pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes

ARTICLE IN PRESS Nitric Oxide ■■ (2014) ■■–■■

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pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes Masaharu Oishi a,1, Tamami Kiyono b,1, Kenji Sato b, Katsuji Tokuhara a, Yoshito Tanaka a, Hirokazu Miki a, Richi Nakatake a, Masaki Kaibori a, Mikio Nishizawa c, Q1 Tadayoshi Okumura a,d,*, Masanori Kon a a

Department of Surgery, Kansai Medical University, Hirakata, Osaka 573-1010, Japan Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Kyoto 606-8522, Japan c Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan d Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan b

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I N F O

Article history: Received 4 July 2014 Revised 6 December 2014 Accepted 9 December 2014 Available online Keywords: Pyroglutamyl leucine Inducible nitric oxide synthase Liver protective effect Primary cultured hepatocytes Nuclear factor-κB iNOS gene antisense transcript

A B S T R A C T

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 pyroGluLeu. 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. © 2014 Published by Elsevier Inc.

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1. Introduction Under the pathological conditions in the liver, the induction of inducible nitric oxide synthase (iNOS) gene expression is upregulated in concomitance with the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1. Excess production of nitric oxide (NO) by iNOS has been implicated as one of the factors in liver injury, although NO has been reported to exert either detrimental or beneficial effects depending on the injuries and cell types involved.

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Abbreviations: pyroGlu-Leu, pyroglutamyl leucine; NO, nitric oxide; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-κB. * Corresponding author. Department of Surgery, Kansai Medical University, 2-5-1 Shinmachi, Hirakata, Osaka 573-1010, Japan. Fax: +81 72 804 2578. E-mail address: [email protected] (T. Okumura). 1 MO and TK contributed equally to this work.

In animal models of liver injury caused by various insults, such as ischemia-reperfusion, partial hepatectomy and endotoxin shock, we have previously reported that clinical drugs such as pirfenidone (antifibrotic agent) [1], edaravone (free radical scavenger) [2], FR183998 (NA+/H+ exchanger inhibitor) [3,4], insulin-like growth factor-I [5] and sivelestate (neutrophil elastase inhibitor) [6] had liver protective effects. These drugs inhibited the induction of iNOS and NO production as well as the decreased production of various inflammatory mediators, including TNF-α. Further, our experiments with primary cultured rat hepatocytes (in vitro inflammation model), where IL-1β stimulates the expression of iNOS and TNF-α [7–9], revealed that these drugs also inhibited the induction of iNOS and NO production [3,10–12]. Thus, downregulating NO production is considered an indicator of liver protection in our in vitro model. Some amino acids [13–16] and enzymatic hydrolysates of food proteins [17,18] have been shown to exert hepatoprotective effects in experiments with animal models. It has been demonstrated that

http://dx.doi.org/10.1016/j.niox.2014.12.005 1089-8603/© 2014 Published by Elsevier Inc.

Please cite this article in press as: Masaharu Oishi, et al., pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.12.005

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the supplementation of an enzymatic hydrolysate of wheat gluten (WGH) decreased serum levels of aminotransferases in patients with hepatitis from different backgrounds without adverse effect [19]. The WGH suppressed rat liver cirrhosis induced by chronic injection of carbon tetrachloride [20]. These results suggested that oral administration of WGH can have a therapeutic effect against acute and chronic liver diseases. As wheat gluten is rich in glutaminyl residues, WGH contains high amounts of peptides with glutaminyl residues at their amino termini, which are converted to pyroglutamyl residues during processing [21,22]. Recently, we found that pyroglutamyl leucine (pyroGlu-Leu) in WGH was identified as a hepatoprotective peptide in D-galactosamine-induced acute hepatitis of rats [23]. However, the mechanisms of pyroGlu-Leu involved in liver protective effects have not been fully explored. In this study, we examined IL-1β-treated cultured hepatocytes as a simple in vitro inflammation model for animal models and investigated whether pyroGlu-Leu directly inhibits the induction of iNOS and NO production, and if so, the mechanisms involved in its inhibitory effects.

minor modifications [27,28]. Ethanol-soluble fractions of culture medium or cells were subjected to solid phase extraction using a spin column packed with a strong cation exchanger (AG50W × 8; Bio-Rad Laboratories, Hercules, CA, USA). The eluents were injected onto an Inertsil ODS-3 column (2 mm i.d. × 250 mm; GL Science, Tokyo, Japan), and fractions corresponding to pyroGlu or pyroGlu-Leu were derivatized with 2-nitrophenyl hydrazine and were determined by reversed-phase high-performance liquid chromatography (C18-MSII).

