Inhibitory effect of glycyrrhizin on lipopolysaccharide and d-galactosamine-induced mouse liver injury

https://doi.org/10.1016/j.ejphar.2007.08.012Get rights and content

Abstract

The effects of glycyrrhizin isolated from licorice root were investigated on acute hepatitis induced by lipopolysaccharide (LPS) and d-galactosamine in mice. Serum alanine aminotransferase (ALT) activity was markedly increased 6 h to 8 h after administration of LPS/d-galactosamine. Levels in serum of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 reached a maximum by 2 h, whereas levels of IL-18, as well as of ALT, were maximal at 8 h. Glycyrrhizin (ED50: 14.3 mg/kg) inhibited the increase in ALT levels when it was given to mice at 30 min before administration of LPS/d-galactosamine. Inflammatory responses, including infiltration of neutrophils and macrophages in the liver injury, were modulated by glycyrrhizin. Increases in ALT levels were reduced by an administration of glycyrrhizin at 10 min and 60 min but not 3 h, even after LPS/d-galactosamine treatment. However, glycyrrhizin had no effect on the production of TNF-α, IL-6, IL-10 and IL-12, whereas it significantly inhibited IL-18 production. Exogenous IL-18 further increased the elevation in ALT levels in mice treated with LPS/d-galactosamine. Glycyrrhizin completely suppressed the effect of IL-18 of increasing ALT levels. IL-18 was detected by immunohistochemistry in inflammatory cells such neutrophils and macrophages in liver injury. Glycyrrhizin reduced the responsiveness of cells to IL-18 in the liver injury. These results suggest that glycyrrhizin inhibits the LPS/d-galactosamine-induced liver injury through preventing inflammatory responses and IL-18 production. Furthermore, it seems that glycyrrhizin prevents IL-18-mediated inflammation in liver injury.

Introduction

Glycyrrhizin, a saponin isolated from the licorice root (Glycyrrhiza spp), is known to be effective as an anti-inflammatory (Ohuchi et al., 1981, Okimasu et al., 1983) and an anti-allergic agent (Kuroyanagi and Saito, 1966), and has a steroid-like action (Kumagai et al., 1957). Some other effects of glycyrrhizin have been found, such as inhibition of virus growth (Pompei et al., 1979, Ito et al., 1988) and interferon (IFN)-inducing activity (Abe et al., 1982). Furthermore, glycyrrhizin inhibits liver injuries in animal models (Nose et al., 1994, Okamoto and Kanda, 1999, Okamoto, 2000) and induces hepatocyte proliferation in rats (Kimura et al., 2001). In Japan, Stronger Neo-Minophagen C, the active ingredient of which is glycyrrhizin, has been used as a treatment for over 25 years for patients with chronic hepatitis. The intravenous administration of Stronger Neo-Minophagen C decreases elevated serum alanine aminotransferase (ALT) activity in patients with chronic viral hepatitis C and reduces the progression of liver disease to hepatocelluar carcinoma (Arase et al., 1997, Ikeda et al., 2006). The effects and safety of Stronger Neo-Minophagen C treatment in patients with chronic hepatitis C have also been evaluated in Europe (van Rossum et al., 1999, van Rossum et al., 2001). However, the mechanisms by which glycyrrhizin inhibits hepatitis have not been clearly identified.

Endotoxemia occurs frequently in cases of liver failure (Nolan, 1975, Nakao et al., 1994) and is thought to play a role in the pathogenesis of liver diseases (Nolan, 1989). d-Galactosamine is an amino sugar selectively metabolized by hepatocytes, which induces a depletion of the uridine triphosphate pool and thereby an inhibition of macromolecule (RNA, protein and glycogen) synthesis in the liver (Decker and Keppler, 1974). The combination of lipopolysaccharide (LPS) and d-galactosamine causes hepatic failure in rodents (Galanos et al., 1979). Upon stimulation with LPS, liver macrophages secrete various pro-inflammatory cytokines including tumor necrosis factor (TNF)-α which is a terminal mediator for apoptosis, subsequently leading to hepatic necrosis (Tiegs et al., 1989, Leist et al., 1995, Sass et al., 2002). Furthermore, this model provides a practical tool for the evaluation of drugs or compounds that interfere with hepatic apoptosis as well as with inflammatory liver injury (Tiegs, 1994).

