Oxidative Medicine and Cellular Longevity / 2014 / Article / Fig 4

Research Article

Subanesthetic Isoflurane Reduces Zymosan-Induced Inflammation in Murine Kupffer Cells by Inhibiting ROS-Activated p38 MAPK/NF-κB Signaling

Figure 4

0.7% ISO reduces ZY-induced ROS generation; ROS is essential to ZY-induced activation of p38 MAPK and NF-κB, as well as PGE2 production. (a) At 0.5 h after KCs (1 × 106 cells/well in six-well culture plates) were treated with ZY or CM (Ctrl) for 0.5 h with or without MCI-186 (50 μM) pretreatment for 0.5 h, the cells were exposed to RA with or without 0.7% ISO for another 0.5 h. The KCs were continuously treated with ZY (0.5 mg/mL) or Ctrl for 24 h. RIA was performed to detect PGE2 production. (b) The KCs with or without MCI-186 (50 μM) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 1 h. Western blot was performed to determine the phosphorylation of p38 MAPK (Thr180/Tyr182) and NF-κB (Ser536). β-Actin and lamin B were used as the internal controls. The ratios from p-p38 MAPK to p38 MAPK and from pNF-κB to NF-κB are indicated above the bands. (c) The KCs (5 × 104 cells/well in 96-well culture plates) with or without MCI-186 (50 μM) pretreatment for 0.5 h were stimulated with ZY (0.5 mg/mL) for 1 h with or without 0.7% ISO posttreatment for 0.5 h. DCFH-DA was used to assess the production of intracellular ROS. Representative data are from three independent experiments and expressed as mean ± SD. versus Ctrl + RA or Ctrl + ISO; versus ZY + RA. “Time” in the figure represents the time periods of Ctrl or ZY treatment. ZY: zymosan; ISO: isoflurane; Ctrl: control; RA: room air.