Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca(2+) fluxes

Shikonin, an anti-inflammatory compound of “Shikon”, inhibits the neutrophil superoxide (O(2)(•−)) 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 Ca(2+) concentration ([Ca(2+)](i)) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca(2+)](i) (by fluorescence) and O(2)(•−) 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 [Ca(2+)](i) via fluxes such as inositol 1,4,5-trisphosphate-induced Ca(2+) release (IICR) and store-operated Ca(2+) entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca(2+)](i) and O(2)(•−) generation (IC(50) values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca(2+) fluxes showed that shikonin inhibits IICR and IICR-linked O(2)(•−) generation (IC(50) values: 0.28 and 0.31 µM for [Ca(2+)](i) and O(2)(•−), respectively), as well as SOCE and SOCE-linked O(2)(•−) generation (IC(50) values: 0.39 and 0.25 µM for [Ca(2+)](i) and O(2)(•−), respectively). These results suggested that shikonin inhibits the O(2)(•−) generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca(2+) fluxes such as IICR and SOCE.