Utilization of IκB–EGFP Chimeric Gene as an Indicator to Identify Microbial Metabolites with NF-κB Inhibitor Activity

NF-κB is an inducible transcription factor involved in the regulation of gene expression for cytokine release, anti-apoptosis, adhesion molecule, and cell cycle regulation [1]. There are five members in the NF-κB family composed of p50/p105, p52/p100, c-Rel, Rel A, and Rel B [2]. These components all have a consensus amino acid domain that is Rel homology domain (RHD). RHD, a regulatory element for activity of NF-κB, is involved in NF-κB dimerization and IκB binding. Without intracellular stimulation, IκB associates with RHD of NF-κB to inhibit NF-κB migration into the nucleus and retain NF-κB in cytosol. As extracellular signals of NF-κB activation transduce into cells, IκB will be phosphorylated and results in degradation. The degradation of IκB can abolish the inhibitory effect on the activity of NF-κB. Therefore, the amount of intracellular IκB can be an indicator for the activity of NF-κB.The microbial metabolite is an important source of certain medical reagents. Particularly, many investigators indicated that microbial metabolites provide a source of abundant antioxidants. Previous investigations revealed that antioxidants have the ability to inhibit activity of NF-κB [3-5]. Antioxidants, such as β-catenin and bortezomib, have been reported to have a cytotoxic ability against tumor cells by inhibiting the activity of NF-κB [6,7]. Therefore, utilizing the principle of NF-κB activity regulation to develop a drug discovery system may help to rapidly find new drugs.In this study, we have focused on the establishment of a rapid and precise screening system with NF-κB inhibitory activity, and the metabolite products of microbes and the natural extract propolis were tested in this system. The plasmid pIκB–EGFP, which contained a chimeric gene that encodes a fusion protein, green fluorescent protein (EGFP) fused with IκB-α (IκB–EGFP), was used to establish the screening system. The pIκB–EGFP was transfected into cells and the transfected cells were treated with micro