Decompensated MASH-Cirrhosis Model by Acute and Toxic Effects of Phenobarbital

Abstract

Metabolic dysfunction-associated Steatohepatitis (MASH), is a prominent cause for liver cirrhosis. MASH-cirrhosis is responsible for liver complications and there is no specific treatment. To develop new therapeutic approaches, animal models are needed. The aim of this study was to develop a fast animal model of MASH-cirrhosis in rats reflecting the human disease. Carbon tetrachloride (CCl₄) injections in combination with a high-fat Western diet (WD) were used to induce MASH-cirrhosis. To accelerate liver injury, animals received phenobarbital (PB) in their drinking water using two different regimens. Rats developed advanced MASH-cirrhosis characterized by portal hypertension, blood biochemistry, hepatic ballooning, steatosis, inflammation and fibrosis. Importantly, rats receiving low-dose PB for the long term (LT) showed ascites after 6 weeks, whereas rats with high-dose short-term (ST) PB developed ascites after 8 weeks. ST- and LT-treated rats showed increased portal pressure (PP) and decreased mean arterial pressure (MAP). Of note, hepatocyte ballooning was only observed in the LT group. The LT administration of low-dose PB with CCl₄ intoxication and WD represents a fast and reproducible rat model mimicking decompensated MASH-cirrhosis in humans. Thus, CCl₄ + WD with LT low-dose phenobarbital treatment might be the preferred rat animal model for drug development in MASH-cirrhosis.

Keywords: carbon tetrachloride (CCl4); high-fat and high-cholesterol Western diet (WD); long-term (LT); metabolic dysfunction-associated steatohepatitis (MASH); phenobarbital (PB); short-term (ST).

Figure 1

Development of portal hypertension and disease-dependent mortality. Schematic design of in vivo experiments in SD rats showing 5 groups of rats which were used. Besides healthy control rats, carbon tetrachloride (CCl₄)-intoxicated rats and MASH rats (CCl4 + WD) without phenobarbital in their drinking water were used. Two groups of phenobarbital-treated rats were analyzed. One group with short-term (ST) and high-dose phenobarbital (0.3 g/L) treatment for 3 days and one group with long-term and low dose of phenobarbital (0.06 g/L) were used. CCl₄ was intraperitoneally injected (i.p.) twice per week until ascites was present (A). Kapplan–Meier curve of all rat groups showing the survival of ST-treated rats during the first week, the second week and the third week in comparison to healthy, CCl₄- and CCl₄ + WD-treated rats (B). Survival curve of LT, ST (data pooled), healthy, CCl₄ and CCl₄ + WD rats (C). Cumulative incidence of ascites development in LT, ST, CCl₄- and CCl₄ + WD-treated animals in days (D). CCl₄ + WD rats were not treated until the highest incidence of ascites to obtain a better comparison to phenobarbital-treated animals. Table of the models and the amount of ascites produced in the rat groups (E). Portal pressure (PP) of all rat groups (F). Mean arterial pressure (MAP) of all rat groups (G). Results are expressed as means + SEM; n = 5 per group. (*/** p < 0.05/p <0.001 vs. Healthy; #### p < 0.00001 vs. CCl₄; ++ p < 0.005 vs. ST).

Figure 2

Characterization of chronic liver injury. Hematoxylin and Eosin (H&E) stainings in livers of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (A). Illustrations of the Glisson’s triad to show hepatocyte ballooning and inflammation in LT-treated livers. Inflammation and hepatocyte ballooning is indicated by arrows in 40× magnification (PV = portal vein; BD = bile duct). Hepatic fibrosis score of all rat models from grade F0–F4. Fibrosis is scored into the following groups: F0 = no fibrosis; F1 = perisinusoidal or periportal fibrosis; F2 = perisinusoidal and periportal fibrosis; F3 = bridging fibrosis; F4 = cirrhosis (B). Hepatic Sirius red staining and its quantification of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (C,D). Amount of hepatic hydroxyproline (HP) in healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (E). Gene expression level of Col1a1 in livers of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (F). Hepatic protein expression of Col1a1 (G). Immunohistochemistry stainings of α-smooth muscle actin (α-SMA) and its quantification in all rat groups (H,I). Gene expression level of hepatic α-SMA in the rat groups (J). Representative Western blot of hepatic α-SMA in healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (K). Gene expression levels of Glial fibrillary acidic protein (GFAP) in the livers of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (L). The scale bar is 200 µm in the 10× magnifications and 100 µm in the 20× magnifications. Results are expressed as means + SEM; n = 5 per group. (*/** p < 0.05/ p < 0.001 vs. Healthy; #/## p < 0.05/ p < 0.001 vs. CCl₄; °/°° p < 0.05/p < 0.001 vs. control; + p < 0.05 vs. ST).