2. Materials and methods

Culture medium was used for measurements of nitrite (a stable metabolite of NO) to reflect NO production by the Griess method [29]. Culture medium was also used for measurements of LDH activity to reflect cell viability using a commercial kit (Roche Diagnostics, Mannheim, Germany).

2.1. Materials Pyroglutamyl leucine (pyroGlu-Leu) was generously provided by Nissin Pharma Inc. (Fujimino, Saitama, Japan). Recombinant human IL-1β (2 × 107 U/mg protein) was purchased from MyBioSource (San Diego, CA, USA). [γ-32P]Adenosine-5′-triphosphate (ATP; -222 TBq/ mmol) was obtained from DuPont-New England Nuclear Japan (Tokyo, Japan). Rats were kept at 22 °C under a 12 h/12 h light/ dark cycle, and received food and water ad libitum. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and approved by the Animal Care Committee of Kansai Medical University. 2.2. Primary cultures of hepatocytes Hepatocytes were isolated from male Wistar strain rats (200– 220 g; Charles River, Tokyo, Japan) by collagenase (Wako Pure Chemicals, Osaka, Japan) perfusion [24,25]. The hepatocytes were determined to consist of at least 98% hepatocytes by microscopic observation in primary cultures of rat hepatocytes (data not shown). Isolated hepatocytes were suspended in culture medium at 6 × 105 cells/ml, seeded into 35 mm plastic dishes (2 ml/dish; Falcon Plastic, Oxnard, CA, USA) and cultured at 37 °C in a CO2 incubator under a humidified atmosphere of 5% CO2 in air. The culture medium was Williams’ medium E (WE, Sigma Chemical Co., St. Louis, MO, USA), supplemented with 10% newborn calf serum, Hepes (5 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), dexamethasone (10 nM) and insulin (10 nM). After 5 h, the medium was replaced with fresh serum- and hormone-free WE, and the cells were cultured overnight before use in experiments. The numbers of cells attached to the dishes was calculated by counting the nuclei [26] and using a ratio of 1.37 ± 0.04 nuclei/cell (mean ± SE, n = 7 experiments). 2.3. Determination of pyroglutamate (pyroGlu) and pyroGlu-Leu in primary cultures of hepatocytes The culture medium (100 μl) was mixed with three volumes of ethanol (final 75%). Cells (106/dish) were washed (1 ml of PBS() × 2 times), scraped (rubberpoliceman) with 100 μl of PBS(-) and mixed with ethanol (final 75%). pyroGlu and pyroGlu-Leu were determined by precolumn derivatization as described previously with

2.4. Treatment of cells with pyroGlu-Leu On day 1, the cells were washed with fresh serum- and hormonefree WE, and incubated with IL-1β (1 nM; 347 U/ml) in the same medium in the presence or absence of pyroGlu-Leu for the indicated times. The doses of sample are indicated in the appropriate figures and their legends. 2.5. Determinations of NO production and lactate dehydrogenase (LDH)

