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Nepalolide A inhibits the expression of inducible nitric oxide synthase by modulating the degradation of IκB‐α and IκB‐β in C6 glioma cells and rat primary astrocytes
Author(s) -
Wang ChuenNeu,
Shiao YoungJi,
Lin YunLian,
Chen ChiehFu
Publication year - 1999
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0702785
Subject(s) - nitric oxide synthase , lipopolysaccharide , microbiology and biotechnology , nitric oxide , tumor necrosis factor alpha , nf κb , chemistry , proinflammatory cytokine , interferon , astrocyte , nfkb1 , electrophoretic mobility shift assay , omega n methylarginine , biology , biochemistry , signal transduction , inflammation , immunology , endocrinology , gene expression , transcription factor , central nervous system , gene
The effects of nepalolide A on the expression of inducible nitric oxide synthase (iNOS) caused by incubation with lipopolysaccharide/interferon‐γ (LPS/IFN‐γ) or tumour necrosis factor‐α/interleukin‐1β/IFN‐γ (TNF‐α/IL‐1β/IFN‐γ, mixed cytokines) in C6 glioma cells and primary astrocytes of rat were investigated. The mechanisms by which nepalolide A confers its effect on iNOS expression were also elucidated. Treatment with LPS/IFN‐γ and mixed cytokines for 24 h elicited the induction of iNOS activity as determined by nitrite accumulation in the culture medium and assay of enzyme activity. Nepalolide A at 10 μ M abrogated the LPS/IFN‐γ‐ and mixed cytokines‐mediated induction of iNOS by more than 90% in C6 glioma cells, and by 80% for mixed cytokines‐induced induction of iNOS in primary astrocytes. The effect of nepalolide A (2–10 μ M ) was concentration‐dependent. The inhibition of iNOS induction by nepalolide A was attributed to decreases in the content of iNOS protein and the level of iNOS mRNA, as measured by immunoblotting and reverse transcriptase‐polymerase chain reaction. Electrophoretic mobility shift assay was used to evaluate the effect of nepalolide A on the activation of nuclear factor‐κB (NF‐κB). Results showed that nepalolide A diminished the LPS/IFN‐γ‐mediated association of NF‐κB with consensus oligonucleotide in a concentration‐dependent manner. The activation of NF‐κB by mixed cytokines was modulated both in the extent of activation and in its time‐course by nepalolide A. The ability of nepalolide A to inhibit NF‐κB activation was further confirmed by studies on the degradation of the inhibitor of NF‐κB, IκB, as measured by immunoblotting. The present study demonstrates that the attenuation of NF‐κB activation by nepalolide A was mediated by blockade of the degradation of IκB, leading to suppression of the expression of iNOS.British Journal of Pharmacology (1999) 128 , 345–356; doi: 10.1038/sj.bjp.0702785