Establishment of reporter platforms capable of detecting NF-£eB mediated immuno-modulatory activity
Journal
Journal of Agricultural and Food Chemistry
Journal Volume
61
Journal Issue
51
Pages
12582-12587
Date Issued
2013
Author(s)
Wang C.-H.
Lin J.-H.
Chiang A.-N.
Chiou S.-T.
Chen Y.-A.
Pan M.-H.
Abstract
Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are transcriptional targets of nuclear factor kappa B (NF-£eB) that are involved in inflammatory responses. The aim of this study is to develop a method for efficiently detecting inflammation modulatory activities. Here we established RAW264.7 macrophage cells stably expressing a luciferase reporter gene directed by iNOS or COX-2 promoter. Lipopolysaccharide (LPS) treatment stimulated the luciferase activity which paralleled with increased iNOS and COX-2 mRNA levels determined by RT-q-PCR. The LPS-stimulated luciferase activity was blocked by NF-£eB inhibitor CAPE and by nobiletin, an anti-inflammatory natural product from citrus peels. We have applied the platforms to screen various mushroom species; analysis by scatter plot revealed a strong correlation to the results obtained by ELISA-based detection of TNF-£\. Together we have established luciferase reporter systems sensitive to NF-£eB-dependent iNOS and COX-2 activation, which provides an alternative screening method for identifying food components with immune-modulatory activities. ? 2013 American Chemical Society.
Subjects
cell platform
COX-2
iNOS
luciferase assay
RAW264.7
Type
journal article