Background/Aims: Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated as proinflammatory biomarker in liver injury. The application of active hexose correlated compound (AHCC) as a functional food in complementary and alternative medicine has increased. The possibility that AHCC might inhibit iNOS induction was investigated as a potential liver-protective effect. Methods: Hepatocytes were isolated from rats by collagenase perfusion and cultured. Primary cultured hepatocytes were treated with interleukin-1β in the presence or absence of AHCC-sugar fraction (AHCC-SF). Results and Conclusion: AHCC-SF inhibited the production of NO and reduced expressions of iNOS mRNA and its protein. AHCC-SF had no effects on either IĸB degradation or nuclear factor-ĸB (NF-ĸB) activation. In contrast, AHCC-SF inhibited the upregulation of type I interleukin-1 receptor (IL-1RI) through the inhibition of Akt phosphorylation. Transfection experiments with iNOS promoter-luciferase constructs revealed that AHCC-SF reduced the levels of iNOS mRNA at both promoter transactivation and mRNA stabilization steps. AHCC-SF inhibited the expression of iNOS gene antisense transcript, which is involved in iNOS mRNA stabilization. These findings demonstrate that AHCC-SF suppresses iNOS gene expression through a IĸB/NF-ĸB-independent but Akt/IL-1RI-dependent pathway, resulting in the reduction of NO production. AHCC-SF may have therapeutic potential for various liver injuries.

Keywords:
Type I interleukin-1 receptor, Active hexose correlated compound, Interleukin-1β, iNOS, Nuclear factor-ĸB, iNOS gene antisense transcript
1.
Tsuchiya H, Kaibori M, Yanagida H, Yokoigawa N, Kwon AH, Okumura T, Kamiyama Y: Pirfenidone prevents endotoxin-induced liver injury after partial hepatectomy in rats. J Hepatol 2004;40:94–101.
2.
Tsuji K, Kwon AH, Yoshida H, Qiu Z, Kaibori M, Okumura T, Kamiyama Y: Free radical scavenger (edaravone) prevents endotoxin-induced liver injury after partial hepatectomy in rats. J Hepatol 2005;42:94–101.
3.
Tanaka T, Uchida Y, Kaibori M, Hijikawa T, Ishizaki M, Yamada M, Matsui K, Ozaki T, Tokuhara K, Kamiyama Y, Nishizawa M, Ito S, Okumura T: Na+/H+ exchanger inhibitor, FR183998, has protective effect in lethal acute liver failure and prevents iNOS induction in rats. J Hepatol 2008;48:289–299.
4.
Hijikawa T, Kaibori M, Uchida Y, Yamada M, Matsui K, Ozaki T, Kamiyama Y, Nishizawa M, Okumura T: Insulin-like growth factor-I prevents liver injury through the inhibition of TNF-α and iNOS induction in D-galactosamine and lipopolysaccharide-treated rats. Shock 2008;29:740–747.
5.
Ishizaki M, Kaibori M, Uchida Y, Hijikawa T, Tanaka H, Ozaki T, Tokuhara K, Matsui K, Kwon AH, Kamiyama Y, Nishizawa M, Okumura T: Protective effect of FR183998, a Na+/H+ exchanger inhibitor, and its inhibition of iNOS induction in hepatic ischemia-reperfusion injury in rats. Shock 2008;30:311–317.
6.
Nakanishi H, Kaibori M, Teshima S, Yoshida H, Kwon AH, Kamiyama Y, Nishizawa M, Ito S, Okumura T: Pirfenidone inhibits the induction of iNOS stimulated by interleukin-1β at a step of NF-kappaB DNA binding in hepatocytes. J Hepatol 2004;41:730–736.
7.
Yoshida H, Kwon AH, Kaibori M, Tsuji K, Habara K, Yamada M, Kamiyama Y, Nishizawa M, Ito S, Okumura T: Edaravone prevents iNOS expression by inhibiting its promoter transactivation and mRNA stability in cytokine-stimulated hepatocytes. Nitric Oxide 2008;18:105–112.
8.
Geller DA, de Vera ME, Russell DA, Shapiro RA, Nussler AK, Simmons RL, Billiar TR: A central role for IL-1 beta in the in vitro and in vivo regulation of hepatic inducible nitric oxide synthase. IL-1 beta induces hepatic nitric oxide synthesis. J Immunol 1995;155:4890–4898.
