Fig. 4
Fifty-micromolar H2O2 preconditioning improved the redox state of the mitochondria; 50 μM H2O2 attenuated oxidative stress (300 μM)-induced mitochondrial dysfunction by inhibiting mtROS generation in BMSCs. Cells were pretreated with 50 μM H2O2 for 12 h, washed, and then incubated with fresh medium in the presence or absence of 300 μM H2O2 for an additional 24 h. a The mitochondrial O2•− production was determined using MitoSOX Red staining and observed under the inverted fluorescence microscopy. Scale bar, 20 μm. b The mitochondrial O2•− levels were measured using MitoSOX Red staining, and the fluorescence values were read at an excitation wavelength of 510 nm and emission wavelength of 579 nm (n = 4). The absorbance values were expressed as the mitochondrial O2•− levels. c Fifty-micromolar H2O2 suppressed oxidative stress-induced collapse of mitochondrial membrane potential in BMSCs. Changes of Δψm was observed using the inverted fluorescence microscopy. Red fluorescence was emitted by JC-1 aggregates in healthy mitochondria with polarized inner mitochondrial membranes, whereas green fluorescence was emitted by cytosolic JC-1 monomers, indicating Δψm dissipation. Scale bar, 20 μm. Merged images indicate the co-localization of JC-1 aggregates and monomers. d Δψm in each group was calculated as the ratio of red to green fluorescence, indicating changes in mitochondrial membrane potential. All results are presented as mean ± SEM of at least four independent experiments (n = 4). *P < 0.05, **P < 0.01 vs. the control group; ##P < 0.01, vs. the 300 μM H2O2 group