Figure 4

ROS scavengers block MAPK activation and pro-inflammatory cytokine production in s-Mtb-stimulated microglia. A) Effect of ROS scavengers on p38 and ERK1/2 activation. After pre-treatment for 30 min with NAC (10, 20, or 30 mM), DPI (10, 20, or 50 μM), or rotenone (1, 10, or 100 μM), BV-2 cells were stimulated with 1% s-Mtb for 30 min. The cells were then harvested and subjected to Western blot analysis to detect phosphorylated p38 and ERK1/2. The same blots were washed and blotted for α-actin as a loading control. B) Effect of ROS scavengers on TNF-α, IL-6, and IL-12p40 production. Cells were pre-treated with NAC, DPI, and rotenone as described in A. Culture supernatants were harvested after stimulation with 1% s-Mtb for 18 h, and TNF-α, IL-6, and IL-12p40 expression was measured by ELISA. The mean amount ± SD of TNF-α, IL-6, or IL-12p40 from cultures treated with 1% s-Mtb or 0.1% DMSO was set at 100, and the relative drop in cytokine production in the presence of each inhibitor is indicated. Significant differences compared to cultures incubated with 1% s-Mtb alone: **, P < 0.01; ***, P < 0.001. M, media only; D, 0.1% DMSO as a solvent control.