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

摘要

class="enumerated" style="list-style-type:decimal">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 μM 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 μM) 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.