Thymoquinone attenuates proinflammatory responses in lipopolysaccharide-activated mast cells by modulating NF-kappaB nuclear transactivation

Activated mast cells play an important role in the development and maintenance of chronic inflammation by releasing proinflammatory cytokines such as Tumor necrosis factor alpha (TNF alpha). TNFa is a key mediator of immune and inflammatory responses as it controls the expression of inflammatory genes network and its overproduction contributes significantly to the pathological complications observed in many inflammatory diseases. We have previously shown that thymoquinone (TQ), which has broad anti-inflammatory activities, attenuates allergic inflammation in mice. In the present study, we investigated the effect of TQ on LPS-induced TNFa production in the rat basophil cell line, RBL-2H3. Stimulation of RBL-2H3 cells with LPS markedly increased TNF alpha production. TQ treatment significantly inhibited LPS-induced TNF alpha mRNA expression and protein production. To understand the mechanism by which TQ inhibited TNF alpha production, we examined its effects on activation of NF-kappa B transcription factor, which has been shown to be involved in regulating TNFa responses. LPS activated the NF-kappa B pathway, resulting in accumulation of NF-kappa B p65 and p50 subunits in the nucleus and activation of TNF alpha promoter. TQ administration to LPS-stimulated cells did not noticeably alter NF-kappa B cytosolic activation or nuclear expression as demonstrated by western blot analysis. Instead, TQ significantly increased the amount of the repressive NF-kappa B p50 homodimer, and simultaneously decreased the amount of transactivating NF-kappa B p65:p50 heterodimer, bound to the TNF alpha promoter as revealed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Transient transfection of RBL-2H3 cells with TNF alpha promoter-driven luciferase gene constructs demonstrated that one of the three NF-kappa B binding sites in the TNF alpha promoter, the kappa B3 site, played a major role in the induction of TNF alpha promoter-driven luciferase gene expression by LPS, as well as in mediating the inhibitory effects of TQ on TNF alpha production, as TQ had minimal effect on the TNFa promoter-luciferase construct that lacks the kappa B3 site. Together, these results suggest that TQ attenuates the proinflammatory response in LPS-stimulated mast cells by modulating nuclear transactivation of NF-kappa B and TNFa production. (c) 2007 Elsevier B.V All rights reserved.