[Psoralen promotes osteogenic differentiation of MC3T3-E1 cells by regulating mitophagy via PINK1/Parkin pathway]

This study aims to investigate the protective effect of psoralen against hydrogen peroxide(H_2O_2)-induced oxidative stress injury in mouse cranial apical osteoblasts(MC3T3-E1) and explore its molecular mechanism in promoting osteogenic differentiation through PTEN-induced putative kinase 1(PINK1)/Parkin-mediated mitophagy regulation. An oxidative injury model in MC3T3-E1 cells was established with 200 μmol·L~(-1) H_2O_2. The experiment was conducted with four groups: normal, model, psoralen, and inhibitor groups. Cell viability and proliferation were assessed by the CCK-8 assay and EdU incorporation assay, respectively. Apoptosis was analyzed by Annexin V-FITC/PI double-staining flow cytometry. Mitochondrial membrane potential was evaluated with the JC-1 probe, and reactive oxygen species(ROS) levels were measured by the DCFH-DA probe. Early osteogenic differentiation markers and late mineralized nodule formation were observed by alkaline phosphatase(ALP) staining and alizarin S staining, respectively. Transmission electron microscopy was employed to examine mitochondrial ultrastructure changes. Immunofluorescence staining and Western blot were employed to determine the expression levels of PINK1, Parkin, p62, Runt-related transcription factor 2(Runx2), and Osterix. Molecular docking and 100 ns molecular dynamics simulations were conducted to validate the binding mode and stability of psoralen with PINK1. The results showed that psoralen ameliorated H_2O_2-induced cell damage in a concentration-dependent manner, with the optimal concentration being 80 μmol·L~(-1). Psoralen significantly promoted cell proliferation, upregulated the expression of Runx2, Osterix, PINK1, and Parkin, enhanced mitochondrial membrane potential, increased mitophagy levels and osteogenic differentiation capacity, and enlarged mineralized nodule area. Simultaneously, it inhibited apoptosis, reduced intracellular ROS content, and downregulated the expression of autophagy substrate p62. Molecular docking results showed that the binding energy between psoralen and PINK1 was-7.024 kcal·mol~(-1), and 100 ns molecular dynamics simulations further confirmed the structural stability of the formed complex with persistent hydrogen bond interactions. In conclusion, psoralen directly targets and activates the PINK1/Parkin pathway to mediate mitophagy, clear damaged mitochondria, improve the oxidative stress microenvironment, promote the expression of osteogenic marker proteins and mineralization, thereby enhancing osteogenic differentiation of MC3T3-E1 cells, providing a new target and candidate drug for the prevention and treatment of osteoporosis.