Repeated whiskey binges promote liver injury in rats fed a choline-deficient diet

Abstract

Background/aims: Alcoholic liver disease is associated with nutritional deficiency and it may aggravate within the context of fatty liver. We investigated the relationship between alcohol intake (whiskey binge drinking) and a choline-deficient diet (CD) and assessed whether stellate cells could contribute to liver injury in this model.

Results: Rats fed the CD diet plus whiskey showed increased liver damage compared to rats fed the CD diet, as demonstrated by H&E staining, elevated transaminases, steatosis, TNF-alpha levels, enhanced CYP2E1 activity, impaired antioxidant defense, elevated lipid peroxidation, and protein carbonyls. The combined treatment triggered an apoptotic response as determined by elevated Bax, caspase-3 activity, cytochrome-c release, and decreased Bcl-2 and Bcl-XL. Stellate cells were activated as increased expression of alpha-Sma was observed over that by the CD diet alone. The combined treatment shifted extracellular matrix remodeling towards a pro-fibrogenic response due to up-regulation of collagen I, TIMP1, and Hsp47 proteins, along with down-regulation of MMP13, MMP2, and MMP9 expression, proteases which degrade collagen I. These events were accompanied by increased phosphorylation of p38, a kinase that elevates collagen I.

Conclusions: Repeated alcohol binges in the context of mild steatosis may promote activation of stellate cells and contribute to liver injury.

Figure 1

H&E staining (A). Rats fed the chow diet and given repeated alcohol binges showed minimal steatosis, while rats fed the CD diet and given repeated whiskey binges showed periportal and pericentral microvesicular steatosis (original magnification=200x). ALT and AST (B). The activity of ALT and AST are presented in U/l as means±S.E.M. (n=8). *p<0.05 for CD+W vs. CH+W, •p<0.05 for CD+W vs. CD. CH: chow diet, CD: choline-deficient diet, W: whiskey.

Figure 2

Hepatic Non-Sterified Fatty Acids (A) are expressed as mEq/mg protein and refer to means±S.E.M. (n=8). **p<0.01 for CD vs. CH, ***p<0.001 for CD+W vs. CH+W; •••p<0.001 for CD+W vs. CD. Hepatic Triglycerides (B) are expressed as μg/mg and refer to means±S.E.M. (n=8), **p<0.01 for CD vs. CH, ***p<0.001 for CD+W vs. CH+W; •••p<0.001 for CD+W vs. CD. Leptin Expression (C) was evaluated by Western blot analysis. Results are expressed as AU of densitometry under the blots and are average values of n=3 per group. Leptin expression in rats fed the CH diet was assigned a value of 1. ***p<0.001 for CD vs. CH or CD+W vs. CH+W. Hepatic Leptin Expression (D) was assessed by qRT-PCR. Results are expressed as fold-induction over the CH diet which was assigned a value of 1. ***p<0.001 for CD vs. CH or CD+W vs. CH+W. Plasma Leptin Levels (E) are expressed as ng/ml. ***p<0.001 for CD vs. CH or CD+W vs. CH+W. CH: chow diet, CD: choline-deficient diet, W: whiskey.

Figure 3

Extracellular Matrix Proteins. Liver tissue was stained (right panels) and quantified (upper left panel) for collagenous proteins using the Sirius red/fast green method (A). Results are given as average values±S.E.M. (n=8) and are expressed in μg of collagenous protein per mg of total protein; *p<0.05 for CD vs. CH and **p<0.01 for CD+W vs. CH+W, •p<0.05 for CD+W vs. CD. The expression of COL1A1 mRNA was assessed by qRT-PCR and is expressed as fold change over the CH group which was assigned a value of 1. **p<0.01 for CD vs. CH, ***p<0.001 for CD+W vs. CH+W, and •••p<0.001 for CD+W vs. CD. Western Blot Analysis (B) was carried out for collagen I, α-smooth muscle actin (α-Sma), MMP13, TIMP1, MMP2, MMP9, Hsp47, and β-tubulin. Results are expressed as means±S.E.M (n=3) and are given in AU of densitometry. The expression of the indicated protein in the CH group was assigned a value of 1. *p<0.05, **p<0.01, ***p<0.001 for CD vs. CH and for CD+W vs. CH+W; •p<0.05, ••p<0.01 for CD+W vs. CD. CH: chow diet, CD: choline-deficient diet, W: whiskey.

