DIO&CCL4 Induced MASH(NASH) Mouse Model

Metabolic dysfunction-associated steatohepatitis (MASH), previously referred to as nonalcoholic steatohepatitis (NASH), is a chronic liver disease. This condition frequently arises from metabolic irregularities like obesity or diabetes, leading to an accumulation of fat in the liver, which can become toxic and cause inflammation. This inflammation, combined with fibrosis, can progress to cirrhosis or hepatocellular carcinoma (HCC). MASH stands as the most severe manifestation within the spectrum of metabolic dysfunction-associated steatotic liver disease (MASLD), formerly nonalcoholic fatty liver disease (NAFLD).¹

The aim of this study is to develop a MASH (NASH) model with fibrosis characteristics that can meet pre-clinical needs. We use the first FDA-approved MASH drug, Rezdiffra (resmetirom), to assess the efficacy of the MASH model in treatment.

Figure 1. Study outline for Composite MASH Mouse Model experiment.

Composite MASH Mouse Model Study Methods:

Forty C57BL/6J mice (4-5 weeks, male) were received and raised under a standard 12-hour light/dark cycle (lights off at 7:00 pm). Thirty-five mice were fed a high fat diet (HFD; D12492, 60% of kcal from fat), and the other five mice were fed a chow diet for 12 weeks. Carbon tetrachloride (CCl4) can cause liver damage and be administered alone or with a high-fat diet to induce fatty liver or liver fibrosis, a method which has a relatively short induction period. Mice were randomized and divided into 4 groups at the beginning of the intervention: Group 1, chow diet control (n=5); Group 2, HFD control (n=5); Group 3, HFD+Carbon tetrachloride (CCL4) (n=5); Group 4, HFD+CCL4+resmetirom at a daily dose of 3 mg/kg p.o. (n=5). Treatments started on the thirteenth week, after 12 weeks of high-fat diet (HFD), and lasted 4 weeks. Body weight was measured once daily during treatment. All mice were euthanized after treatment, with blood and livers collected for further validation.

Figure 2. Effect of resmetirom on body weight and liver weight. (A) Body weight development over the intervention period, (B) Liver weight at the endpoint of study.  *P <0.05, significantly different from HFD control. Data are presents as Mean±SEM, n = 5 per group. Values of *p < 0.05 were considered statistically significant by one-way ANOVA analysis.

Figure 3. Resmetirom ameliorated serum TC, HDL-C and LDL-C levels. (A) Total cholesterol (TC) level, (B) High-density lipoprotein cholesterol (HDL-C) level, (C) Low-density lipoprotein cholesterol (LDL-C) level. Data are presented as Mean±SEM, n=5 per group. # p < 0.05, ## p < 0.005 and ####p < 0.001 comparing with WT group; * p < 0.05, ** p < 0.005 comparing with HFD+CCL4 group by one-way ANOVA analysis.

Figure 4. Resmetirom protected serum ALT and AST levels induced by CCL4 on Day 14 and at the endpoint, respectively. (A) Alanine aminotransferase (ALT) level, (B) Aspartate aminotransferase (AST) level. Data are presented as Mean±SEM, n=5 per group. # p < 0.05, ## p < 0.005 comparing with WT group; * p < 0.05, ** p < 0.005 comparing with HFD+CCL4 group by one-way ANOVA analysis

Figure 5. Histopathology in mice fed chow diet (WT), high-fat diet (HFD), HFD treated with CCL4 (0.125mL/kg), HFD treated with CCL4 (0.125mL/kg) and resmetirom (3mg/kg) for 4 weeks. Resmetirom protected HFD-induced steatosis and hepatic ballooning in the liver. (A) Representative images of H&E, (B) Representative images of ORO staining, (C) Representative images of PSR staining, (D) NAS score. Blue arrows indicate steatosis, green arrows indicate lobular inflammation, black arrows indicate hepatocyte ballooning in the figure A. Pathology scores of steatosis (0–3), lobular inflammation (0–3), ballooning (0–2), NAS Activity (0–8). Data are presented as Mean±SEM, n=5 per group.

Figure 6. Resmetirom inhibited mRNA levels of liver fibrosis markers. Representative mRNA levels and quantitative analysis of (A) liver α-SMA (Acta2) expression, (B) hepatic collagen 1α1 expression. Data are presented as Mean±SEM, n=5 per group. * p < 0.05, ** p < 0.005, ****p < 0.001 by one-way ANOVA analysis.

Conclusion:

A high-fat diet (HFD) can increase body weight, liver weight, serum TC, HDL-C, LDL-C, AST, and ALT levels in DIO mice after 12 weeks of feeding. CCL4  reduces the induction time and exacerbates liver fibrosis in DIO mice, accelerating and aggravating the progression of MASH in mice. Mice treated with CCL4 can develop obesity, steatosis, and fibrosis, meeting the clinical criteria of MASH. This model is widely utilized in the pharmaceutical industry for evaluating new potential drugs for MASH. Treatment with resmetirom significantly lowers body weight and liver weight while decreasing serum TC, HDL-C, and LDL-C levels. Moreover, resmetirom shows efficacy in the DIO+CCL4 composite mouse model of MASH by reducing steatosis and fibrosis.

Reference:

1. Shuang Wang and Scott L. Found in Translation – Fibrosis in Metabolic Dysfunction-Associated Steatohepatitis (MASH). Sci Transl Med. 2023 Oct 4; 15(716).