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* Department of Biochemistry and Molecular Genetics,
Pathology Laboratory and
Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona School of Medicine, Barcelona, Spain; and
Department of Pharmacology, New York Medical College, Valhalla, New York, USA
1Correspondence: Department of Biochemistry and Molecular Genetics, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. E-mail: jclaria{at}clinic.ub.es
SPECIFIC AIM
A new family of bioactive lipid mediators generated from docosahexaenoic acid (DHA) that carry potent anti-inflammatory properties has recently been identified. The aim of the current study was to assess the formation of these DHA-derived products in the liver and to explore whether these lipid signals protect liver cells from necroinflammatory injury.
PRINCIPAL FINDINGS
1. Supplementation of hepatocyte cultures with DHA significantly reduces genotoxic and oxidative damage
To test in vitro the potential protective effects of 
-3 essential fatty acids on liver cells, hepatocytes were grown in medium supplemented with DHA; DNA damage was assessed by a single-cell gel electrophoresis assay, the so-called comet assay. Results indicate that the length of comet tails, and therefore the extent of DNA strand breaks induced by hydrogen peroxide, was significantly lower in hepatocytes growing in medium supplemented with DHA than in hepatocytes growing in medium alone. Oxidative stress levels [i.e., malondialdehyde (MDA) levels measured by HPLC in cell lysates] were significantly decreased in hepatocytes growing in a DHA-rich medium.
2. Dietary supplementation of mice with DHA blunts carbon tetrachloride (CCl4) -induced necroinflammatory damage
To extend the protective actions of DHA observed in hepatocytes at the in vivo level, we administered diets supplemented with DHA to mice submitted to the experimental model of CCl4-induced liver injury and compared the hepatic necroinflammatory damage with that of mice receiving a control diet. Histological examination of livers from mice treated with CCl4 for 5 wk revealed massive and severe hepatocyte necrosis, inflammation, and ballooning at the centrilobular zone with bridging of necrosis that severely disrupted the sinusoidal and lobular architecture of the liver (Fig. 1
A, upper panels). A significant improvement in hepatic pathology occurred in mice fed experimental diets enriched with DHA (Fig. 1A
, middle and lower panels). The scores for necroinflammatory liver injury and hepatocyte ballooning were significantly lower in mice fed -3-enriched experimental diets than in those receiving a control diet (Fig. 1B
).
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3. Detection of the novel bioactive lipid mediators 17S-hydroxy-DHA (17S-HDHA) and protectin D1 in the liver of mice fed a DHA-enriched diet
We next assessed the profile of bioactive lipid mediators generated in livers from mice fed DHA-enriched diets. We first monitored changes in the proinflammatory cyclooxygenase (COX)-2 pathway and found that administration of diets enriched with -3 essential fatty acids led to significant decreases in hepatic COX-2 mRNA expression and prostaglandin (PG) E2 levels. When we analyzed the profile of lipid mediators in the liver of mice fed a DHA-enriched diet by HPLC gas chromatography/mass spectrometry, we identified thepotent anti-inflammatory and proresolution DHA-derived lipid signals, protectin D1 and 17S-HDHA. These unique products were identified by coelution with the biosynthetic standards and by their characteristic UV spectra consistent with the presence of a conjugated triene double bond structure for protectin D1 and a conjugated diene chromophore for 17S-HDHA. The identity of these DHA-derived products was further confirmed by mass spectrometry analysis, which displayed ions at m/z 503, 413, and 323 (diagnostic for a dihydroxy DHA structure) and at m/z 415 and 325.
4. Lipid signals identified in the liver of mice supplemented with DHA display potent biological activity on hepatocytes and macrophages
To explore whether DHA-derived lipid signals could mediate the hepatoprotective effects associated with DHA-enriched diets, we set out a series of experiments in hepatocytes and macrophages, the cell type that plays a major role in initiating the cascade of events leading to liver inflammation and injury. As shown in Fig. 2
, 17-HDHA, a lipid signal identified in the liver of mice fed a DHA-enriched diet and a marker of activation of the protectin D1 pathway, displayed potent biological activity in these cells. This lipid mediator abrogated genotoxic damage (Fig. 2A
), improved oxidative stress status (Fig. 2B
) in hepatocytes, and significantly inhibited TNF-
release by murine macrophages (Fig. 2C
). In addition, 17-HDHA decreased macrophage 5-LO expression. In a cell-based PPAR
and luciferase reporter transactivation assay, synthetic 17-HDHA acted in a concentration-dependent manner as an agonist of PPAR, a central inhibitor of inflammatory and fibrogenic responses in the liver.
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CONCLUSIONS AND SIGNIFICANCE
Virtually every type of liver disease has an inflammatory component as its underlying cause. Although beneficial as a limited wound-healing process, if the inflammatory response is not resolved it results in tissue damage, scar accumulation, fibrosis, and eventually in the life-threatening condition of liver cirrhosis. Therefore, developing novel strategies to modulate the factors that govern hepatic inflammation represents a primary focus for disrupting the sequence of events leading to liver injury.
Long chain -3 essential fatty acids display potent anti-inflammatory properties. Dietary interventions rich in DHA and/or eicosapentaenoic acid (EPA) have been shown to keep inflammation under control without side effects and are used as preventive measures against illnesses such as rheumatoid arthritis, cystic fibrosis, ulcerative colitis, asthma, atherosclerosis, cancer, and cardiovascular disease. Although the molecular mechanisms underlying the beneficial actions of -3 essential fatty acids remain to be clearly defined, a new series of bioactive lipid mediators generated from these fatty acids has recently been identified. During the resolution phase of acute inflammatory response, cell-cell interactions and transcellular biosynthesis lead to production of novel bioactive lipid mediators from DHA and EPA, termed resolvins (resolution phase interaction products) and protectin D1. Protectin D1, a bioactive lipid mediator derived from DHA, potently regulates critical events associated with inflammation and its resolution, including inhibition of PMN infiltration and T cell migration, and reduction of cytokine and chemokine formation and IL-1-induced NF
B activation.
Results of this study demonstrate that dietary interventions rich in DHA modulate hepatic inflammation and may help prevent necroinflammatory liver injury. The decrease in COX-2 expression and PGE2 levels noted in livers from animals receiving a DHA-enriched diet was associated with an increase in the formation of DHA-derived anti-inflammatory mediators, protectin D1 and 17S-HDHA. The potential protective actions of these DHA-derived lipid signals in the liver was further supported by experiments in vitro demonstrating that synthetic 17-HDHA attenuated DNA damage and oxidative stress in hepatocytes, and key markers of inflammation in macrophages (Fig. 3
). 17-HDHA inhibited TNF- release in murine macrophages. Since an increased production of TNF- by liver macrophages is implicated in the pathogenesis of alcoholic liver disease and liver fibrosis, down-regulation of this key proinflammatory cytokine in macrophages treated with 17-HDHA may in part account for the protective effects of dietary DHA in necroinflammatory liver injury. We noted a decrease in 5-LO protein expression in macrophages treated with 17-HDHA. Earlier studies established that inhibition of 5-LO attenuates necroinflammatory liver injury and fibrosis.
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Using a well-established animal model of liver injury, we demonstrate that dietary -3 fatty acids are protective by reducing the incidence and severity of liver damage. We demonstrate for the first time that dietary supplementation amplifies formation of DHA-derived anti-inflammatory lipid signals, namely protectin D1 and 17S-HDHA in the liver. These findings strongly support the concept that patients with liver disease might benefit from a dietary supplementation with -3 essential fatty acids.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6250fje
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).