Japanese Kampo Medicine, Ninjinyoeito, Inhibits the Induction of iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes

Abstract

Background: Japanese Kampo medicine, ninjinyoeito (NYT), is used for the treatment of complaints in patients with general fatigue, anorexia and anemia, and is recently applied in patients with chronic hepatitis C. However, there is little scientific evidence to demonstrate the liver-protective effects of NYT. We examined proinflammatory cytokine stimulated hepatocytes as a simple in vitro liver injury model to determine liver-protective effects of NYT and to clarify the mechanisms involved in. Methods: Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of NYT. Inflammatory biomarkers including inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α were analyzed. Results: IL-1β stimulated the induction of iNOS and enhanced the production of NO. Simultaneous addition of NYT decreased the levels of iNOS protein and its mRNA expression, resulting in the reduction of NO production. NYT also reduced the expression of TNF-α mRNA and enhanced the expression of IL-10 mRNA. NYT inhibited two essential signaling pathways for iNOS induction, the activation of nuclear factor (NF)-κB and the upregulation of type I IL-1 receptor. Experiments with transfection and iNOS gene antisense transcript revealed that NYT reduced the levels of iNOS mRNA at both the promoter activation and mRNA stabilization steps. The delayed administration of NYT after IL-1β addition also inhibited iNOS induction. Conclusions: Results demonstrate that NYT can influence the induction of inflammatory mediators, such as iNOS and TNF-α, in part through the inhibition of NF-κB activation in hepatocytes. NYT may have therapeutic potential for various acute liver injuries.