Background, aims: We have shown that shikonin, the major active substance of 'Shikon', has antiinflammatory, antioxidant and wound healing effects through its multiple targets like key protein kinases in the inflammation cascade, nitric oxide synthase, NADPH oxidase 2 (Nox2), as well as expression of pro- and/or antiinflammatory genes in various cell types. Presently, by simultaneous monitoring of intracellular Ca2+ level ([Ca2+]i) and superoxide (O2-) generation by fluorescence and chemiluminescence, respectively, we aimed to elucidate whether shikonin targets Ca2+ fluxes essential for activation of the O2--generating Nox2 in fMLP-stimulated neutrophils. Results: Shikonin inhibited the fMLP-induced [Ca2+]i elevation and O2- generation in a synchronized, dose-dependent manner (IC50s: 1.45 and 1.12 μM, respectively). It inhibited the inositol 1,4,5-trisphosphate-induced Ca2+ release (IICR) and the store-operated Ca2+ entry (SOCE) that are both required for Nox2 activation by fMLP: IC50s for changes in [Ca2+]i and O2- generation showed submicromolar values of around 0.3 μM in average. Conclusion: It is shown for the first time that shikonin inhibits the fMLP-stimulated Nox2 activity by targeting Ca2+ fluxes like IICR and SOCE.
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