Fig. 3.
Involvement of p38 MAPK/NF-κB pathway in the suppression of ethanol-induced Nox2 and p22phox expression by gAcrp in RAW 264.7 macrophages. (A, B) Cells were pretreated with Bay 11-7082 (5 μM), a selective inhibitor of IKK, for 1 hour, followed by stimulation with 100 mM ethanol for 24 hours. Messenger RNA levels of Nox2 (A) and p22phox (B) were assessed by qRT-PCR as described previously. Values are expressed as mean ± S.E.M. (n = 8). *P < 0.05 compared with control; #P < 0.05 compared with cells treated with ethanol. (C, D) Cells were pretreated with SB203580 (10 μM), a selective inhibitor of p38 MAPK, for 1 hour followed by treatment with 100 mM ethanol for additional 24 hours. Nox2 (C) and p22phox (D) mRNA levels were measured by qRT-PCR as described previously. Values represent fold change relative to control cells and are expressed as mean ± S.E.M. (n = 4). *P < 0.05 compared with cells not treated with ethanol; #P < 0.05 compared with cells treated with ethanol. (E) Cells were transiently cotransfected with pNFκB-luc and Renilla reporter gene as described in Materials and Methods. After 24 hours of transfection, cells were then pretreated with gAcrp (0.1 μg/ml) for 18 hours, followed by treatment with 100 mM ethanol for an additional 24 hours. Transcriptional activity of NF-κB was measured by luciferase reporter assay. Values are presented as fold increase compared with the control samples and are expressed as mean ± S.E.M. (n = 5). *P < 0.05 compared with cells not treated with ethanol; #P < 0.05 compared with cells treated with ethanol.