Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells by andrographolide

1 Andrographolide, an active component found in leaves of Andrographis pani culata, has been reported to exhibit nitric oxide (NO) inhibitory property in endotoxin-stimulated macrophages, however, the detailed mechanisms remai n unclear. In the present study we investigated the effect of andrographoli de on the expression of inducible NO synthase (iNOS) mRNA, protein, and enz yme activity in RAW 264.7 macrophages stimulated with lipopolysaccharide (L PS) plus interferon-gamma (IFN-gamma). 2 RAW 264.7 cells stimulated with LPS/IFN-gamma activated NO production; in this condition andrographolide (1-100 mu M) inhibited NO production in a d ose-dependent manner with an IC50 value of 17.4 +/- 1.1 mu M Andrographolid e also reduces the expression of iNOS protein level but without a significa nt effect on iNOS mRNA. The reduction of iNOS activity is thought to be cau sed by decreased expression of iNOS protein. 3 In a protein stability assay, andrographolide moderately but significantl y reduced the amount of iNOS protein as suggested by accelerating degradati on. Furthermore, andrographolide also inhibited total protein de novo synth esis as demonstrated by [S-35]-methionine incorporation. 4 As a whole, these data suggest that andrographolide inhibits NO synthesis in RAW 264.7 cells by reducing the expression of iNOS protein and the redu ction could occur through two additional mechanisms: prevention of the de n ovo protein synthesis and decreasing the protein stability via a post-trans criptional mechanism. It is also possible that inhibition of iNOS protein e xpression and NO production under immune stimulation and/or bacteria infect ion may explain, in part, the beneficial effects of andrographolide as an a nti-inflammatory agent.