Figure 7.

SIRT1/FoxO1 activation by melatonin attenuates H₂O₂-induced mitochondrial injury and apoptosis in bovine ovarian granulosa cells. (A) Mean fluorescence intensity was measured to detect ROS level by flow cytometry assay (n = 3). (B,C) ΔΨm was detected using the dye JC-1 (n = 3). The conversion of polymers (red) and monomers (green), and the fluorescence ratio, were measured to analyze ΔΨm. The color from red to blue indicates a decrease in cell density. (D) The concentrations of intracellular Ca²⁺ were detected by flow cytometry assay with Fluo-4 AM. (E) The release of Cyt-c was measured by ELISA. (F) The relative mRNA levels of BAX, CASPASE-3, and BCL-2 were detected by qRT-PCR (n = 3). (G,H) The relative protein levels of BAX, CASPASE-3, and BCL-2 were detected by Western blot (n = 3). (I,J) The apoptosis level of granulosa cells was measured by flow cytometry (n = 3). Apoptosis rate was expressed as the sum of the values of Q2-2 (late apoptotic cells) and Q2-4 (early apoptotic cells) in the four quadrants. The color from red to blue indicates a decrease in cell density. (K,L) The ratio of TUNEL-positive cells was detected by double staining of TUNEL (green) and DAPI (blue). The values are shown as mean ± SD. * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. MT+H₂O₂ group.