Triptolide Inhibits NADPH Oxidase Activity and iNOS Expression in LPS+IFNγ‐Stimulated Microvascular Endothelial Cells

Abstract

Triptolide is the active component of Chinese herb Tripterygium wilfordii hook. This herb has been used in China for centuries to treat autoimmune diseases, suggesting that triptolide may have anti-inflammatory actions. The enzymes NADPH oxidase and inducible nitric oxide synthase (iNOS) produce free radicals that may cause tissue damage during inflammation. The present study investigated whether triptolide inhibits NADPH oxidase activity and iNOS expression. Primary cultures of microvascular endothelial cells were prepared from mouse skeletal muscle and then stimulated with E. coli lipopolysaccharide (LPS, 25 ng/ml) and interferon-γ (IFNγ, 100 U/ml) to model inflammatory stimuli. The cells responded to LPS+IFNγ with increased production of the superoxide free radical. This increase was inhibited by NADPH oxidase inhibitors diphenyleneiodonium (10 μM) and apocynin (1 mM) but not by xanthine oxidase inhibitor allopurinol (100 μM), mitochondrial respiration blocker rotenone (10 μM) or NOS inhibitor L-NAME (1 mM). LPS+IFNγ-stimulated protein expression of NADPH oxidase subunits Nox1 and p47phox as well as iNOS. Triptolide (250 nM added with LPS+IFNγ) prevented the LPS+IFNγ-induced increases in cellular production of superoxide and expression of Nox1, p47phox and iNOS. In conclusion, these data are consistent with hypothesis that triptolide inhibits the NADPH oxidase activity and iNOS expression induced by LPS+IFNγ in microvascular endothelial cells.