Elsevier

Biochimie

Volume 93, Issue 5, May 2011, Pages 854-866
Biochimie

Research paper
Downregulation of matrix metalloproteinase-9 by melatonin during prevention of alcohol-induced liver injury in mice

https://doi.org/10.1016/j.biochi.2011.02.007Get rights and content

Abstract

Matrix metalloproteinases (MMPs) have been implicated in inflammatory and degradative processes in several diseases. The study aims to explore the mechanism of MMP-9 regulation in alcohol-induced acute liver injury and its protection by melatonin in mice. Alcohol-induced acute liver injury was induced in female Balb/C mice by ethanol administration and protection studies were carried out with a well-known antioxidant molecule, melatonin. Degree of liver injury was monitored by histological and biochemical analysis of liver tissues. Oral administration of ethanol in mouse caused significant increase in alanine amino transferase (ALT) activity in serum. Depletion of glutathione and enhancement of lipid peroxidation as well as protein oxidation was observed in liver tissues following ethanol treatment. However, melatonin exhibited potent hepatoprotective activity by inhibiting ALT activity and oxidative stress. Additionally, MMP-9 expression was increased by ethanol in a dose and time dependent manner in liver tissue and serum. Increased secretion of proMMP-9 was strongly correlated with the expression of proinflammatory cytokines e.g., tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL6. Melatonin showed hepatoprotective role by downregulation of MMP-9 and upregulation of tissue inhibitor of metalloproteases (TIMP-1) expression in liver tissue. Nuclear factor (NF)-κB, plays an important role in inducing inflammatory genes during oxidative stress, thus the role of NF-κB in ethanol-induced liver injury was investigated. Ethanol induced nuclear translocation of NF-κB and increased degradation of inhibitor of NF-κB (IκBα) in liver tissues. Moreover, ethanol-induced NF-κB translocation into nucleus was inhibited significantly by melatonin. This is the first study to elucidate the induction of MMP-9 expression by NF-κB-dependent pathway in ethanol-induced acute liver injury in mice. This study also identifies the novel role of melatonin in hepatoprotection via MMP-9 down regulation.

Highlights

► Mouse model of alcohol-induced hepatic injury and protection studies. ► Induction of MMP-9 in liver tissue and serum with alcohol administration. ► Attenuation of MMP-9 expression by melatonin and subsequent healing of liver injury.

Introduction

Alcohol consumption is a leading cause of illness and death from liver disease throughout the world and presently there are no effective therapeutic regimens [1], [2]. Binge alcohol drinking is a more potent health hazard caused due to bulk intoxication of alcohol at a single go. Alcohol-induced acute liver injury, which presents initially as acute inflammation then progresses to fatty liver, alcoholic hepatitis and ultimately to fibrosis and cirrhosis [3].

A number of hypotheses regarding the mechanisms by which alcohol causes hepatic injury have been suggested with oxidative stress and proinflammatory cytokine production being leading putative etiological factors [4], [5]. However, the mechanisms by which the extracellular matrix (ECM) is altered during alcohol-induced acute liver injury are largely unknown. Recent evidence suggests that degradation of the ECM by specific enzymes known as matrix metalloproteinases (MMPs) may contribute to the development of alcohol-induced liver injury [6]. MMPs are zinc containing endopeptidase, synthesized initially as zymogen and activated in an uncontrolled way under different pathophysiological conditions [7]. According to their substrate specificity MMPs can be subdivided into several groups like gelatinases, collagenase, stromelysin, matrilysin and membrane type MMPs [8]. Their role in the pathogenesis of alcohol-induced liver damage has been suggested by the observation that several MMPs, including MMP-1 (collagenase-1), MMP-2 (gelatinase A), MMP-9 (gelatinase B), MMP-3 (stromelysin-1) and MMP-11 (stromelysin-3) mRNA are expressed in stellate cells and hepatocytes [9].

MMP-2 and MMP-9 seem to have particular relevance to the liver, as they are critically involved in the degradation of components of the basement membrane such as collagen IV and fibronectin (two major components of the space of Disse) [10], [11]. Humans with chronic hepatitis C demonstrate elevated MMP-9 mRNA expression in liver tissues [12]. However; it has been argued that MMP-9 upregulation in alcohol-induced liver damage is indicative of an inflammatory challenge rather than a necessity for degradative processes. MMPs activities are regulated at several levels e.g., at the level of synthesis, secretion and activation [13].

