Inhibition of NF-kB Activity Decreases iNOS Expression in ACCs Cells

Objective: To investigate the NF-kB activity and expression levels of VEGF and iNOS of ACCs cells which exposed to the different concentrations and time points of SNAP. Methods: Two ACCs cell lines were exposed to SNAP with the concentration 0, 31.25, 62.5, 125, 250 or 500uM for 4 hours, respectively; or cell lines pretreated with 100ng/ml LPS, then exposed to SNAP with the concentration 500uM for 0, 1, 2 and 4 hours. EMSA was used to detect NF-kB activity. Semi-quantitative reverse transcription-PCR assay was carried out to quantify the mRNA level of iNOS and VEGF. Immuofluorescence double staining and confocal laser scanning were carried out to detect NF-kB nuclear localization and staining intensity of iNOS and VEGF. Results: In ACCs cell lines, NF-kB was significantly activated after stimulation with 31.25uM SNAP for 4 hours. With the same concentration, the mRNA expression of iNOS and VEGF were significantly increased (P<0.01), and the protein levels of iNOS and VEGF were also increased. The NF-kB activity and expression levels of VEGF and iNOS significantly decreased when treated with 500uM SNAP. Moreover, NF-kB activity and expression levels of VEGF and iNOS up-regulated by 100ng/ml LPS significantly reduced in a time dependent manner when treated with 500uM SNAP. Conclusion: In ACCs cells, high concentrations of NO may inhibit NF-kB activity and expressions of iNOS and VEGF while low concentrations of NO may promote NF-kB activity and expressions of iNOS and VEGF.