Yonsei Med J.  2015 Nov;56(6):1572-1581. 10.3349/ymj.2015.56.6.1572.

Thalidomide Accelerates the Degradation of Extracellular Matrix in Rat Hepatic Cirrhosis via Down-Regulation of Transforming Growth Factor-beta1

• Lv P
• Meng Q
• Liu J
• Wang C

• KMID: 2345885
• DOI: http://doi.org/10.3349/ymj.2015.56.6.1572

Abstract

PURPOSE
The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis.
MATERIALS AND METHODS
Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-smooth muscle actin (alpha-SMA) protein in the liver, transforming growth factor beta1 (TGF-beta1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-beta1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction.
RESULTS
Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-beta1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group.
CONCLUSION
Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-beta1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.

Keyword

Thalidomide; cirrhosis; extracellular matrix; matrix metalloproteinase-13; tissue inhibitor of metalloproteinase-1; transforming growth factor-beta1

MeSH Terms

Actins
Animals
Carbon Tetrachloride/toxicity
Collagen Type III/metabolism
Down-Regulation
Extracellular Matrix/metabolism
Immunohistochemistry
Immunosuppressive Agents/*pharmacology
Liver Cirrhosis, Experimental/chemically induced/*metabolism/pathology/*prevention & control
Male
RNA, Messenger/analysis/metabolism
Rats
Rats, Wistar
Thalidomide/*pharmacology
Tissue Inhibitor of Metalloproteinase-1/biosynthesis/*drug effects
Transcription Factor RelA/biosynthesis/drug effects
Transforming Growth Factor beta1/biosynthesis/*drug effects
Transforming Growth Factors/metabolism
Actins
Carbon Tetrachloride
Collagen Type III
Immunosuppressive Agents
RNA, Messenger
Thalidomide
Tissue Inhibitor of Metalloproteinase-1
Transcription Factor RelA
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  Fig. 1 Appearance of rat livers. Cirrhosis was much more severe in rats in the M and S than in T. N, normal control group; M, model group; S, spontaneous recovery group; T, thalidomide-treated group.

• 

  Fig. 2 Liver histopathology of the rat. (A) Histopathology of liver tissues in study groups was determined using hematoxylin and eosin staining (×100). N indicates the normal control group; M indicates the model group, in which septa and collagen deposition formed pseudolobule; S indicates the spontaneous recovery group, in which the changes are less pronounced; T indicates the thalidomide-treated group, in which architecture is similar to that of the normal control group. (B) Comparison of total Scheuer scores of the liver. The number of cases in each group was analyzed statistically: (N) 14, (M) 11, (S) 12, and (T) 14. Data were analyzed with ANOVA. *p<0.01 versus N, †p<0.01 versus M, ‡p<0.01 T versus S. ANOVA, analysis of variance.

• 

  Fig. 3 Immunohistochemical staining for MMP-13 in rat livers. (A) Positive MMP-13 cells were mainly distributed at the fibro-septa band, area of necrosis and inflammatory cell infiltration in hepatic lobules (original magnification N-T, ×400, immunohistochemical staining). (B) Comparison of the expression of MMP-13 using immunohistochemical analysis. The number of cases in each group was analyzed statistically: (N) 14, (M) 11, (S) 12, and (T) 14. Data were analyzed with ANOVA. *p<0.01 versus N, †p<0.01 versus M, ‡p<0.01 T versus S. N, normal control group; M, model group; S, spontaneous recovery group; T, thalidomide-treated group; MMP-13, matrix metalloproteinase-13; ANOVA, analysis of variance.

• 

  Fig. 4 Immunohistochemical staining for TIMP-1 in rat livers. (A) Positive TIMP-1 cells were mainly distributed at the fibro-septa band, area of necrosis and inflammatory cell infiltration in hepatic lobules (original magnification N-T, ×400, immunohistochemical staining). (B) Comparison of the expression of TIMP-1 using immunohistochemical analysis. The number of cases in each group was analyzed statistically: (N) 14, (M) 11, (S) 12, and (T) 14. Data were analyzed with ANOVA. *p<0.01 versus N, †p<0.01 versus M, ‡p<0.01 T versus S. N, normal control group; M, model group; S, spontaneous recovery group; T, thalidomide-treated group; TIMP-1, tissue inhibitor of metalloproteinase-1; ANOVA, analysis of variance.

• 

  Fig. 5 Immunohistochemical staining for α-SMA in rat livers. (A) Positive α-SMA cells were mainly distributed at the fibro-septa band, area of necrosis and inflammatory cell infiltration in hepatic lobules, and they were also distributed at the periantral HSC (original magnification N-T, ×400, immunohistochemical staining). (B) Comparison of the expression of α-SMA using immunohistochemical analysis. The number of cases in each group was analyzed statistically: (N) 14, (M) 11, (S) 12, and (T) 14. Data were analyzed with ANOVA. *p<0.01 versus N, †p<0.01 versus M, ‡p<0.01 T versus S. N, normal control group; M, model group; S, spontaneous recovery group; T, thalidomide-treated group; α-SMA, α-smooth muscle actin; HSC, hepatic stellate cell; ANOVA, analysis of variance.

• 

  Fig. 6 Expression of TGF-β1 protein in rat livers using Western blot analysis. (A) Lane 1 shows the N; lane 2 shows the M; lane 3 shows the S; lane 4 shows the T. β-actin served as an internal control. (B) Comparison of the expression of TGF-β1 protein using Western blot analysis. The number of cases in each group was analyzed statistically: (N) 14, (M) 11, (S) 12, and (T) 14. Data were analyzed with ANOVA. *p<0.01 versus N, †p<0.01, ‡p<0.05 versus M, §p<0.01 T versus S. N, normal control group; M, model group; S, spontaneous recovery group; T, thalidomide-treated group; TGF-β1, transforming growth factor β1; ANOVA, analysis of variance.

• 

  Fig. 7 Expression of MMP-13, TIMP-1, and TGF-β1 mRNA in rat livers as indicated by RT-PCR analysis. (A) Lane 1 shows the 100 bp marker; lane 2 shows the N; lane 3 shows the M; lane 4 shows the S; lane 5 shows the T. GAPDH served as an internal control. (B) Comparison of the expression of MMP-13, TIMP-1, and TGF-β1 mRNA using RT-PCR analysis. The number of cases in each group was analyzed statistically: (N) 14, (M) 11, (S) 12, and (T) 14. Data were analyzed with ANOVA. *p<0.01 versus N, †p<0.01, ‡p<0.05 versus M, §p<0.01 T versus S. N, normal control group; M, model group; S, spontaneous recovery group; T, thalidomide-treated group; MMP-13, matrix metalloproteinase-13; TIMP-1, tissue inhibitor of metalloproteinase-1; TGF-β1, transforming growth factor β1; mRNA, messenger ribonucleic acid; RT-PCR, reverse transcriptase polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TGF-β1, transforming growth factor β1; ANOVA, analysis of variance.

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