Effect of natsudaidain isolated from Citrus plants on TNF-α and cyclooxygenase-2 expression in RBL-2H3 cells

Abstract

Abstract Objectives Flavonoids inhibit the activity of chemical mediators released from mast cells. Our aim was to investigate the effects of natsudaidain, a polymethoxyflavone isolated from Citrus plants, on mast cells. Methods We investigated the inhibitory effects of natsudaidain, which is a polymethoxy-flavone isolated from Citrus plants, on histamine release, tumour necrosis factor-α production and cyclooxygenase-2 expression in Ca ionophore-stimulated rat basophilic leukemia cells (A23187-stimulated RBL-2H3 cells) by spectrofluorometric, ELISA and immunoblotting methods. Key findings The percent of histamine release from A23187-stimulated RBL-2H3 cells pretreated with natsudaidain at 5, 25 and 50 μM was not changed as compared with non-treated A23187-stimulated cells. At 100 and 200 μM, natsudaidain pretreatment resulted in slightly reduced histamine release (% histamine release, 89.8 ± 3.5% and 71.5 ± 5.6% at 100 and 200 μM). Thus, natsudaidain hardly affects histamine release from RBL-2H3 cells, except at high concentrations. On the other hand, natsudaidain dose-dependently inhibited tumour necrosis factor-α protein and mRNA levels in A23187-stimulated RBL-2H3 cells; a concentration of 6.8 μM was required for a 50% reduction. In addition, all concentrations of this compound that we tested also inhibited cyclooxygenase-2 protein expression. The mRNA levels of cyclooxygenase-2 in A23187-stimulated RBL-2H3 cells treated with natsudaidain were also markedly decreased. The phosphorylated-p38 MAPK protein levels in A23187-stimulated RBL-2H3 cells treated with natsudaidain were lower than in the non-treated cells. Conclusions These findings suggest that natsudaidain inhibits tumour necrosis factor-α and cyclooxygenase-2 production by suppressing p38 MAPK phosphorylation but not p65 NFKB phosphorylation, and that natsudaidain might alleviate inflammatory diseases.