Fig 2: Pharmacologic inhibition of sEH attenuates hepatic ethanol-induced injury, inflammation, steatosis, and stress signaling. (A) Structure of the sEH pharmacologic inhibitor TPPU. (B) Timeline of pharmacologic inhibition of sEH in the chronic plus binge model of ALD. (C) Serum TPPU (n = 4–5/group), (D) body weight (n = 5–6/group), (E) liver/body weight ratio (n = 5–6/group), (F) hepatic triglycerides (TGs, n = 5–6/group), (G) serum ALT (n = 4–5/group), (H) hepatic total cholesterol (TC, n = 4–5/group), (I) hepatic mRNA expression of monocyte chemotactic protein 1 (MCP1), TNFa, sterol regulatory element-binding protein 1c (SREBP1c), and PPAR? (n = 4/group), and (J) hepatic immunoblots of phospho-PERK-T981, PERK, phospho-eIF2a-S51, eIF2a, phospho-IRE1a, IRE1a, phospho-NF-?B p65-S536, NF-?B p65, phospho-eNOS-S1177, eNOS, NOX2, NOX4, SOD-1, cleaved Caspase 3 (cCasp3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 4–5/group) in Ephx2fl/fl mice without (EtOH) and with sEH inhibitor (EtOH + TPPU). Phosphorylation of PERK, eIF2a, IRE1a, NF-?B p65, and eNOS were normalized with their respective protein, and protein expression of NOX2, NOX4, SOD-1, and cCasp3 were normalized with GAPDH. *P < .05, **P < .01 EtOH vs EtOH + TPPU. AU, arbitrary unit.

Fig 3: Hepatic PTP1B disruption is associated with decreased ethanol-induced oxidative stress. Ctrl (Ptpn1fl/fl) and KO (Ptpn1fl/fl, Alb-Cre) female mice were used in the chronic plus binge model. Pair: control diet + maltose gavage, EtOH: ethanol diet + ethanol gavage. Immunoblots of (A) CYP2E1, pAKT, AKT, pERK, ERK and GAPDH, and (B) peNOS, eNOS, iNOS, NOX2, NOX4, SOD1, GPx1/2, and GAPDH in mouse liver lysates (left panels). Each lane represents an independent animal. Protein expression of CYP2E1, iNOS, NOX2, NOX4, SOD1, and GPx1/2 was normalized with GAPDH. Phosphorylation of AKT, ERK, and eNOS was normalized with their respective protein. C) Lipid peroxidation was determined by immunoblotting liver lysates with 4-hydroxynonenal (4-HNE) then quantitated (full lane intensity of 4-HNE immunoblot was normalized with Actin), as well as immunostaining of liver sections (D). All quantitative results were plotted as means + SEM (n = 5 pair-fed Ctrl mice and n = 6 for each of the remaining groups). *p < 0.05, **p < 0.01 Pair vs. EtOH and †p < 0.05, ††p < 0.01 Ctrl vs. KO by a two-tailed t-test (A), and One-way ANOVA with post-hoc Tukey's test (B, C). A.U.: arbitrary unit. Scale bar: 50 µm.

Fig 4: Effects of AC on signaling molecules that regulate TJ structure and function, on the upregulation of ileum NADPH oxidases and NOS2, and on protein oxidation induced by HFD consumption in mice. A- Phosphorylation levels of p65 (Ser536), ERK1/2 (Thr202/Tyr204) and MLC (Ser19) in total ileum homogenates were measured by Western blot. Bands were quantified and values normalized to non-phosphorylated protein levels. B- Total protein levels of NOX1, NOX4, NOS2 and 4-HNE-protein adducts (MW: 40 kDa). Proteins were measured by Western blot in the ileum of mice fed a control diet (empty bars), the control diet supplemented with 40 mg AC/kg body weight (dashed bars), a HFD (black bars), or the HFD supplemented with 40 mg AC /kg body weight (blue bars) for 14 weeks. Bands were quantified and values normalized to HSC70 levels (loading control). Results were referred to those of the control group (C). Results are shown as mean ± SE of 6–8 animals/treatment. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Effects of AC on TNFa-induced upregulation of signals that regulate TJ structure and the associated upregulation of NADPH oxidases and protein/lipid oxidation in Caco-2 cells. Caco-2 cells were incubated for 6 h at 37 °C in the absence of additions (control, C) (empty bars); or after addition of 5 ng/ml TNFa in the absence (TNFa) (black bars) or the presence of 0.5 µM PCA (grey bars); 1 µM cyanidin-3-O-glucoside (dotted bars), 0.5 µM delphinidin-3-O-glucoside (dashed bars), 0.1 µM peonidin (white bars) or 5 µg AC/ml (light blue bars). A- Phosphorylation levels of p65 (Ser536), ERK1/2 (Thr202/Tyr204), and MLC (Ser19), and B- total protein levels of NOX1, NOX4, and 4-HNE-protein adducts (MW: 40 kDa) were measured by Western blot. Bands were quantified and values normalized to (A) the non-phosphorylated protein and B- ß-actin levels. Results were referred to control group values (C). Results are shown as mean ± SE of 6 independent experiments. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)