Figure 4.

L6H21 inhibited LPS-induced MAPK phosphorylation and NF-κB activation. (A and B) MPMs were pretreated with vehicle control (DMSO) or L6H21 (2.5, 5 or 10 μM) for 2 h followed by incubation with LPS (0.5 μg·mL⁻¹) for 30 min. The protein levels of p-ERK, ERK, p-p38, p38, p-JNK, JNK, I-κB, β-actin and GAPDH were examined by Western blot respectively. Values below the Western blots represent the mean optical density ratio in three independent experiments, and the column figure data (A) were normalized to β-actin and then expressed versus total level of MAPKs (*P < 0.05, **P < 0.01). (C) MPMs were pretreated with vehicle control (DMSO) or L6H21 (2, 2.5 or 10 μM) for 2 h, and then stimulated with LPS (0.5 μg·mL⁻¹) for 1 h. Nuclear extracts were analysed for NF-κB activity by EMSA. (D) MPMs were pretreated with L6H21 (10 μM) or vehicle control (DMSO) for 2 h, and then stimulated with LPS (0.5 μg·mL⁻¹) for 1 h. The cells were incubated with p65 antibody and then Cy3 fluorescein-conjugated secondary antibody, and the nuclei were stained with DAPI. The images (200×) were obtained by fluorescence microscopy and overlay. Similar results were obtained in three independent experiments.