Effect of Agkistroden blomhoffi (mamushi) on superoxide generation in human neutrophils

Background The purpose of this study is to investigate the effect of Agkistroden blomhoffi (mamushi) aqueous extract on stimulus-induced superoxide generation in human neutrophils. Methods Superoxide generation was assayed by measuring the reduction of ferricytochrome C (cyt c) using a dual-beam spectrophotometer. The phosphorylation of neutrophil proteins and the translocation of cytosolic compounds to the cell membrane were detected using specific monoclonal antibodies. Results Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in a concentration-dependent manner. fMLP-induced translocation of cytosolic compounds p47phox, p67phox and Rac to the cell membrane was suppressed in parallel to the suppression of superoxide generation. The fractions with MW below 10 kDa dose-dependently suppressed tyrosyl phosphorylation of 45.0, 77.6 and 99.5 kDa proteins in fMLP-treated human neutrophils; the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced translocation of p47phox to the cell membrane and superoxide generation. Conclusions Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in human neutrophils and also suppressed the translocation of cytosolic compounds to the cell membrane and tyrosyl phosphorylation of proteins. The efficacious components are the fractions with MW below 10 kDa. O-phosphoserylethanolamine, a new dipeptide isolated from mamushi, also suppressed fMLP-induced superoxide generation in human neutrophils in a dose-dependent manner.