Epigallocatechin-3-gallate inhibits interleukin-1beta-induced expression of nitric oxide synthase and production of nitric oxide in human chondrocytes: suppression of nuclear factor kappaB activation by degradation of the inhibitor of nuclear factor kappaB.
OBJECTIVE
The proinflammatory cytokine interleukin-1beta (IL-1beta) induces the production of high levels of nitric oxide (NO) in human chondrocytes. Green tea (Camellia sinensis) polyphenols are potent antiinflammatory agents and have been shown to inhibit NO production in tumor cell lines. In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG), a green tea polyphenol, on IL-1beta-induced production of NO in primary human osteoarthritis (OA) chondrocytes.


METHODS
Human chondrocytes were derived from OA cartilage and were treated with EGCG (100 microM) and IL-1beta (2 ng/ml) for different periods, and inducible nitric oxide synthase (iNOS) messenger RNA and protein expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Production of NO was determined as nitrite in culture supernatant. Activation and translocation of nuclear factor kappaB (NF-kappaB), levels of inhibitor of nuclear factor kappaB (IkappaB), and NF-kappaB DNA binding activity were determined by Western blotting and a highly sensitive and specific enzyme-linked immunosorbent assay. Activity of IkappaB kinase was determined using in vitro kinase assay.


RESULTS
Human chondrocytes cotreated with EGCG produced significantly less NO compared with chondrocytes stimulated with IL-1beta alone (P < 0.005). The inhibition of NO production correlated with the suppression of induction and expression of NF-kappaB-dependent gene iNOS. EGCG inhibited the activation and translocation of NF-kappaB to the nucleus by suppressing the degradation of its inhibitory protein IkappaBalpha in the cytoplasm.


CONCLUSIONS
Our results indicate that EGCG inhibits the IL-1beta-induced production of NO in human chondrocytes by interfering with the activation of NF-kappaB through a novel mechanism. Our data further suggest that EGCG may be a therapeutically effective inhibitor of IL-1beta-induced inflammatory effects that are dependent on NF-kappaB activation in human OA chondrocytes.
Singh
Rashmi
R
Case Western Reserve University, Cleveland, Ohio 44106-4946, USA.
Ahmed
Salahuddin
S
Islam
Najmul
N
Goldberg
Victor M
VM
Haqqi
Tariq M
TM
D000975:Antioxidants / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D020370:Osteoarthritis, Knee / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism
D000975:Antioxidants; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D012333:RNA, Messenger; D009569:Nitric Oxide; D002392:Catechin; D004247:DNA; C045651:epigallocatechin gallate; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II