Carbon tetrachloride (CCl4) accelerated development of non-alcoholic fatty liver disease (NAFLD)/steatohepatitis (NASH) in MS-NASH mice fed western diet supplemented with fructose (WDF)

Abstract

Background: Multiple murine models of nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) have been established by using obesogenic diets and/or chemical induction. MS-NASH mouse (formally FATZO) is a spontaneously developed dysmetabolic strain that can progress from hepatosteatosis to moderate fibrosis when fed a western diet supplemented with 5% fructose (WDF). This study aimed to use carbon tetrachloride (CCl₄) to accelerate and aggravate progression of NAFLD/NASH in MS-NASH mouse.

Methods: Male MS-NASH mice at 8 weeks of age were fed WDF for the entire study. Starting at 16 weeks of age, CCl₄ was intraperitoneally administered twice weekly at a dose of 0.2 mL/kg for 3 weeks or 0.08 mL/kg for 8 weeks. Obeticholic acid (OCA, 30 mg/kg, QD) was administered in both MS-NASH and C57Bl/6 mice fed WDF and treated with CCl₄ (0.08 mL/kg).

Results: WDF enhanced obesity and hepatosteatosis, as well as induced moderate fibrosis in MS-NASH mice similar to previous reports. Administration of CCl₄ accelerated liver fibrosis with increased bridging and liver hydroxyproline contents, but had no significant impact on liver steatosis and lipid contents. High dose CCl₄ caused high mortality and dramatic elevation of ALT and ASL, while low dose CCl₄ resulted in a moderate elevation of ALT and AST with low mortality. Compared to C57BI/6 mice with WDF and CCl₄ (0.08 mL/kg), MS-NASH mice had more prominent hepatosteatosis and fibrosis. OCA treatment significantly lowered liver triglycerides, steatosis and fibrosis in both MS-NASH and C57Bl/6 mice fed WDF with CCl₄ treatment.

Conclusions: CCl₄ reduced induction time and exacerbated liver fibrosis in MS-NASH mice on WDF, proving a superior NASH model with more prominent liver pathology, which has been used favorably in pharmaceutical industry for testing novel NASH therapeutics.

Keywords: FATZO; Hepatosteatosis; High fat diet; Inflammation; Liver fibrosis; NAFLD; NASH.

Fig. 1

Effects of high dose CCL₄ (0.2 mL/kg) twice weekly for 3 weeks in MS-NASH mice fed Control diet (CD) or Western diet supplemented with fructose (WDF). (a) Body weight; (b) daily food and (c) caloric intake; serum (d) ALT and (e) AST before and after repeated high dose CCl₄. (f) Time course of acute response of ALT and AST to a single high dose CCl₄ in mice on control diet (CD). Data presented as mean ± SEM

Fig. 2

Histopathology in MS-NASH mice fed Control diet (CD) or Western diet supplemented with Fructose (WDF) treated high dose CCl₄ (0.2 mL/kg) twice weekly for 3 weeks. Left panel: Representative images of H&E and PSR staining in animals fed (a and b) CD; and WDF treated (c, d) without or (e, f) with CCl₄, respectively. Red arrows indicate fibrosis. Right panel: (g) Pathology scores of steatoses (0–3), lobular inflammation (0–3), ballooning (0–2), NAFLD Activity (0–8). (h) left: fibrosis score (0–4) and right: Percentage fibrosis area, quantitatively analyzed as total PSR positive staining area over total liver section area scanned and processed by HALO software. Data presented as mean ± SEM. # p < 0.05, ### p < 0.005 comparing with CD group; * p < 0.05, *** p < 0.005 comparing with WDF group by one-way ANOVA analysis