MMP activity is tightly regulated by its inhibitor, tissue inhibitor of metalloproteinases (TIMPs). The significant role of proinflammatory cytokines such as tumor necrosis factor (TNF) -α and interleukin (IL)-1β has been indicated in both clinical and experimental alcoholic liver diseases [14], [15].

Three major pathways for alcohol metabolism exist in the liver e.g., alcohol dehydrogenase in the cytosol, microsomal ethanol oxidizing system in the endoplasmic reticulum and aldehyde oxidase in the mitochondria. Dysregulation of above pathways lead to over production of reactive oxygen species (ROS), including superoxide, peroxide and hydroxyl radical which has been evidenced to play an important role in various alcohol-induced liver injury e.g., alcoholic liver disease, cirrhosis and fibrosis of the liver [14], [16].

Endotoxin generates due to alcohol injury activates the redox sensitive transcription factor, NF-κB, in Kupffer cells resulting in the induction of certain TNFα that triggers a cascade of biochemical signals [17]. NF-κB a dimeric transcription factor regulates the expression of a spectrum of genes involved in cell survival, inflammation as well as proliferation. Several MMPs including MMP-9,-3 contain the NF-κB binding site in their promoter region that regulates their expression [7]. NF-κB exists mainly as RelA (p65) and p50 heterodimer and is retained in cytoplasm by inhibitor protein inhibitor of NF-κB (IκBα) in unstimulated cells [18].

Melatonin (N-acetyl-5-methoxytryptamine), the principle hormone of pineal gland plays an important role in pulsatility, circadian, and seasonal rhythmicity [19]. It is evidenced that melatonin has protective effect against experimental liver injury induced by various chemical insults including CCl4 [20], [21]. The hepatoprotective mechanisms of melatonin involves the clearance of ROS, regulation of cytokine secretion, and inhibition of apoptosis [22], [23]. We herein studied the role of melatonin on MMP expressions during protection of alcohol-induced acute liver injury. We also investigated the mechanism of MMP-9 regulation by proinflammatory cytokines during liver injury and the action of melatonin thereon. In our knowledge this is the first report that describes the regulation of MMP-9 by melatonin in protection of alcohol-induced acute liver damage in mouse model.

Section snippets

Chemicals

Gelatin from porcine skin, 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB), GSH, thiobarbituric acid (TBA), trichloroaceticacid (TCA), tetraethoxypropane, dinitrophenylhydrazine, guanidine hydrochloride, trifluoroacetic acid, TritonX-100 (TX) and EDTA free protease inhibitors cocktail (cat no- P8340) and melatonin were obtained from Sigma (St. Louis, USA). Standard MMP-9 and MMP-2 were purchased from Chemicon (Hampshire, UK, cat no-CC073). Polyclonal anti-MMP-9 (sc-6841), anti-MMP-2 (sc-10736),

Histological analysis of liver tissue during alcohol-induced liver injury and effect of melatonin thereon

In order to determine the effects of ethanol on acute liver injury, we developed liver injury in animal model by oral administration of ethanol in mice. This approach permits the influence of melatonin to be tested on mouse liver tissues during hepatoprotection. Mice were sacrificed 3 days after ethanol administration and histopathological as well as biochemical analysis were performed in liver tissues and in serum. To check the effect of ethanol on liver function in mice, we first examined the

Discussion

Alcoholic liver disease (ALD) affects millions of patients world-wide every year. Alcoholic liver injury is more severe and prevalent in woman than in man [33]. Herein, the ethanol gavaging in mice is a simple experimental model that mimics key aspects of alcoholic diseases in humans and, is useful for exploring the mechanism and treatment of alcoholic liver diseases. The mice developed pathological changes in liver function after 3 days, such as elevated level of ALT, AST in serum that

Author contributions

AM and SP performed all the experiments and wrote the manuscript. SS designed the study, analyzed the data and edited the manuscript.

Acknowledgements

AM and SP acknowledge the receipt of Senior Research Fellowship from Council of Scientific and Industrial Research, New Delhi, India. Work is supported by grants NBA2007 of DBT, IAP001 and CLP 261 of NTRF, India.

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