9.
Kitade H, Sakitani K, Inoue K, Masu Y, Kawada N, Hiramatsu Y, Kamiyama Y, Ito S, Okumura T: Interleukin-1β markedly stimulates nitric oxide formation in the absence of other cytokines or lipopolysaccharide, in primary cultured rat hepatocytes, but not in Kupffer cells. Hepatology 1996;23:797–802.
10.
Kleinert H, Pautz A, Linker K, Schwarz PM: Regulation of the expression of inducible nitric oxide synthase. Eur J Pharmacol 2004;500:255–266.
11.
Teshima S, Nakanishi H, Nishizawa M, Kitagawa K, Kaibori M, Yamada M, Habara K, Kwon AH, Kamiyama Y, Ito S, Okumura T: Up-regulation of IL-1 receptor through PI3K/Akt is essential for the induction of iNOS gene expression in hepatocytes. J Hepatol 2004;40:616–623.
12.
Yamada M, Nishizawa M, Nakatake R, Habara K, Yoshida H, Ozaki T, Matsui K, Hamada Y, Kamiyama Y, Ito S, Okumura T: Characterization of alternatively spliced isoforms of the type I interleukin-1 receptor on iNOS induction in rat hepatocytes. Nitric Oxide 2007;17:98–105.
13.
Habara K, Hamada Y, Yamada M, Tokuhara K, Tanaka H, Kaibori M, Kamiyama Y, Nishizawa M, Ito S, Okumura T: Pitavastatin up-regulates the induction of iNOS through enhanced stabilization of its mRNA in proinflammatory cytokine-stimulated hepatocytes. Nitric Oxide 2008;18:19–27.
14.
Matsui K, Nishizawa M, Ozaki T, Kimura T, Hashimoto I, Yamada M, Kaibori M, Kamiyama Y, Ito S, Okumura T: Natural antisense transcript stabilizes inducible nitric oxide synthase mRNA in rat hepatocytes. Hepatology 2008;47:686–697.
15.
Aviles H, O’Donnell P, Sun B, Sonnenfeld G: Active hexose correlated compound (AHCC) enhances resistance to infection in a mouse model of surgical wound infection. Surg Infect (Larchmt) 2006;7:527–535.
16.
Ritz BW, Nogusa S, Ackerman EA, Gardner EM: Supplementation with active hexose correlated compound increases the innate immune response of young mice to primary influenza infection. J Nutr 2006;136:2868–2873.
17.
Daddaoua A, Martínez-Plata E, López-Posadas R, Vieites JM, González M, Requena P, Zarzuelo A, Suárez MD, de Medina FS, Martínez-Augustin O: Active hexose correlated compound acts as a prebiotic and is antiinflammatory in rats with hapten-induced colitis. J Nutr 2007;137:1222–1228.
18.
Fujii H, Nishioka H, Wakame K, Sun B: Nutritional food active hexose correlated compound (AHCC) enhances resistance against bird flu. Nihonhokandaigaeiryougakkaishi (Journal of Japanese Complementary and Alternative Medicine) 2007;4:37–40.
19.
Aviles H, O’Donnell P, Orshal J, Fujii H, Sun B, Sonnenfeld G: Active hexose correlated compound activates immune function to decrease bacterial load in a murine model of intramuscular infection. Am J Surg 2008;195:537–545.
20.
Terakawa N, Matsui Y, Satoi S, Yanagimoto H, Takahashi K, Yamamoto T, Yamao J, Takai S, Kwon AH, Kamiyama Y: Immunological effect of active hexose correlated compound (AHCC) in healthy volunteers: a double-blind, placebo-controlled trial. Nutr Cancer 2008;60:643–651.
21.
Sakagami H, Aoki T: Induction of immunopotentiation activity by a protein-bound polysaccharide, PSK. Anticancer Res 1991;11:993–1000.
22.
Ye SF, Ichimura K, Wakame K, Ohe M: Suppressive effects of Active Hexose Correlated Compound on the increased activity of hepatic and renal ornithine decarboxylase induced by oxidative stress. Life Sci 2003;74:593–602.
23.
