Effects of AF on LPS-stimulated NO production in RAW 264.7 macrophage cells.

<p>Cells (5 X 10⁵ cells/well in 24-well pate) were treated with LPS (100 ng/mL) only and with different concentrations of AF for 24 h. The amount of nitrite in the medium was measured using Griess assays. The values are expressed as the means ± SED. of three individual experiments. *p < 0.05 and **p < 0.01 indicate significant differences from the LPS alone-treated cells.</p

AF inhibits iNOS and COX-2 mRNA and protein levels in RAW 264.7 macrophage cells.

<p>RAW264.7 macrophage cells were treated with 0–50 mM AF and then co-treated with LPS for 24 h. Protein (A) and mRNA (B) levels were determined by RT-PCR and Western blot analysis, respectively. The data shown are representative of three independent experiments and indicate the mean ± SEM. *p < 0.05 and **p < 0.01 indicate significant differences from the LPS alone-treated cells.</p

Effects of AF on LPS-induced nuclear translocation of NF-κB and AP-1 in RAW 264.7 macrophage cells.

<p>Cells were treated in the presence or absence of various concentrations (0–50 μg/ml) of AF for 30 min and further incubated with or without 100 ng/ml of LPS for 15 min. Then, the cells were harvested and separated into nuclear (A) and cytosolic extracts (B), as described in the Methods section. The protein extracts were separated on SDS-PAGE gels and immunoblotted using Western blot analysis. The antibodies used were anti-NF-κB, -p-c-Jun, -hnRNP, -IκB, and -β-actin. hnRNP and β-actin were used as controls for nuclear- and cytosol-specific proteins, respectively. (C), (D) EMSA showing the reduction in NF-κB and AP-1 DNA binding activity in nuclear proteins. Cells were prepared by stimulation with 100 ng/mL LPS for 15 min and pre-treated with 50 μM AF for 30 min. (E), (F) The translocation of NF-κB (p65) and AP-1 to the nucleus was analyzed by confocal microscopy. Macrophages were immunostained using FITC for NF-κB and AP-1 and Hoechst to label nuclei. White scale bars, 5 μm. NT, no treatment; A, AF; L, LPS; L+A, LPS with AF.</p

A schematic illustration of the anti-inflammatory activity of AF.

<p>AF suppressed the inflammatory response in RAW264.7 macrophages through suppression of NF-κB, AP-1(c-Jun), p-ERK1/2, TNF-a, IL-6, and IL-1B.</p

Effects of AF on the levels of LPS-induced cytokines TNF-α, IL-6, and IL-1β in RAW 264.7 macrophages.

<p>The mRNA levels of TNF-α, IL-6, and IL-1β were determined by RT-PCR in cells treated with 100 ng/mL of LPS only or with 1–50 μM of AF for 24 h. Band density in Fig 4 (versus β-Actin) is indicated as the mean ± SEM of three independent experiments. **p < 0.01 indicates significant differences from the LPS alone-treated cells.</p

Chemical structure and effects of AF on cell viability in RAW 264.7 macrophage cells.

<p><b>(</b>A) Chemical structure of AF. (B), (C) Cells (1 × 10⁴ cell/well) were treated with the indicated concentrations of AF in the absence or presence of LPS for 24 h, and cellular viability was measured by the MTT assay. The data shown are representative of three independent experiments and indicate the mean ± SEM. NT indicates “no treatment”.</p

Inhibitory effects of AF on MAPK phosphorylation in RAW 264.7 macrophage cells.

<p>Cells were pre-treated with 50 μM AF for 30 min prior to LPS treatment. After treatment with 100 ng/mL LPS for 15 min, the levels of phosphorylated ERK, p38, and JNK were analyzed by immunoblotting. The levels of ERK, JNK, and p38 were estimated by the loaded protein as each control. The data shown are representative of three independent experiments and indicate the mean ± SEM. **p < 0.01 indicates significant differences from the LPS alone-treated cells. Probes: NT, no treatment.</p