IL-18 was originally identified as an INF-γ-inducing factor (Okamura et al., 1995) and has been suggested to be a potent inflammatory cytokine that regulates autoimmune and inflammatory diseases (Nakanishi et al., 2001, Dinarello and Fantuzzi, 2003, Gracie et al., 2003). On LPS stimulation, IL-18 is produced by Kupffer cells (liver macrophages), B-cells and dendritic cells (Ghayur et al., 1997). There is evidence that IL-18 plays a critical role in Propionibacterium acnes and LPS-induced liver injury (Okamura et al., 1995, Sakano et al., 1999). IL-18 stimulates gene expression and synthesis of TNF-α, IL-1, Fas ligand and several chemokines (Dinarello, 1999, Nakanishi et al., 2001, Nakahira et al., 2002). IL-18 deficient mice are resistant to LPS-induced liver injury (Sakano et al., 1999). In humans, IL-18 is involved in the pathogenesis of acute hepatic injury (Yumoto et al., 2002) and of chronic hepatitis C (Jia et al., 2002, Ludwiczek et al., 2002). Treatment with interferon and ribavirin induces a significant decrease in serum IL-18 concentration (Marin-Serrano et al., 2006). However, it has been unclear whether IL-18 participates in LPS/d-galactosamine-induced mouse liver injury.

In the present study, we have examined the effect of glycyrrhizin on LPS/d-galactosamine-induced liver injury and the involvement of IL-18 in this model.

Section snippets

Experimental animals

Six-week-old male BALB/c mice weighing 23–25 g were purchased from Japan SLC (Hamamatsu, Japan). The animals were kept in an environmentally controlled room (24 ± 1 °C, 55 ± 10% humidity) and allowed free access to food and water. This study was performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society.

Materials

Lipopolysaccharide (Escherichia coli, O55:B5) and d-galactosamine were purchased from Sigma (St. Louis, MO, USA).

Changes in serum ALT and cytokine levels in mice treated with LPS/d-galactosamine

Intravenous administration of LPS/d-galactosamine in the mice stimulated inflammation of the liver associated with alteration in cytokine levels. Serum ALT levels were markedly increased 6 h to 8 h after LPS/d-galactosamine treatment (Fig. 1). Levels of TNF-α, IL-6, IL-10 and IL-12 became maximal by 2 h after treatment, while an elevation in serum concentrations of IL-18, as well as ALT, was significantly greater by 8 h.

Effect of glycyrrhizin on LPS/d-galactosamine-induced liver injury

Glycyrrhizin with an ED50 value of 14.3 mg/kg dose-dependently inhibited

Discussion

The induction of liver injury in mice by LPS/d-galactosamine represents a promising animal model for elucidating the mechanism of clinical dysfunction and for evaluating the efficacy of hepatoprotectives. TNF-α is a key factor that contributes to the triggering of an inflammatory cascade involving the induction of cytokines such as INF-γ and IL-1β (Tiegs et al., 1989, Oku et al., 2002). Furthermore, liver injury induced by LPS has been reported to be abrogated by treatment with anti-TNF-α mAb

Acknowledgments

The authors acknowledge Dr. A.F. Walls, Southampton General Hospital, UK, for reading the manuscript in preparation. We also thank Ms. Chieko Sasagawa, Pharmacological Research Department, for technical assistance.

References (45)

  • TiegsG.

    Immunotoxicology of host-response-mediated experimental liver injury

    J. Hepatol.

    (1994)
  • TiegsG. et al.

    Tumor necrosis factor is a terminal mediator in galactosamine/endotoxin-induced hepatitis in mice

    Biochem. Pharmacol.

    (1989)
  • van RossumT.G.J. et al.

    Glycyrrhizin induced reduction of ALT in European patients with chronic hepatitis C

    Am. J. Gastroenterol.

    (2001)
  • AbeN. et al.

    Interferon induction by glycyrrhizin and glycyrrhetinic acid in mice

    Microbiol. Immunol.

    (1982)
  • AraseY. et al.

    The long term efficacy and glycyrrhizin in chronic hepatitis C patients

    Cancer

    (1997)
  • BeutlerB. et al.

    Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin

    Science

    (1985)
  • ChosayJ.S. et al.

    Neutrophil margination and extravasation in sinusoids and venules of liver during endotoxin-induced injury

    Am. J. Physiol.

    (1997)
  • DeckerK. et al.

    Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death

    Rev. Physiol., Biochem. Pharmacol.

    (1974)
  • DinarelloC.A. et al.

    Interleukin-18 and host defense against infection

    J. Infect. Dis.

    (2003)
  • GalanosC. et al.

    Galactosamine-induced sensitization to the lethal effects of endotoxin

    Proc. Natl. Acad. Sci. U. S. A.

    (1979)
  • GhayurY. et al.

    Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production

    Nature

    (1997)
  • GracieJ.A. et al.

    Interleukin-18

    J. Leukoc. Biol.

    (2003)
  • Cited by (0)

    View full text