Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation
Figure 5
Effect of compound 1h on ROS production and MAPK phosphorylation in LPS-activated macrophages.
In (A), RAW 264.7 macrophages (5 × 105/ml; 1 ml) were incubated for 30 min with compound 1h (20 µM), N-acetyl cysteine (NAC; 10 mM), or DMSO (vehicle), then 2’, 7’-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by LPS (1 µg/ml) stimulation for the indicated time, then ROS levels were measured by detection of the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In (B), RAW 264.7 macrophages (5 × 105/ml; 1 ml) were incubated for 30 min with compound 1h (20 µM) or DMSO, then LPS (1 µg/ml) was added and incubation continued for 0-60 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed by Western blotting and expressed relative to actin expression and as a fold increase compared to the control group at 0 time. In (C), J774A.1 macrophages (5 × 105/ml; 1 ml) were incubated for 30 min with 10-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed as in B. In (A), the data are expressed as the mean ± SD for three separate experiments, while, in (B) and (C), the results are representative of those obtained in three different experiments. * indicates a significant difference at the level of p < 0.05 compared to the DMSO/LPS group.