Hepatic Transcriptome and Its Regulation Following Soluble Epoxide Hydrolase Inhibition in Alcohol-Associated Liver Disease

Abstract

Alcohol-associated liver disease (ALD) is a serious public health problem with limited pharmacologic options. The goal of the current study was to investigate the efficacy of pharmacologic inhibition of soluble epoxide hydrolase (sEH), an enzyme involved in lipid metabolism, in experimental ALD, and to examine the underlying mechanisms. C57BL/6J male mice were subjected to acute-on-chronic ethanol (EtOH) feeding with or without the sEH inhibitor 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB). Liver injury was assessed by multiple end points. Liver epoxy fatty acids and dihydroxy fatty acids were measured by targeted metabolomics. Whole-liver RNA sequencing was performed, and free modified RNA bases were measured by mass spectrometry. EtOH-induced liver injury was ameliorated by TUCB treatment as evidenced by reduced plasma alanine aminotransferase levels and was associated with attenuated alcohol-induced endoplasmic reticulum stress, reduced neutrophil infiltration, and increased numbers of hepatic M2 macrophages. TUCB altered liver epoxy and dihydroxy fatty acids and led to a unique hepatic transcriptional profile characterized by decreased expression of genes involved in apoptosis, inflammation, fibrosis, and carcinogenesis. Several modified RNA bases were robustly changed by TUCB, including N⁶-methyladenosine and 2-methylthio-N⁶-threonylcarbamoyladenosine. These findings show the beneficial effects of sEH inhibition by TUCB in experimental EtOH-induced liver injury, warranting further mechanistic studies to explore the underlying mechanisms, and highlighting the translational potential of sEH as a drug target for this disease.

Graphical abstract

Figure 1

Inhibition of soluble epoxide hydrolase by 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB) attenuates acute-on-chronic ethanol (EtOH)-associated liver injury in mice. A: Plasma alanine aminotransferase (ALT) activity. B: Representative liver hematoxylin and eosin (H&E)-stained sections. C: Liver triglyceride (TG) levels. D: Chloroacetate esterase (CAE) staining for neutrophils (positive cells indicated with arrows). E: Quantification of CAE-positive cells. F: Myeloperoxidase (MPO) activity in liver lysates. G–H: Quantitative analysis [percent frequencies of total major histocompatibility complex class II receptor (MHCII⁺)CD206⁺ cells] and representative contour plots indicating mean frequencies of the hepatic MHCII⁺CD206⁺ cells assessed by using flow cytometry. I–J: Western blot analysis and quantitation of hepatic CHOP expression, normalized to β-actin expression. Data are expressed as means ± SEM. n = 5 to 15 animals per group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (comparisons with no asterisk were not statistically significant). Scale bars: 50 μm (B and D). Total magnification: ×400 (B and D). ns, not significant; PF, pair-fed.

Figure 2

A: Heatmap with Ward clustering showing trends in individual hepatic polyunsaturated fatty acid metabolites (color scale represents Z score). B: Principal component analysis (PCA) of polyunsaturated fatty acid metabolites. n = 5 animals per group. EtOH, ethanol; PC1, first principal component; PC2, second principal component; PF, pair-fed; TUCB, 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid.

Figure 3

Liver RNA sequencing analysis. A: Principal component analysis (PCA) of all mRNA across the four groups. B: Summary of differentially expressed mRNAs across experimental groups. C: Heatmap of ethanol (EtOH)-responsive mRNAs unique with 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB) and graphical representation of patterns of expression. n = 3 animals per group. DE, differentially expressed; PC1, first principal component; PC2, second principal component; PC3, third principal component; PF, pair-fed.

Figure 4

Epitranscriptomic modification of the liver RNA bases. A: Principal component analysis (PCA) of 16 detected RNA base modifications. B: Heatmap of all 16 base modifications with Ward clustering (left side, color scale represents Z score) showing two distinct groups of metabolites: C: Metabolites increased by 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB) in the ethanol (EtOH) versus EtOH + TUCB comparison. D: Metabolites decreased by TUCB in the EtOH versus EtOH + TUCB comparison. Data are expressed as mean fold change [compared with the pair-fed (PF) group] ± SEM. n = 6 animals per group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 (comparisons with no asterisk were not statistically significant). m⁶Am, N⁶, 2′-O-dimethyladenosine; ms²t⁶A, 2-methylthio-N⁶-threonylcarbamoyladenosine; m⁵U, 5-methyluridine.

Figure 5

Gene expression of epitranscriptomic writers, erasers, and readers in the mRNA sequencing database. A: Heatmap with Ward clustering (left side, color scale represents Z score) for each category. B: Principal component analysis (PCA) for writers, erasers, and readers. n = 6 animals per group. EtOH, ethanol; PC1, first principal component; PC2, second principal component; PF, pair-fed; TUCB, 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid.