Elsevier

Journal of Hepatology

Volume 50, Issue 6, June 2009, Pages 1236-1246
Journal of Hepatology

Polyenephosphatidylcholine prevents alcoholic liver disease in PPARα-null mice through attenuation of increases in oxidative stress

https://doi.org/10.1016/j.jhep.2009.01.025Get rights and content

Background/Aims

Alcoholic liver disease (ALD) is one of the leading causes of cirrhosis and yet efficient therapeutic strategies are lacking. Polyenephosphatidylcholine (PPC), a major component of essential phospholipids, prevented alcoholic liver fibrosis in baboons, but its precise mechanism remains uncertain. We aimed to explore the effects of PPC on ALD using ethanol-fed peroxisome proliferator-activated receptor α (Ppara)-null mice, showing several similarities to human ALD.

Methods

Male wild-type and Ppara-null mice were pair-fed a Lieber-DeCarli control or 4% ethanol-containing diet with or without PPC (30 mg/kg/day) for 6 months.

Results

PPC significantly ameliorated ethanol-induced hepatocyte damage and hepatitis in Ppara-null mice. These effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ROS)-generating enzymes, including cytochrome P450 2E1, acyl-CoA oxidase, and NADPH oxidases, in addition to restoration of increases in Toll-like receptor 4 and CD14. PPC also decreased Bax and truncated Bid, thus inhibiting apoptosis. Furthermore, PPC suppressed increases in transforming growth factor-β1 expression and hepatic stellate cell activation, which retarded hepatic fibrogenesis.

Conclusions

PPC exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ALD as a result of inhibition of the overexpression of ROS-generating enzymes. Our results demonstrate detailed molecular mechanisms of the anti-oxidant action of PPC.

Introduction

Chronic alcohol consumption can cause a wide spectrum of liver abnormalities that ranges from simple steatosis to hepatitis, cirrhosis, and hepatocellular carcinoma. It has been reported that alcoholic liver disease (ALD) remains the most common cause of liver cirrhosis in Western countries [1]. Since the appearance of hepatitis is a predictor of progression to cirrhosis and liver cancer, appropriate therapeutic intervention at this point is important in treating ALD.

Numerous data on the pathogenesis of ALD have been obtained from animal studies [1], [2], [3]. Chronic alcohol consumption induces hepatic oxidative stress due to increased generation of reactive oxygen species (ROS) and/or reduced anti-oxidant capacity. Oxidative stress causes further lipid peroxidation, which can directly damage the membranes of cells and organelles and lead to release of reactive aldehydes with potent pro-inflammatory and pro-fibrotic properties. Chronic alcohol intake also increases gut-derived lipopolysaccaride (LPS) concentration in portal blood, which binds to Toll-like receptor 4 (TLR4)/CD14 complexes and activates nuclear factor-kappa B (NF-κB), triggering pro-inflammatory responses such as induction of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Furthermore, ethanol is singularly so toxic that it can induce hepatocyte apoptosis by itself. These mechanisms are all presumed to contribute to human ALD to varying degrees.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily. Three isoforms of PPARs exist, designated as PPARα, PPARβ/δ, and PPARγ. Of these, PPARα is associated with the control of fatty acid transport and metabolism primarily in the liver [4]. A close relationship between PPARα and the development of ALD is believed to exist since chronic alcohol consumption decreases hepatic PPARα expression and suppresses the transcriptional activity of PPARα-regulated genes [5], [6]. We previously reported that PPARα-null (Ppara−/−) mice fed a 4% ethanol-containing Lieber-DeCarli diet for 6 months exhibited hepatomegaly, macrovesicular steatosis, hepatocyte apoptosis, mitochondrial swelling, hepatitis, and hepatic fibrosis, all of which resembled the clinical and pathological features of patients with ALD [7]. These abnormalities appeared with very high reproducibility and stressful surgical procedures to increase alcohol levels, such as gastric tube insertion, were not required. Therefore, Ppara−/− mice are regarded as a useful animal model to investigate the pathogenesis of human ALD.

Essential phospholipids are highly-purified phosphatidylcholine fractions containing linoleic acid and other unsaturated fatty acids. Polyenephosphatidylcholine (PPC), a major active ingredient in essential phospholipids, has a high bioavailability and affinity for cellular and subcellular membranes and maintains membrane fluidity and function. Several experiments have demonstrated the hepatoprotective effects of PPC [8], [9], [10]. However, the precise molecular mechanism of PPC action against ethanol toxicity has not been fully elucidated in vivo, which prompted us to evaluate the effects of PPC on ALD in greater detail using Ppara−/− mice.

Section snippets

Mice and treatment

Generation of Ppara−/− mice on a Sv/129 genetic background was described previously [11]. The mice were housed in an environment controlled for temperature, humidity, and light (25 °C, 12-h light/dark cycle) and maintained with standard laboratory chow and tap water ad libitum until 12 weeks of age. Male 12-week-old Sv/129 wild-type or Ppara−/− mice (n = 24 in each genotype) weighing 31–35 g were selected, randomly divided into 4 groups, and pair-fed the following diet for 6 months: (1) control

General effect of PPC in 4% ethanol-fed mice

All mice survived treatment and the body weights of mice did not differ in each genotype (Fig. 1A). Although liver TG contents were increased in wild-type and Ppara−/− mice fed a 4% ethanol-containing diet, hepatomegaly and significant elevation of serum AST and ALT levels were observed only in ethanol-treated Ppara−/− mice (Fig. 1B, D, and E), which were consistent with the previous report [7]. In histological examinations, focal necrosis of hepatocytes and infiltration of inflammatory cells,

Discussion

The present study characterized the diverse hepatoprotective effects of PPC on ALD in Ppara−/− mice. Namely, we identified a novel and unique mechanism involving attenuation of hepatic ROS generation through down-regulation of CYP2E1, AOX, NOX-2, and NOX-4. Such action differs from that of well-known anti-oxidants tocopherol and S-adenosyl methionine, which restores glutathione content [1], [20]. The peculiar properties of PPC to inhibit ROS production are considered to be essential in the

Acknowledgement

We thank Trevor Ralph for his editorial assistance.

References (36)

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The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

These authors contributed equally to this work.

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