Progression and Regression of Hepatic Lesions in a Mouse Model of NASH Induced by Dietary Intervention and Its Implications in Pharmacotherapy

Abstract

Understanding of the temporal changes of hepatic lesions in the progression and regression of non-alcoholic steatohepatitis (NASH) is vital to elucidation of the pathogenesis of NASH, and critical to the development of a strategy for NASH pharmacotherapy. There are challenges in studying hepatic lesion progression and regression in NASH patients due to the slow development of NASH in humans, one being the requirement for multiple biopsies during the longitudinal follow-up. Here we studied lesion progression and regression in the diet-induced animal model of NASH by application or removal of the pathogenic diet for multiple time periods. Male C57BL/6 mice fed Western diet developed progressive hepatic steatosis/macrovesicular vacuolation, inflammation, and hepatocyte degeneration, as well as perisinusoidal fibrosis and occasionally portal fibrosis as early as 2 months after initiation of the Western diet. In the same period, the mice exhibited elevated ALT (alanine aminotransferase) and AST (aspartate aminotransferase) enzyme activities, CK18 (cytokeratin-18), PIIINP (N-terminal propeptide of type III collagen), and TIMP-1 (tissue inhibitor of metalloproteinase-1). Hepatic steatosis diminished rapidly when the Western diet was replaced by normal rodent chow diet and hepatic inflammation and hepatocyte degeneration were also reduced. Interestingly, perisinusoidal fibrosis and portal fibrosis regressed 8 months after chow diet replacement. To understand pharmacotherapy for NASH, mice with established NASH hepatic lesions were treated with either FXR agonist obeticholic acid (Ocaliva), or CCR2/5 antagonist Cenicriviroc. Similar to the diet replacement, metabolic modulator Ocaliva markedly reduced steatosis/macrovesicular vacuolation, hepatic inflammation, and hepatocyte degeneration effectively, but exhibited no significant effect on liver fibrosis. Anti-inflammation drug Cenicriviroc, on the other hand, markedly decreased inflammation and hepatocyte degeneration, and mildly decreased liver fibrosis, but exhibited no effect on hepatic steatosis/macrovesicular vacuolation. In conclusion, we found the progression of NASH hepatic steatosis/macrovesicular vacuolation, and inflammation eventually lead to hepatocyte death and fibrosis. Life style change and current pharmacotherapies in development may be effective in treating NASH, but their effects on NASH-induced fibrosis may be mild. Since fibrosis is known to be an independent risk for decompensated cirrhosis, cardiovascular events, and mortality, our study suggests that effective anti-fibrosis therapy should be an essential component of the combined pharmacotherapy for advanced NASH.

Keywords: CCR2/5; fibrosis; inflammation; non-alcoholic steatohepatitis; obeticholic acid; pathogenesis; steatosis.

Figure 1

Changes in body and liver weight after the treatment with Western diet. Male C57BL/6J mice were fed Western diet as described in (A). Their body weight (B) was recorded. Their liver weight (C) was measured immediately after euthanasia. Two-tail T-test was used to calculated P-values of body weight or liver weight between mice fed on chow or Western diet for the same time periods (*P < 0.05; ***P < 0.001).

Figure 2

Progression of hepatic lesions in the NASH mice fed Western diet. Male C57BL/6J mice were fed chow or Western diet for different durations. The right and left lateral lobes were collected, and analyzed. The pathological scores from both lobes of an animal were pooled when performing categorical analysis. Hepatic inflammation (A), macrovesicular vacuolation (B), microvesicular vacuolation (C), hepatocyte degeneration (D), perisinusoidal fibrosis (E), and portal fibrosis (F) were graded.

Figure 3

Hepatic lesions of the NASH Mice at Different Age. The representative images were taken from the liver sections of the mice fed either rodent chow (A,B) or Western diet for 60 (C,D), 150 (E,F) and 245 (G,H), and 352 (I,J) days respectively. (A,C,E,G,I) are the images of the liver sections stained with H&E; Panels (B,D,F,H,J) are the images of the liver sections stained with Sirius Red.

Figure 4

Changes in body and liver weight of the NASH mice after the Western diet was replaced by chow. Male C57BL/6J mice were fed Western diet for 240 days. The Western diet was then replaced by rodent chow as described in (A). Their body weight (B) was recorded. Their liver weight (C) was measured immediately after euthanasia. The statistical analysis (One way ANOVA) was performed and statistical significance was indicated in the figure (P < 0.01, ***P < 0.001).

Figure 5

Regression of hepatic lesions in the NASH mice after the Western diet was replaced by chow. At different time after the dietary replacement, the right and left lateral lobes of the NASH mice were collected, and analyzed. The pathological scores from both lobes of an animal were pooled when performing categorical analysis. Hepatic inflammation (A), macrovesicular vacuolation (B), microvesicular vacuolation (C), hepatocyte degeneration (D), perisinusoidal fibrosis (E), and portal fibrosis (F) were graded. Bridging fibrosis was also analyzed, but absent in all the animals.

Figure 6

Hepatic lesion regression after the Western diet was replaced by chow. The representative images were taken from the liver sections of the NASH mice at baseline (A,B), or after their Western diet was replaced by rodent chow for 42 (C,D), 84 (E,F), and 112 (G,H) days, respectively. (A,C,E,G) are the images of the liver sections stained with H&E; Panels (B,D,F,H) are the images of the liver sections stained with Sirius Red.

Figure 7

Pharmacological studies of Ocaliva (OCA) and Cenicriviroc (CVC) in the NASH Mice induced by Western diet. NASH mice fed Western diet for 150 days were treated with vehicle, OCA or CVC as described in (A). Their body weight (B) recorded before euthanasia, and liver weight (C) were measured immediately after euthanasia. The statistical analysis (One way ANOVA) was performed and no statistical significance was found.

Figure 8

Hepatic lesions of the NASH MIce treated with Ocaliva (OCA) and Cenicriviroc (CVC). After 77 days of drug treatment, the right and left lateral lobes of the NASH mice were collected, and analyzed. The pathological scores from both lobes of an animal were pooled when performing categorical analysis. Hepatic inflammation (A), macrovesicular vacuolation (B), microvesicular vacuolation (C), hepatocyte degeneration (D), perisinusoidal fibrosis (E), and portal fibrosis (F) were graded. Bridging fibrosis was also analyzed, but absent in all the animals.

Figure 9

Liver images of the NASH mice treated with Ocaliva (OCA) and Cenicriviroc (CVC). The representative images were taken from the liver sections of the NASH mice treated with vehicle (A,B), 20 mg/kg/day (C,D), and 40 mg/kg/day (E,F) of OCA. (A,C,E) are the images of the liver sections stained with H&E; (B,D,F) are the images of the liver sections stained with Sirius Red.