Establishment of Reporter Platforms Capable of Detecting NF-κB Mediated Immuno-Modulatory Activity

• Published in: Journal of agricultural and food chemistry Vol. 61; no. 51; pp. 12582 - 12587
• Main Authors: Wang, Chung-Huang, Lin, Ju-Hwa, Lu, Ting-Jang, Chiang, An-Na, Chiou, Shih-Ting, Chen, Yi-An, Pan, Min-Hsiung, Hsieh, Shu-Chen
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 26.12.2013
• Subjects: Animals; Anti-Inflammatory Agents - analysis; Anti-Inflammatory Agents - immunology; Biological and medical sciences; Biological Assay - instrumentation; Biological Assay - methods; Citrus - chemistry; citrus peels; Cyclooxygenase 2 - genetics; Cyclooxygenase 2 - immunology; Fundamental and applied biological sciences. Psychology; Genes, Reporter; inflammation; lipopolysaccharides; luciferase; macrophages; Macrophages - drug effects; Macrophages - enzymology; Macrophages - immunology; messenger RNA; Mice; mushrooms; NF-kappa B - analysis; NF-kappa B - immunology; nitric oxide synthase; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - immunology; Plant Extracts - pharmacology; prostaglandin synthase; reporter genes; screening; transcription (genetics); transcription factor NF-kappa B; tumor necrosis factor-alpha; iNOS; COX-2; luciferase assay; RAW264.7; cell platform; Enzyme; Luciferase; Oxidoreductases
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf404887u

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are transcriptional targets of nuclear factor kappa B (NF-κB) 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-κB 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-α. Together we have established luciferase reporter systems sensitive to NF-κB-dependent iNOS and COX-2 activation, which provides an alternative screening method for identifying food components with immune-modulatory activities.