2.6. Western blot analysis Total cell lysates were obtained from cultured cells as described previously with minor modifications as follows. Cells (1 × 106 cells/35-mm dish) were lysed in 100–200 μl of solubilizing buffer (10 mM Tris-HCl, pH 7.4, containing 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete protease inhibitor cocktail (Roche Diagnostics), passed through a 26-gauge needle, allowed to stand on ice for 30 min and then centrifuged (16,000 × g for 15 min). The supernatant (total cell lysate) was mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (final: 125 mM Tris-HCl, pH 6.8, containing 5% glycerol, 2% SDS and 1% 2-mercaptoethanol), subjected to SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). Immunostaining was performed using primary antibodies against mouse iNOS (Affinity BioReagents, Golden, CO, USA), human IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rat β-tubulin (internal control; Clone TUB2.1; Sigma), followed by visualization with an ECL blotting detection reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA). 2.7. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was extracted from cultured hepatocytes using a guanidinium-phenol-chloroform method [30] with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For strand-specific RT-PCR analysis, cDNAs were synthesized from total RNA with strand-specific primers, and step-down PCR or quantitative RT-PCR was performed using PC708 (Astec, Fukuoka, Japan) or a Rotor-Gene Q 2plex HRM (Qiagen, Tokyo, Japan), respectively. For iNOS (257 bp), TNF-α (275 bp), the type I IL-1 receptor (IL-1RI, 327 bp), cytokine-induced neutrophil chemoattractant (CINC)-1 (231 bp) and elongation factor1α (EF; internal control, 332 bp) mRNA, an oligo(dT) primer was used for RT and the primer sets 5′-CCAACCTGCAGGTCTTCGATG-3′ and 5′-GTCGATGCACAACTGGGTGAAC-3′ (257-bp product), 5′TCCCAACAAGGAGGAGAAGTTCC-3′ and 5′-GGCAGCCTTGTCCC TTGAAGAGA-3′ (275-bp product), 5′-CGAAGACTATCAGTTTT

Please cite this article in press as: Masaharu Oishi, et al., pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.12.005

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TGGAAC-3′ and 5′-GTCTTTCCATCTGAAGCTTTTGG-3′ (327-bp product), 5-GCCAAGCCACAGGGGCGCCCGT-3′ and 5-ACTTG GGGACACCCTTTAGCATC-3′ (231-bp product), and 5′-TCTGGTTGGA ATGGTGACAACATGC-3′ and 5′-CCAGGAAGAGCTTCACTCAAAGCTT3′ (332-bp product) were used for PCR, respectively. For the antisense transcript of iNOS (iNOS-AST, 211 bp), the sense primer 5′TGCCCCTCCCCCACATTCTCT-3′ was used for RT and the primer set 5′-CCTTTGCCTCATACTTCCTCAGA-3′ and 5′-ATCTTCATCAAGGAATT ATACACGG-3′ (211-bp product) were used for PCR. The stepdown PCR protocols for iNOS and EF were: 10 cycles of (94 °C, 1 min; 72 °C, 2 min); 15 cycles of (94 °C, 1 min; 65 °C, 1 min 30 s; 72 °C, 20 s); and 5 cycles of (94 °C, 1 min; 60 °C, 1 min 30 s; 72 °C, 20 s). The amplified products were analyzed by 3% agarose gel electrophoresis with ethidium bromide, and the levels of iNOS and EF were semi-quantified using a UV transilluminator. The levels of iNOS, TNFα, IL-1RI, CINC-1, iNOS-AST and EF were measured by quantitative RT-PCR. Rotor-Gene SYBR Green PCR Kit (Qiagen) was included in reaction mixture, and the following touchdown protocol was applied: 1 cycle at 95 °C for 5 min and 45 cycles at 95 °C for 5 s and 60 °C for 10 s. The cDNAs for the rat iNOS mRNA and iNOS-AST were deposited in DDBJ/EMBL/GenBank under Accession Nos. AB250951 and AB250952, respectively.

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(submitted to DDBJ/EMBL/GenBank under Accession No. AB250951) was used to replace the SV40 polyadenylation signal (SVpA) of pRiNOS-Luc to create pRiNOS-Luc-3′UTR. 2.10. Transfection and luciferase assay Transfection of cultured hepatocytes was performed as described previously [8,34]. Briefly, hepatocytes were cultured at 4 × 105 cells/dish (35 mm) in WE supplemented with serum, dexamethasone and insulin for 7 h, before being subjected to magnet-assisted transfection (MATra). Reporter plasmids pRiNOS-Luc-SVpA or pRiNOS-Luc-3’UTR (1 μg) and the CMV promoter-driven β-galactosidase plasmid pCMV-LacZ (1 ng) as an internal control were mixed with MATra-A reagent (1 μl; IBA GmbH, Göttingen, Germany). After incubation for 15 min on a magnetic plate at room temperature, the medium was replaced with fresh WE containing serum. The cells were cultured overnight, and then treated with IL-1β in the presence or absence of adenosine. The luciferase and β-galactosidase activities of cell extracts were measured using PicaGene (Wako Pure Chemicals) and Beta-Glo (Promega) kits, respectively. 2.11. Statistical analysis

2.8. Electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared according to Schreiber et al. [31] with minor modifications [32]. Briefly, the dishes were placed on ice, washed with Tris-HCl-buffered saline, harvested with the same buffer using a rubber policeman and centrifuged (1,840 × g for 1 min). The precipitate (2 × 106 cells from two 35 mm dishes) was suspended in 400 μl of lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 500 U/ml trasylol, 0.5 mM PMSF and 1 mM dithiothreitol) and incubated on ice for 15 min. After addition of Nonidet P-40 (final: 0.625%), the cells were lysed by vortexing (2–3 times for 1 min each) and centrifuged (15,000 × g for 1 min). The nuclear pellet was resuspended with extraction buffer (10 mM Hepes, pH 7.9, 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 500 U/ml trasylol, 0.5 mM PMSF and 1 mM dithiothreitol), followed by continuous mixing for 20 min and centrifugation (15,000 × g for 5 min). Aliquots of the supernatant (nuclear extract) were frozen in liquid nitrogen and stored at −80 °C until use. Binding reactions (total: 15 μl) were performed by incubating nuclear extract aliquots (4 μg of protein) in reaction buffer (20 mM Hepes, pH 7.9, 1 mM EDTA, 60 mM KCl, 10% glycerol and 1 mg of poly (dI-dC)) with the probe (approximately 40,000 cpm) for 20 min at room temperature. The products were electrophoresed at 100 V in a 4.8% polyacrylamide gel in high ionic strength buffer (50 mM Tris-HCl, 380 mM glycine, 2 mM EDTA, pH 8.5) and the dried gels were analyzed by autoradiography. An NF-κB consensus oligonucleotide (5′-AGTTGAGGGGA-CTTTCCCAGGC-3′) from the mouse immunoglobulin κ light chain was purchased (Promega, Madison, WI, USA) and labeled with [γ-32P]ATP and T4 polynucleotide kinase. The protein concentration was measured by the method of Bradford [33] with a binding assay kit (Bio-Rad) using bovine serum albumin as a standard. 2.9. Construction of luciferase reporter plasmids and expression plasmids The 1.2-kb 5′-flanking region including the TATA box of the rat iNOS gene was inserted into the pGL3-Basic vector (Promega) to create pRiNOS-Luc-SVpA [32]. A rat cDNA for the 3′-untranslated region (UTR) of the iNOS mRNA was amplified with the primers 5′tgctctaGACAGTGAGGGGTTTGGAGAGA-3′ and 5′-gcggatccttta TTCTTGATCAAACACTCATTTT-3′, and the resultant cDNA was digested with BamH I and Xba I. This cDNA for the iNOS 3′-UTR

The results shown in the figures are representative of 3–4 independent experiments yielding similar findings. Differences were analyzed by the Bonferroni–Dunn test, and values of P < 0.05 were considered to indicate significant differences. 3. Results 3.1. pyroGlu-Leu inhibits the induction of iNOS in IL-1β-stimulated hepatocytes Proinflammatory cytokine IL-1β stimulates the induction of iNOS gene expression, which is followed by the expression of iNOS protein and production of NO, in primary cultures of rat hepatocytes as we reported previously [7,8]. The addition of pyroGlu-Leu with IL-1β decreased the production of NO time-dependently in the culture medium (Fig. 1A). pyroGlu-Leu (0.4–1.6 mg/ml) also decreased NO production and iNOS protein expression dose-dependently (Fig. 1B). pyroGlu-Leu showed practically no cellular cytotoxicity as evaluated by release of lactate dehydrogenase into the medium (Fig. 2) and Trypan blue exclusion in hepatocytes (data not shown). 3.2. pyroGlu-Leu is incorporated in hepatocytes We examined whether pyroGlu-Leu is stable in the medium and is incorporated in cells. Experiments with column chromatography revealed that pyroGlu-Leu was not degraded to pyroGlutamate in the medium during the incubation periods (Fig. 3A) and was incorporated into cells dose-dependently (Fig. 3B). The levels of pyroGlu-Leu in cells were approximately 0.1% of total pyroGluLeu added to the medium. 3.3. pyroGlu-Leu decreases the expression of iNOS mRNA and its antisense transcript, and TNF-α and IL-1RI mRNA RT-PCR experiments revealed that pyroGlu-Leu decreased the expression of iNOS mRNA time-dependently (Fig. 4A and 4B). pyroGluLeu also decreased the expression of iNOS antisense transcript (Fig. 4C), which is involved in the stabilization of iNOS mRNA [35]. Results indicated that pyroGlu-Leu inhibited the induction of iNOS gene expression at a transcriptional and/or post-transcriptional step. Further, quantitative RT-PCR experiments revealed that pyroGlu-Leu reduced the expression of TNF-α and IL-1RI mRNA, but

Please cite this article in press as: Masaharu Oishi, et al., pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.12.005

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Fig. 3. Incorporation of pyroGlu-Leu in hepatocytes. Cells were treated with IL-1β (1 nM) in the presence or absence of pyroGlu-Leu (pEL, 0.4–1.6 mg/ml) for 8 h. (A) The degradation of pEL in culture medium. The levels of pEL (open bars) and its degradate pyroglutamate (pGlu, filled bars) were measured in the medium. (B) Incorporation of pEL into cells. The levels of pEL (open bars) in cells and the ratios of cellular pEL/medium pEL (filled bars) were measured. Each bar shows an average of two independent experiments. Error bars (within 10%) were omitted.

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Fig. 1. Effects of pyroGlu-Leu on the induction of NO production and iNOS in hepatocytes. Cultured hepatocytes were treated with IL-1β (1 nM) in the presence or absence of pyroGlu-Leu (pEL). (A) Effects of pEL (1.6 mg/ml) addition for the indicated times on NO production (IL-1β, open circles; IL-1β + pEL, filled circles; pEL, filled triangles; controls (without IL-1β and pEL), open triangles). Error bars within symbol size were omitted. (B) Effects of addition with various doses of pEL (0.4– 1.6 mg/ml) for 8 h on NO production (upper) or iNOS protein (lower). The levels of nitrite were measured in the culture medium (data are means ± SD for n = 3 dishes/ point; *P < 0.05 vs. IL-1β alone). In the western blotting panels, cell lysates (15 μg of protein) were subjected to SDS-PAGE in a 7.5% gel, and immunoblotted with an anti-iNOS or anti-β-tubulin antibody.

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not of CINC-1 (human IL-8 analog) mRNA (Fig. 5A, 5B and 5C, respectively). 3.4. pyroGlu-Leu decreases iNOS mRNA synthesis and stabilization We carried out transfection experiments with iNOS promoterfirefly luciferase constructs, namely pRiNOS-Luc-SVpA and pRiNOS-

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Luc-3’UTR (Fig. 6A), which detect the activities of iNOS promoter transactivation (iNOS mRNA synthesis) and iNOS mRNA stabilization, respectively [35,36]. IL-1β increased the luciferase activities of these constructs, and pyroGlu-Leu significantly reduced both of these luciferase activities (Fig. 6B and 6C).

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3.5. pyroGlu-Leu has no effect on IκB degradation and NF-κB activation We examined the mechanisms involved in the inhibition of iNOS induction. iNOS induction is regulated in part by a IκB/NF-κB signaling pathway, where IL-1β stimulates the degradation of IκB proteins after the phosphorylation by IκB kinase, which is followed by the activation of NF-κB. Western blotting with cell lysates and electrophoretic mobility shift assay with nuclear extracts revealed that pyroGlu-Leu influenced neither the degradation of IκBα protein (Fig. 7A) nor the activation of NF-κB (its translocation from the cytoplasm to the nucleus and DNA binding) (Fig. 7B), respectively.

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4. Discussion

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Fig. 2. Effects of pyroGlu-Leu on cellular cytotoxicity. Cells were treated with IL1β (1 nM) in the presence or absence of pyroGlu-Leu (pEL, 0.4–1.6 mg/ml) for 8 h. The lactate dehydrogenase (LDH) activities were measured in the culture medium (data are means ± SD for n = 3 dishes/point).

