Fig. 4.

Inhibitory effects of NSC23766 on high glucose-induced mitochondrial dysfunction. (a) INS-1 cells were treated with NSC23766 (50 μM) for 2 h and then exposed to high concentrations of glucose (30 mM) for 24 h and then mitochondrial membrane potentials was analyzed by FACs using DiO6 dye; a representative FACs data shown in the upper panel and the quantification of the M1 ratio from three independent experiments shown in the lower panel. ^*P<0.0001 vs. control, ^**P<0.0001 vs. HG. (b, c) INS-1 cells were treated with NSC23766 (50 μM) for 2 h and then exposed to high concentrations of glucose (30 mM) for 24 h. Cytoplasmic extract were fractionated using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit. Cytosolic cytochrome c and cleaved caspase-3 protein expression were analyzed by immunoblotting. ^*P<0.0001 vs. control, ^#P<0.001 vs. HG, ^**P<0.005 vs. HG. Data represents three independent experiments and equal loading was confirmed with β-actin. (d) Inhibition of Rac1 activity blocks high glucose induced ROS production. INS-1 cells were treated with NSC23766 (50 μM) for 2 h or transfected with 100 nmol/L Rac1-siRNA and then exposed to high concentrations of glucose (30 mM) for 24 h. Cellular ROS production was analyzed by fluorescence microscopy using 10 μM/L DCF-DA. ^*P<0.0001 vs. control, ^**P<0.005 vs. HG, ^***P<0.001 vs. HG. (e) Inhibition of Rac1 activity blocks high glucose induced INS-1 cell apoptosis. INS-1 cells were treated with NSC23766 (50 μM) for 2 h and then exposed to high concentrations of glucose (30 mM) for 24 h. INS-1 cell apoptosis was assessed by TUNEL-assay using fluorescence microscopy. ^*P<0.001 vs. control, ^*P<0.001 vs. HG.