Figure 5.

ACM suppresses IFN-γ-induced ROS production and iNOS expression/NO release. Primary microglia were treated with 20 ng/ml IFN-γ for the indicated time periods (A, B) or pretreated (P) with ACM for the indicated times and then treated with 20 ng/ml IFN-γ for 5 min (C, D). Intracellular ROS levels were assayed using 10 μm DCF as in Figure 1. A, C, Fluorescence (DCF) images. Scale bar, 50 μm. B, D, DCF intensities of cells in A and C were counted using LSM 5 Image Browser (Zeiss). Values are mean ± SEM of 10–15 cells. *p < 0.01 compared with IFN-γ. E, Primary microglia were treated with 20 ng/ml IFN-γ for the indicated times (left) or 72 h (right) in the absence or presence of ACM. F, Primary microglia were treated with 20 ng/ml IFN-γ for 12 h (left) or 72 h (right) in the absence or presence of the ROS scavenging agents trolox (Trol) or butylated hydroxyanisole (BHA). iNOS protein expression was detected by Western blotting (E, F, left). The amounts of nitrite converted from NO in the media were measured using Griess reagents, as described in Materials and Methods (E, F, right). Values in E and F are mean ± SEM of three samples. *p < 0.01 compared with IFN-γ; ^#p < 0.05 compared with control. The data are representative of three independent experiments.