Figure 3. Isoflurane and zymosan regulate NF-κB activation via ROS/p38 MAPK signaling.

A., B. Neutrophils were sequentially treated with zymosan (10 μg/ml) for 15 min, 0.7% isoflurane for 15 min where indicated, and zymosan for 6 h in the presence or absence of NAC (50 mM). Cells were subjected to fluorescence measurement of H₂O₂ levels (A) and Western blot analysis (B). Scale bar = 10 μM. C., D. Neutrophils were treated with zymosan for 6 h in the presence of the p38 MAPK inhibitor SB202190 (10 mM) where indicated, followed by immunofluorescent staining of p65 (C) and Western blot analysis of nuclear p65 levels (D). Scale bar = 10 μM. E., F. Neutrophils were treated with zymosan for indicated time periods, followed by measurement of NADPH oxidase activity (E) or Western blot assay using the membrane and cytosol fractions of cell lysates (F). Gas and β-actin were used as inner controls for membrane and cytosolic fractions, respectively. G. Neutrophils were treated with zymosan for 12 h in the presense of NAC (50 mM), SB202190 (10 mM) or NADPH oxidase inhibitor DPI (10 mM) where indicated, followed by measurement of NO and ONOO⁻ production. Data are represented as the mean ± SEM of 3 replicates or representative of 3 independent experiments. *P < 0.05, ^#P < 0.01, as compared with the cells exposed to zymosan alone (B, C, E, H). *P < 0.05, ^#P < 0.01, as compared with the cells exposed to vehicle alone (F, G).