In the inflamed liver, the activation of Kupffer cells (liver nonparenchymal cells, resident macrophages) represents a central mechanism of inflammatory liver injury involving the production of inflammatory mediators including TNF-α and NO. It had been presumed that hepatocytes (liver parenchymal cells) do not produce TNF-α. However, we have found that hepatocytes also produced high levels of TNF-α in response to IL-1β, in addition to the induction of iNOS expression [9]. IL-1β is an endogenous signal against infection and inflammation and activates the NF-κB signaling system in hepatocytes [37]. These observations prompted us to investigate whether pyroGlu-Leu influences the iNOS induction, and if so, TNF-α and other cytokines such as CINC-1 are involved in the liver protective effects of pyroGlu-Leu in our in vitro model.

Please cite this article in press as: Masaharu Oishi, et al., pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.12.005

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ARTICLE IN PRESS In this study, we found that pyroGlu-Leu inhibited the induction of iNOS and NO production in our in vitro liver inflammation model with primary cultured rat hepatocytes (Fig. 1). pyroGluLeu also inhibited the expression of TNF-α mRNA (Fig. 5A). It suggested that pyroGlu-Leu has liver protective effects through the suppression of inflammatory mediators such as iNOS and TNF-α. Thus, our simple in vitro system with cultured hepatocytes may be adequate for screening of liver protective components with iNOS (and/or TNF-α) as an inflammatory biomarker, because it is rapid and inexpensive compared with in vivo animal models of liver injury. However, the liver-protective effects of drugs and foods deduced from this model need to be examined and supported in in vivo animal models. The induction of iNOS gene expression is regulated by transactivation of the iNOS promoter and posttranscriptional modifications involving mRNA stability [38]. There are two essential signaling pathways for iNOS induction, the activation of NF-κB through the degradation of IκB and the upregulation of IL-1RI through the activation of phosphatidylinositol 3-kinase (PI3K)/ Akt [39]. In experiments with iNOS promoter constructs, pyroGluLeu was found to inhibit iNOS induction at the steps of its mRNA synthesis and stabilization (Fig. 6). In the former process, pyroGluLeu likely reduced the transactivation of the iNOS promoter (Fig. 6B). However, pyroGlu-Leu inhibited neither IκBα degradation (Fig. 7A) nor NF-κB activation (Fig. 7B), indicating that pyroGlu-Leu cannot

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Fig. 5. Effects of pyroGlu-Leu on the expression of TNF-α, IL-1RI and CINC-1 mRNA. Cells were treated with IL-1β (1 nM) in the presence or absence of pyroGlu-Leu (pEL, 1.6 mg/ml) for the indicated times. Total RNA was analyzed by strand-specific quantitative RT-PCR to detect (A) TNF-α, (B) IL-1RI and (C) CINC-1 by normalizing the copy number by that of elongation factor-1α. Data are calculated as fold change versus normal control cells. Data represent the mean ± SD (n = 3 dishes/time point/group). IL-1β (open bars) and IL-1β ± pEL (filled bars). *P < 0.05 versus IL-1β alone.

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Fig. 4. Effects of pyroGlu-Leu on the expression of iNOS mRNA and its antisense transcript. Cells were treated with IL-1β (1 nM) in the presence or absence of pyroGluLeu (pEL, 1.6 mg/ml) for the indicated times. Total RNA was analyzed by strandspecific RT-PCR to detect (A) iNOS and elongation factor-1α (EF) using as an internal control, and by quantitative RT-PCR to detect (B) iNOS by normalizing the copy number by that of EF and (C) the iNOS gene antisense transcript (iNOS-AST) by normalizing the copy number by that of normal control cells. Data are calculated as fold change versus normal control cells. Data represent the mean ± SD (n = 3 dishes/time point/ group). IL-1β (open bars) and IL-1β ± pEL (filled bars). *P < 0.05 versus IL-1β alone.

influence the nuclear translocation of NF-κB from the cytoplasm through IκB degradation. In concert with NF-κB translocation, the upregulation of IL-1RI, which stimulates the phosphorylation of NFκB subunit p65, is required for transcriptional activation of the iNOS gene, as we reported previously [39]. In the present study, we found that pyroGlu-Leu decreased the expression of IL-1RI mRNA (Fig. 5B), presumably leading to the inhibition of p65 phosphorylation and decreased DNA binding of NF-κB, and resulting in decreased activities of iNOS promoter transactivation. However, it cannot negate the possibility that pyroGlu-Leu may affect iNOS mRNA synthesis through other transcription factors rather than NF-κB.

