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. 2007;2007:45673.
doi: 10.1155/2007/45673.

Anti-inflammatory Effects of Flavonoids: Genistein, Kaempferol, Quercetin, and Daidzein Inhibit STAT-1 and NF-kappaB Activations, Whereas Flavone, Isorhamnetin, Naringenin, and Pelargonidin Inhibit Only NF-kappaB Activation Along With Their Inhibitory Effect on iNOS Expression and NO Production in Activated Macrophages

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Free PMC article

Anti-inflammatory Effects of Flavonoids: Genistein, Kaempferol, Quercetin, and Daidzein Inhibit STAT-1 and NF-kappaB Activations, Whereas Flavone, Isorhamnetin, Naringenin, and Pelargonidin Inhibit Only NF-kappaB Activation Along With Their Inhibitory Effect on iNOS Expression and NO Production in Activated Macrophages

Mari Hämäläinen et al. Mediators Inflamm. .
Free PMC article

Abstract

In inflammation, bacterial products and proinflammatory cytokines induce the formation of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS), and compounds that inhibit NO production have anti-inflammatory effects. In the present study, we systematically investigated the effects of 36 naturally occurring flavonoids and related compounds on NO production in macrophages exposed to an inflammatory stimulus (lipopolysaccharide, LPS), and evaluated the mechanisms of action of the effective compounds. Flavone, the isoflavones daidzein and genistein, the flavonols isorhamnetin, kaempferol and quercetin, the flavanone naringenin, and the anthocyanin pelargonidin inhibited iNOS protein and mRNA expression and also NO production in a dose-dependent manner. All eight active compounds inhibited the activation of nuclear factor-kappaB (NF-kappaB), which is a significant transcription factor for iNOS. Genistein, kaempferol, quercetin, and daidzein also inhibited the activation of the signal transducer and activator of transcription 1 (STAT-1), another important transcription factor for iNOS. The present study characterises the effects and mechanisms of naturally occurring phenolic compounds on iNOS expression and NO production in activated macrophages. The results partially explain the pharmacological efficacy of flavonoids as anti-inflammatory compounds.

Figures

Figure 1
Figure 1
Basic chemical structures of the phenolic compounds used in the present study.
Figure 2
Figure 2
(a) Effects of increasing concentrations of flavone, daidzein, and genistein on LPS (100 ng/ml)-induced NO production in J774 cells during a 24-hour incubation time. (b) Effects of increasing concentrations of quercetin, isorhamnetin, and kaempferol on LPS (100 ng/ml)-induced NO production in J774 cells during a 24-hour incubation time. (c) Effects of increasing concentrations of naringenin and pelargonidin on LPS (100 ng/ml)-induced NO production in J774 cells during a 24-hour incubation time. NO production was determined by measuring nitrite accumulation in the culture medium by Griess reaction. Mean + SEM, n=6.
Figure 3
Figure 3
(a) Effects of flavone, daidzein, and genistein on LPS (100 ng/ml)-induced iNOS protein expression in J774 cells during a 24-hour incubation time. (b) Effects of quercetin, isorhamnetin, and kaempferol on LPS (100 ng/ml)-induced iNOS protein expression in J774 cells during a 24-hour incubation time. (c) Effects of naringenin and pelargonidin on LPS (100 ng/ml)-induced iNOS protein expression in J774 cells during a 24-hour incubation time. iNOS protein expression was detected by Western blot. Mean + SEM, n=3, **P<.01.
Figure 4
Figure 4
Effects of flavonoids on iNOS mRNA expression. Cells were treated with LPS (100 ng/ml) or with LPS and the tested compound (100 μM) and RNA was extracted after 6 hours. iNOS and GAPDH mRNA were measured by real-time RT-PCR and iNOS mRNA levels were normalised against GAPDH. Mean + SEM, n=3, **P<.01.
Figure 5
Figure 5
Effects of flavonoids on LPS-induced NF-κB activation determined as nuclear translocation of NF-κB. J774 cells were incubated for 30 minutes with LPS (100 ng/ml) or with LPS and the flavonoid (100 μM), and nuclear proteins were extracted. Western blot was used to detect the p65 subunits of NF-κB in the nuclear extracts. p65 levels in LPS-treated cells were set at 100% and the other values were related to that. The dotted line represents the nuclear p65 levels in untreated control cells. Mean + SEM, n=46, **P<.01 as compared to LPS-induced level.
Figure 6
Figure 6
(a)–(c) Effects of flavonoids on LPS-induced STAT-1 activation determined as nuclear translocation of STAT-1α. J774 cells were incubated for 6 hours with LPS (100 ng/ml) or with LPS and the flavonoid (100 μM), and nuclear proteins were extracted. Western blot was used to detect STAT-1α in the nuclear extracts. STAT-1α levels in LPS-treated cells were set at 100% and the other values were related to that. The dotted line represents the nuclear STAT-1α levels in untreated control cells. Mean + SEM, n=3, ** P<.01, * P<.05 as compared to LPS-induced level.

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