Matsui Y, Uhara J, Satoi S, Kaibori M, Yamada H, Kitade H, Imamura A, Takai S, Kawaguchi Y, Kwon AH, Kamiyama Y: Improved prognosis of postoperative hepatocellular carcinoma patients when treated with functional foods: a prospective cohort study. J Hepatol 2002;37:78–86.
24.
Seglen PO: Preparation of isolated rat liver cells. Methods Cell Biol 1976;13:29–83.
25.
Kanemaki T, Kitade H, Hiramatsu Y, Kamiyama Y, Okumura T: Stimulation of glycogen degradation by prostaglandin E2 in primary cultured rat hepatocytes. Prostaglandins 1993;45:459–474.
26.
Horiuti Y, Ogishima M, Yano K, Shibuya Y: Quantification of cell nuclei isolated from hepatocytes by cell lysis with nonionic detergent in citric acid. Cell Struct Funct 1991;16:203–207.
27.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR: Analysis of nitrate, nitrite and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131–138.
28.
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–159.
29.
Nunokawa Y, Ishida N, Tanaka S: Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem Biophys Res Commun 1993;191:89–94.
30.
Hart RP, Liu C, Shadiack AM, McCormack RJ, Jonakait GM: An mRNA homologous to interleukin-1 receptor type I is expressed in cultured rat sympathetic ganglia. J Neuroimmunol 1993;44:49–56.
31.
Sabath DE, Broome HE, Prystowsky MB: Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned T-helper lymphocyte. Gene 1990;91:185–191.
32.
Nishizawa M, Nakajima T, Yasuda K, Kanzaki H, Sasaguri Y, Watanabe K, Ito S: Close kinship of human 20alpha-hydroxysteroid dehydrogenase gene with three aldo-keto reductase genes. Genes Cells 2000;5:111–125.
33.
Unezaki S, Nishizawa M, Okuda-Ashitaka E, Masu Y, Mukai M, Kobayashi S, Sawamoto K, Okano H, Ito S: Characterization of the isoforms of MOVO zinc finger protein, a mouse homologue of Drosophila Ovo, as transcription factors. Gene 2004;336:47–58.
34.
Schreiber E, Matthias P, Müller MM, Schaffner W: Rapid detection of octamer binding proteins with mini-extracts, prepared from a small number of cells. Nucleic Acids Res 1989;17:6419.
35.
Oda M, Sakitani K, Kaibori M, Inoue T, Kamiyama Y, Okumura T: Vicinal dithiol-binding agent, phenylarsine oxide, inhibits iNOS gene expression at a step of NF-ĸB DNA binding in hepatocytes. J Biol Chem 2000;275:4369–4373.
36.
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.
37.
Sakitani K, Kitade H, Inoue K, Kamiyama Y, Nishizawa M, Ito S, Okumura T: Anti-inflammatory drug sodium salicylate inhibits nitric oxide formation induced by interleukin-1β at a translational step, but not a transcriptional step, in hepatocytes. Hepatology 1997;25:416–420.
38.
Inoue T, Kwon AH, Oda M, Kaibori M, Kamiyama Y, Nishizawa M, Ito S, Okumura T: Hypoxia and heat inhibit inducible nitric oxide synthase gene expression by different mechanisms in rat hepatocytes. Hepatology 2000;32:1037–1044.
39.
Matsui K, Kawaguchi Y, Ozaki T, Tokuhara K, Tanaka H, Kaibori M, Matsui Y, Kamiyama Y, Wakame K, Miura T, Nishizawa M, Okumura T: Effect of active hexose correlated compound on the production of nitric oxide in hepatocytes. JPEN J Parenter Enteral Nutr 2007;31:373–381.
40.
Pautz A, Linker K, Hubrich R, Korhonen R, Altenhofer S, Kleivert H: The polypyrimidine tract-binding protein (PTB) is involved in the post-transcriptional regulation of human inducible nitric oxide synthase expression. J Biol Chem 2006;281:32294–32302.
41.
Ozaki T, Habara K, Matsui K, Kaibori M, Kwon AH, Ito S, Nishizawa M, Okumura T: Dexamethasone inhibits the induction of iNOS gene expression through destabilization of its mRNA in proinflammatory cytokine-stimulated hepatocytes. Shock 2010;33:64–69.
© 2011 S. Karger AG, Basel
2011
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.