An adenosine derivative compound, IFC305, reverses fibrosis and alters gene expression in a pre-established CCl4-induced rat cirrhosis

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Abstract

Cirrhosis is a complex process that involves a dynamic modification of liver cell phenotype associated to gene expression changes. This study investigates the reversing capacity of an adenosine derivative compound (IFC305) on a rat model of liver cirrhosis and gene expression changes associated with it. Rats were treated with IFC305 or saline for 5 or 10 weeks after cirrhosis induction (CCl4 treatment for 10 weeks). Fibrosis score, collagenase activity and amount of hepatic stellate cells (HSC, activated and with a lipid-storing phenotype) were measured in livers. In addition, gene expression analysis was performed using 5K DNA microarrays and quantitative RT-PCR. Treatment of cirrhotic rats with IFC305 for 5 or 10 weeks compared to saline control, induced: (1) reduction of fibrosis (50–70%) and of collagen, of α-SMA and desmin proteins, as well as of activated HSCs in liver, (2) increased collagenase activity and cell number of lipid-storing HSC, (3) improved serum parameters of liver function, such as reduced activity of aminotransferases and bilirubin. Expression of 413 differential genes, deregulated in cirrhotic samples, tended to be normalized by IFC305 treatment. Some genes modulated at transcript level by IFC305 were Tgfb1, Fn1, Col1a1, C9, Apoa1, Ass1, Cps1, and Pparg. The present study shows that IFC305 reverses liver fibrosis through modulation of adipogenic and fibrosis-related genes and by ameliorating hepatic function. Thus, understanding of the anti-cirrhotic effect of IFC305 might have therapeutical potential in patients with cirrhosis.

Introduction

Cirrhosis or extensive liver fibrosis in patients is still an important cause of death worldwide. Although mortality due to cirrhosis has decreased in several countries during the last two decades, it is still extremely high in developing countries like Mexico (Bosetti et al., 2007). Cirrhosis is a highly dynamic pathological state associated to hepatic cells differentiation, phenotype changes, and cellular behavior, these events are induced by chronic liver injury that produces an inflammation process that lead to liver fibrogenesis, for review (Bataller and Brenner, 2005). Chronic damage of the liver produces hepatocyte-nodular lesions due to high cell turnover, whereas hepatic stellate cells (HSC), with a quiescent retinoid-storing phenotype, are activated to fibrogenic proliferative cell type that lack the retinoid-storing ability, these activated HSC also resemble a myofibroblast phenotype characterized by overproduction of extracellular matrix proteins (ECM), such as collagen, fibronectin, tenasin, and ondulin. Thus, parenchymal liver tissue is replaced by accumulated scar fibrous tissue, leading to deterioration of hepatic function (Friedman, 2005). Liver failure is associated, for example, with a disturbed urea cycle producing excessive blood ammonia and hepatic encephalopathy (Blei, 2004).

Chronic CCl4-intoxication of rats has been extensively used as model of liver fibrosis, resulting in cirrhosis when prolonged for more than 8–12 weeks (Iredale, 2007). We reported previously the fibrosis-reversing capacity of adenosine treatment on CCl4-induced cirrhotic rats. This effect has been associated with enhanced collagenolytic activity, diminution of tissue inhibitor of metalloproteinases (TIMPs) 1 and 2, modulation of cell redox state, maintenance of liver energy availability, and preservation of an adequate mitochondrial function of hepatic cells (Hernandez-Munoz et al., 1994, Hernandez-Munoz et al., 1997, Hernandez-Munoz et al., 2001). Adenosine has been proposed as a promising hepatoprotector substance for hepatic cirrhosis.

In this study, we used IFC305, an adenosine-aspartate derivative, which hypothetically includes the hepatoprotective effect of adenosine and the additional possible effect of aspartate favoring urea cycle in the liver as reported (Yassuda Filho et al., 2003). The results presented here, in CCl4-model rats, demonstrate that treatment with IFC305 reversed hepatic fibrosis, reduced presence of activated HSCs, increased the number of lipid-storing HSCs, and modified global gene expression of fibrogenesis and lipid metabolism-related genes, as well as the rate-limiting enzymes of the urea cycle (Yamamoto and Sugihara, 1988). Our findings suggest that reversal of fibrosis by IFC305 could be therapeutically efficacious in the treatment of cirrhosis.

