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Abstract: In animal models of liver injury, proinflammatory cytokines are implicated in inducing iNOS, which is followed by the production of NO in hepatocytes. Previously we have reported that the up-regulation of type I IL-1 receptor (IL-1RI) is required for the transcriptional activation of iNOS gene, in concert with the activation of transcription factor NF-κB. In this study, we found three alternatively spliced isoforms of IL-1RI in primary cultured rat hepatocytes: two (long and short) membrane-bound and one soluble IL-1RI. Interleukin (IL)-1β markedly augmented the mRNA levels of long and short IL-1RI with time, but was less effective for soluble IL-1RI. Two membrane-bound IL-1RI were localized in the intracellular fraction, whereas soluble IL-1RI was released into the culture medium. Cotransfection experiments with iNOS promoter-luciferase constructs revealed that the overexpression of long and short IL-1RI, but not soluble IL-1RI, significantly increased the transactivation of iNOS promoter and the stabilization of its mRNA. In contrast, the addition of conditioned medium containing soluble IL-1RI reduced the induction of iNOS and NO production stimulated by IL-1β. These results further suggest that the enhancement of IL-1RI isoforms may contribute to the regulation of iNOS induction in hepatocytes. [Copyright &y& Elsevier]

Abstract: Studies have indicated that protective effects of statins (HMG-CoA reductase inhibitor) are associated with the regulation of endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS) in heart and liver diseases. Statins have been reported to enhance hepatic NO production and decrease the vascular tone in patients with cirrhosis. However, it is unclear which NOS contributes to the increased NO production. We hypothesized that statins are involved in the up-regulation of iNOS in inflammatory liver, resulting in decreased hepatic resistance. Primary cultured rat hepatocytes were treated with pro-inflammatory cytokine interleukin (IL)-1β in the presence or absence of pitavastatin. Pretreatment of cells with pitavastatin resulted in up-regulation of iNOS induction by IL-1β, followed by increased NO production. Pitavastatin had no effects on the degradation of IκB or activation of NF-κB. However, pitavastatin super-induced the up-regulation of type I IL-1 receptor (IL-1RI), which is essential for iNOS induction in addition to the IκB/NF-κB pathway. Mevalonate and geranylgeranylpyrophosphate blocked the stimulatory effects of pitavastatin on iNOS and IL-1RI induction. Transfection experiments revealed that pitavastatin increased the stability of iNOS mRNA rather than its promoter transactivation. In support of this observation, pitavastatin increased the antisense-transcript corresponding to the 3′-UTR of iNOS mRNA, which stabilizes iNOS mRNA by interacting with the 3′-UTR- and RNA-binding proteins. These findings demonstrate that pitavastatin up-regulates iNOS by the stabilization of its mRNA, presumably through the super-induction of IL-1RI and antisense-transcript. This implies that statins may contribute to a novel potentiated treatment in liver injuries including cirrhosis. [Copyright &y& Elsevier]

Abstract: Edaravone has an anti-inflammatory effect in experimental models of various organ injuries. We reported that edaravone reduces the induction of inducible nitric oxide synthase (iNOS) as well as pro-inflammatory cytokines in endotoxin-treated rats with partial hepatectomy, leading to the prevention of liver injury. Studies were performed to investigate the mechanisms involved in the inhibition of iNOS expression by edaravone in hepatocytes. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of edaravone, and iNOS and its signal were analyzed. Edaravone decreased the expression of iNOS mRNA and its protein stimulated by IL-1β, resulting in the reduction of NO production. Edaravone inhibited the activation of transcription factor NF-κB through IκB degradation and the up-regulation of type I IL-1 receptor through PI3K/Akt activation, which are essential signals for iNOS induction. Further transfection experiments with iNOS promoter-luciferase construct having iNOS 3′-UTR revealed that edaravone decreased the stability of iNOS mRNA. In support of this observation, edaravone decreased the expression of iNOS antisense-transcript, which stabilizes iNOS mRNA by interacting with its 3′-UTR and RNA-binding protein. Edaravone may inhibit the induction of iNOS gene expression at steps of promoter transactivation and mRNA stabilization in cytokine-stimulated hepatocytes. [Copyright &y& Elsevier]

Abstract: Nitric oxide (NO) generated from inducible NO synthase (iNOS) during hepatic injury has been reported to contribute to cytoprotection or cellular damage. Rebamipide, anti-gastric ulcer drug, has protective effects in a variety of tissue and organ injury. However, it remains unknown whether rebamipide is involved in the regulation of iNOS gene expression under pathological conditions. We examined whether rebamipide influences the induction of iNOS in hepatocytes exposed to pro-inflammatory cytokine. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of rebamipide. Pretreatment of cells with rebamipide resulted in up-regulation of iNOS induction by IL-1β, followed by increased NO production. Rebamipide enhanced the degradation of IκBα and the activation of NF-κB. Further, rebamipide super-induced the up-regulation of type I IL-1 receptor (IL-1RI), which is essential for iNOS induction in addition to the IκB/NF-κB pathway. Transfection experiments revealed that rebamipide increased the transactivation of iNOS promoter and the stability of iNOS mRNA. In the latter, rebamipide increased the antisense-transcript corresponding to the 3′-UTR of iNOS mRNA, which stabilizes iNOS mRNA by interacting with the 3′-UTR and RNA-binding proteins. These findings demonstrate that rebamipide up-regulates iNOS by iNOS promoter activation through NF-κB, and by its mRNA stabilization presumably through the super-induction of IL-1RI and antisense-transcript. Rebamipide may contribute to a novel potentiated treatment in liver injuries. [Copyright &y& Elsevier]