Prevention of hepatic fibrosis in a murine model of metabolic syndrome with nonalcoholic steatohepatitis

Abstract

The endocannabinoid pathway plays an important role in the regulation of appetite and body weight, hepatic lipid metabolism, and fibrosis. Blockade of the endocannabinoid receptor CB1 with SR141716 promotes weight loss, reduces hepatocyte fatty acid synthesis, and is antifibrotic. D-4F, an apolipoprotein A-1 mimetic with antioxidant properties, is currently in clinical trials for the treatment of atherosclerosis. C57BL/6J mice were fed a high-fat diet for 7 months, followed by a 2.5-month treatment with either SR141716 or D-4F. SR141716 markedly improved body weight, liver weight, serum transaminases, insulin resistance, hyperglycemia, hypercholesterolemia, hyperleptinemia, and oxidative stress, accompanied by the significant prevention of fibrosis progression. D-4F improved hypercholesterolemia and hyperleptinemia without improvement in body weight, steatohepatitis, insulin resistance, or oxidative stress, and yet, there was significant prevention of fibrosis. D-4F prevented culture-induced activation of stellate cells in vitro. In summary, C57BL/6J mice given a high-fat diet developed features of metabolic syndrome with nonalcoholic steatohepatitis and fibrosis. Both SR141716 and D-4F prevented progression of fibrosis after onset of steatohepatitis, ie, a situation comparable to a common clinical scenario, with D-4F seeming to have a more general antifibrotic effect. Either compound therefore has the potential to be of clinical benefit.

Figure 1

Protocol for the six treatment groups.

Figure 2

Liver histology. Representative sections of liver after 9.5 months. A: H&E stain; B–G: Sirius red stain. A: Liver after 9.5 months of Western diet (group 1) demonstrates steatohepatitis; B: liver after 9.5 months of Western diet (group 1) demonstrates fibrosis; C: 9.5 months of Western diet with D-4F during last 2.5 months (group 3); D: 9.5 months of Western diet with SR141716 during last 2.5 months (group 2); E: 7 months of Western diet followed by 2.5 months of standard chow (group 4); F: 7 months of Western diet followed by 2.5 months of standard chow plus D-4F (group 6); G: 7 months of Western diet followed by 2.5 months of standard chow plus SR141716 (group 5).

Figure 3

Morphometric image analysis. The figure demonstrates whole section-scanning morphometric image analysis of slides stained with Sirius red to determine extent of fibrosis. Control groups: 6-month control (open bar), 6 months of feeding with the Western diet (checked bars). Treatment groups received 7 months of Western diet followed by: 2.5 months of Western diet or chow as indicated (black bar), 2.5 months of D-4F and either Western diet or chow as indicated (hatched bar), or 2.5 months of SR141716 and either Western diet or chow as indicated (dotted bar). Single factor analysis of variance for all groups P < 0.01; *P < 0.005 by least significant difference compared to Western diet; ^§P < 0.001 compared to Western diet and P = 0.05 compared to chow for the last 2.5 months by least significant difference; ¶no significant difference versus 9.5 months of Western diet.

Figure 4

α-SMA staining. A: Control; B: 9.5 months of Western diet; C: 7 months of Western diet followed by 2.5 months of chow; D: 9.5 months of Western diet with addition of SR141716 during the last 2.5 months; E: 9.5 months of Western diet with addition of D-4F during the last 2.5 months. α-SMA (green) stains activated HSCs. Erythrocytes (red) are visible in venules and sinusoids.

Figure 5

Transmission electron micrograph of the liver of a mouse that received 6 months of the Western diet, illustrating thickened sinusoidal endothelium (E), and a subendothelial basal lamina (arrow) and collagen deposition (CF) in the space of Disse. S, sinusoid lumen. Original magnification, ×6000.

Figure 6

Scanning electron micrographs of livers from lean mice fed a normal chow diet (A, C) compared to obese mice fed the Western diet for 6 months (B, D). Compare the loss of fenestrae in D versus C. Note the sinusoids and their pattern distorted by the enlarged, fat-laden parenchymal cells in B versus A. The arrow in D points to the compression of the sinusoid caused by a ballooning hepatocyte containing a large fat droplet. Original magnifications: ×1000 (A, B); ×8000 (C, D).

Figure 7

Effect of D-4F on primary murine HSCs. HSCs were cultured for 7 days in the presence (B, C, F) or absence (A, E) of D-4F. α-SMA staining (α-SMA antibody, 1:250 dilution) demonstrates that 87% of cells cultured in the absence of D-4F are positive for α-SMA (A), whereas none of the stellate cells cultured in the presence of D-4F express α-SMA fibers (B). C: Higher concentrations of α-SMA antibody (α-SMA antibody, 1:50 dilution) demonstrate the characteristic compact morphology of quiescent stellate cells after 7 days culture in the presence of D-4F. D: Oil red O staining of stellate cells after 1 day in culture demonstrates the characteristic lipid droplets. Stellate cells cultured for 7 days without D-4F demonstrate few lipid droplets (E), whereas stellate cells cultured in the presence of D-4F maintain significantly more lipid droplets (F).