Effect of Epigallocatechin-3-Gallate on Expression of iNOS mRNA and Production of No in HaCaT Cell

Background: Nitric oxide (NO) plays an important role in inflammation and multiple stages of carcinogenesis. 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 the expression of inducible NO synthase (iNOS) mRNA and generation of NO in HaCaT cells. Methods: HaCaT cells were treated with EGCG and NAC for 1 hour. 1 hour later, they were irradiated with 50mJ/ UVB and treated with 200 /ml LPS for 12 hours, respectively. The iNOS mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) and NO production was assessed by spectrophotometric method based on Griess reaction. Nuclear factor B (NF-B) binding activity was determined by electrophoretic mobility shift assay (EMSA). Results: The results were as follows 1. EGCG inhibited UVB and LPS induced expression of inducible nitric oxide synthase. 2. HaCaT cells cotreated with EGCG produced significantly less iNOS mRNA and NO compared with HaCaT cells stimulated with UVB irradiation or LPS. 3. The inhibition of iNOS mRNA and NO production correlated with the suppression of expression of NF-B dependent gene iNOS. 4. EGCG inhibited the activation and translocation of NF-B to the nucleus Conclusion: Our data suggests that EGCG inhibits the UVB and LPS-induced production of NO in HaCaT cells by interfering with the activation of NF-B through a novel mechanism. Our results further suggest that EGCG may be therapeutically effective in UVB and cytokine induced cutaneous inflammation.