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Japanese herbal medicine, inchinkoto, inhibits inducible nitric oxide synthase induction in interleukin-1β-stimulated hepatocytes

Authors


Dr Tadayoshi Okumura, Department of Surgery, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8506, Japan. Email: okumura@takii.kmu.ac.jp

Abstract

Aim:  A herbal medicine, kampo inchinkoto (TJ-135), is used to treat jaundice and liver fibrosis in patients with cirrhosis. In the inflamed liver, proinflammatory cytokines stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression. Over-production of nitric oxide (NO) by iNOS has been implicated as a factor in liver injury. We examined interleukin (IL)-1β-stimulated hepatocytes as a simple in vitro injury model to determine liver-protective effects of TJ-135. The objective was to investigate whether TJ-135 influences iNOS induction and to determine its mechanism.

Methods:  Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of TJ-135. The induction of iNOS and its signaling pathway were analyzed.

Results:  IL-1β produced increased levels of NO. This effect was inhibited by TJ-135, which exerted its maximal effects at 3 mg/mL. TJ-135 decreased the levels of iNOS protein and its mRNA expression. Experiments with nuclear extracts revealed that TJ-135 inhibited the translocation of nuclear factor-κB (NF-κB) to the nucleus and its DNA binding. TJ-135 also inhibited the activation of Akt, resulting in the reduction of type I IL-1 receptor mRNA and protein expression. Transfection experiments with iNOS promoter-luciferase constructs demonstrated that TJ-135 suppressed iNOS induction by inhibition of promoter transactivation and mRNA stabilization. TJ-135 reduced the expression of an iNOS gene antisense-transcript. Delayed administration or withdrawal of TJ-135 after IL-1β addition also inhibited iNOS induction.

Conclusions:  Results indicate that TJ-135 inhibits the induction of iNOS at both transcriptional and post-transcriptional steps, leading to the prevention of NO production. TJ-135 may have therapeutic potential for various liver injuries through the suppression of iNOS induction.

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