Development of a mouse iron overload-induced liver injury model and evaluation of the beneficial effects of placenta extract on iron metabolism

Abstract

Hepatic iron deposition is seen in cases of chronic hepatitis and cirrhosis, and is a hallmark of a poorer prognosis. Iron deposition is also found in non-alcoholic steatohepatitis (NASH) patients. We have now developed a mouse model of NASH with hepatic iron deposition by combining a methione- and choline-deficient (MCD) diet with an iron-overload diet. Using this model, we evaluated the effects of human placenta extract (HPE), which has been shown to ameliorate the pathology of NASH. Four-week-old male C57BL/6 mice were fed the MCD diet with 2% iron for 12 weeks. In liver sections, iron deposition was first detected around the portal vein after 1 week. From there it spread throughout the parenchyma. Biliary iron concentrations were continuously elevated throughout the entire 12-week diet. As a compensatory response, the diet caused elevation of serum hepcidin, which accelerates excretion of iron from the body. Accumulation of F4/80-positive macrophages was detected within the sinusoids from the first week onward, and real-time PCR analysis revealed elevated hepatic expression of genes related inflammation and oxidative stress. In the model mice, HPE treatment led to a marked reduction of hepatic iron deposition with a corresponding increase in biliary iron excretion. Macrophage accumulation was much reduced by HPE treatment, as was the serum oxidation-reduction potential, an index of oxidative stress. These data indicate that by suppressing inflammation, oxidative stress and iron deposition, and enhancing iron excretion, HPE effectively ameliorates iron overload-induced liver injury. HPE administration may thus be an effective strategy for treating NASH.

Keywords: Molecular biology.

Fig. 1

Protocol used for the methione- and choline-deficient (MCD) with iron-overload diet. A, Schematic depiction of the protocol. After the 4 weeks of a normal diet, the mice were divided into MCD +2% iron (MCD-Fe) diet and normal diet groups. The two diets were then administered for 12 weeks. B, Body weight changes on the MCD-Fe and normal diets. Body weights were measured in 4- to 13-week-old mice. Symbols depict means ±SEM; n = 6.

Fig. 2

Evaluation of iron deposition in the liver. A, Liver sections from mice fed the MCD-Fe diet. Shown is Berlin blue staining of the sections. Iron deposits appear blue. Note the progressive increase in blue staining. Scale bars = 100 μm. B, Percent Berlin blue-positive area within the liver sections. Symbols depict means ±SEM; n = 6.

Fig. 3

Evaluation of iron excretion and serum hepcidin level. A, B, Time-dependent changes in urinary (A) and biliary (B) iron concentrations in mice fed the MCD-Fe or normal diet. Symbols depict means ±SEM; n = 5–9. **p < 0.01, MCD-Fe vs. control. C, Serum hepcidin concentrations measured during the first 4 weeks of the MCD-Fe and normal diets. Symbols depict means ±SEM; n = 9 in the MCD-Fe group, and n = 2 in the normal group.

Fig. 4

Evaluation of macrophage accumulation in the liver. A, Immunostaining of F4/80 in sections of liver samples collected from mice on the MCD-Fe diet for the indicated times. Green; F4/80, Blue; DAPI. Scale bars = 100 μm. Note the time-dependent increase in green fluorescent staining. B, Calculation of F4/80-positive area per 200× microscope field. Symbols depict means ±SEM; n = 6.

Fig. 5

Inflammation-, fibrosis- and oxidative stress-related gene expression in liver. A, Time courses of the relative hepatic expression levels of genes encoding the proinflammatory cytokines IL-1β and IL-6 and the fibrosis-related molecule collagen-α1 in mice on the MCD-Fe diet. B, Time courses of the relative hepatic gene expression of NADPH oxidase subunits. Symbols depict means ±SEM; n = 4.

Fig. 6

Effect of HPE treatment on body weights in mice on the MCD-Fe diet. Body weights were measured in 4- to 13-week-old mice fed the MCD-Fe diet with or without HPE treatment. Symbols depict means ±SEM. n = 6.

Fig. 7

Schematic depiction of the protocol used for HPE administration to mice on the MCD-Fe diet. After 4 weeks of normal diet, the mice were administered the MCD +2% iron diet for 4 weeks. Mice were intramuscularly administered HPE (0.1 ml of Laennec (3.6 mg/kg)) or control saline twice a week during the 4 weeks.

Fig. 8

Effect of HPE treatment on iron deposition in the liver. A, Sections of liver samples collected from mice on the MCD-Fe diet for the indicated times, with or without HPE treatment. The sections are stained with Berlin blue. Scale bars = 100 μm. B, Percent Berlin blue-positive area within the liver sections. Symbols depict means ±SEM; n = 6. *p < 0.05.

Fig. 9

Effect of HPE treatment on urinary and biliary iron excretion. Time-dependent changes in urinary (A) and biliary (B) iron concentration in mice on the MCD-Fe diet with or without HPE treatment. Symbols depict means ±SEM. n = 5.

Fig. 10

Effect of HPE-treatment on macrophage accumulation in the live. A, Immunostaining of F4/80 in sections of liver samples collected from mice on the MCD-Fe diet for the indicated times, with or without HPE treatment. Green, F4/80; Blue, DAPI. Scale bars = 100 μm. B, Calculation of F4/80-positive area per 200× microscope field. Symbols depict means ±SEM; n = 6. *p < 0.05, **p < 0.01, HPE treated vs. untreated.

Fig. 11

Effect of HPE treatment on oxidative stress. A, Immunostaining of 4HNE in sections of liver samples collected from mice on the MCD-Fe diet for the indicated times, with or without HPE treatment. Scale bars = 100 μm. B, Serum oxidation-reduction potentials (ORP) were measured as an index of oxidative stress using a RedoxSYS Analyzer. Bars depict means ±SEM; n = 4–5. *p < 0.05, ***p < 0.001, HPE treated vs. untreated.