Inhibition of Inducible Nitric-Oxide Synthase Expression by(5R)-5-Hydroxytriptolide in Interferon-gamma-and Bacterial Lipopolysaccharide-Stimulated Macrophages

摘要

(5R)-5-Hydroxytriptolide(LLDT-8)is a novel analog of triptolide that has antiarthritic,hepatoprotective,and antiallogenic trans-plantation-rejective effects.In the present study,we report that LLDT-8 inhibited nitric oxide(NO)production and inducible nitric-oxide synthase(iNOS)expression in macrophages.LLDT-8 significantly attenuated NO production,in a dose-dependent manner,in primary peritoneal macrophages and a macrophage cell line of Raw 264.7 cells following stimulation with interferon(IFN)-gamma,lipopolysaccharide(LPS),and IFN-gamma plus LPS.It also reduced the production of tumor necrosis factor-alpha from LPS-stimulated Raw 264.7 cells.To further elucidate the mechanism responsible for the inhibition of NO,we examined the effect of LLDT-8 on IFN-gamma and LPS-induced iNOS expression.Indeed,LLDT-8 prevented NO generation by inhibiting iNOS expression at mRNA level and protein level,rather than by interfering its enzymatic activity.In IFN-gamma-stimulated Raw 264.7 cells,LLDT-8 suppressed the gene transcription of signal transducer and activator of transcription 1 alpha and interferon regulatory factor(IRF)-1,but it displayed no apparent effect on IFN-gamma receptor level on cell surface.After LPS challenge,LLDT-8 further abrogated the expression of LPS receptor complex,including CD14,Toll-like receptor 4,and myeloid differentiation protein-2;decreased the LPS-induced phos-phorylation of stress-activated protein kinase/c-Jun NH_2-termi-nal kinase,extracellular signal-regulated kinase 1/2,and p38 mitogen-activated protein kinase(MAPK);retarded the degradation of I kappa B alpha;and ameliorated the DNA binding activity of nuclear factor-kappa B(NF-kappa B)to nuclear proteins that accounts for transcriptional regulation of iNOS.Taken together,these results suggest that LLDT-8 reduces NO production and iNOS expression by inhibiting IFN-gamma-triggered IRF-1 expression and LPS-triggered MAPK phosphorylation and NF-kappa B activation.