Beneficial effects of an endogenous enrichment in n3-PUFAs on Wnt signaling are associated with attenuation of alcohol-mediated liver disease in mice

Abstract

Alcohol-associated liver disease (ALD) is a major human health issue for which there are limited treatment options. Experimental evidence suggests that nutrition plays an important role in ALD pathogenesis, and specific dietary fatty acids, for example, n6 or n3-PUFAs, may exacerbate or attenuate ALD, respectively. The purpose of the current study was to determine whether the beneficial effects of n3-PUFA enrichment in ALD were mediated, in part, by improvement in Wnt signaling. Wild-type (WT) and fat-1 transgenic mice (that endogenously convert n6-PUFAs to n3) were fed ethanol (EtOH) for 6 weeks followed by a single LPS challenge. fat-1 mice had less severe liver damage than WT littermates as evidenced by reduced plasma alanine aminotransferase, hepatic steatosis, liver tissue neutrophil infiltration, and pro-inflammatory cytokine expression. WT mice had a greater downregulation of Axin2, a key gene in the Wnt pathway, than fat-1 mice in response to EtOH and LPS. Further, there were significant differences between WT and fat-1 EtOH+LPS-challenged mice in the expression of five additional genes linked to the Wnt signaling pathway, including Apc, Fosl1/Fra-1, Mapk8/Jnk-1, Porcn, and Nkd1. Compared to WT, primary hepatocytes isolated from fat-1 mice exhibited more effective Wnt signaling and were more resistant to EtOH-, palmitic acid-, or TNFα-induced cell death. Further, we demonstrated that the n3-PUFA-derived lipid mediators, resolvins D1 and E1, can regulate hepatocyte expression of several Wnt-related genes that were differentially expressed between WT and fat-1 mice. These data demonstrate a novel mechanism by which n3-PUFAs can ameliorate ALD.

Keywords: Wnt signaling; alcohol-associated liver disease; n3-PUFAs; n6/n3-PUFA ratio.

FIGURE 1

Evaluation of liver injury in experimental animals. A, Plasma ALT activity demonstrating decreased levels in EtOH-fed, LPS-challenged fat-1 vs WT mice. B, H&E-stained liver sections, C-D, Oil Red O staining and quantitation, respectively. Scale bar is 20 μm. E, Total n6-PUFA levels. F, Total n3-PUFA levels. G, Ratio of hepatic n6/n3-PUFAs. Values are expressed as mean ± SEM, (n = 7–10), *P < .05, one-way ANOVA

FIGURE 2

Hepatic neutrophils and macrophages in fat-1 and WT mice. A, Photomicrograph of immunohistochemistry for F4/80 (macrophages) expression (arrows indicate F4/80-positive cells) and (B) qPCR quantitation for F4/80 mRNA expression. C, Photomicrographs of immunohistochemistry for MPO expression (neutrophils) in liver sections (arrows indicate MPO-positive cells). D, Quantitation of MPO-positive cells. E-F, Hepatic expression Ly6g, and Pai-1, respectively. Six-10 animals/per group; *P < .05, one-way ANOVA. Scale bars are 50 μm

FIGURE 3

Hepatic expression of TNF-α, IL-6, IL-1β, and IL-10 following chronic EtOH exposure and LPS challenge in WT and fat-1 mice. The expression of Tnf-α, Il-6, Il-1β, and Il-10 was measured by qPCR (panels A, C, E, and G, respectively) and by immunoassay [TNF-α (B), IL-6 (D), and IL-10 (H)] or Western blotting/densitometry [cleaved, 17 kDa form of IL-1β (F)]. *P < .05, one-way ANOVA, n = 3–10 animals/per group

FIGURE 4

PCR array analysis of the hepatic expression of Wnt pathway genes. A, Venn diagram summarizing gene expression changes induced by EtOH+LPS in fat-1 and WT mice (Red, upregulated; Blue, downregulated). B, Volcano plot demonstrating gene expression changes between EtOH+LPS-treated fat-1 and WT mice. n = 4 (PF) or n = 3 (EtOH+LPS-treated)

FIGURE 5

Differential expression of Wnt pathway genes between EtOH+LPS-treated WT and fat-1 mice. PCR array data for genes whose expression is significantly different between treated groups, expressed as mean 2^−ΔCt ± SEM (panels A-D and F). Western blots and quantitative analysis of FOSL1/FRA-1 (E) and phospho-JNK1 and JNK-1 (G). *P < .05, one-way ANOVA, n = 6–10/group

FIGURE 6

Axin2 expression in EtOH+LPS-treated mice and in primary hepatocytes from WT and fat-1 mice. A, PCR array data demonstrating larger downregulation of hepatic Axin2 expression in WT vs fat-1 mice following EtOH+LPS treatment. B, Immunohistochemistry of AXIN2 expression demonstrating expression around the central vein (CV). C, Axin2 expression via qPCR of primary hepatocytes treated with 50 mM of EtOH for 24 hours or with 200 ng/mL Wnt-9b for 4 hours. D, Axin2 expression in primary hepatocytes treated with 10 mM of LiCl for 4 hours. Data are presented as the mean ± SEM, *P < .05, one-way ANOVA

FIGURE 7

Effect of RvD1 and RvE1 on gene expression in primary hepatocytes. Primary hepatocyte cultures from WT mice were treated with 100 nM of RvD1 or 100 nM of RvE1 for 3 hours followed by qPCR analysis for the expression of genes within the Wnt signaling pathway, (A) Axin2, (B) Apc, and (C) Porcn and the Wnt target gene, (D) Fosl1/Fra-1. Data are presented as the mean ± SEM, *P < .05, one-way ANOVA

FIGURE 8

Schematic of the hepatic effects of EtOH+LPS on Wnt gene expression in WT and fat-1 mice. WT mice have a high n6/n3 ratio and when treated with EtOH+LPS have reduced Wnt signaling and increased expression of pro-inflammatory genes (Mapk8/Jnk1 and Fosl1/Fra-1). fat-1 mice have a decreased n6/n3 ratio and partially rescued Wnt signaling following EtOH+LPS treatment, decreased expression of pro-inflammatory genes, and therefore, exhibit less liver injury than WT mice