Alcoholic Liver Disease: A Mouse Model Reveals Protection by Lactobacillus fermentum

Abstract

Objectives: Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment.

Methods: The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications.

Results: Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake.

Conclusions: We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease.

Figure 1

Representative photomicrographs of liver sections stained with hematoxylin–eosin. (a) Liver histology of mice fed control (Con), ethanol (EtOH) diets, and ethanol and probiotic diet (EtOH-P). Bar=100 μm for all panels. (b) Histogram representing the degree of hepatic steatosis as determined by severity scores from I to III. The score is represented as percentage of steatosis (grade I=5–33% grade II=34–66% grade III=67–100%). Results are expressed as mean±s.d. In b, the horizontal lines on top of the histograms indicate a significant difference within the groups (P<0.001). For further explanation of abbreviations and model features see Table 1. AU, arbitrary unit; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks; wks, weeks.

Figure 2

Aspartate transaminase (AST) and alanine transaminase (ALT) levels in the serum of mice. Histograms showing (a) AST and (b) ALT levels. Each value is expressed as mean±s.d. *P<0.001 vs. Con, 4EtOH, 8EtOH, 12EtOH-P, and 8EtOH-4P; ^◊P<0.001 vs. Con, 4EtOH-P, and 8EtOH-P; ^#P<0.001 vs. Con; ^§P<0.001 vs. all other groups; ^¥P<0.05 vs. 4EtOH and 8EtOH; ⁺P<0.001 vs. 12EtOH-P. For the meaning of the abbreviations in a and b, indicating each mouse group, see legend for Figure 1 and Table 1. Con, control; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks.

Figure 3

Inducible form of NOS (iNOS) levels in the liver of control (Con) and ethanol (EtOH) fed mice. (a) The bars indicate the degree of the iNOS gene expression normalized for the reference genes, according to the Livak method (2^−ΔΔCT), in the liver of control (Con), ethanol diets, and ethanol and probiotic diet mice. (b) Ratio iNOS levels/β-actin levels as a reflection of iNOS increase and decrease (mean±s.d.). One-way analysis of variance, Bonferroni post hoc test. (c) Representative cropped blots for iNOS in Con and EtOH-fed mice. The gels were run under the same experimental conditions and β-actin was used as an internal control. *P<0.01 vs. 8EtOH and 12EtOH; ^#P<0.01 vs. 4EtOH; ^ΔP<0.01 vs. 4EtOH, 12EtOH-P, and 8EtOH-4P. For the meaning of the abbreviations in a and b, indicating each mouse group, see legend for Figure 1 and Table 1. AU, arbitrary unit; Con, control; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks.

Figure 4

Hepatocellular distribution of the inducible form of NOS (iNOS). (a) Representative photomicrographs of liver sections immunohistochemically stained for iNOS showing iNOS distribution both in the nucleus and the cytoplasm of hepatocytes. Strong staining for iNOS was present in the liver cells after 4, 8, and 12 weeks of alcohol intake, but when alcohol intake was accompanied with the probiotic, the staining intensity decreased. Bar=100 μm for all panels. (b) Histogram showing the percentages of hepatocytes positive for iNOS. The analysis was conducted in a blind manner (using coded slides with the observer not knowing the source of them) and the results are expressed as mean±s.d. *P<0.001 vs. all other groups; ^§P<0.01 vs. 4EtOH-P; ^¥ P<0.001 vs. 8EtOH-4P. For the meaning of the abbreviations in a and b, indicating each mouse group, see legend for Figure 1 and Table 1. Con, control; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks; wks, weeks.

Figure 5

Levels of nitrotyrosine-positive cells in liver sections. (a) Representative photomicrographs of 3-nitrotyrosine (3-NT) immunoreactivity. Alcohol intake induced oxidative stress and protein nitration, while simultaneous intake of alcohol and the probiotic for 8 weeks reduced this effect. Bar=100 μm for all panels. (b) Histogram showing the percentages of hepatocytes positive to 3-NT in the various groups studied. The analysis was conducted in a blind manner (using coded slides with the observer not knowing the source of them). *P<0.001 vs. all other groups; ^¥ P<0.001 vs. 4EtOH-P, 8EtOH, and 12EtOH-P; ^#P<0.05 vs. 8EtOH, 8EtOH-4P. For the meaning of the abbreviations in a and b, indicating each mouse group, see legend for Figure 1 and Table 1. Con, control; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks; wks, weeks.

Figure 6

hsp60 gene expression and Hsp60 protein levels in the liver. (a) The bars show the levels of hsp60 gene expression normalized for the reference genes, according to the Livak method (2^−ΔΔCT), in the liver of control (Con), ethanol diets, and ethanol and probiotic diet mice. (b) Western blotting results. Ratio Hsp60 levels/β-actin levels as a reflection of Hsp60 increase (mean±s.d.). (c) Representative cropped western blots for Hsp60 in control and ethanol-fed mice. All gels were run under the same experimental conditions and β-actin was used as an internal control. *P<0.01 vs. 4EtOH-P, 8EtOH; ^#P<0.05 vs. 12EtOH-P; ^§P<0.05 vs. Con and 4EtOH; ^¥ P<0.05 vs. Con, 4EtOH-P. For the meaning of the abbreviations in a and b, indicating each mouse group, see legend for Figure 1 and Table 1. AU, arbitrary unit; Con, control; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks; wks, weeks.

Figure 7

Levels and preferential localization of Hsp60 in control and ethanol-fed mice. (a) Liver sections stained with a monoclonal antibody specific for Hp60. Bar=100 μm for all panels. (b) Histogram showing the percentages of hepatocytes positive to Hsp60. The analysis was conducted in a blind manner (using coded slides with the observer not knowing the source of them). *P<0.001 vs. all other groups (except for 4EtOH); ^ΔP<0.001 vs. 4EtOH; §P<0.05 vs. 4EtOH-P and 12EtOH-P. For the meaning of the abbreviations in a and b, indicating each mouse group, see legend for Figure 1 and Table 1. Con, Control; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks; wks, weeks.

Figure 8

Ethanol induced Hsp60 nitration. Representative western blots are shown below the densitometry histogram. *P<0.05 vs. 8EtOH; ^◊P<0.01 vs. 12EtOH. For the meaning of the abbreviations indicating each mouse group, see legend for Figure 1 and Table 1. AU, arbitrary unit; Con, control; 4EtOH, ethanol-fed mice for 4 weeks; 8EtOH, ethanol-fed mice for 8 weeks; 12EtOH, ethanol-fed mice for 12 weeks; 4EtOH-P, ethanol and probiotic-fed mice for 4 weeks; 8EtOH-P, ethanol and probiotic-fed mice for 8 weeks; 12EtOH-P, ethanol and probiotic-fed mice for 12 weeks; 8EtOH-4P, ethanol-fed mice for 8 weeks and then given probiotic for 4 weeks; wks, weeks.