Fig. 4.

The promotional effects of Mg²⁺ on BMP-2 resulted from its stimulatory effects on cellular metabolism via the Akt-glycolysis-Mrs2-mitochondrial axis. A) Representative images of intracellular Mg²⁺ dynamics as detected by the Mag-green probe (red fluorescence represents mitochondria). B) Dynamic detection of Mg-green intensity in the mitochondrial region. C) Seahorse analysis of the OCRs and ECARs alterations in BMSCs induced by 20 ng mL⁻¹ BMP-2 with or without Mg²⁺ for 7 days. BMSCs cultured in normal medium served as the control. D) Key parameters calculated from the above Seahorse assay curves. E) Heatmap depicting the expression profile of metabolic genes. F) Mg²⁺ addition stimulated the phosphorylation of Akt. G) Analysis of ΔΨm by flow cytometry. Cells in the upper left region exhibited a high ΔΨm, while cells in the lower right region had a low ΔΨm. H) Charts depicting the relative ΔΨm as determined by measuring the mean fluorescence intensity, the activities of metabolic enzymes, and ATP synthesis in the different groups (* and # represent p < 0.05, while ** and ## represent p < 0.01 in comparison with the control and 20 ng mL⁻¹ groups, respectively). I) Changes in the Mg-green intensity in the mitochondria of normal and Mrs2 knockout BMSCs. J) Mrs2 knockout markedly suppressed ATP production in BMSCs treated with BMP-2 and Mg²⁺. (** represents p < 0.01). K) Alp staining of normal and mutant BMSCs. L) Mechanism of the stimulatory effects of Mg²⁺ on the efficacy of BMP-2.