The influence of acetylshikonin, a natural naphthoquinone, on the production of leukotriene B4 and thromboxane A2 in rat neutrophils

Both A23187 and formyl-Met-Leu-Phe (fMLP) induced the release of arachidonic acid and the production of thromboxane B2 and leukotriene B4 from rat neutrophils that were inhibited by acetylshikonin in a concentration-dependent manner. Acetylshikonin blocked exogenous arachidonic acid-induced leukotriene B4 and thromboxane B2 production in neutrophils and inhibited the enzymatic activity of ram seminal vesicles cyclooxygenase and human recombinant 5-lipoxygenase, whereas it had no effect on cytosolic phospholipase A2 activity, in cell-free systems. 3-Morpholinosydnonimine- and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE)-mediated dihydrorhodamine 123 oxidation (to assess the lipid peroxide and peroxynitrite scavenging activity) was reduced by acetylshikonin. The membrane recruitment of cytosolic phospholipase A2 was inhibited, but the phosphorylation of cytosolic phospholipase A2 was enhanced, by acetylshikonin in the A23187-induced response. Acetylshikonin alone stimulated extracellular signal regulated kinase (ERK) phosphorylation and enhanced this response in cells stimulated with A23187 and fMLP. The phosphorylation of ERKs and cytosolic phospholipase A2 was attenuated by U0126, a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Acetylshikonin facilitated both A23187- and fMLP-mediated translocation of 5-lipoxygenase to the membrane. Acetylshikonin attenuated both fMLP- and ionomycin-mediated [Ca2+]i elevation. These results indicate that the inhibition of eicosanoid production by acetylshikonin is due to the attenuation of cytosolic phospholipase A2 membrane recruitment via the decrease in [Ca2+]i and to the blockade of cyclooxygenase and 5-lipoxygenase activity.