*2.3. Effects of Wistin on Pro-Inflammatory Enzymes and Cytokine Gene Expression in LPS-Induced RAW 264.7 Cells*

Next, we investigated the involvement of wistin in the modulation of mRNA levels of inflammatory enzymes (iNOS and COX-2) and pro-inflammatory cytokines (IL-1β and IL-6) at indicated time points after LPS treatment [38]. Wistin significantly decreased the mRNA expression levels of inflammatory enzymes (iNOS and COX-2) (Figure 3a,b). The expression levels of pro-inflammatory cytokines (IL-1β and IL-6) were not detected at 0 h after LPS treatment and were the highest at 12 h after LPS treatment. In addition, wistin-treated groups showed a significant decrease in the mRNA expression levels of proinflammatory cytokines at each time point compared to LPS treatment groups. Therefore, these results suggest that wistin could modulate pro-inflammatory enzymes and cytokine gene expression induced by LPS.

**Figure 2.** The effect of wistin on NO and ROS production in LPS-stimulated RAW264.7. The cells were treated with the indicated concentrations of wistin for 30 min prior to treatment with LPS (0.1 μg/mL) for 24 h. (**a**) The NO production was measured in treated wistin in the culture supernatant. ROS production was measured on a microplate reader (**b**), FACS (**c**,**d**), and fluorescence microscope (**e**). (**f**) Quantitative analysis of ROS production using ImageJ software. The scale bar represents 100 μm. \* *p* < 0.05 compared with the LPS-treated group. MFI: mean fluorescence intensity. The data are presented as the means ± SD; *n* = 3.
