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 γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), 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-γ was the most important inducer of iNOS in Caco-2 cells. Treating Caco-2 cells with both IFN-γ and PMA using an optimal mixture of 8000 U/ml IFN-γ and 0.1 μg/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-γ 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).