A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer

Abstract

Background and aims: Although the majority of patients with non-alcoholic fatty liver disease (NAFLD) have only steatosis without progression, a sizeable fraction develop non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis and hepatocellular carcinoma (HCC). Many established diet-induced mouse models for NASH require 24-52 weeks, which makes testing for drug response costly and time consuming.

Methods: We have sought to establish a murine NASH model with rapid progression of extensive fibrosis and HCC by using a western diet (WD), which is high-fat, high-fructose and high-cholesterol, combined with low weekly dose of intraperitoneal carbon tetrachloride (CCl₄), which serves as an accelerator.

Results: C57BL/6J mice were fed a normal chow diet ± CCl₄ or WD ± CCl₄ for 12 and 24 weeks. Addition of CCl₄ exacerbated histological features of NASH, fibrosis, and tumor development induced by WD, which resulted in stage 3 fibrosis at 12 weeks and HCC development at 24 weeks. Furthermore, whole liver transcriptomic analysis indicated that dysregulated molecular pathways in WD/CCl₄ mice and immunologic features were similar to those of human NASH.

Conclusions: Our mouse NASH model exhibits rapid progression of advanced fibrosis and HCC, and mimics histological, immunological and transcriptomic features of human NASH, suggesting that it will be a useful experimental tool for preclinical drug testing.

Lay summary: A carefully characterized model has been developed in mice that recapitulates the progressive stages of human fatty liver disease, from simple steatosis, to inflammation, fibrosis and cancer. The functional pathways of gene expression and immune abnormalities in this model closely resemble human disease. The ease and reproducibility of this model make it ideal to study disease pathogenesis and test new treatments.

Keywords: Fatty liver disease models; Fibrosis; Hepatic stellate cells; Hepatocellular carcinoma; Insulin resistance; NAFLD; NASH; Steatohepatitis.

Figure 1. Metabolic profile of mice treated with diet and CCl₄

Mice were treated with ND/Oil, WD/Oil, ND/CCl₄, and WD/CCl₄ for up to 24 weeks. Body weight change for 24 weeks (A). From 0 to 11 weeks, ND/Oil: n = 10, WD/Oil: n = 20, ND/CCl₄:n = 20, WD/CCl₄: n = 19. From 12 to 24 weeks, ND/Oil: n = 5, WD/Oil: n = 10, ND/CCl₄: n = 10, WD/CCl₄: n = 10. Liver weight (B), liver per body weight ratio (C), serum ALT (D), AST (E), blood glucose (F), plasma insulin (G), and total cholesterol (H) were measured at 12 and 24 weeks. ND/Oil: n = 5, WD/Oil: n = 10, ND/CCl₄: n = 10, WD/CCl₄: n = 9 animals at 12 weeks. ND/Oil: n = 5, WD/Oil: n = 10, ND/CCl₄: n = 10, WD/CCl₄: n = 10 animals at 24 weeks. Results were expressed as mean ± SEM, and were compared by two-way ANOVA with Bonferroni post-hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. ND, normal diet; WD, western diet; CCl₄, carbon etrachloride.

Figure 2. Histological features of mice treated with diet and CCl₄

H&E (A) and Sirius Red (B) staining of representative mice treated with ND/Oil, WD/Oil, ND/CCl₄, and WD/CCl₄ for 12 and 24 weeks. Original magnification x 100. Quantification of Sirius Red-positive area at 12 (C) and 24 (D) weeks. Results were expressed as mean ± SEM, and were compared by two-way ANOVA with Bonferroni post-hoc test. ND/Oil: n = 5, WD/Oil: n = 10, ND/CCl₄: n = 10, WD/CCl₄: n = 9 animals at 12 weeks. ND/Oil: n = 5, WD/Oil: n = 10, ND/CCl₄: n = 10, WD/CCl₄: n = 10 animals at 24 weeks. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Figure 3. Histological comparison of WD and CCl₄ –treated mice with human NASH

H&E staining of liver sections from representative mice treated with WD/CCl₄ for 24 weeks (left) or human NASH (right) showing steatosis (A), lobular inflammation (B), hepatocyte ballooning with Mallory-Denk bodies (C). Original magnification x 1000.

Figure 4. Hepatic stellate cell activation in mice treated with diet and CCl₄

Immunostaining for desmin (A) and αSMA (B) in liver sections from representative mice treated with ND/Oil, WD/Oil, ND/CCl₄, and WD/CCl₄ for 12 and 24 weeks. Original magnification x 100. Quantification of desmin-positive tissue area (C) and αSMA-positive tissue area (D). Results were expressed as mean ± SEM, and were compared by two-way ANOVA with Bonferroni post-hoc test. ND/Oil: n = 3, WD/Oil: n = 5–8, ND/CCl₄: n = 5–6, WD/CCl₄: n = 5–10 animals at 12 and 24 weeks. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 5. Ductular reaction and hepatocyte proliferation in mice treated with diet and CCl₄

Immunostaining for CK-19 (A) and Ki67 (B) in liver sections from representative mice treated with ND/Oil, WD/Oil, ND/CCl4, and WD/CCl4 for 12 and 24 weeks. Original magnification x 200. Quantification of CK-19-positive area (C) and the number of Ki67-positive hepatocytes per high power field (HPF) (D). Results were expressed as mean ± SEM, and were compared by two-way ANOVA with Bonferroni post-hoc test. ND/Oil: n = 3, WD/Oil: n = 5, ND/CCl₄: n = 5, WD/CCl₄: n = 5–6 animals at 12 and 24 weeks. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 6. Tumor development in mice treated with diet and CCl₄

Tumor numbers per mouse (A) and largest tumor size (B) in mice treated with ND/Oil, WD/Oil, ND/CCl₄, and WD/CCl₄ for 24 weeks. Representative pictures of dysplastic nodules (C–F), HCC with fat and ballooning (G–J), clear cell HCC of lipid rich variant (K–N) in mice treated with WD/CCl₄ for 24 weeks. Gross pictures (C, G, K). H&E staining (D, H, L, original magnification x 10). H&E staining showing interface between tumor and adjacent parenchyma (E, I, M, original magnification x 200). H&E staining showing tumor region of dysplastic nodules (F, J, N, original magnification x 50).

Figure 7. Cross-species comparison of global transcriptome dysregulation between human NASH and mouse NASH models

Dysregulated molecular pathways in either of two human NASH series were compared to 16 previously published diet, chemical, and/or genetic NASH mouse models together with our mouse models (see Methods and Supplementary Table 2 for details). The mouse models are ordered according to similarity to human NASH compared to healthy liver based on Euclidean distance. Orange and green colors in the heat map indicate statistical significance of induction and suppression of each gene set, respectively. HFChSuD: high fat/cholesterol/sugar diet, HFChD: high fat/cholesterol diet, HFD: high fat diet, MCD: methionine/choline-deficient diet, CFD: choline/folate-deficient diet (see Supplementary Table 2 for detailed references of datasets used).

Figure 8. Immunostaining for T lymphocyte subtypes in mice treated with WD and CCl₄

Immunostaining was performed on liver sections from mice fed a WD/CCl₄ (12 weeks) for T lymphocyte subtypes CD3, CD4 and CD8 (black arrows). CK8/18 staining showed cells with CK8/18⁺ inclusions typical for human NASH (Black arrows). White arrows indicate ballooning degeneration of hepatocytes without CK8/18⁺ staining. Original magnification x 400. Average T lymphocyte subtype numbers were assessed by counting positive cells per field (200x magnification) in 20 random fields per liver section. Lymphocytes were identified by the dark staining nuclei with minimal cytoplasm (see Supplementary Table 3).