Dietary Linoleic Acid and Its Oxidized Metabolites Exacerbate Liver Injury Caused by Ethanol via Induction of Hepatic Proinflammatory Response in Mice

Abstract

Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines in vitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis.

Figure 1

The experimental animal model of alcoholic liver disease. A: Schematic representation of the chronic-binge–ethanol (EtOH) exposure protocol. C57BL/J mice were fed control or ethanol diets for 10 days, followed by single gavage of maltose dextrose or ethanol, respectively. Animals were euthanized 9 hours after gavage. B: Serum ALT. C: Representative images of hepatic hematoxylin and eosin (H&E) staining. D: Liver triglyceride (TG) levels. E: Representative images of Oil Red O staining. F and G: Liver (F) and plasma (G) levels of 9- and 13-HODEs. Data are expressed as means ± SEM. Each experiment is a representative or the average of 6–10 mice in each group. ^∗P < 0.05. Original magnification: ×400 (C); ×200 (E). HODE, hydroxyoctadecadienoic acid; SF, saturated fat; USF, unsaturated fat.

Figure 2

Effect of different types of dietary lipids and chronic ethanol (EtOH) administration on hepatic oxidative stress. A: Cytochrome p450 2E1 (CYP2E1) protein expression analyzed by Western blotting with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (Ctrl). B: The intensity of protein bands (from A) was quantified by densitometry using the ImageJ software. C and D: Representative images and quantification of hepatic 4-hydroxy-2-nonenal staining (4-HNE). Quantification is presented as percentage of 4-HNE–positive area over the entire area of the image slide, and was performed using ImageJ by manually chosen identical thresholds applied to all images being analyzed. E: Hepatic thiobarbituric acid reactive substance (TBARS) levels. Data are expressed as means ± SEM . Each experiment is a representative of the average of 8–10 mice in each group. ^∗P < 0.05. Original magnification, ×400 (C). SF, saturated fat; USF, unsaturated fat.

Figure 3

Effect of different types of dietary lipids and chronic-binge–ethanol (EtOH) administration on hepatic inflammation. A and B: Representative images of chloroacetate esterase staining (CAE). Arrows indicate CAE-positive neutrophils. C: Quantification of CAE staining performed by counting CAE-positive neutrophils in a random series of five digital images per animal. D: Tumor necrosis factor-α (TNF-α). E: IL-1β. F: Monocyte chemotactic protein 1 (MCP-1). G: Inducible nitric oxide synthase (iNOS). H: Arginase 1 (ARG-1). I: Transforming growth factor-β1 (TGF-β1). D–I: Hepatic mRNA levels were measured by RT-PCR. Genes were normalized to 18S rRNA as an internal control. Results are presented as fold changes relative to the SF pair-fed group. Data are expressed as means ± SEM. Each experiment is a representative or the average of 8–10 mice per group (D–I). n = 6 to 8 animals per group (C). ^∗P < 0.05. Original magnification: ×400 (A); ×1000 (B). SF, saturated fat; USF, unsaturated fat.

Figure 4

The 9- and 13-HODE–induced cytokine expression in RAW264.7 macrophages. A–D: RT-PCR analysis of the expression of M1 macrophage markers: A: Tumor necrosis factor-α (TNF-α). B: Macrophage inflammatory protein-2α (MIP-2α). C: Monocyte chemoattractant protein-1 (MCP-1). D: Inducible nitric oxide synthase (iNOS). E and F: M2 macrophage markers: arginase-1 (ARG-1) (E) and transforming growth factor-β (TGF-β1) (F). mRNA levels were measured by RT-PCR. Genes were normalized to 18S rRNA as an internal control. Results are presented as fold change relative to vehicle/control (25 mmol/L ethanol). Data are expressed as means ± SEM. n = 3 (A–F). ^∗P < 0.05. HODE, hydroxyoctadecadienoic acid.

Figure 5

Genetic deletion of Alox15 modestly attenuates chronic-binge–ethanol (EtOH)-induced liver injury with no effect of hepatic steatosis. A and B: Liver levels of 9- and 13-HODEs. C and D: Liver 15- and 12-HETE levels. E: Representative images of hepatic hematoxylin and eosin (H&E) staining. F: Biochemical assessment of liver triglycerides (TGs). G: Serum ALT levels (data are from two independent experiments). Data are expressed as means ± SEM. Each experiment is a representative or the average of 5–8 mice per group. ^∗P < 0.05. Original magnification, ×400 (E). WT, wild-type. HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid.

Figure 6

Effect of Alox15 deficiency on cytochrome p450 2E1 (CYP2E1) expression and markers of liver inflammation. A: CYP2E1 protein expression analyzed by Western blotting with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (Ctrl). B: The intensity of protein bands (from A) was quantified by densitometry using the ImageJ software. C: Tumor necrosis factor-α (TNF-α) mRNA. D: Macrophage inflammatory protein 2-α (MIP-2α) mRNA. C and D: Hepatic mRNA levels were measured by RT-PCR. Genes were normalized to 18S rRNA as an internal control. E: Representative images of chloroacetate esterase staining (CAE; arrows indicate CAE-positive neutrophils). F: Quantification of CAE staining performed by counting CAE-positive neutrophils in a random series of five digital images per animal. Results are presented as fold changes relative to the WT pair-fed group. Data are expressed as means ± SEM. Each experiment is a representative or the average of 5–8 mice per group. ^∗P < 0.05. Original magnification, ×400 (E). EtOH, ethanol; WT, wild-type.