Figure 5. Nrf2-mediated inhibitions of proinflammatory cytokine genes are independent of ROS elimination.

(a) Evaluation of the ROS level in DEM- or NAC-treated BMDMs. WT BMDMs were stimulated with 5-ng ml⁻¹ LPS or vehicle, with or without 100-μM DEM or 1-mM NAC, for 4 h. The ROS level was evaluated by fluorescence after treatment with CellROX reagent during the last 30 min of 4 h stimulation. Data are mean±s.d. from four mice (*P<0.05, paired t-test). (b–d) Evaluation of the ROS-mediated effect. The relative expressions of IL6 (b), IL1b (c) and IL1a (d) were examined by RT-qPCR after the same stimulations as (a). Note that NAC-treatment does not affect these cytokine mRNA expressions. (e–h) Time-course change of inflammatory cytokine gene expression after the chemical Nrf2 inducer treatment. Relative mRNA expression of IL6 (e), IL1b (f), IL1a (g) and Nqo1 (h) in WT BMDMs were examined at indicated times from the start of M1 stimulation in the presence of 100-μM DEM or vehicle. DEM and vehicle were added in the culture media at the same time as the M1 stimulus. The value of M1+Vehicle at 6 h is set to 1. *P<0.05 (unpaired t-test) against M1+Vehicle; ^#P<0.05 (unpaired t-test) against Con+Vehicle. Note that DEM inhibits the expression of the inflammatory cytokine mRNAs, but induces the expression of Nqo1 mRNA.