Anti-Inflammatory Potential of Carpomitra costata Ethanolic Extracts via Inhibition of NF-κB and AP-1 Activation in LPS-Stimulated RAW264.7 Macrophages

Figure 5

Effect of CCE on NF-κB and AP-1 activation in LPS-induced RAW264.7 macrophages. (a) RAW264.7 cells were pretreated with CCE (30, 50, or 100 μg/mL) for 1 h and then stimulated with LPS for 2 h. Nuclear extracts were prepared and evaluated for NF-κB and AP-1 using EMSA. (b, c, and d) Cells were pretreated with NF-κB inhibitors (BAY 11-7082 and parthenolide) and AP-1 inhibitor (SR-11032) for 30 min and were then stimulated with LPS (100 ng/mL) for 24 h. Levels of NO and PGE₂ in the supernatant were detected using the Griess reaction assay (b) and ELISA (c). (d) The expression levels of TNF-α, IL-6, and IL-1β in the culture medium were determined using ELISA. Each value indicates the mean ± SEM and is representative of results obtained from three independent experiments. versus control group; versus LPS-stimulated group. (e) Cells were pretreated with SP600125 (a JNK inhibitor) and LY294002 (an Akt inhibitor) 30 min prior to LPS stimulation for 2 h. Nuclear extracts were prepared and evaluated for NF-κB and AP-1 using EMSA. All experiments were repeated three times and representative results are shown. CP: cold (unlabeled) probe, PT: parthenolide, BAY: BAY 11-7082, SR: SR-11032, SP: SP600125, and LY: LY294002.