5,7-dihydroxy-3,4,6-trimethoxyflavone inhibits the inflammatory effects induced by Bacteroides fragilis enterotoxin via dissociating the complex of heat shock protein 90 and I kappa B alpha and I kappa B kinase-gamma in intestinal epithelial cell culture

Enterotoxin produced by enterotoxigenic Bacteroides fragilis (BFT) has been associated with mucosal inflammation and diarrhoeal diseases. In this study, the anti-inflammatory molecular mechanism of 5,7-dihydroxy-3,4,6-trimethoxyflavone (eupatilin) was characterized in an HT-29 intestinal epithelial cell line stimulated with BFT. Pre-treatment of HT-29 cells with eupatilin decreased the production significantly of both interleukin (IL)-8 and prostaglandin E(2) induced by BFT in a dose-dependent manner. BFT-activated nuclear factor-kappaB (NF-kappa B) signals in HT-29 cells and pretreatment with eupatilin suppressed NF-kappa B activation that resulted in the significant inhibition of IL-8 and cyclo-oxygenase-2 expression. BFT-induced phosphorylation of both I kappa B alpha and I kappa B kinase (IKK) signals was prevented in eupatilin-pretreated HT-29 cells. Transfection of siRNA for IKK-alpha and IKK-beta decreased the production of IL-8 and prostaglandin E(2); however, the transfection of IKK-beta siRNA showed a more significant reduction of BFT-induced I kappa B alpha phosphorylation compared with that of IKK-alpha siRNA. In addition, herbimycin A, a specific inhibitor of heat shock protein 90 (Hsp90), decreased the BFT-induced activation of IKK and NF-kappa B, suggesting that Hsp90 is associated with a pathway of IKK-NF-kappa B-IL-8/cyclo-oxygenase-2 gene signalling. Furthermore, eupatilin dissociated the complex between Hsp90 and IKK-gamma in BFT-stimulated HT-29 cells. These results suggest that eupatilin can suppress the NF-kappa B signalling pathway by targeting the Hsp90-IKK-gamma complex in intestinal epithelial cells and may attenuate BFT-induced inflammatory responses.J. M. Kim and D. H. Lee contributed equally to this work. We thank Dr Martin F. Kagnoff and Dr Joseph A. DiDonato for providing the reporter gene plasmids and Sung-Hee Yang, Jung Sang Youn and Han Jin Lee for their expert technical assistance. This work was supported by a grant from Seoul R&BD Program and the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R11-2008-044-01004-0)