Shikonin reduces oedema induced by phorbol ester by interfering with IkappaBalpha degradation thus inhibiting translocation of NF-kappaB to the nucleus.
OBJECTIVE
In the present paper we studied the effect of shikonin on ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and determined the mechanisms through which shikonin might exert its topical anti-inflammatory action.


METHODS
Acute ear oedema was induced in mice by topical application of TPA. The in vitro assays used macrophages RAW 264.7 cells stimulated with lipopolysaccharide. Cyclooxygenase-2, inducible nitric oxide synthase, protein kinase Calpha, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (pERK), c-Jun N-terminal kinase (JNK), pJNK, p38, p-p38, p65, p-p65, inhibitor protein of nuclear factor-kappaB (NF-kappaB) (IkappaBalpha) and pIkappaBalpha were measured by Western blotting, activation and binding of NF-kappaB to DNA was detected by reporter gene and electrophoretic mobility shift assay, respectively, and NF-kappaB p65 localization was detected by immunocytochemistry.


RESULTS
Shikonin reduced the oedema (inhibitory dose 50 = 1.0 mg per ear), the expression of cyclooxygenase-2 (70%) and of inducible nitric oxide synthase (100%) in vivo. It significantly decreased TPA-induced translocation of protein kinase Calpha, the phosphorylation and activation of ERK, the nuclear translocation of NF-kappaB and the TPA-induced NF-kappaB-DNA-binding activity in mouse skin. Moreover, in RAW 264.7 cells, shikonin significantly inhibited the binding of NF-kappaB to DNA in a dose-dependent manner and the nuclear translocation of p65.


CONCLUSIONS
Shikonin exerted its topical anti-inflammatory action by interfering with the degradation of IkappaBalpha, thus inhibiting the activation of NF-kappaB.
Andújar
I
I
Departament de Farmacologia, Universitat de València, Burjassot, Spain.
Recio
M C
MC
Bacelli
T
T
Giner
R M
RM
Ríos
J L
JL
D000287:Administration, Topical; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000008:administration & dosage / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004487:Edema / Q000188:drug therapy* / Q000503:physiopathology; D005260:Female; D020672:I-kappa B Proteins / Q000187:drug effects / Q000378:metabolism; D007249:Inflammation / Q000188:drug therapy* / Q000503:physiopathology; D020128:Inhibitory Concentration 50; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D009285:Naphthoquinones / Q000008:administration & dosage / Q000494:pharmacology*; D010766:Phosphorylation; D021381:Protein Transport / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate
D000894:Anti-Inflammatory Agents, Non-Steroidal; D020672:I-kappa B Proteins; D016328:NF-kappa B; D009285:Naphthoquinones; C000606205:Nfkbia protein, mouse; D000072000:NF-KappaB Inhibitor alpha; C016101:shikonin; D013755:Tetradecanoylphorbol Acetate