Article date: August 1996
By: Markus Hecker, Christiane Preiß, Peter Klemm, Rudi Busse, in Volume 118, Issue 8, pages 2178-2184
In view of the potential deleterious effects of high amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) in inflammation, the prevention of the expression of this enzyme represents an important therapeutic goal. In cytokine‐stimulated cells, activation of nuclear factor kB (NF‐kB) is crucial for the increase in iNOS gene expression. Since NF‐kB activation appears to involve a redox‐sensitive step, we have investigated whether three structurally unrelated antioxidants, 5,7‐dihydroxyflavone (chrysin), 3,4‐dichloroisocoumarin (DCI) and N‐acetyl 5‐hydroxytryptamine (N‐acetylserotonin, NAS), affect iNOS expression in cultured RAW 264.7 monocyte/macrophages stimulated with bacterial lipopolysaccharide (LPS, 140 ng ml−1) and interferon‐γ (IFNγ, 5 u ml−1).
During a 6 h incubation period neither LPS nor IFNγ alone exerted a significant effect but when combined, caused a prominent increase in nitrite formation, iNOS mRNA and protein abundance. Co‐incubation with chrysin (50 μm), DCI (50 μm) or NAS (1 mM) markedly attenuated this increase in iNOS gene expression.
DCI, but not chrysin or NAS, prevented the activation of NF‐kB in cells exposed to LPS plus IFNγ for 30 min. In contrast, all three antioxidants significantly blunted the DNA‐binding activity of interferon regulatory factor 1 (IRF‐1), which mediates the synergistic effect of IFNγ on iNOS gene expression in cells treated for 2 h with LPS plus IFNγ.
DCI thus appears to inhibit iNOS gene expression at the transcriptional level by preventing the activation of both NF‐kB and IRF‐1. The inhibitory effect of DCI on NF‐kB activation, however, does not seem to be related to its antioxidative properties, since DCI, unlike chrysin or NAS, is a potent serine protease inihbitor which stabilizes the inactive NF‐kB complex by protecting the inhibitory IkB‐α subunit from proteolytic degradation.
The virtually identical inhibitory effect of chrysin, DCI and NAS on the activation of IRF‐1 points to a redox‐sensitive step in the activation of this transcription factor, which in contrast to NF‐kB requires de novo protein synthesis.
Since iNOS gene expression in human cells and tissues usually requires the combination of several cytokines, antioxidants such as chrysin and NAS which do not interfere with the activation of NF‐kB may be of therapeutic value for selectively inhibiting the enhanced expression of this enzyme in inflammation.
In view of the potential deleterious effects of high amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) in inflammation, the prevention of the expression of this enzyme represents an important therapeutic goal. In cytokine‐stimulated cells, activation of nuclear factor kB (NF‐kB) is crucial for the increase in iNOS gene expression. Since NF‐kB activation appears to involve a redox‐sensitive step, we have investigated whether three structurally unrelated antioxidants, 5,7‐dihydroxyflavone (chrysin), 3,4‐dichloroisocoumarin (DCI) and N‐acetyl 5‐hydroxytryptamine (N‐acetylserotonin, NAS), affect iNOS expression in cultured RAW 264.7 monocyte/macrophages stimulated with bacterial lipopolysaccharide (LPS, 140 ng ml−1) and interferon‐γ (IFNγ, 5 u ml−1).
During a 6 h incubation period neither LPS nor IFNγ alone exerted a significant effect but when combined, caused a prominent increase in nitrite formation, iNOS mRNA and protein abundance. Co‐incubation with chrysin (50 μm), DCI (50 μm) or NAS (1 mM) markedly attenuated this increase in iNOS gene expression.
DCI, but not chrysin or NAS, prevented the activation of NF‐kB in cells exposed to LPS plus IFNγ for 30 min. In contrast, all three antioxidants significantly blunted the DNA‐binding activity of interferon regulatory factor 1 (IRF‐1), which mediates the synergistic effect of IFNγ on iNOS gene expression in cells treated for 2 h with LPS plus IFNγ.
DCI thus appears to inhibit iNOS gene expression at the transcriptional level by preventing the activation of both NF‐kB and IRF‐1. The inhibitory effect of DCI on NF‐kB activation, however, does not seem to be related to its antioxidative properties, since DCI, unlike chrysin or NAS, is a potent serine protease inihbitor which stabilizes the inactive NF‐kB complex by protecting the inhibitory IkB‐α subunit from proteolytic degradation.
The virtually identical inhibitory effect of chrysin, DCI and NAS on the activation of IRF‐1 points to a redox‐sensitive step in the activation of this transcription factor, which in contrast to NF‐kB requires de novo protein synthesis.
Since iNOS gene expression in human cells and tissues usually requires the combination of several cytokines, antioxidants such as chrysin and NAS which do not interfere with the activation of NF‐kB may be of therapeutic value for selectively inhibiting the enhanced expression of this enzyme in inflammation.
DOI: 10.1111/j.1476-5381.1996.tb15660.x
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