Figure 2

Characterization of chronic liver injury. Hematoxylin and Eosin (H&E) stainings in livers of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (A). Illustrations of the Glisson’s triad to show hepatocyte ballooning and inflammation in LT-treated livers. Inflammation and hepatocyte ballooning is indicated by arrows in 40× magnification (PV = portal vein; BD = bile duct). Hepatic fibrosis score of all rat models from grade F0–F4. Fibrosis is scored into the following groups: F0 = no fibrosis; F1 = perisinusoidal or periportal fibrosis; F2 = perisinusoidal and periportal fibrosis; F3 = bridging fibrosis; F4 = cirrhosis (B). Hepatic Sirius red staining and its quantification of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (C,D). Amount of hepatic hydroxyproline (HP) in healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (E). Gene expression level of Col1a1 in livers of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (F). Hepatic protein expression of Col1a1 (G). Immunohistochemistry stainings of α-smooth muscle actin (α-SMA) and its quantification in all rat groups (H,I). Gene expression level of hepatic α-SMA in the rat groups (J). Representative Western blot of hepatic α-SMA in healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (K). Gene expression levels of Glial fibrillary acidic protein (GFAP) in the livers of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats (L). The scale bar is 200 µm in the 10× magnifications and 100 µm in the 20× magnifications. Results are expressed as means + SEM; n = 5 per group. (*/** p < 0.05/ p < 0.001 vs. Healthy; #/## p < 0.05/ p < 0.001 vs. CCl₄; °/°° p < 0.05/p < 0.001 vs. control; + p < 0.05 vs. ST).

Figure 3

Hepatic steatosis and inflammation after ST and LT Phenobarbital treatment. Effect of carbon tetrachloride (CCl₄), CCl₄ + WD and ST or LT Phenobarbital treatment on liver steatosis in SD rats. Hepatic Oil red O staining of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats and its quantification (A). Plasma triglyceride levels in all rat groups (B). Representative Western blot of the lipogenesis marker Sterol regulatory element-binding protein-1 (SREBP1) and its quantification (C). Plasma transaminase levels (ALT; AST) (D,E) and the deRitis ratio (F) in healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats. Gamma-Glutamyl Transferase (GGT) levels in all rat groups (G). Gene expression of pro-inflammatory markers IL6, EMR1 and CCL2 (H–J) in the livers of healthy, CCl₄, control (CCl₄ + WD), ST- and LT-treated rats. The scale bar is 100 µm. Results are expressed as means + SEM; n = 5 per group. (*/** p < 0.05/p < 0.005 vs. Healthy; #/## p < 0.05/p < 0.005 vs. CCl₄; °/°° p < 0.05/p < 0.005 vs. control; ++ p < 0.005 vs. ST).

Figure 4

Phenobarbital-induced organ dysfunction of liver and brain in experimental cirrhosis. Effect of carbon tetrachloride (CCl₄), CCl₄ + WD and Phenobarbital on total Bilirubin (A), Albumin (B) and INR (C) in plasmas of SD rats. Extrahepatic impairment of the brain shown by the neurological behavior test (D) and the brain water content (E) in all rats. Results are expressed as means + SEM; n = 5 per group. (*/** p < 0.05/p < 0.005 vs. Healthy; #/## p < 0.05/p < 0.005 vs. CCl₄; °/°° p < 0.05/p < 0.005 vs. control; ++ p < 0.005 vs. ST).