JNK pathway is involved in the inhibition of inflammatory target gene expression and NF-kappaB activation by melittin

BACKGROUND: Bee venom therapy has been used to treat inflammatory diseases including rheumatoid arthritis in humans and in experimental animals. We previously found that bee venom and melittin (a major component of bee venom) have anti-inflammatory effect by reacting with the sulfhydryl group of p50 of nuclear factor-kappa B (NF-κB) and IκB kinases (IKKs). Since mitogen activated protein (MAP) kinase family is implicated in the NF-κB activation and inflammatory reaction, we further investigated whether activation of MAP kinase may be also involved in the anti-inflammatory effect of melittin and bee venom. METHODS: The anti-inflammatory effects of melittin and bee venom were investigated in cultured Raw 264.7 cells, THP-1 human monocytic cells and Synoviocytes. The activation of NF-κB was investigated by electrophoretic mobility shift assay. Nitric oxide (NO) and prostaglandin E(2 )(PGE(2)) were determined either by Enzyme Linked Immuno Sorbent Assay or by biochemical assay. Expression of IκB, p50, p65, inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) as well as phosphorylation of MAP kinase family was determined by Western blot. RESULTS: Melittin (0.5–5 μg/ml) and bee venom (5 and 10 μg/ml) inhibited lipopolysaccharide (LPS, 1 μg/ml) and sodium nitroprusside (SNP, 200 μM)-induced activation of c-Jun NH2-terminal kinase (JNK) in RAW 264.7 cells in a dose dependent manner. However, JNK inhibitor, anthra [1,9-cd]pyrazole-6 (2H)-one (SP600215, 10–50 μM) dose dependently suppressed the inhibitory effects of melittin and bee venom on NF-κB dependent luciferase and DNA binding activity via suppression of the inhibitory effect of melittin and bee venom on the LPS and SNP-induced translocation of p65 and p50 into nucleus as well as cytosolic release of IκB. Moreover, JNK inhibitor suppressed the inhibitory effects of melittin and bee venom on iNOS and COX-2 expression, and on NO and PGE(2 )generation. CONCLUSION: These data show that melittin and bee venom prevent LPS and SNP-induced NO and PGE(2 )production via JNK pathway dependent inactivation of NF-κB, and suggest that inactivation of JNK pathways may also contribute to the anti-inflammatory and anti-arthritis effects of melittin and bee venom.