem cells (BMSCs) is a key mechanism of osteoporosis (OP). This study sought to elucidate how kinsenoside (Kin) regulates ferroptosis and enhances BMSCs' osteogenic differentiation.

METHODS

Network pharmacology, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, as well as molecular docking were employed for the identification and analysis of the intersection targets of Kin and OP. Differential gene expression analysis was conducted to validate the identified core targets. The culture of BMSCs was carried out in high glucose (HG) cell culture medium containing Kin, the ferroptosis agonist erastin, or the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin (DM). The viability of cells was evaluated using the cell counting kit-8 assay. BMSCs' osteogenic differentiation was assessed through alkaline phosphatase and alizarin red S staining. Additionally, the expression of OP-related proteins, ferroptosis-related proteins, p-AMPK, AMPK, and sirtuin 1 (SIRT1) were detected via Western blot. The ferroptosis of BMSCs was evaluated using 2,7-dichlorofluorescein diacetate staining, along with quantification of malondialdehyde, glutathione, and ferroptosis-related proteins.

RESULTS

HG suppressed BMSCs' osteogenic differentiation and enhanced ferroptosis, while Kin promoted BMSCs' osteogenic differentiation and inhibited ferroptosis. Kin and erastin co-treatment reduced osteogenic differentiation and enhanced ferroptosis of BMSCs compared to Kin treatment alone. Network pharmacology, molecular docking, differential gene expression, and KEGG enrichment analyses, suggested AMPK/SIRT1 as a potential key pathway underlying Kin's therapeutic effect on OP. Kin activated AMPK/SIRT1 signaling pathway, while co-treatment with DM reversed this activation, accompanied by decreased osteogenic differentiation and increased ferroptosis in BMSCs.

CONCLUSION

Kin enhances BMSCs' osteogenic differentiation under HG conditions by suppressing ferroptosis via activation of the AMPK/SIRT1 signaling pathway.