Sage Journals: Discover world-class research

Abstract

Background: Active hexose correlated compound (AHCC) is a“ complex compound” containing polysaccharides. AHCC has been reported to improve the prognosis of postoperative hepatocellular carcinoma patients. However, the molecular mechanism of this improvement is not fully understood. In the diseased liver, nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) is considered to be a causal factor for various hepatopathies. In this study, the possibility of AHCC regulation of NO production by iNOS was pursued as a potential liver-protecting mechanism. Methods: Primary cultured rat hepatocytes were treated with interleukin-1β (IL-1β) in the presence or absence of AHCC. NO production, iNOS induction, and iNOS signal were analyzed. Results: IL-1β stimulated iNOS induction through the activation of nuclear factorκ B (NFκB), leading to NO production. The addition of AHCC inhibited NO production, showing >80% inhibition at 8 mg/mL. AHCC also decreased the levels of iNOS protein and mRNA. However, AHCC influenced neither the degradation of inhibitory protein κB (IκB) nor the activation of NFκB stimulated by IL-1β. Transfection experiments with an iNOS promoter-luciferase construct (iNOS-Luc) revealed that AHCC had no effect on the transactivation activity of the iNOS promoter. By contrast, AHCC inhibited the activity of iNOS-Luc containing a 3′untranslated region (UTR) with adenosine and uridine (AU)–rich elements, which shows the stabilizing activity of iNOS mRNA. Conclusions: Results indicated that AHCC inhibits the induction of iNOS at the level of transcription, causing a decrease in NO production in hepatocytes. AHCC seems to decrease the levels of iNOS mRNA by reducing mRNA stabilization rather than inhibiting its synthesis.

It is possible that the inhibitory effect of active hexose correlated compound (AHCC) on the production of nitric oxide through the inhibition of inducible nitric oxide synthase induction is associated with AHCC-induced protection against liver failure.

Get full access to this article

View all access options for this article.

References

Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993;392:2002– 2012.

Harbrecht BG, Billiar TR, Stadler J, et al. Inhibition of nitric oxide synthesis during endotoxemia promotes intrahepatic thrombosis and an oxygen radical-mediated hepatic injury. J Leukoc Biol. 1992 ;52:390– 394.

Bohlinger I, Leist M, Barsig J, Uhlig S, Tiegs G, Wendel A. Interleukin-1 and nitric oxide protect against tumor necrosis factorα -induced liver injury through distinct pathways.Hepatology. 1995;22:1829– 1837.

Saavedra JE, Billiar TR, Williams DL, et al. Targeting nitric oxide (NO) delivery in vivo: design of a liver-selective NO donor prodrug that blocks tumor necrosis factor-α-induced apoptosis and toxicity in the liver. J Med Chem. 1997 ;40:1947– 1954.

Mellouk S, Hoffman SL, Liu ZZ, de la Vega P, Billiar TR, Nussler AK. Nitric oxide-mediated antiplasmodial activity in human and murine hepatocytes induced by gamma interferon and the parasite itself: enhancement by exogenous tetrahydrobiopterin. Infect Immun. 1994 ;62:4043– 4046.

Thiemermann C, Ruetten H, Wu C, Vane JR. The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase. Br J Pharmacol. 1995;116:2845– 2851.

Stadler J, Billiar TR, Curran RD, Stuehr DJ, Ochoa JB, Simmons RL. Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am J Physiol. 1991 ;260:C910– C916.

Tu W, Kitade H, Kaibori M, et al. An enhancement of nitric oxide production regulates energy metabolism in rat hepatocytes after a partial hepatectomy. J Hepatol. 1999 ;30:944– 950.

Tu W, Kitade H, Satoi S, et al. Increased nitric oxide production in hepatocytes is involved in liver dysfunction following obstructive jaundice. J Surg Res. 2002 ;106:31– 36.

10.

Tu W, Satoi S, Zhang Z, et al. Hepatocellular dysfunction induced by nitric oxide production in hepatocytes isolated from rats with sepsis.Shock. 2003;19:373– 377.

11.

Li J, Billiar TR. Nitric oxide, IV: determinants of nitric oxide protection and toxicity in liver. Am J Physiol. 1999 ;276:G1069– G1073.

12.

Chen T, Zamora R, Zuckerbraun B, Billiar TR. Role of nitric oxide in liver injury. Curr Mol Med. 2003 ;3:519– 526.

13.

Milner JA. Functional foods: the US perspective. Am J Clin Nutr. 2000;71(6 Suppl): 1654S–1659S.

14.

Roberfroid MB. Concepts and strategy of functional food science: the European perspective. Am J Clin Nutr. 2000 ;71(6 Suppl):1660S– 1664S.

15.

Matsui Y, Uhara J, Satoi S, et al. Improved prognosis of postoperative hepatocellular carcinoma patients when treated with functional foods: a prospective cohort study. J Hepatol. 2002 ;37:78– 86.

16.

Sun B, Wakame K, Mukoda T, Toyoshima A, Kanazawa T, Kosuna K. Protective effects of AHCC on carbon tetrachloride induced liver injury in mice. Natural Medicines. 1997 ;51:310– 315.

17.

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.

18.

Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29– 83.

19.

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.

20.

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.

21.

Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156– 159.

22.

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.

23.

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.

24.

Nishizawa M, Nakajima T, Yasuda K, et al. Close kinship of human 20alpha-hydroxysteroid dehydrogenase gene with three aldo-keto reductase genes. Genes Cells. 2000;5:111– 125.

25.

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 .

26.

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-kB DNA binding in hepatocytes. J Biol Chem. 2000;275:4369– 4373.

27.

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.

28.

Inoue T, Kwon A-H, Oda M, et al. Hypoxia and heat inhibit inducible nitric oxide synthase gene expression by different mechanisms in rat hepatocytes. Hepatology. 2000 ;32:1037– 1044.

29.

Sakitani K, Kitade H, Inoue K, et al. The 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.

30.

Kitade H, Sakitani K, Inoue K, et al. 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.

31.

Eberhardt W, Kunz D, Hummel R, Pfeilschifter J. Molecular cloning of the rat inducible nitric oxide synthase gene promoter. Biochem Biophys Res Commun. 1996;223:752– 756.

32.

Xie Q-W, Whisnant R, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferonγ and bacterial lipopolysaccharide. J Exp Med. 1993 ;177:1779– 1784.

33.

Nunokawa Y, Oikawa S, Tanaka S. Human inducible nitric oxide synthase gene is transcriptionally regulated by nuclear factor-κB dependent mechanism. Biochem Biophys Res Commun. 1996 ;223:347– 352.

34.

Akira S, Kishimoto T. NF-IL6 and NF-κB in cytokine gene regulation. Adv Immunol. 1997 ;65:1– 46.

35.

Kleinert H, Pautz A, Linker K, Schwarz PM. Regulation of the expression of inducible nitric oxide synthase. Eur J Pharmacol. 2004 ;500:255– 266.

36.

Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A. Identification of a common nucleotide sequence in the 3′-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci U S A. 1986;83:1670– 1674.

Effect of Active Hexose Correlated Compound on the Production of Nitric Oxide in Hepatocytes

Abstract

Get full access to this article

References