Inhibitory effects of polymethoxy flavones isolated from Citrus reticulate on degranulation in rat basophilic leukemia RBL-2H3: Enhanced inhibition by their combination Original Research Article

Polymethoxy flavones (PMFs) are present in fruit tissues of Citrus species. It has been reported that flavonoids isolated from several Citrus have been shown to suppress the degranulation as inferred by histamine release in rat basophilic leukemia RBL-2H3 cells. In this study, we examined the effect of PMFs (PMF-1: 6,7,4′,5′-tetramethoxy-5-monohydroxyflavone, PMF-2: 5,6,8,3′,6′-pentamethoxy flavone, PMF-3: 5,6,7,3′,4′,5′-hexamethoxy flavone) on the degranulation in RBL-2H3 cells. All the PMFs suppressed the degranulation from Ag-stimulated RBL-2H3 cells. Interestingly, PMF-combination (PMF-1 + PMF-2; PMF-1 + PMF-3) treatment enhanced the inhibition of degranulation compared with PMF-single treatment. In order to clarify the inhibitory mechanism of degranulation by PMFs, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCγs. All the PMFs significantly suppressed the activation of Syk and PLCγs. In Ag-mediated activation of FcεRI on mast cells, three major subfamilies of mitogen-activated protein kinases, especially ERK44/42, were activated. These PMFs reduced the level of phospho-ERKs. The intracellular free Ca2+ concentration ([Ca2+]i) was elevated by FcεRI activation, and PMF treatment reduced the elevation of [Ca2+]i by suppressing Ca2+ influx. Thus, it was suggested that the suppression of Ag-stimulated degranulation by these PMFs mainly is due to the Syk/PLCγs/PKC pathway and Ca2+ influx.