Immunopharmacology and Inflammation
Melatonin protects against alcoholic liver injury by attenuating oxidative stress, inflammatory response, and apoptosis

https://doi.org/10.1016/j.ejphar.2009.06.044Get rights and content

Abstract

Melatonin is reported to exhibit a wide variety of biological effects, including antioxidant and anti-inflammatory. Previous studies show that melatonin has a protective role in different types of liver injury and fibrosis. But its role in the pathogenesis of alcoholic liver injury remains obscure. The present investigation was designed to determine the effects of melatonin on alcohol-induced hepatic injury in mice. The degree of alcoholic liver injury was evaluated by measuring serum markers and pathological examination. Treatment with melatonin significantly attenuated the increased level of serum aminotransferase, reduced the severe extent of hepatic cell damage, steatosis and the immigration of inflammatory cells, but had no effects on hepatic expression of lipogenic genes. Furthermore, melatonin decreased serum and tissue inflammatory cytokines levels, tissue lipid peroxidation, neutrophil infiltration and inhibited the apoptosis of hepatocytes. Kupffer cells isolated from ethanol-fed mice produced high amounts of reactive oxygen species and tumor necrosis factor alpha, whereas Kupffer cells from melatonin treatment mice produced less reactive oxygen species and tumor necrosis factor alpha compared with model alcohol-feeding mice. These findings suggest that melatonin may represent a novel, protective strategy against alcoholic liver injury by attenuating oxidative stress, inflammatory response and apoptosis.

Introduction

Alcohol drinking is a major etiologic factor in chronic liver disease worldwide, causing fatty liver, alcoholic hepatitis, cirrhosis, and/or hepatocellular carcinoma (Williams, 2006). In spite of the past few decades of research, our understanding of the pathogenesis of alcohol hepatotoxicity is still limited. An important progress in this field has been the appreciation of the role of oxidative stress and inflammatory responses in the pathogenesis of alcohol liver damage (Vidali et al., 2008). These mechanisms include the activation of intrahepatic macrophages, also known as Kupffer cells, releasing proinflammatory cytokines, chemokines and reactive oxygen species, as well as complex interactions between alcohol metabolism, various hepatic cells, multiple cytokines, and the immune system (Arteel, 2003, Dey and Cederbaum, 2006, Hoek and Pastorino, 2002, Nagy, 2003). Studies using animal models suggest that alcohol intake permeabilizes the gut, resulting in increased hepatic endotoxin levels, which is followed by activation of Kupffer cells. Activated Kupffer cells generate oxidative stress and produce a variety of soluble factors and cytokines (Yin et al., 1999). Among them, tumor necrosis factor alpha (TNF-α) is considered as one of the most important cytokines involved in the pathogenesis of alcoholic liver injury (Ji et al., 2004, Yin et al., 1999).

As it is well known, melatonin, the chief secretory product of the pineal gland, participates in many important physiological functions, including anti-inflammatory and antioxidant (Cuzzocrea and Reiter, 2002, Korkmaz et al., 2009). In both in vitro and in vivo experiments, melatonin detoxifies a variety of free radicals and reactive oxygen intermediates, including the hydroxyl radical, singlet oxygen, peroxynitrite anion and nitric oxide. 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., superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (Subramanian et al., 2007, Tan et al., 2007). Moreover, recent studies indicate that melatonin is effective in inhibiting oxidative liver damage. Calvo et al. (2001) found that melatonin protects against alpha-naphthylisothiocyanate-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 alpha-naphthylisothiocyanate-induced liver injury. Melatonin also dose-dependently reduced liver lipid peroxide content in carbon tetrachloride treated rats. This indicated that melatonin exerts a therapeutic effect on carbon tetrachloride-induced acute liver injury in rats, possibly through its antioxidant action (Ohta et al., 2003, Ohta et al., 2000). Wang et al., 2004, Wang et al., 2005a, Wang et al., 2005b reported that melatonin protects immune liver injury and liver fibrosis by inhibiting inflammatory cytokines and oxidative stress.

Although increasing studies show that melatonin has a protective role in different types of liver injury and fibrosis, its role in the pathogenesis of alcoholic liver injury remains obscure. The present investigation was designed to determine the effects of melatonin on alcohol-induced hepatic injury in mice. Our findings suggest that melatonin may represent a novel, protective strategy against alcoholic liver injury by attenuating oxidative stress, inflammatory response, and apoptosis.

Section snippets

Drugs and chemicals

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. 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 (malondialdehyde, MDA), GPx and SOD activity were obtained from the Jiancheng Institute of Biotechnology (Nanjing, China). ELISA kits of tumor necrosis

Melatonin improves alcohol-induced hepatic injury

Alcohol-induced liver injury was indicated by elevated serum ALT, hepatic triglyceride and liver pathological changes characterized by hepatic cellular necrosis, steatosis and inflammation. For assessment of tissue damage of the liver, the serum transaminase ALT and AST activities were measured. There was a slight increase in serum ALT and AST activities after alcohol feeding, treatment with melatonin significantly reduced the ALT and AST level (Fig. 1B). H&E and oil red staining revealed

Discussion

It is widely accepted that chronic alcohol consumption permeabilizes the gut, leading to the accumulation of endotoxins in the liver. These endotoxins activate Kupffer cells produce reactive oxygen species and a variety of soluble factors and cytokines (e.g., TNF-α), precipitating liver injury (Uesugi et al., 2002). The present findings suggest that during alcoholic liver injury, administration of antioxidant melatonin can block this progress by attenuating oxidative stress and inflammatory

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