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Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma- and bacterial lipopolysaccharide-stimulated macrophages.

Abstract

(5R)-5-Hydroxytriptolide (LLDT-8) is a novel analog of triptolide that has antiarthritic, hepatoprotective, and antiallogenic transplantation-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 1alpha 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 phosphorylation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase (MAPK); retarded the degradation of IkappaBalpha; and ameliorated the DNA binding activity of nuclear factor-kappaB (NF-kappaB) 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-kappaB activation.

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  • Authors+Show Affiliations

    ,

    Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Rd., Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China.

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    MeSH

    Animals
    Blotting, Western
    Cell Line
    Diterpenes
    Electrophoretic Mobility Shift Assay
    Enzyme Inhibitors
    Flow Cytometry
    Gene Expression Regulation, Enzymologic
    Glyceraldehyde-3-Phosphate Dehydrogenases
    Interferon-gamma
    Lipopolysaccharides
    Macrophages
    Male
    Mice
    Mice, Inbred C57BL
    Mitogen-Activated Protein Kinases
    NF-kappa B
    Nitric Oxide
    Nitric Oxide Synthase Type II
    Phosphorylation
    Recombinant Proteins
    Reverse Transcriptase Polymerase Chain Reaction
    Tumor Necrosis Factor-alpha
    p38 Mitogen-Activated Protein Kinases

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    16166270

    Citation

    Zhou, Ru, et al. "Inhibition of Inducible Nitric-oxide Synthase Expression By (5R)-5-hydroxytriptolide in Interferon-gamma- and Bacterial Lipopolysaccharide-stimulated Macrophages." The Journal of Pharmacology and Experimental Therapeutics, vol. 316, no. 1, 2006, pp. 121-8.
    Zhou R, Zheng SX, Tang W, et al. Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma- and bacterial lipopolysaccharide-stimulated macrophages. J Pharmacol Exp Ther. 2006;316(1):121-8.
    Zhou, R., Zheng, S. X., Tang, W., He, P. L., Li, X. Y., Yang, Y. F., ... Zuo, J. P. (2006). Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma- and bacterial lipopolysaccharide-stimulated macrophages. The Journal of Pharmacology and Experimental Therapeutics, 316(1), pp. 121-8.
    Zhou R, et al. Inhibition of Inducible Nitric-oxide Synthase Expression By (5R)-5-hydroxytriptolide in Interferon-gamma- and Bacterial Lipopolysaccharide-stimulated Macrophages. J Pharmacol Exp Ther. 2006;316(1):121-8. PubMed PMID: 16166270.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma- and bacterial lipopolysaccharide-stimulated macrophages. AU - Zhou,Ru, AU - Zheng,Shen-Xi, AU - Tang,Wei, AU - He,Pei-Lan, AU - Li,Xiao-Yu, AU - Yang,Yi-Fu, AU - Li,Yuan-Chao, AU - Geng,Jian-Guo, AU - Zuo,Jian-Ping, Y1 - 2005/09/15/ PY - 2005/9/17/pubmed PY - 2006/4/1/medline PY - 2005/9/17/entrez SP - 121 EP - 8 JF - The Journal of pharmacology and experimental therapeutics JO - J. Pharmacol. Exp. Ther. VL - 316 IS - 1 N2 - (5R)-5-Hydroxytriptolide (LLDT-8) is a novel analog of triptolide that has antiarthritic, hepatoprotective, and antiallogenic transplantation-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 1alpha 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 phosphorylation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase (MAPK); retarded the degradation of IkappaBalpha; and ameliorated the DNA binding activity of nuclear factor-kappaB (NF-kappaB) 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-kappaB activation. SN - 0022-3565 UR - https://www.unboundmedicine.com/medline/citation/16166270/Inhibition_of_inducible_nitric_oxide_synthase_expression_by__5R__5_hydroxytriptolide_in_interferon_gamma__and_bacterial_lipopolysaccharide_stimulated_macrophages_ L2 - http://jpet.aspetjournals.org/cgi/pmidlookup?view=long&pmid=16166270 DB - PRIME DP - Unbound Medicine ER -