Theaflavin-3,3′-digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-κB in macrophages

Abstract

Nitric oxide (NO) plays an important role in inflammation and also in multiple stages of carcinogenesis. We investigated the effects of various tea polyphenols, including theaflavin, a mixture of theaflavin-3-gallate and theaflavin-3′-gallate, theaflavin-3,3′-digallate, thearubigin, and (−)-epigallocatechin-3-gallate on the induction of NO synthase in lipopolysaccharide-activated murine macrophages, RAW 264.7 cells. Theaflavin-3,3′-digallate was found to be stronger than (−)-epigallocatechin-3-gallate in inhibiting NO generation and inducible NO synthase protein in activated macrophages, while theaflavin, a mixture of theaflavin-3-gallate and theaflavin-3′-gallate and thearubigin were less effective. Inhibition of NO production was observed when cells were cotreated with theaflavin-3,3′-digallate and lipopolysaccharide. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses demonstrated that significantly reduced 130-kDa protein and mRNA levels of inducible NO synthase were expressed in lipopolysacchride-activated macrophages with theaflavin-3,3′-digallate, compared to those without theaflavin-3,3′-digallate. Electrophoretic mobility shift assay (EMSA) indicated that theaflavin-3,3′-digallate blocked the activation of nuclear factor κB (NF-κB), a transcription factor necessary for inducible NO synthase induction. Theaflavin-3,3′-digallate also blocked phosphorylation of IκB from cytosolic fraction and reduced lipopolysacchride-induced nuclear accumulation of transcription factor NF-κB p65 and p50 subunits. These results suggest that theaflavin-3,3′-digallate decreases the protein levels of inducible NO synthase by reducing the expression of inducible NO synthase mRNA, and the reduction could be via preventing the activation of NF-κB, thereby inhibiting the induction of inducible NO synthase transcription. It was also demonstrated that the gallic acid moiety of theaflavin-3,3′-digallate is essential for their potent anti-inflammation activity.