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.

A Comparison of the Gene Expression Profiles of Non-Alcoholic Fatty Liver Disease between Animal Models of a High-Fat Diet and Methionine-Choline-Deficient Diet

“…Furthermore, NAFLD can be induced by feeding mice different kinds of diets, including a high-fat diet (HFD) [19], high-carbohydrate diet (HCD) [20], methionine-and choline-deficient (MCD) diet [21], and fast food (FF) diet [22]. Furthermore, to reach the NASH and fibrosis stage, some models need to be triggered by a second stimulus or agent ("second hit"), such as tunicamycin [23], dexamethasone [24], and carbon tetrachloride (CCl 4 ) [25]. It was found that male C57BL/6J mice fed a high-fat, -sucrose, and -cholesterol diet with high fructose or glucose water and injected with CCl 4 showed the closest similarity to the human NAFLD pattern [26].…”

Liver Protein Expression in Nash Mice on a High-Fat Diet

“…Even the rise in ALT and AST levels noted here in MS-NASH mice (reaching 300-500 U/mL), while substantial, are not necessarily evidence of serious liver damage. Acute chemical-induced liver toxicity in mice is associated with ALT / AST levels in the range of 3000-6000 U/mL (12).…”

“…Even after 25 weeks, however, these changes are generally mild. Some studies have reported widespread liver damage (beyond steatosis) at earlier time-points but, typically, liver toxins such as carbon tetrachloride, thioacetamide or alcohol are administered along with the high-fat diet (12,13). Animals with severe genetic anomalies (14) or animals maintained on a toxic, nutrient-restricted diet such as the methionine-and choline-deficient diet (15) may develop severe liver anomalies more rapidly.…”

An Efficient Model of Non-alcoholic Fatty Liver Disease (NAFLD) Versus Current Experimental Models: Effects of Fructose, Fat, and Carbon Tetrachloride on NAFLD

“…The intervention was performed for six weeks to induce hepatic steatosis. In this regard, Zhang et al (2020) examined the effect of CCl 4 injection on NAFLD induction; the CCl 4 dose in the present study was based on the results of their study. Besides, because we did not achieve definite results in the groups during the pilot study, we modified the prescribed dose of CCl 4 to an intermediate level (0.1 mL/kg) between the two doses used by Zhang et al (0.2 and 0.08 mL/kg) (9).…”

“…When combined with a Western diet rich in fat and fructose, it can induce oxidative stress, inflammation, and apoptosis. As fibrosis progresses in hepatocytes, fatty liver leads to steatosis; CCl 4 induces NAFLD through this mechanism (7)(8)(9). In previous studies, steatosis and hepatic fibrosis were induced via CCl4 injection at doses of 0.2 mL/kg (8, 10), 0.5 -0.7 mL/kg (11,12), 1 mL/kg (13), and 2 mL/kg (14).…”