Oxidative Medicine and Cellular Longevity / 2014 / Article / Fig 2

Research Article

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

Figure 2

0.7% ISO reduces ZY-induced NF-κB activation in vitro. (a) At 0.5 h after ZY or CM (Ctrl) treatment, the KCs (1 × 106 cells/well in six-well culture plates) were exposed to RA with or without 0.7% ISO for 0.5 h. The cells were continuously stimulated with Ctrl for 0 h and 6 h or with ZY (0.5 mg/mL) for 0.5, 1, 2, 3, and 6 h. Western blot was performed to determine the phosphorylation of IKKβ (Ser180) and NF-κB (Ser536). β-Actin and lamin B were used as the internal controls. The ratios from pIKKβ to IKKβ and from pNF-κB to NF-κB are indicated above the bands. (b) The KCs were treated as the same way to (a) except that they were continuously stimulated with ZY (0.5 mg/mL) or Ctrl for 24 h. Immunocytochemical staining was performed to assess the nuclear translocation of NF-κB p65 in the KCs immunostained with anti-NF-κB p65 antibody. NF-κB p65-positive cells were then calculated and densitometrically quantified. (c) The KCs were treated as the same way to (b). The NF-κB DNA-binding activity was assayed by determining the optical density. (d) The KCs with or without NAI (2 μM) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 24 h. RIA was performed to detect PGE2 production. 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.