Elsevier

Life Sciences

Volume 77, Issue 15, 26 August 2005, Pages 1902-1915
Life Sciences

Melatonin ameliorates carbon tetrachloride-induced hepatic fibrogenesis in rats via inhibition of oxidative stress

https://doi.org/10.1016/j.lfs.2005.04.013Get rights and content

Abstract

Melatonin is reported to exhibit a wide variety of biological effects, including antioxidant and anti-inflammatory. Evidence shows the important role of oxidative stress in the etiopathogenesis of hepatic fibrosis. The aim of this study was to investigate the protective effects of administration of melatonin in rats with carbon tetrachloride-induced fibrosis for 6 weeks. Hepatic fibrotic changes were evaluated biochemically by measuring tissue hydroxyproline levels and histopathogical examinations. Malondialdehyde (MDA), an end product of lipid peroxidation, and glutathione peroxidase (GSH-px) and superoxide dismutase (SOD) levels were evaluated in tissue homogenates by spectrophotometry. The nuclear factor-kappaB (NF-κB) in liver tissue was examined by immunohistochemistry. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) concentrations in Kupffer cells (KCs) culture supernatants were measured with ELISA. The rats injected subcutaneously with CCl4 for 6 weeks resulted in hepatic fibrotic changes increased hydroxyproline and MDA levels, and decreased GSH-px and SOD levels, whereas melatonin reversed these effects. Furthermore, melatonin inhibited the expression of NF-κB in liver tissue and decreasing production of proinflammatory cytokines such as TNF-α and IL-1β from KCs in fibrotic rats. These present results suggest that melatonin ameliorates carbon tetrachloride-induced hepatic fibrogenesis in rats via inhibition of oxidative stress and proinflammatory cytokines production.

Introduction

Hepatic fibrosis is a progressive pathological process involving multiple cellular and molecular events that lead ultimately to the accumulation of collagen and extra cellular matrix proteins in the space of Disse. Chronic injury leading to fibrosis in the liver occurs in response to a variety of insults, including viral hepatitis (especially hepatitis B in China), alcohol abuse, drugs, metabolic diseases, etc. When this injury process is combined with ineffective regeneration and repair, there is increasing distortion of the normal liver architecture, and the end result is cirrhosis (Safadi and Friedman, 2002, Friedman, 1993, Friedman, 2000). Current evidence indicates that hepatic fibrosis even cirrhosis is dynamic and can be bidirectional (involving phases of progression and regression) (Pinzani and Rombouts, 2004). Unfortunately, no effective hepatic antifibrotic therapies are available until now. Several lines of evidence have suggested the important role of oxidative stress in the etiopathogenesis of hepatic fibrosis (Shimizu, 2001, Shimizu, 2003, Parola and Robino, 2001).

Oxidative stress aggravates liver fibrosis via hepatic stellate cell activation (Parola and Robino, 2001, Poli, 2000), and lipid peroxidation stimulates the collagen gene transcription in cell culture of fibroblasts and hepatic stellate cells (HSC) (Lee et al., 1995, Parola et al., 1993). Furthermore, proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1 β) produced by Kupffer cells (KCs) also play an important role in the initiation and perpetuation of HSC activation (Luckey and Petersen, 2001). Thus inhibiting oxidative stress and reducing inflammatory cytokines production were regarded useful antifibrotic strategies in the early stages of hepatic fibrogenesis (Friedman, 2003).

As it is well known, melatonin, the chief secretory product of the pineal gland, participates in many important physiological functions, including anti-inflammatory (Cuzzocrea and Reiter, 2001, Cuzzocrea and Reiter, 2002) and antioxidant (Reiter et al., 1995, Reiter et al., 2000). It detoxifies a variety of free radicals and reactive oxygen intermediates, including the hydroxyl radical, singlet oxygen, peroxynitrite anion and nitric oxide (Tan et al., 1993a, Tan et al., 1993b, Pozo et al., 1994, Allegra et al., 2003). In both in vitro and in vivo experiments, melatonin has been found to protect cells, tissues, and organs against oxidative damage induced by a variety of free-radical-generating agents and processes, e.g., the carcinogen safrole, LPS, carbon tetrachloride (CCl4), ischemia-reperfusion, amyloid-protein, and ionizing radiation. Melatonin also has been reported to stimulate the activities of enzymes and increase gene expression that improve the total antioxidative defense capacity of the organism, i.e., SOD, glutathione peroxidase, and glutathione reductase (Antolin et al., 1996, Pablos et al., 1998, Reiter et al., 2000, Kilanczyk and Bryszewska, 2003, Rodriguez et al., 2004). Moreover, recent studies indicate that melatonin is effective in inhibiting oxidative liver damage. Calvo JR et al. found that melatonin protects against ANIT-induced liver injury with cholestasis in rats, and suggests that this protective effect is likely due to its antioxidative properties and above all to its capacity to inhibit liver neutrophil infiltration, a critical factor in the pathogenesis of ANIT-induced liver injury (Calvo et al., 2001). Melatonin also could dose-dependently reduce liver lipid peroxide content in CCl4 treated rats. This indicated that melatonin exerts a therapeutic effect on CCl4-induced acute liver injury in rats, possibly through its antioxidant action (Ohta et al., 2000, Ohta et al., 2004).

In the present study, the effects of melatonin on CCl4-induced hepatic fibrogenesis during the initial phases in rats were investigated. In addition, the antioxidant and anti-inflammatory properties of melatonin in fibrotic rats were studied.

Section snippets

Drugs and materials

Melatonin, purchased from Sigma Chemical Co. (St. Louis, MO, USA), was dissolved in 0.9% ethanol (≤ 0.01%, v/v) and stored at − 20 °C. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT), RPMI 1640, Hepes buffer, collagenase IV, Pronase E, Dnase, Nycodenz and LPS from Escherichia coli were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Commercial kits used for determining lipid peroxidation (TBARS), glutatione peroxidase (GSH-px) and superoxide dismutase (SOD) activity

Effect of melatonin on liver function

In the model group, the level of ALT and AST increased significantly compared with the control group, whereas the ratio of A / G decreased. Melatonin (5, 10, 20 mg/kg) significantly decreased the elevated serum transaminase activities (Fig. 1), whereas the ratio of A / G had an increasing tendency and had no significance compared with the CCl4 injection only group (Table 1).

Effect of melatonin on hydroxyproline content in liver

Hepatic fibrosis was quantified by the measurement of hepatic hydroxyproline. It was found that the hydroxyproline content of

Discussion

The classic hepatotoxin carbon tetrachloride (CCl4) is widely used to induce liver fibrosis in animal models, as its action on rats partly resembles human liver fibrosis. Oxidative stress has been postulated as a major molecular mechanism involved in this experimental model (Poli, 2000, Reeves and Friedman, 2002, Huang et al., 2003). Furthermore, previous reports suggested that oxidative stresses play an important role for the inactivation of KCs in the initial phase of liver fibrosis in rats

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