title: pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes.
abstract: Pyroglutamyl leucine (pyroGlu-Leu), which is a peptide isolated from wheat gluten hydrolysate, has been reported to be a hepatoprotective compound in acute liver failure. In inflamed liver, proinflammatory cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)-α stimulate the induction of inducible nitric oxide synthase (iNOS). Excess production of nitric oxide (NO) by iNOS is an inflammatory biomarker in liver injury. We examined proinflammatory cytokine-stimulated hepatocytes as a simple "in vitro inflammation model" to determine liver protective effects of pyroGlu-Leu and its mechanisms of action. We hypothesized that pyroGlu-Leu inhibits the induction of iNOS gene expression, resulting in the attenuation of hepatic inflammation. Hepatocytes were isolated from rats by collagenase perfusion and cultured. Primary cultured cells were treated with IL-1β in the presence or absence of pyroGlu-Leu. The induction of iNOS and its signaling pathway were analyzed. IL-1β stimulated the enhancement of NO production in hepatocytes and this effect was inhibited by pyroGlu-Leu. pyroGlu-Leu decreased the expression of iNOS protein and its mRNA. Transfection experiments with iNOS-luciferase constructs revealed that pyroGlu-Leu inhibited both of iNOS promoter transactivation and its mRNA stabilization. pyroGlu-Leu also decreased the expression of an iNOS gene antisense transcript, which is involved in iNOS mRNA stability. However, pyroGlu-Leu had no effects on IκB degradation and NF-κB activation. Results demonstrate that pyroGlu-Leu inhibited the induction of iNOS gene expression at transcriptional and post-transcriptional steps through IκB/NF-κB-independent pathway, leading to the prevention of NO production. pyroGlu-Leu may have therapeutic potential for liver injury through the suppression of iNOS.
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Oishi
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Kyoto 606-8522, Japan.
authors.forename: Tamami
authors.initials: T
authors.lastname: Kiyono
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Kyoto 606-8522, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Sato
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
authors.forename: Katsuji
authors.initials: K
authors.lastname: Tokuhara
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Miki
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
authors.forename: Richi
authors.initials: R
authors.lastname: Nakatake
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kaibori
authors.affiliation: Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
authors.forename: Mikio
authors.initials: M
authors.lastname: Nishizawa
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan; Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan. Electronic address: okumura@hirakata.kmu.ac.jp.
authors.forename: Tadayoshi
authors.initials: T
authors.lastname: Okumura
authors.affiliation: Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Kon
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: D004151:Dipeptides; D053583:Interleukin-1beta; D016328:NF-kappa B; D012333:RNA, Messenger; C041765:pyroglutamylleucine; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011761:Pyrrolidonecarboxylic Acid
keywords: Inducible nitric oxide synthase; Liver protective effect; Nuclear factor-κB; Primary cultured hepatocytes; Pyroglutamyl leucine; iNOS gene antisense transcript