Fig. 6

NOX2 inhibition suppressed endosomal ROS generation and chemokine induction by silica NPs in vitro. A-B. Intracellular levels of ROS signals in Raw264.7 cells that endocytosed silica-NPs. Raw264.7 cells incubated with silica-NPs for 6 h were stained with DCFDA (intracellular ROS indicator), and signals were quantified by flowcytometry. A. Representative histography of fluorescent intensity of RAW cells by DCFDA assay. DCFDA(−) represents vehicle treated cells without staining with DCFDA (negative control). B. The graph shows relative levels of intracellular ROS normalized by signals in untreated cells. C-E. Chemokine gene expression in RAW264.7 cells incubated with silica-NPs in the presence of N-acetylcysteine (NAC). Total RNA was isolated from RAW264.7 cells 6 h after silica-NPs exposure with or without addition of 10 mM of NAC, and gene expression of proinflammatory cytokine genes (MIP1α (C), MIP2 (E), and TNF-α (F)) was determined by qRT-PCR. F-G. Assessment of endosomal ROS levels in RAW264.7 cells with different silica-NPs using OxyBURST. Cells were incubated with silica-NPs (50 nm-plain, 50 nm-NH₂) for 2 h or PMA for 30 min as a positive control. Some experiments were done with cells pre-treated with gp91ds-tat, a specific inhibitor of NOX2. F. Representative images obtained by confocal microscopy. Scale bars = 10 μm G. Average number of puncta indicating the signals of endosomal compartment with increased ROS level per cell. (# p = 0.0001 vs vehicle, 50 nm-NH₂, PMA + gp91ds-tat, 50 nm-plain + gp91ds-tat, and 50 nm-NH₂ + gp91ds-tat groups, respectively by one-way ANOVA with Tukey’s multiple comparison test) H-J. Induction of proinflammatory cytokines in Raw264.7 cells treated with silica-NPs was suppressed by treatment with gp91ds-tat, a specific inhibitor of NOX2 activation. For Figs. B, C, D, E, H, I, and J, * p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison tests