Investigation of the inhibition by acetylshikonin of the respiratory burst in rat neutrophils

Abstract

1 The ability of acetylshikonin to inhibit the respiratory burst in rat neutrophils was characterized and the underlying mechanism of action was also assessed in the present study. 2 Acetylshikonin caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)-and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O 2 .7) generation with IC 50 values of 0.48+0.03 and 0.39+0.03 mM, respectively. Acetylshikonin also inhibited the O 2 consumption in neutrophils in response to fMLP/CB as well as to PMA. 3 Acetylshikonin did not scavenge the generated O 2.7 in the xanthine-xanthine oxidase system or during dihydroxyfumaric acid (DHF) autoxidation but, on the contrary, acetylshikonin enhanced the O 2 .7 generation in these cell-free oxygen radical generating systems. 4 Acetylshikonin inhibited the formation of inositol trisphosphate (IP 3 ) (39.0+7.8% inhibition at 10 mM, P50.05) in neutrophils in response to fMLP. 5 Both the neutrophil cytosolic protein kinase C (PKC) activity and the PMA-induced PKC associated with the membrane were una ected by acetylshikonin. 6 Acetylshikonin did not a ect the porcine heart protein kinase A (PKA) activity. Upon exposure to acetylshikonin, the cellular cyclic AMP level was decreased in neutrophils in response to fMLP. 7 The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP/CB were inhibited by acetylshikonin (60.1+7.3 and 63.2+10.5% inhibition, respectively, at 10 mM, both P50.05). Moreover, acetylshikonin attenuated the fMLP/CB-induced protein tyrosine phosphorylation (about 90% inhibition at 1 mM). 8 In PMA-activated neutrophil particulate NADPH oxidase preparations, acetylshikonin did not inhibit, but enhanced, the O 2 .7 generation in the presence of NADPH. However, acetylshikonin decreased the membrane associated p47 phox in PMA-activated neutrophils (about 60% inhibition at 1 mM). 9 Collectively, these results suggest that the attenuation of protein tyrosine phosphorylation and a failure in the assembly of a functional NADPH oxidase complex probably contribute predominantly to the inhibition of respiratory burst in neutrophils by acetylshikonin. In contrast, the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways play only a minor role in this respect.

Shikonin inhibits IgE-mediated histamine release by human basophils and Syk kinase activity

“…Consequently, shikonin effects based on Syk and Lyn inactivation would be extensive to cells where these kinases are expressed. This might be the case of a previously reported prevention by acetylshikonin of mast cell degranulation in the hind-paw edema model [8], as well as the inhibition of neutrophil respiratory burst caused by impaired tyrosine phosphorylation reactions [14]. Also, the previously reported inhibition of PLC activity in neutrophils by acetylshikonin [15] might involve targeting of Syk, because Syk is the upstream kinase that phosphorylates PLC leading to its activation [23].…”

“…Chen et al showed another possibility, that shikonin blocks chemokine binding to CC chemokine receptor-1 [11]. Other effects that might contribute for the anti-inflammatory activity of shikonins are the prevention of leukotriene B4 biosynthesis in leukocytes [12], inhibition of superoxide-generating activity [13,14] and impairment of phosphatidylinositol signaling in neutrophils [15]. Acetylshikonin was found to suppress histamine release from rat peritoneal mast cells stimulated with compound 48/80 [8], a classical mast cell secretagogue which directly activates GTP-binding protein and induces phospholipase C-related cell signaling [16].…”

Inhibitory effect of acetylshikonin on human gastric carcinoma cell line SGC-7901 in vitro and in vivo

“…It has been shown that acetylshikonin, one of the shikonin derivatives, is effective against sterilized and infected wounds [7,8] . It has been demonstrated that acetylshikonin can inhibit tumor-induced cutaneous angiogenesis [9] and has obvious antitumor effects on S180 sarcoma model [10] .…”

Evaluation of radical scavenging properties of shikonin

“…A finding against reactivity of shikonin with O 2 •− was that acetylshikonin was unable to scavenge O 2 •− produced by the dihydroxyfumaric acid autoxidation as detected by nitroblue tetrazolium reduction. ( 40 ) In this assay, no substantial kinetic competition would be expected from nitroblue tetrazolium having a slower rate constant for the reaction with O 2 •− (5 × 10 4 M −1 s −1 ) ( 41 ) than that estimated for the reaction with shikonin. The reason for the inability of shikonin to prevent this reaction needs further elucidation.…”

“…Previous studies also suggested failure in the assembly of Nox2 system in acetylshikonin-treated neutrophils due to an impaired translocation of cytosolic p47 phox to the membranes. ( 40 ) Whatever the mechanisms of the inhibition, the fact that shikonin inhibits respiratory burst of neutrophils before activation could have implications in its effects in vivo , in modulation of inflammation-related events.…”