Abstract
This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. However, 2',3-dihydroxy-5-methoxybiphenyl (RIR-2) was not found to inhibit PMA-induced respiratory bust and cathepsin G release. Signaling pathways regulated by RIR2 which modulated fMLP-induced respiratory burst and cathepsin G release were evaluated by an interaction between Gβ-protein with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gβ-protein. Briefly, 2',3-dihydroxy-5-methoxybiphenyl (RIR-2) inhibited fMLP-induced superoxide anion production (IC50=2.57±0.22 μM, n=4), cathepsin G release (IC50=18.72±3.76 μM, n=4) and migration in a concentration dependent manner. Further, 2',3-dihydroxy-5-methoxybiphenyl (RIR-2) specifically suppresses fMLP-induced Src family kinases phosphorylation, -Raf/ERK phosphorylation, by inhibiting an interaction between Gβ-protein with Src kinases without inhibiting Src kinases activities. Consequently, 2',3-dihydroxy-5-methoxybiphenyl (RIR2) attenuated the downstream targets of Src kinase, such as Tec translocation from the cytosol to the inner leaflet of the plasma membrane, phosphorylation of AKT, P38, PLCγ2, PKC and membrane localization of p47phox and P40phox, furthermore, 2',3-dihydroxy-5-methoxybiphenyl (RIR-2) attenuated fMLP-induced intracellular calcium mobilization, moreover, 2',3-dihydroxy-5-methoxybiphenyl (RIR-2) was not found to increase cyclicAMP. 2',3-dihydroxy-5-methoxybiphenyl (RIR2) was not a competitive or allosteric antagonist of fMLP. On the other hand, PMA-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47phox and P40phox remained unaffected. In conclusion, 2',3-dihydroxy-5-methoxybiphenyl (RIR2) specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting an interaction between Gβ-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47phox and p40phox.
