Interventional Potential of Recombinant Feline Hepatocyte Growth Factor in a Mouse Model of Non-alcoholic Steatohepatitis

Abstract

Background and Aims: Hepatocyte growth factor (HGF) is a multifunctional pleiotropic protein involved in tissue regeneration, protection, angiogenesis, anti-inflammatory and anti-fibrotic responses, and tumorigenesis, through binding to its receptor MET. Recombinant HGF protein has been shown to mitigate various liver disease models, such as alcohol-induced liver injury, hepatic ischemia-reperfusion injury, and fibrosis. This study aimed to investigate the anti-inflammatory, anti-fibrotic, and anti-lipogenic effects of exogenous administration of feline HGF on a non-alcoholic steatohepatitis (NASH) mouse model. Methods: Wild-type C57BL/6 mice were fed a choline-deficient amino acid defined (CDAA) diet for 3 weeks to create the mouse model of NASH, which displays hepatic steatosis, inflammation, injury, and very mild fibrosis. One mg/kg of recombinant feline HGF was administered intravenously daily in the last 7 days of the total 3 weeks of CDAA diet feeding. Then, hepatic steatosis, inflammation, injury, and fibrogenic gene expression was examined. Results: After 3 weeks of a CDAA diet-feeding, the vehicle-treated mice exhibited evident deposition of lipid droplets in hepatocytes, inflammatory cell infiltration, and hepatocyte ballooning along with increased serum ALT levels whereas recombinant HGF-treated mice showed reduced hepatic steatosis, inflammation, and ballooned hepatocytes with a reduction of serum ALT levels. Recombinant HGF administration promoted hepatocyte proliferation. Increased hepatic lipid accumulation was accompanied by elevated expression of lipogenesis genes Fasn and Dgat1 in vehicle-treated mice. In HGF-treated mice, these genes were reduced with a decrease of lipid accumulation in the liver. Consistent with the anti-inflammatory property of HGF, augmented macrophage infiltration and upregulation of chemokines, Cxcl1, Ccl2, and Ccl5 in the CDAA diet fed mice, were suppressed by the addition of the HGF treatment. Finally, we examined the fibrotic response. The vehicle-treated mice had mild fibrosis with upregulation of Col1a1, Acta2, Timp1, Tgfb1, and Serpine1 expression. Recombinant HGF treatment significantly suppressed fibrogenic gene expression and collagen deposition in the liver. Conclusion: Recombinant feline HGF treatment suppressed the progression of NASH in a CDAA diet feeding mouse model.This suggests that recombinant HGF protein has therapeutic potential for NASH.

Keywords: HGF; NAFLD; NASH; inflammation; recombinant.

Figure 1

Recombinant HGF administration inhibited CDAA diet-induced NASH. Male C57BL/6 mice were fed a CSAA or CDAA diet for 3 weeks. In the last 7 days, vehicle (Veh) or 1 mg/kg of recombinant feline HGF were given intravenously daily (CSAA-Veh, n = 8; CSAA-HGF, n = 10; CDAA-Veh, n = 13; CDAA-HGF, n = 11). (A) H&E staining. Original magnification, x200. (B) NAFLD activity score. Hepatic steatosis, inflammation, hepatocyte ballooning, and fibrosis were evaluated through H&E stains. Data are presented as mean ± S.D. (**P < 0.01, significantly different from CSAA-Veh; ^#P < 0.05, ^##P < 0.01, significantly different from CDAA-Veh).

Figure 2

CDAA diet-mediated liver damage was suppressed by recombinant HGF treatment. Serum ALT level. Data are presented as mean ± SEM. (**P < 0.01, significantly different from CSAA-Veh; ^##P < 0.01, significantly different from CDAA-Veh).

Figure 3

Administration of recombinant HGF promoted hepatocyte proliferation. (A) Representative images of PCNA staining. Original magnification, x200. (B) Quantification of PCNA staining. Data are presented as mean ± SEM.

Figure 4

Recombinant HGF protein attenuated CDAA diet-induced lipogenesis. (A) Oil red O staining of lipid droplets. Original magnification, x200. (B) Quantification of Oil Red O staining. (C) Hepatic mRNA expression of Fasn and Dgat1. Data are presented as mean ± SEM. (*P < 0.05, **P < 0.01, significantly different from CSAA-Veh; ^#P < 0.05, significantly different from CDAA-Veh).

Figure 5

Recombinant HGF protein had anti-inflammatory effect in CDAA diet-induced hepatic steatosis. (A) Immunohistochemistry for F4/80. Original magnification, x200. (B) Quantification of F4/80 staining. (C) Hepatic mRNA expression of Cxcl1, Ccl2, and Ccl5. Data are presented as mean ± SEM. (*P < 0.05, significantly different from CSAA-Veh; ^#P < 0.05, significantly different from CDAA-Veh).

Figure 6

Recombinant HGF ameliorated CDAA diet-induced fibrogenic response. (A) Sirius Red staining. Original magnification, x100. (B) Hepatic mRNA expression of Col1a1, Acta2, Timp1, Tgfb1, and Serpine1. Data are presented as mean ± SEM. (*P < 0.05, **P < 0.01, significantly different from CSAA-Veh; ^#P < 0.05, significantly different from CDAA-Veh).