Fig. 4.

Effect of nitrones on H₂O₂-induced mitochondrial dysfunction, caspase-3 activation and modulation of pro- and anti-apoptotic proteins in BAEC. (A) Determination of mitochondrial membrane potential (MMP). BAEC were treated with 100 µM H₂O₂ in the absence or presence of nitrone (0–50 µM) for 24 h. After treatment cells were stained with rhodamine 123 and incubated for 60 min, and MMP was measured using a FACS Calibur flow cytometer (BD) with excitation at 488 and emission at 535 nm. Results are expressed as a representative histogram analysis. (B) Time-dependent activation of caspase-3 by H₂O₂. To determine time-dependent activation of caspase-3 by H₂O₂, BAEC were treated with 100 µM H₂O₂ at various time points (0–24 h) and caspase-3 activity was determined by using the 7-amino-4-trifluoromethyl coumarin assay (AFC) linked to the tetrapeptide DEVD as described in “materials and methods” section. Results are represented as mean ± SEM, *p < 0.05 vs. control (untreated cells). (C) Inhibition of H₂O₂-induced caspase-3 activation by nitrones. To determine nitrone-induced inhibition of caspase-3 activity, BAEC were preincubated with nitrone (0–50 µM) and then treated with 100 µM H₂O₂ for 24 h. After treatment cells were incubated with DEVD-AFC. Finally the release of free AFC was monitored using Cytofluor 4000 fluorimeter (excitation, 400 nm; emission, 508 nm). Results are represented as mean ± SEM, *p < 0.05 vs. control (untreated cells), and #p < 0.05 vs. H₂O₂-treated cells, where n = 3. (D) Modulation of pro-and anti-apoptotic proteins by the nitrones in H₂O₂-treated BAEC. Expression levels of p53, Bax, Bcl-2 and p-Bad in BAEC treated with H₂O₂ in the absence and presence of nitrone were monitored by western blotting. (E), (F) Densitometric analysis of the expressions of p53, p-Bad and Bax/ Bcl-2 ratio. Results are represented as mean ± SEM, *p < 0.05 vs. control (untreated cells), and #p < 0.05 vs. H₂O₂-treated cells, where n = 3.