Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IκB kinase

We previously found that bee venom (BV) and melittin (a major component of BV) has anti-inflammatory effect by reacting with the sulfhydryl group of p50 of NF-κB. Since the sulfhydryl group is present in IκB kinase (IKKα and IKKβ), anti-inflammatory effect of melittin via interaction with IKKs was investigated. We first examined binding of melittin to IKKs using surface plasmon resonance analyzer. Melittin binds to IKKα (Kd = 1.34 × 10−9 M) and IKKβ (Kd = 1.01 × 10−9 M). Consistent with the high binding affinity, melittin (5 and 10 μg/ml) and BV (0.5, 1 and 5 μg/ml) suppressed sodium nitroprusside, TNF-α and LPS induced-IKKβ and IKKβ activities, IκB release, and NF-κB activity as well as the expressions of iNOS and COX-2, and the generation of nitric oxide (NO) and prostaglandin E2 (PGE2) in Raw 264.7 mouse macrophages and synoviocytes obtained from rheumatoid arthritis patients. The binding affinities of melittin to mutant IKKs, was reduced, and the inhibitory effect of melittin on IKK and NF-κB activities, and NO and PGE2 generation were abrogated by the reducing agents or in Raw 264.7 transfected with mutant plasmid IKKα (C178A) or IKKβ (C179A). These results suggest that melittin binding to the sulfhydryl group of IKKs resulted in reduced IKK activities, IκB release, NF-κB activity and generation of inflammatory mediators, indicating that IKKs may be also anti-inflammatory targets of BV.