Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca2+ fluxes　

Shikonin, an anti-inflammatory compound of “Shikon”, inhibits the neutrophil superoxide (O2.-) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2
activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca2+ concentration ([Ca2+]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca2+]i (by fluorescence) and O2.- generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca2+]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca2+ release (IICR) and store-operated Ca2+ entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE.
Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca2+]i and O2.- generation (IC50 values of 1.45 and 1.12 M, respectively) in a synchronized manner. Experiments for specific Ca2+ fluxes showed that shikonin inhibits IICR and IICR-linked O2.- generation (IC50 values: 0.28 and 0.31 M for [Ca2+]i and O2.-, respectively), as well as SOCE and SOCE-linked O2.- generation (IC50 values: 0.39 and 0.25 M for [Ca2+]i and O2.-, respectively). These results suggested that shikonin inhibits the O2.- generation by Nox2 in fMLP-stimulated
neutrophils by targeting Ca2+ fluxes such as IICR and SOCE.
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Journal of Clinical Biochemistry and Nutrition, Published online.