Sivelestat Suppresses iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes

Abstract

Recent evidence has indicated that sivelestat, a neutrophil elastase inhibitor, has liver-protective effects in a variety of liver injuries. Proinflammatory cytokines including interleukin (IL)-1β stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, leading to excess production of NO and resulting in liver damage. We hypothesized that inhibition of iNOS induction underlies the protective effects of sivelestat on the liver. The objective of this study was to investigate whether sivelestat directly influences iNOS induction in cultured hepatocytes, which is used as a simple in vitro injury model, and to determine the mechanism involved. Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of sivelestat. The induction of iNOS and its signaling pathway were analyzed. Sivelestat inhibited the induction of iNOS mRNA and its protein, followed by decreased production of NO. Transfection and iNOS gene antisense-transcript experiments revealed that sivelestat reduced the levels of iNOS mRNA at both the promoter activation and mRNA stabilization steps. However, sivelestat had no effects on the degradation of IκB and nuclear translocation of NF-κB subunit p65, although it moderately blocked the activation of NF-κB. In contrast, sivelestat blocked the upregulation of IL-1 receptor I through the inactivation of phosphatidylinositol 3-kinase/Akt. Delayed sivelestat addition experiments demonstrated that the destabilization of the iNOS mRNA contributed more significantly to the inhibitory effects of sivelestat than the reduction in iNOS mRNA synthesis. Sivelestat may provide useful therapeutic effects through the suppression of iNOS induction involved in liver injury.