Please cite this article in press as: Masaharu Oishi, et al., pyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.12.005

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A kB kB TATA iNOS Gene

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Fig. 6. Effects of pyroGlu-Leu on the transactivation of iNOS promoter. (A) Schematic representation of the promoter region of the iNOS gene. Two reporter constructs are shown beneath the iNOS gene and mRNA. The constructs consist of the rat iNOS promoter (1.2 kb), a luciferase gene and the SV40 poly(A) region (pRiNOS-LucSVpA) or iNOS 3′-UTR (pRiNOS-Luc-3’UTR). ‘An’ indicates the presence of a poly(A) tail. The iNOS 3′-UTR contains AREs (AUUU(U)A × 6), which contribute to mRNA stabilization. (B, C) Each construct was introduced into hepatocytes, and the cells were treated with IL-1β (1 nM) in the presence or absence of pyroGlu-Leu (pEL, 1.6 mg/ ml) for 8 h for pRiNOS-Luc-SVpA (B) and 5 h for pRiNOS-Luc-3’UTR (C). The luciferase activities were normalized by the β-galactosidase activity. The fold activation was calculated by dividing the luciferase activity by the control activity (without IL-1β and pEL). Data are means ± SD for n = 4 dishes. *P < 0.05 versus IL-1β alone.

Regarding the iNOS mRNA stabilization, the 3′-UTR of the iNOS mRNA in rats has six AREs (AUUU(U)A), which are associated with ARE-binding proteins such as HuR and heterogeneous nuclear ribonucleoproteins L/I (PTB), thus contributing to the stabilization of the mRNA [40]. Recently, we have reported that the antisense strand corresponding to the 3′-UTR of iNOS mRNA is transcribed from the iNOS gene, and that the iNOS mRNA antisense transcript plays a key role in stabilizing the iNOS mRNA by interacting with the 3′-UTR and ARE-binding proteins [35]. Drugs such as edaravone [11], FR183998 [3,4], insulin-like growth factor I [5] and sivelestate [12] were found to inhibit iNOS induction partly by suppressing iNOS antisense transcript production in primary cultured hepatocytes or/ and damaged livers. In the present study, pyroGlu-Leu also destabilized the iNOS mRNA through the inhibition of iNOS gene antisense transcript expression (Fig. 4). In our previous study [23], the orally administered pyroGluLeu was absorbed into portal blood at the concentration of 5 μM 30 min after the ingestion in rats (20 mg/kg), showing that pyroGluLeu is resistant to peptidase digestion in the gastrointestinal tract and blood. In the present in vitro model with cultured hepatocytes, pyroGlu-Leu was also not degraded (Fig. 3A) and was incorporated into cells (Fig. 3B), indicating that pyroGlu-Leu, but not its degradates, directly can inhibit the induction of iNOS and NO production. Horiguchi et al. [41] reported that WGH increased NK cell activity on the immune system in healthy human subjects. The administration of wheat protein hydrolysate (glutamine peptide) resulted in the improvement in hepatitis and reductions in liver fat accumulation in steatohepatitic patients [19]. In conclusion, pyroGlu-Leu inhibited the expression iNOS and TNF-α in in vitro liver inflammation model, demonstrating that pyroGlu-Leu can prevent the induction of inflammatory mediators including iNOS in various liver injuries, which is involved in its liver protective effects.

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Acknowledgments This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 24657121, and by grants from the Science Research Promotion Fund of the Japan Q2 Private School Promotion Foundation.

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A Time (min)

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Fig. 7. Effects of pyroGlu-Leu on the degradation of IκBα protein and activation of NF-κB. Cells were treated with IL-1β (1 nM) in the presence or absence of pyroGluLeu (pEL, 1.6 mg/ml) for the indicated times. (A) Cell lysates (20 μg of protein) were subjected to SDS-PAGE in a 12.5% gel, followed by immunoblotting with anti-IκBα or anti-β-tubulin antibody. (B) Activation of NF-κB. Nuclear extracts (4 μg of protein) were analyzed by electrophoretic mobility shift assay. Representative results of three independent experiments are shown.

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