Fig. 3

Effects of low dose CCL₄ (0.08 mL/kg) twice weekly for 8 weeks in MS-NASH mice fed Western diet supplemented with fructose (WDF). Top panel: (a) Body weights; serum (b) ALT and (c) AST levels before and after low dose CCL₄. In-life AST and AST levels at weeks 11, 12 and 14 were measured ~ 72 h; and the terminal one at week 16 measured ~ 24 h, after CCl₄ administration. Bottom Panel: Liver (d) weight; (e) cholesterol; and (f) triglycerides measured at the end of the study. Data presented as mean ± SEM. * p < 0.05, *** p < 0.005, WDF vs. WDF + CCl₄ group by Holm-Sidak t-test

Fig. 4

Histopathology in Western diet supplemented with fructose (WDF) fed MS-NASH mice treated with low dose CCL₄ (0.08 mL/kg) for 8 weeks. Left panel: Representative images of H&E and PSR staining from MS-NASH mice on WDF. Yellow arrows indicate macrovesicular vacuolation steatosis; blue arrows indicate typical microvesicular ballooning within the blue square; and red arrows indicate fibrosis. (a and b) Without CCl₄. (c and d) With CCl₄. Bottom panel: Hepatosteatosis analysis from H&E images. (e) Semi quantification of pathology scores (0–3), lobular inflammation (0–3), ballooning (0–2), and NAFLD activity (0–8). (f) Quantitative histology analyzed as percentage of steatosis area, and cell counts of inflammation and hepatic ballooning by Reveal ImageDx software. Right panel: Fibrosis analysis from PSR images and live contents. (g) Fibrosis score by pathologist (0–4). (h) Fibrosis area by ImageDx. (i) Live hydroxyproline contents by biochemistry. Data presented as mean ± SEM. *** p < 0.005, WDF vs. WDF + CCL₄ group using Holm-Sidak t-test

Fig. 5

Therapeutic effects of Obeticholic acid (OCA, 30 mg/kg, QD) in Western diet supplemented with fructose (WDF) fed MS-NASH or C57BI/6 mice treated low dose CCl₄ (0.08 mL/kg) twice weekly for 8 weeks. Top panel: (a) Body weight; serum (b) ALT and (c) AST. Bottom panel: (d) Terminal liver weight; and liver contents of (e) triglycerides and (f) cholesterol. Data represented as mean ± SEM. * p < 0.05, *** p < 0.005, Veh. vs OCA groups by Holm-Sidak t-test

Fig. 6

Histopathology of Obeticholic acid (OCA, 30 mg/kg, QD) treatment on Western diet supplemented with fructose (WDF) fed MS-NASH or C57BI/6 mice under low dose CCl₄ (0.08 mL/kg) twice weekly for 8 weeks. Left panel: Representative images of H&E and PSR staining in mice on WDF treated with CCl₄. Yellow arrows indicate steatosis, blue arrows indicate typical microvesicular ballooning within the blue square, and red arrows indicate fibrosis. MS-NASH mice with (a and b) vehicle or (c and d) OCA. C57Bl/6 mice with (e and f) vehicle or (g and h) OCA. Bottom panel: Hepatosteatosis analysis from H&E images. (i) Semi quantification of Pathology scores (0–3), lobular inflammation (0–3), ballooning (0–2), and NAFLD activity (0–8). (j) Quantitative histology analyzed as percentage of steatosis area, and cell counts of inflammation and hepatic ballooning by Reveal ImageDx software. Right panel: Fibrosis analysis from PSR images and liver contents. (k) Fibrosis score by pathologist (0–4). (l) Fibrosis area by ImageDx. (m) Live hydroxyproline contents by biochemistry. Data represented as mean ± SEM. * p < 0.05, *** p < 0.005, Veh VS OCA group using Holm-Sidak t-test

Fig. 7

Comparison of survival rates in Western diet supplemented with fructose (WDF) fed MS-NASH or C57BI/6 mice under CCl₄ twice weekly

Fig. 8

Correlation between Pathology scores and Reveal ImageDx analysis. Correlations between pathology scores for (a) steatosis; (b) lobular inflammation; (c) hepatocyte ballooning; and (d) fibrosis and Reveal ImageDx quantification by simple linear correlation with Pearson’s coefficients. All the Pearson correlation coefficient r values are statistically significant