A new method to induce nonalcoholic steatohepatitis (NASH) in mice

Abstract

Background: General overnutrition is one of the key factors involved in the development of nonalcoholic fatty liver disease (NAFLD) as the most common liver disease occur by two steps of liver injury ranges from steatosis to nonalcoholic steatohepatitis (NASH). Here the effect of fructose, fat-rich and western diet (WD) feeding was studied along with aggravative effect of cigarette smoking on liver status in mice.

Methods: Sixty-four male NMRI mice were included in this study and assigned into 4 groups that fed standard, fructose-rich, high fat-, and western-diet for 8 weeks and then each group divided in two smoker and nonsmoker subgroups according to smoke exposing in the last 4 weeks of feeding time (n = 8). Histopathological studies, serum biochemical analyses and hepatic TNF-α level were evaluated in mice to compare alone or combination effects of dietary regimen and cigarette smoking.

Results: Serum liver enzymes and lipid profile levels in WD fed mice were significantly higher than in other studied diets. Exposing to cigarette smoke led to more elevation of serum biochemical parameters that was also accompanied by a significant increase in hepatic damage shown as more severe fat accumulation, hepatocyte ballooning and inflammation infiltrate. Elevated TNF-α level confirmed incidence of liver injury.

Conclusion: The finding of this study demonstrated that a combination of cigarette smoke exposure and WD (rich in fat, fructose, and cholesterol) could induce a more reliable mouse model of NASH.

Keywords: Cigarette smoke; Fructose; High fat diet; Mice; NAFLD; NASH; Western diet.

Fig. 1

The comparative effects of different diets on liver histology to evaluate fatty change (a), inflammation (b), ballooning degeneration (c) (kruskal-wallis followed by mann-whitney’s test), and NAS score (d) (one-way ANOVA followed by tukey’s test) in hepatic tissues of mice fed with SD, Fru, HFD, and WD with or without cigarette exposure after 8 weeks with hematoxylin and eosin staining using the semiquantitative NAS System. Data are depicted using box and whisker plots showing median, minimum and maximum values, n = 8 in each group. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 significant difference compared to mice fed standard diet as control. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001significant difference compared to smoker mice fed WD. c: control; Cs: Cigarette smoke; Fru: fructose; HFD: high fat diet; WD: western diet; NAS: NAFLD Activity Scoring

Fig. 2

Representative microscopic images (Bar: 20 μm) of H&E stained liver sections from Control (a), Control + CS (b), Fructose fed diet (c), Fru + Cs (d, e: Congo Red), HFD fed (f), HFD + Cs (g), WD fed (h), WD + Cs (i) mice show histopathological changes including: fatty change, hepatocyte ballooning (as indicated by arrow) and inflammatory cell infiltration. Fru + Cs (E) image represents congo-red stain of liver section

Fig. 3

The comparative effects of different diets on hepatic inflammatory cytokine level, TNF-α after 8 weeks. Data are depicted using box and whisker plots showing median, minimum and maximum values, n = 8 in each group. ^*P < 0.05 and ^***P < 0.001 significant difference compared to mice fed standard diet as control. ^#P < 0.05 and ^###P < 0.001significant difference compared to smoker mice fed WD. C: control; Cs: Cigarette smoke; Fru: fructose; HFD: high fat diet; WD: western diet; TNF-α: tumor necrosis factor- α