Epigallocatechin‐3‐gallate inhibits interleukin‐1β–induced expression of nitric oxide synthase and production of nitric oxide in human chondrocytes: Suppression of nuclear factor κB activation by degradation of the inhibitor of nuclear factor κB

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Abstract
Objective The proinflammatory cytokine interleukin-1β (IL-1β) 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-1β–induced production of NO in primary human osteoarthritis (OA) chondrocytes. Methods Human chondrocytes were derived from OA cartilage and were treated with EGCG (100 μM) and IL-1β (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 κB (NF-κB), levels of inhibitor of nuclear factor κB (IκB), and NF-κB DNA binding activity were determined by Western blotting and a highly sensitive and specific enzyme-linked immunosorbent assay. Activity of IκB kinase was determined using in vitro kinase assay. Results Human chondrocytes cotreated with EGCG produced significantly less NO compared with chondrocytes stimulated with IL-1β alone (P < 0.005). The inhibition of NO production correlated with the suppression of induction and expression of NF-κB–dependent gene iNOS. EGCG inhibited the activation and translocation of NF-κB to the nucleus by suppressing the degradation of its inhibitory protein IκBα in the cytoplasm. Conclusion Our results indicate that EGCG inhibits the IL-1β–induced production of NO in human chondrocytes by interfering with the activation of NF-κB through a novel mechanism. Our data further suggest that EGCG may be a therapeutically effective inhibitor of IL-1β–induced inflammatory effects that are dependent on NF-κB activation in human OA chondrocytes.
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