Sorafenib prevents liver fibrosis in a non-alcoholic steatohepatitis (NASH) rodent model

Abstract

Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg(-1)·day(-1) by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH.

Figure 1. Analysis of liver extracellular matrix by morphometry of collagen fibers stained in red with Sirius Red. A, Control animal (n=4); B, nonalcoholic steatohepatitis (NASH) animal that received only vehicle (n=10); C, NASH animal treated with 2.5 mg·kg^-1·day^-1 of sorafenib for 6 weeks (n=10); D, graph demonstrating that NASH animals treated with sorafenib presented a decrease in the area occupied by collagen fibers in liver parenchyma compared with NASH animals that received only vehicle (P<0.001, Tukey test).

Figure 2. A, B, C, Graphic representation of TIMP1, TIMP2, and MMP9 mRNA expression in control (n=4), nonalcoholic steatohepatitis (NASH; n=10), and sorafenib-treated animals (n=10). A, TIMP1 mRNA expression was significantly lower in NASH animals compared to control (P= 0.036, Tukey test) and further impaired in sorafenib-treated rats (P<0.001 vs control and P=0.015 vs NASH, Tukey test). B, TIMP2 was significantly lower in the sorafenib group compared to control and NASH groups (P<0.001, Tukey test). C, MMP9 mRNA content was increased in animals treated with sorafenib (P<0.001 vs control and NASH, non-parametric Tukey test). There was no statistical difference in MMP9 mRNA transcription levels in the liver from NASH animals compared to control rats (P= 0.834, non-parametric Tukey test). D, E, F, Graphic representation of IL-6, IL-10, and TNF-α in control, NASH, and sorafenib-treated animals. D, Protein levels of IL-6 were similar in the NASH and control groups (P=0.55, Tukey test). IL-6 levels were significantly lower in sorafenib animals compared to NASH animals (P=0.002, Tukey test). E, IL-10 protein levels were also lower in the sorafenib group compared to the NASH (P<0.001, non-parametric Tukey test) and control groups (P=0.039, non-parametric Tukey test). F, No differences in TNF-α protein levels were detected among the three groups (non-parametric Tukey test).

Figure 3. Graphic representation of HSP60, HSP90, and GST mRNA expression in control (n=4), nonalcoholic steatohepatitis (NASH; n=10), and sorafenib-treated animals (n=10). The mRNA expression of HSP60, HSP90, and GST did not differ among the three groups.

Figure 4. A, Stages of mitochondrial respiration in liver homogenates of control (CTRL; n=4), nonalcoholic steatohepatitis (NASH; n=10), and sorafenib-treated animals (n=10). All stages of mitochondrial respiration in liver homogenates from NASH animals were markedly impaired compared to controls. Sorafenib treatment improved mitochondrial function, restoring the parameters to almost the levels observed in control animals. RCR: respiratory control rate; S3: activated state; S4: basal state respiration. *P<0.05 compared to CTRL group; ^#P<0.05 compared to NASH group (Tukey test). B, Graphic representation of PGC1α mRNA expression in control, NASH, and sorafenib-treated animals (P=0.99, control vs sorafenib group; P<0.001, control vs NASH; P<0.001, NASH vs sorafenib group; non-parametric Tukey test).