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)-Epigallocatechin-3-gallate Blocks the Induction of Nitric
Oxide Synthase by Down-Regulating Lipopolysaccharide-Induced Activity
of Transcription Factor Nuclear Factor-
B
Institute of Biochemistry, College of Medicine, National Taiwan
University, Taipei, Taiwan, Republic of China
Nitric oxide (NO) plays an important role in inflammation and multiple
stages of carcinogenesis. We investigated the effect of various tea
polyphenols and caffeine on the induction of NO synthase (NOS) in
thioglycollate-elicited and lipopolysaccharide (LPS)-activated
peritoneal macrophages. Gallic acid (GA), (
)-epigallocatechin (EGC),
and ()-epigallocatechin-3-gallate (EGCG), the major tea catechin,
were found to inhibit inducible NOS (iNOS) protein in activated
macrophages. EGCG, a potent antitumor agent with anti-inflammatory and
antioxidant properties, inhibited NO generation, as measured by the
amount of nitrite released into the culture medium. Inhibition of NO
production was observed when cells were cotreated with EGCG and LPS.
iNOS activity in soluble extracts of lipopolysaccharide-activated macrophages treated with EGCG (5 and 10 µM) for 6-24 hr
was significantly lower than that in macrophages without EGCG
treatment. Western blot, reverse transcription-polymerase chain
reaction, and Northern blot analyses demonstrated that significantly
reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS were expressed
in lipopolysaccharide-activated macrophages with EGCG compared with
those without EGCG. Electrophoretic mobility shift assay indicated that
EGCG blocked the activation of nuclear factor-
B, a transcription
factor necessary for iNOS induction. EGCG also blocked disappearance of
inhibitor B from cytosolic fraction. These results suggest that EGCG
decreases the activity and protein levels of iNOS by reducing the
expression of iNOS mRNA and the reduction could occur through
prevention of the binding of nuclear factor-B to the iNOS promoter,
thereby inhibiting the induction of iNOS transcription.
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