Figure 4

Serum TNFα Levels are average values of n=8 per group and are expressed as pg/ml. ***p<0.001 for CD vs. CH, **p<0.01 for CD+W vs. CH+W; •••p<0.001 for CD+W vs. CD. CH: chow diet, CD: choline-deficient diet, W: whiskey.

Figure 5

CYP2E1 Activity and Expression. The catalytic activity of CYP2E1 was assessed by assaying the rate of oxidation of p-nitrophenol to p-nitrocatechol (A). Results are expressed as pmol/min/mg of microsomal protein and are average values±S.E.M. (n=8). **p<0.01 for CD+W vs. CH+W; ••p<0.01 for CD+W vs. CD. Western blot analysis showing the expression of CYP2E1 in liver microsomes (B). Results are expressed as AU of densitometry under the blots and are average values of n=3 per group. CYP2E1 expression in rats fed with the CH diet was assigned a value of 1. *p<0.05 for CD+W vs. CH+W; •p<0.01 for CD+W vs. CD. CH: chow diet, CD: choline-deficient diet, W: whiskey.

Figure 6

Lipid Peroxidation (A). Lipid peroxidation by-products in the liver were estimated from the malondialdehyde levels (TBARS). Results are expressed as pmol per mg of protein and are means±S.E.M. (n=8). ***p<0.001 for CD+W vs. the CH+W; •••p<0.001 for CD+W vs. CD. Protein Carbonyls (B) are expressed as nmol per mg of protein and are means±S.E.M. (n=8). ***p<0.001 for CD+W vs. CH+W; •••p<0.001 for CD+W vs. CD. CH: chow diet, CD: choline-deficient diet, W: whiskey.

Figure 7

Western Blot Analysis for Bcl-2, Bax, Bcl-X_L and Cytochrome c Release (A). 50 μg of liver protein were electrophoresed in either 10% or 12% SDS-PAGE and immunoblotted for Bcl-2, Bax, and Bcl-X_L. Twenty-five μg of cytosolic fraction were electrophoresed in a 12% SDS-PAGE and immunoblotted for cytochrome-c. AU of densitometry corrected for β-tubulin expression are indicated under the blots and refer to means±S.E.M. (n=3). ***p<0.001 for CD vs. CH and for CD+W vs. CH+W; •••p<0.001 for CD+W vs. CD. Caspase-3 (B) and Caspase-8 Activity (C). In (B) and (C) the cleaved AFC product was determined by fluorimetry and results are expressed as AU of fluorescence/mg protein and are means±S.E.M. (n=8). **p<0.01 for CD vs. CH and ***p<0.001 for CD+W vs. CH+W; ••p<0.01 for CH+W vs. CH and •••p<0.001 for CD+W vs. CD. The Number of Apoptotic Cells (D) per high power field were determined by TUNEL staining, **p<0.01 for CD vs. CH, ***p<0.001 for CD+W vs. CH+W, and •••p<0.001 for CD+W vs. CD. CH: chow diet, CD: choline-deficient diet, W: whiskey.

Figure 8

Protein Kinases. The expression of p38 and phosphorylated p38, ERK1/2, and Akt were analyzed by Western blot. Results are expressed as means±S.E.M (n=3) and are given as AU of densitometry. The expression of the indicated kinase in the CH group was assigned a value of 1. ***p<0.001 for CD vs. CH, ***p<0.001 for CD+W vs. CH+W; ••p<0.01 for CD+W vs. CD. CH: chow diet, CD: choline-deficient diet, W: whiskey.