Fig. 4

The upregulation of VDR signaling protects BMSCs from senescence caused by palmitic acid or a high-fat diet. a Representative images following SA-β-gal staining and alizarin red staining after the osteogenic induction of BMSCs (scale bars = 50 μm). The positive SA-β-gal cells were stained green (black arrow). b The addition of 1,25(OH)₂D or NAC to BMSCs decreased the increase in the intracellular ROS level induced by palmitic acid. ROS levels were analyzed by flow cytometry. c The addition of 1,25(OH)₂D or NAC improved the palmitic acid-induced decrease in the proliferation of BMSCs. Proliferation was analyzed by a CCK8 assay. d Western blot results demonstrating the effect of 1,25(OH)₂D or NAC on VDR and P21 expression in BMSCs. e Quantification of the percentage of SA-β-gal-positive cells, ROS levels, and the expression of VDR and P21 in BMSCs from each group. f Quantification of ARS staining in BMSCs from each group. g The expression of Vdr, p21, p53, and p16 in BMSCs from mice in the Veh and 1,25VD groups was detected by RT‒qPCR. h CCK8 assay demonstrating that the addition of 1,25(OH)₂D improved the proliferation of BMSCs, which was inhibited by HFD feeding. i Representative images of anti-γH2A.X, anti-P21, and EdU immunofluorescence staining as well as SA-β-gal and alizarin red staining in BMSCs from mice in the Veh and 1,25VD groups (scale bars = 50 μm). j Intracellular ROS levels, as indicated by DHE staining, in BMSCs that were detected by flow cytometry. k Quantification of the SA-β-gal-, γH2A.X-, P21-, EdU-positive cell ratios, and ARS staining in BMSCs from mice in the Veh and 1,25VD groups. l Osteogenesis-related gene expression was quantified by RT‒qPCR after osteogenic induction. The data were shown as the mean ± SEM. N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.000 1