Thymoquinone suppressses in vitro production of IL-5 and IL-13 by mast cells in response to lipopolysaccharide stimulation

Inflamm Res. 2007 Aug;56(8):345-51. doi: 10.1007/s00011-007-7051-0.

Abstract

Objective: Activated mast cells produce Th2 cytokines that regulate allergic inflammation. We have previously shown that thymoquinone (TQ) attenuated airway inflammation in a mouse model of allergic airway inflammation. The present study investigated whether TQ affects Th2 cytokine response in vitro in lipopolysaccharide (LPS)-activated rat mast cells, RBL-2H3.

Materials and methods: RBL-2H3 cells were stimulated for 12 h with 0.1 microg/ml LPS in the presence or absence of 10 microM TQ. Th2 cytokine production was measured in the culture supernatants by ELISA. The mRNA expression of IL-5, IL- 13 and GATA transcription factors was determined by RT-PCR. The expression of the transcription proteins c-Fos, c- Jun and phospho-c-Jun were determined by western blotting. The in vivo binding of GATA, AP-1 and NF-AT transcription factors to IL-5 promoter was assessed by chromatin immunoprecipitation analysis.

Results: TQ significantly (p <0.05) inhibited LPS-induced IL-5 and IL-13 mRNA expression and protein production. However, TQ did not affect IL-10 production. GATA transcription factors are involved in the transcription of IL-5 and IL-13. TQ had no effect on the expression of AP-1 protein subunits, c-Jun and c-Fos, but markedly reduced the transcription of GATA-1 and -2 genes. Chromatin immunoprecipitation revealed that GATA, AP-1 and NF-AT binding to IL-5 promoter was induced by LPS stimulation and that TQ inhibited GATA binding at the IL-5 promoter but did not affect AP-1 and NF-AT binding.

Conclusions: These results suggest that TQ inhibits LPS-induced proinflammatory cytokine production in RBL-2H3 cells by blocking GATA transcription factor expression and promoter binding which demonstrates the anti-inflammatory effect of TQ.

MeSH terms

  • Animals
  • Anti-Inflammatory Agents / pharmacology
  • Benzoquinones / pharmacology*
  • Cells, Cultured
  • GATA1 Transcription Factor / genetics
  • GATA2 Transcription Factor / genetics
  • Interleukin-13 / biosynthesis*
  • Interleukin-5 / biosynthesis*
  • Interleukin-5 / genetics
  • Lipopolysaccharides / pharmacology*
  • Mast Cells / drug effects
  • Mast Cells / metabolism*
  • NF-kappa B / metabolism
  • Promoter Regions, Genetic
  • Rats
  • Transcription Factor AP-1 / metabolism

Substances

  • Anti-Inflammatory Agents
  • Benzoquinones
  • GATA1 Transcription Factor
  • GATA2 Transcription Factor
  • Gata1 protein, rat
  • Gata2 protein, rat
  • Interleukin-13
  • Interleukin-5
  • Lipopolysaccharides
  • NF-kappa B
  • Transcription Factor AP-1
  • thymoquinone