Genistein suppresses LPS-induced inflammatory response through inhibiting NF-κB following AMP kinase activation in RAW 264.7 macrophages.

Genistein, the major isoflavone in soybean, was recently reported to exert beneficial effects in metabolic disorders and inflammatory diseases. In the present study, we investigated the effects and mechanisms of a dietary concentration of genistein on the inflammatory response in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results demonstrated that genistein effectively inhibited the LPS-induced overproduction of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), as well as LPS-induced nuclear factor kappa B (NF-κB) activation. In addition, the data also showed that genistein prevented LPS-induced decrease in adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. These effects were obviously attenuated by an AMPK inhibitor. Taken together, our results suggest that the dietary concentration of genistein is able to attenuate inflammatory responses via inhibition of NF-κB activation following AMPK stimulation. The data provide direct evidence for the potential application of low concentrations of genistein in the prevention and treatment of inflammatory diseases

Effect of genistein on TNF-α and IL-6 production in LPS-treated RAW 264.7 cells.

<p>Values are the mean ± S.E.M (n = 3) of three independent experiments.</p>*<p><i>P</i><0.05, **<i>P</i><0.01 compared to the control group.</p>#<p><i>P</i><0.05, ^##<i>P</i><0.01 compared to the LPS group.</p

Primer sequences used to amplify cDNA for qRT-PCR.

<p>Primer sequences used to amplify cDNA for qRT-PCR.</p

AMPK activation decreases the inhibitory effect of genistein on NF-κB activation in LPS-treated RAW 264.7 macrophages.

<p>(A) Cells were pretreated with or without 10 µM genistein or 1 mM AICAR for 1 h and then incubated with 1 µg/mL LPS for 24 h. (B) Cells were pretreated with or without 20 µM Compound C for 30 min, then with or without 10 µM genistein for 1 h and stimulated with LPS for 24 h. Cytoplasm and nuclear extracts were collected for determination of AMPK, p-AMPK, I-κBα, p-IKKα/β, GAPDH, p65, and histone by western blotting. GAPDH or histones was used as an internal control. Experiments were repeated three times, and representative blots are shown here.</p

Effect of genistein on NF-κB activation in LPS-treated RAW 264.7 macrophages.

<p>(A) Cells were pretreated with 1, 5,or 10 µM genistein for 1 h, and then incubated with 1 µg/mL LPS for 24 h. Cells were pretreated with 10 µM genistein for 1 h, and then incubated with 1 µg/mL LPS for 0.25, 0.5, 4, or 24 h. (B) Cell lysates were prepared and analyzed for IκB-α, p-IKKα/β, or GAPDH by western blotting. The nuclear fraction was collected for assessment of NF-κB p65 and histones. GAPDH or histone was used as an internal control. Experiments were repeated three times, and representative blots are shown here.</p

Effect of genistein and LPS on RAW 264.7 macrophage viability.

<p>Cells were pretreated with genistein (0 µM to 100 µM) for 1 h, then incubated with or without 1 µg/mL LPS for 12 h, 24 h, or 48 h respectively. Cell viability was determined by MTT assay. Data are the mean ± S.E.M (n = 3 ) of three independent experiments.</p

Effect of genistein, AMPK activator, and AMPK inhibitor on TNF-α and IL-6 production.

<p>LPS+AICAR: cells were pretreated with 1 mM AICAR for 1 h and then incubated with 1 µg/mL LPS for 24 h; LPS+Gen 10 µM: cells were pretreated with 10 µM genistein for 1 h and then incubated with LPS for 24 h; LPS+Com C: cells were pretreated with 20 µM Compound C for 30 min and then incubated with LPS for 24 h; LPS+Gen 10 µM+Com C: cells were pretreated with 20 µM Compound C for 30 min, incubated with 10 µM genistein for 1 h, and then co-cultured with LPS for 24 h. Values are mean ± S.E.M of three independent experiments.</p>*<p><i>P</i><0.05,^**<i>P</i><0.05 compared to the control group.</p>#<p><i>P</i><0.05, ^##<i>P</i><0.01 compared to the LPS group.</p>△<p><i>P</i><0.05 compared to the LPS+Gen 10 µM group.</p>△<p><i>P</i><0.05 compared to the LPS+Gen 10 µM group.</p