α-Lipoic Acid Prevents the Induction of iNOS Gene Expression Through Destabilization of Its mRNA in Proinflammatory Cytokine-Stimulated Hepatocytes

Abstract

α-Lipoic acid (α-LA) has been reported to reduce ischemia-reperfusion injury (IRI). Proinflammatory cytokines stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, leading to excess production of NO and resulting in liver injury including IRI. We hypothesized that inhibition of iNOS induction underlies the protective effects of α-LA on the liver. The objective was to investigate whether α-LA directly influences iNOS induction in cultured hepatocytes, which is used as a simple in vitro injury model, and the mechanism involved. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of α-LA. The induction of iNOS and NO production and its signal were analyzed. α-LA inhibited the expression of iNOS mRNA and protein dose- and time-dependently, resulting in decreases in NO production. α-LA had no effects on the degradation of IκB proteins and activation of NF-κB. In contrast, α-LA inhibited the upregulation of type I IL-1 receptor stimulated by IL-1β, although α-LA had no effect on Akt activation. Transfection experiments with iNOS promoter-luciferase constructs revealed that α-LA had no effect on the transactivation of the iNOS promoter, but decreased the stabilization of iNOS mRNA. Further, α-LA inhibited the expression of an iNOS gene antisense-transcript, which is involved in iNOS mRNA stability. Results indicate that α-LA inhibits the induction of iNOS gene expression at a posttranscriptional step via iNOS mRNA stabilization, rather than promoter activation. It may provide useful therapeutic effects through the suppression of iNOS induction involved in liver injury.