J Korean Med Sci.  2010 Apr;25(4):570-576. 10.3346/jkms.2010.25.4.570.

Differential Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Thioacetamide-Induced Chronic Liver Injury

• Park SY
• Shin HW
• Lee KB
• Lee MJ
• Jang JJ

• KMID: 1792927
• DOI: http://doi.org/10.3346/jkms.2010.25.4.570

Abstract

Hepatic fibrogenesis, a complex process that involves a marked accumulation of extracellular matrix components, activation of cells capable of producing matrix materials, cytokine release, and tissue remodeling, is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The MMP-TIMP balance can regulate liver fibrogenesis. The aim of this study was to evaluate the expression patterns of MMPs and TIMPs during thioacetamide (TAA)-induced liver fibrogenesis. Chronic liver injury was induced with TAA (200 mg/kg i.p.) for 4 or 7 weeks in male Sprague-Dawley rats. Hepatic injury and fibrosis were assessed by hematoxylin-eosin (H&E) staining, and collagen deposition was confirmed by Sirius Red staining. The level of hepatic injury was quantified by serological analysis. The transcriptional and translational levels of alpha-smooth muscle actin (alpha-SMA), MMPs, and TIMPs in the liver were measured by Western blotting, RT-PCR, and immunohistochemistry. MMP, TIMP, and alpha-SMA were observed along fibrotic septa and portal spaces around the lobules. TAA treatment increased transcription of both MMPs and TIMPs, but only TIMPs showed increased translation. The dominant expression of TIMPs may regulate the function of MMPs to maintain liver fibrosis induced by TAA.

Keyword

Liver Cirrhosis; Thioacetamide; Matrix Metalloproteinases; Tissue Inhibitors of Metalloproteinases

MeSH Terms

Animals
Collagen/metabolism
Extracellular Matrix/chemistry/metabolism
*Liver Cirrhosis/chemically induced/metabolism/pathology
Male
Matrix Metalloproteinases/genetics/*metabolism
Rats
Rats, Sprague-Dawley
Thioacetamide/*toxicity
Tissue Inhibitor of Metalloproteinases/genetics/*metabolism
Tissue Inhibitor of Metalloproteinases
Thioacetamide
Collagen
Matrix Metalloproteinases

Figure

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  Fig. 1 Experimental Design. Animals were treated weekly intraperitoneal injections of TAA at 200 mg/kg (▼), which was replaced by saline in untreated animals (▽). Total experimental period was 4 or 7 weeks. Rats were killed at the end of 4 or 7 weeks after the last injection (▲).

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  Fig. 2 Growth curves and relative liver weight in TAA-treated rats. (A) Each group (n=10) was given repeated injection with TAA or saline for 4 or 7 weeks, and body weights were measured every week. The graph shows the mean±SD of body weight. (B) Relative liver weight was calculated by the ratio to body weight. TAA treatment significantly increases the liver/body weight ratio. Data are expressed as the mean±SD, *P<0.05, TAA treated vs. untreated rats.

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  Fig. 3 Histologic features and collagen deposition of livers after 4 or 7 weeks TAA treatment. (A) TAA significantly induced hepatic fibrosis, especially at week 7 (H&E, ×100). (B) Collagen deposition (red) is prominent in TAA treated rats (Sirius Red, ×100) at week 4 and 7. (C) Fibrosis scores expressed as the average grade±SD. *P<0.001, TAA treated vs. untreated rats.

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  Fig. 4 Protein expressions of MMPs and TIMPs in TAA induced liver fibrosis. (A) MMP-2 activity is increased in TAA treated rats using zymography. (B) Protein expression pattern for MT1-MMP, MMP-2, MMP-13, TIMP-1, and TIMP-2 in TAA treated rats at week 4 and 7. (C) TIMP-1 and TIMP-2 protein levels are significantly increased in the TAA-treated group. β-tubulin was used as the internal standard. *P<0.05, TAA treated vs. untreated rat. All data are representative of three or four experiments performed with each group.

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  Fig. 5 Expression of α-SMA, MT1-MMP, MMP-2, MMP-13, TIMP-1, and TIMP-2 mRNA in TAA-induced liver fibrosis. TAA treatment increases mRNA level for α-SMA, MMPs, and TIMPs. RT-PCR products were obtained with specific primers for each gene and β-actin as described in Table 1.

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  Fig. 6 Immunohistochemical staining for MMPs, TIMPs, and α-SMA in TAA-treated rats. Positive cells are increased in the portal area and central vein in TAA-treated rats (×200). Black head arrows indicate positively stained cells.

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