*2.2. Effects of Wistin on the Production of Pro-Inflammatory Mediators in LPS-Induced RAW 264.7 Cells*

Prolonged inflammatory processes increase the production of nitric oxide (NO) and reactive oxygen species (ROS), leading to tissue dysfunction [36]. ROS can regulate proinflammatory gene expression, and NO is an important pro-inflammatory mediator in inflammatory signaling [37]. To examine the anti-inflammatory effects of wistin, we investigated the production of NO and ROS. Wistin showed a significant decrease in LPSinduced NO production in a dose-dependent manner compared to that in the control group (Figure 2a). In addition, dose-dependent inhibition of intracellular ROS generation by wistin was identified using a microplate reader, fluorescence-activated cell sorting (FACS), and fluorescence microscopy (Figure 2b–e). These data suggest that wistin can reduce the production of pro-inflammatory mediators (NO and ROS) induced by LPS.
