Suppression of nitric oxide synthase and the down-regulation of the activation of NFkappaB in macrophages by resveratrol

Abstract

Resveratrol, naringenin and naringin are naturally occurring flavonoids in grapes and grapefruits. The anti-inflammatory effects of these flavonoids have been well documented, but the mechanism is poorly characterized. High concentration of NO are produced by inducible NO synthase (iNOS) in inflammation, and the prevention of the expression of iNOS may be an important anti-inflammatory mechanism. In this study, the effects of these flavonoids on the induction of NO synthase (NOS) in RAW 264.7 cells activated with bacterial lipopolysaccharide (LPS, 50 ng ml(-1)) were investigated. Resveratrol was found strongly to inhibit NO generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic iNOS protein and steady state mRNA levels. However, the inhibitory abilities of naringenin were lower, and the inhibitory abilities of naringin were almost negligible. In electrophoretic mobility shift assays, the activation of NFkappaB induced by LPS for 1 h was inhibited by resveratrol (30 microM). Furthermore, in immunoblotting analysis, cells treated with LPS plus resveratrol showed an inhibition of phosphorylation as well as degradation of IkappaBalpha, and a reduced nuclear content of NFkappaB subunits. The flavonoids may be of value for inhibiting the enhanced expression of iNOS in inflammation through down-regulation of NFkappaB binding activity.

Figure 1

Structures of resveratrol, naringin and naringenin.

Figure 2

Effects of various concentrations of resveratrol, naringin and naringenin on nitrite release into the culture medium of activated macrophages. RAW 264.7 cells were treated with or without LPS (50 ng ml⁻¹) and various flavonoids or DMSO (0.03%) solvent ((A) naringin; (B) naringenin; (C) resveratrol) for 18 h. At the end of the incubation time, the culture medium was collected for nitrite assay. Each data point is mean±s.e.mean for three determinations. (D) Rates of nitrite release were measured in activated macrophages in the presence of the indicated concentrations of various flavonoids, and the data was normalized (LPS=100%).

Figure 3

Immuno-blotting analysis of inducible NO systhase in activated macrophages with various flavonoids or solvent only at 18 h ((A) naringin; (B) naringenin; (C) resveratrol). At the end of the incubation time, the total protein was extracted for iNOS protein and α-tubulin analysis by using iNOS and α-tubulin-specific antibodies as described in Methods. Quantification of the iNOS protein expression was performed by densitometric analysis (IS-1000 Digital System) of the immunoblot. Data are expressed as the means±s.e.mean of the ratio of maximal protein expression observed with LPS as determined by three independent experiments. The ratio of iNOS to α-tubulin protein expression observed with LPS alone is set at 1. The relative level was calculated as the ratio of iNOS to α-tubulin protein expression, which was performed by densitometric analysis of the immunoblot.

Figure 4

RT–PCR analysis of the expression of iNOS mRNA. (A) RAW 264.7 cells were treated with no flavonoid (lane 1), DMSO (0.03%, lane 2), naringin (30 μM, lane 3), naringenin (30 μM, lane 4), or resveratrol (30 μM, lane 5) before stimulation with LPS (50 ng ml⁻¹) for 5 h. Total RNA was extracted from treated cells and the iNOS mRNA expression was determinated as described in Methods. G3PDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Quantification of the iNOS RNA expression was performed by densitometric analysis (IS-1000 Digital System) of the RT–PCR analysis. Data are expressed as the means±s.e.mean of the ratio of maximal RNA expression observed with LPS in three independent experiments. The ratio of iNOS to G3PDH RNA expression observed with LPS alone is set at 1. The relative level was calculated as the ratio of iNOS to G3PDH RNA expression. (C) Total RNA was extracted from treated cells and assayed for iNOS mRNA expression by Northern blot analysis. Blots were hybridized to ³²P-labelled iNOS probe as described in Methods. Signals for GAPDH mRNA for each lane are shown as controls.

Figure 5

Electrophoretic mobility shift assay using a 5′-end-labelled consensus oligonucleotide for NFκB binding and nuclear extracts from RAW 264.7 cells. (A) RAW 264.7 cells were treated with LPS (50 ng ml⁻¹) without or with different flavonoids (30 μM) or DMSO (0.03%) solvent and then incubated for 1 h. (B) shows competition assay for the identification of NFκB-binding specificity. Lane 1: NFκB complex in LPS-treated RAW 264.7 cells; lane 2: addition of a 25 fold molar excess of the nonradioactive NFκB before adding the [³²P]-NFκB element; lane 3: addition of a 50 fold molar excess of the nonradioactive NFκB before adding the [³²P]-NFκB element. (C) the actual radioactivity (c.p.m) present in respective bands. Data are expressed as the means±s.e.mean of the radioactivity (c.p.m.) in three independent experiments.

Figure 6

The inhibition by flavonoids of LPS-mediated IκBα phosphorylation and degradation. RAW 264.7 cells were treated with LPS (50 ng ml⁻¹) without or with different flavonoids (30 μM) or DMSO (0.03%) solvent and then incubated for 30 min or 1 h. Cytosolic fractions were prepared and analysed for the content of IκBα protein by Western blot. (A) After 30 min activation, the phosphorylated IκBα was detected by Ser32-phospho-specific antibody. Quantification of the phosphorylated IκBα protein expression was performed by densitometric analysis (IS-1000 Digital System) of the Western blot. Data are expressed as the means±s.e.mean of the ratio of maximal phosphorylated IκBα observed with LPS in three independent experiments. The ratio of phosphorylated IκBα to α-tubulin protein expression observed with LPS alone is set at 1. The relative level was calculated as the ratio of phosphorylated IκBα to α-tubulin protein expression. (B) The content of IκBα protein was detected after 1 h activation. Quantification of the IκBα protein expression was performed by densitometric analysis (IS-1000 Digital System) of the Western blot. Data are expressed as the means ±s.e.mean of the ratio of maximal IκBα protein expression in three independent experiments. The ratio of IκBα to α-tubulin protein expression observed with control is set at 1. The relative level was calculated as the ratio of IκBα to α-tubulin protein expression.

Figure 7

Flavonoids reduced nuclear NFκB levels. RAW 264.7 cells were treated with LPS (50 ng ml⁻¹) without or with different flavonoids (30 μM) and then incubated for 1 h. (A) Cytosolic and nuclear fractions were prepared and analysed for the content of c-Rel, p65, p50 and Sp1 proteins. (B) Quantification of the NFκB (c-Rel, p65 and p50) protein expression was performed by densitometric analysis (IS-1000 Digital System) of the Western blot. Data are expressed as the means±s.e.mean of the ratio of maximal NFκB nuclear translocation observed with LPS in three independent experiments. The ratio of NFκB nuclear translocation observed with LPS is set at 1. The relative level was calculated as the ratio of nuclear to cytoplasmic NFκB.