Determining conditions for nitric oxide synthesis in Caco-2 cells using Taguchi and factorial experimental designs.
Intestinal inflammation correlates well with the increased synthesis of nitric oxide (NO), which is attributed mainly to the up-regulation of inducible nitric oxide synthase (iNOS). We optimized the use of interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) as inducers to stimulate NO synthesis in Caco-2 cells using a Taguchi design. The results indicated that IFN-gamma was the most important inducer of iNOS in Caco-2 cells. Treating Caco-2 cells with both IFN-gamma and PMA using an optimal mixture of 8000 U/ml IFN-gamma and 0.1 microg/ml of PMA resulted in a synergistic induction of NO synthesis. Further experiments using a 5-factor/2-level factorial design including Caco-2 growth conditions such as cell passage, culture medium composition, cell seeding time and density, and stimulation time were also performed. Cell seeding and stimulation times significantly (P<0.05) affected NO synthesis, whereas culture medium and seeding density did not appreciably affect NO synthesis in Caco-2 cells. Western blotting and RT-PCR findings confirmed that the optimal mixture of IFN-gamma and PMA effectively up-regulated iNOS mRNA and protein. The induced NO, iNOS mRNA, and protein were all inhibited by the iNOS selective inhibitor, aminoguanidine (AG).
Chen
Xiu-Min
XM
Food, Nutrition, and Health, Faculty of Land and Food Systems, University of British Columbia, 2205 East Mall, Vancouver, BC, Canada, V6T 1Z4.
Kitts
David D
DD
D000704:Analysis of Variance; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D018938:Caco-2 Cells; D002452:Cell Count; D003470:Culture Media; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006146:Guanidines / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation
D015415:Biomarkers; D003470:Culture Media; D004791:Enzyme Inhibitors; D006146:Guanidines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; D013755:Tetradecanoylphorbol Acetate; C004479:pimagedine