Section snippets

Chemicals

CCl4 was from Merck Mexico (Mexico), all other reagents were from Sigma Chemical Co. (St. Louis, MO). IFC305 is the aspartate salt of adenosine 2-aminosuccinic acid–2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (1:1). It was obtained as a white solid with the following properties; molecular Weight 400.34, melting point 285 °C, water-soluble. Synthesized by Probiomed S.A. de C.V. (http://www.probiomed.com.mx, Mexico City, Mexico).

Animals treatment and induction of cirrhosis with CCl4

Male Wistar rats (n = 25) weighing 100–110 g

Effects of IFC305 treatment on rat cirrhosis

Accumulated liver fibrosis of CCl4-treated rats associated to reduction of hepatic parenchymal tissue was clearly denoted by Masson's trichrome staining (Fig. 2A), decreased fibrosis was evident in liver of IFC305-treated animals (Fig. 2B). Quantitative analysis of histological sections shows that liver fibrosis corresponded to 16% of the area in Ci-0 group and remained at 8% at 10 weeks after CCl4 withdrawal (Fig. 2C). Presence of liver fibrosis was associated to a reduced liver parenchyma

Discussion

This in vivo study was designed to analyze the effect of an adenosine derivative compound, IFC305, on the reversibility of CCl4-induced liver cirrhosis in rats. Fibrogenic events, such as increased collagen deposition, decreased number of quiescent HSCs and increased presence of fibroblastic α-SMA-positive cells persisted in cirrhotic samples at 5 and 10 weeks after CCl4 cessation. Parameters indicative of liver dysfunction (AST, ALT, and bilirubin) were increased in rat sera. Other indicators

Acknowledgments

We thank S. Guzmán, J. Santillán and L. Chávez from our institutional Microarray facility, G. Coello, G. Corral and A. Gómez for GenArise software assistance. R. Paredes for microscopy studies, and J. Barbosa for image design. Grant support from Consejo Nacional de Ciencia y Tecnología CONACYT 41433-Q

References (52)

  • T. Knittel et al.

    Transforming growth factor beta 1-regulated gene expression of Ito cells

    Hepatology

    (1996)
  • D. Luna-Moreno et al.

    Food restricted schedules promote differential lipoperoxidative activity in rat hepatic subcellular fractions

    Comp Biochem Physiol A Mol Integr Physiol

    (2007)
  • J. Mann et al.

    Transcriptional regulation of hepatic stellate cells

    Adv Drug Deliv Rev

    (2009)
  • G. Marchesini et al.

    Effect of S-adenosyl-l-methionine administration on plasma levels of sulphur-containing amino acids in patients with liver cirrhosis

    Clin Nutr

    (1992)
  • M.L. Martinez-Chantar et al.

    Importance of a deficiency in S-adenosyl-l-methionine synthesis in the pathogenesis of liver injury

    Am J Clin Nutr

    (2002)
  • K. Maruyama et al.

    Mammalian collagenase increases in early alcoholic liver disease and decreases with cirrhosis

    Life Sci

    (1982)
  • P. Mathurin et al.

    Quantification of apolipoprotein A-I and B messenger RNA in heavy drinkers according to liver disease

    Hepatology

    (1996)
  • G. Ramadori et al.

    Synthesis of cellular fibronectin by rat liver fat-storing (Ito) cells: regulation by cytokines

    Gastroenterology

    (1992)
  • M. Rojkind et al.

    An improved method for determining specific radioactivities of proline-14C and hydroxyproline-14C in collagen and in noncollagenous proteins

    Anal Biochem

    (1974)
  • S. Tsukada et al.

    SMAD and p38 MAPK signaling pathways independently regulate alpha1(I) collagen gene expression in unstimulated and transforming growth factor-beta-stimulated hepatic stellate cells

    J Biol Chem

    (2005)
  • H. Yamamoto et al.

    Blood ammonia levels and hepatic encephalopathy induced by CCl4 in rats

    Toxicol Appl Pharmacol

    (1987)
  • H.A. Yamamoto et al.

    Hepatic ATP content and hyperammonemia induced by CCl4 in rats

    Toxicology

    (1988)
  • M.D. Abramoff et al.

    Image processing with image

    J Biophoton Int

    (2004)
  • R. Bataller et al.

    Liver fibrosis

    J Clin Invest

    (2005)
  • R. Bruck et al.

    Additive inhibitory effect of experimentally induced hepatic cirrhosis by agonists of peroxisome proliferator activator receptor gamma and retinoic acid receptor

    Dig Dis Sci

    (2009)
  • V. Chagoya de Sanchez

    Circadian variations of adenosine and of its metabolism. Could adenosine be a molecular oscillator for circadian rhythms?

    Can J Physiol Pharmacol

    (1995)
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