An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis

Abstract

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine-/choline-deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long-term feeding with a high-fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1-14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Masson's trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.

Keywords: fibrosis; high-fat diet; methionine-restricted diet; mouse model; non-alcoholic steatohepatitis.

Figure 1

Body weight and alanine aminotransferase (ALT) levels in C57BL/6J mice fed standard diet (SD), low-fat diet (LFD) or high-fat diet (HFD) for 24 weeks. (a) Body weight gain. (b) ALT levels. Data are means ± SEM (n = 3). *P < 0.05 vs. SD (control).

Figure 2

Representative liver histopathology in C57BL/6J mice fed standard diet (SD) or high-fat diet (HFD) for 24 weeks. Haematoxylin and eosin (H&E) staining and Masson's trichrome staining of liver in SD-fed mice (a, H&E staining; b, Masson's trichrome staining, 200× magnification). The HFD-fed mice showed steatosis without fibrosis (c, H&E staining; d, Masson's trichrome staining, 200× magnification). Scale bars = 100 μm.

Figure 3

Body weight, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and representative liver histopathology in C57BL/6J mice fed standard diet (SD) or choline-deficient, L-amino acid-defined (CDAA) diet for 6 weeks. (a) Changes in body weight of mice fed SD or the CDAA diet and (b) ALT and AST levels in mice fed the CDAA diet. Data are means ± SEM (n = 3). (c–f) Liver sections were stained with haematoxylin and eosin (H&E) and Masson's trichrome (200× magnification). Scale bars = 100 μm.

Figure 4

Body weight, alanine aminotransferase (ALT) levels and representative liver histopathology in two inbred mouse strains fed choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 1, 3 or 6 weeks. (a) Changes in body weight. (b) ALT levels. Data are means ± SEM (n = 3). Histological findings in the livers of C57BL/6J (left column) and A/J (right column) mice. Haematoxylin and eosin (H&E) staining (c–h) and Masson's trichrome staining (i, j) of liver sections are shown (200 × magnification). Scale bars = 100 μm.

Figure 5

Quantitative real-time PCR of collagen genes (a) and Western blot analysis of alpha-smooth muscle actin (α-SMA; b) of the livers in C57BL/6J mice fed standard diet (SD) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 3 and 6 weeks. Collagen mRNA expression levels were normalized to glutaraldehyde-3-phosphate dehydrogenase (GAPDH). Data are means ± SEM (SD, n = 4; CDAHFD, n = 10). **P < 0.01 vs. SD (control).

Figure 6

C57BL/6J mice were fed standard diet (SD) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 14 weeks. (a) Changes in body weight of mice fed SD or CDAHFD, (b) accumulation of hepatic triglycerides in mice fed CDAHFD, (c) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice fed CDAHFD, (d) MCP-1 levels in mice fed CDAHFD, and (e) hydroxyproline (OH-Pro) content of the livers of mice fed SD or CDAHFD. Data are means ± SEM (n = 3). *P < 0.05 and **P < 0.01 vs. Basal or SD (control).

Figure 7

Histopathological evaluation of liver injury in C57BL/6J mice fed standard diet (SD) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 3, 6, 9 and 12 weeks (n = 3). Fibrosis (a), ballooning (b), inflammation (c) and steatosis (d) scores were evaluated as detailed in Materials and Methods.

Figure 8

Representative liver histopathology in C57BL/6J mice fed standard diet (SD) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 12 weeks. Liver sections were stained with haematoxylin and eosin (H&E) and Masson's trichrome (200 × magnification). Scale bars = 100 μm.