Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats

Abstract

Background: At present there is no effective and accepted therapy for hepatic fibrosis. Transforming growth factor (TGF)-β1 signaling pathway contributes greatly to hepatic fibrosis. Reducing TGF-β synthesis or inhibiting components of its complex signaling pathway represent important therapeutic targets. The aim of the study was to investigate the effect of curcumin on liver fibrosis and whether curcumin attenuates the TGF-β1 signaling pathway.

Methods: Sprague-Dawley rat was induced liver fibrosis by carbon tetrachloride (CCl4) for six weeks together with or without curcumin, and hepatic histopathology and collagen content were employed to quantify liver necro-inflammation and fibrosis. Moreover, the mRNA and protein expression levels of TGF-β1, Smad2, phosphorylated Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) were determined by quantitative real time-PCR, Western blot, or immunohistochemistry.

Results: Rats treated with curcumin improved liver necro-inflammation, and reduced liver fibrosis in association with decreased α-smooth muscle actin expression, and decreased collagen deposition. Furthermore, curcumin significantly attenuated expressions of TGFβ1, Smad2, phosphorylated Smad2, Smad3, and CTGF and induced expression of the Smad7.

Conclusions: Curcumin significantly attenuated the severity of CCl4-induced liver inflammation and fibrosis through inhibition of TGF-β1/Smad signalling pathway and CTGF expression. These data suggest that curcumin might be an effective antifibrotic drug in the prevention of liver disease progression.

Figure 1

Curcumin (Cur) administration ameliorates hepatic inflammation and fibrosis induced by CCl₄administration. (A) Haematoxylin and eosin (H&E) staining of the livers (original magnification, ×200). (B) Evaluation of liver fibrosis by Sirius red staining of liver sections (original magnification, ×200). (C) Semiquantitative evaluation of liver necro-inflammatory activity according to the METAVIR scoring system. *P < 0.05 for Cur/CCl₄ vs. PBS/CCl₄ (n = 8/group). (D) Quantitative image analysis of hepatic Sirius red staining using a computerized image analysis system; **P < 0.001 for Cur/CCl₄ vs. PBS/CCl₄ (n = 6-8/group).

Figure 2

Effects of curcumin on activation of hepatic stellate cells (HSCs) and collagen type I protein expression in the liver. (A, B) Immunohistochemical staining of type I collagen and alpha-smooth muscle actin (α-SMA) in the liver (original magnification, ×200). (C, D) Western blotting analysis of hepatic collagen-I and α-SMA expression. Liver tissue was lysed and subjected to western blot analysis for collagen-I and α-SMA, and the loading control GAPDH. Blots obtained from several experiments were analysed using densitometry, and the densitometric values were pooled from four animals per group and shown as means ± SD in the bar graph. *P < 0.01 for Cur/CCl₄ vs. PBS/CCl₄.

Figure 3

Effects of curcumin (Cur) on transforming growth factor beta1 (TGF-β1) expression in the liver. (A) Immunohistochemical assessment of TGF-β1 in the liver (original magnification, ×200). (B) Western blotting analysis of hepatic TGF-β1 expression. Liver tissue was lysed and subjected to western blot analysis for TGF-β1, and the loading control GAPDH. Blots obtained from several experiments were analysed using densitometry, and the densitometric values were pooled from four animals per group and are shown as means ± SD in the bar graph. *P < 0.05 vs. rats treated with CCl₄ alone. (C) Analysis of hepatic TGF-β1 mRNA expression by quantitative PCR. TGF-β1 mRNA levels were normalized relative to those of GAPDH mRNA in each sample, and values are expressed as the mean ± SD fold increase compared with the normal control group (n=6). *P < 0.01 for Cur/CCl₄ vs. PBS/CCl₄.

Figure 4

Effects of curcumin (Cur) on the expression of Smad2 and Smad3 proteins in rat liver tissue. (A, B) Immunohistochemical staining for Smad2 and Smad3 in the liver (original magnification, ×200). (C, D) Western blot analysis of hepatic Smad2 and Smad3 expression. Liver tissue was lysed and subjected to western blot analysis for Smad2 and Smad3 and the loading control GAPDH. Blots obtained from several experiments were analysed using densitometry, and the densitometric values were pooled from four animals per group and shown as means ± SD in the bar graph. *P < 0.05 for Cur/CCl₄ vs. PBS/CCl₄, **P < 0.01 for Cur/CCl₄ vs. PBS/CCl₄.

Figure 5

Curcumin induces Smad7 and inhibits Smad2, P-Smad2, and Smad3 mRNA and protein expression in liver tissue of CCl₄injected rats. (A-C) The levels of Smad2 (A), Smad3 (B) and Smad7 (C) mRNA in liver tissue were determined by quantitative real-time RT-PCR. Smad2, Smad3 and Smad7 mRNA levels were normalized relative to GAPDH mRNA expression in each sample, and values are expressed as the mean ± SD fold increase over normal control group (n=6). *P < 0.01 for Cur/CCl₄ vs. PBS/CCl₄. (D-E) Western blots analysis of hepatic P-Smad2 and Smad7 expression, with GAPDH as the loading control. Blots obtained from several experiments were analysed using densitometry, and the densitometric values were pooled from four animals per group and are shown as means ± SD in the bar graph. *P < 0.05 for Cur/CCl₄ vs. PBS/CCl₄.

Figure 6

Curcumin inhibits connective tissue growth factor (CTGF) expression in liver tissue of CCl₄treated rats. (A) Immunohistochemical staining for CTGF in rat liver (original magnification, ×200). (B) Western blots analysis of hepatic CTGF expression and GAPDH as loading control. Blots obtained from several experiments were analysed using densitometry, and the densitometric values were pooled from four animals per group and are shown as means ± SD in the bar graph. *P < 0.05 for Cur/CCl₄ vs. PBS/CCl₄. (C) The levels of CTGF mRNA in liver tissue were determined by quantitative real-time RT-PCR and normalized relative to GAPDH mRNA expression in each sample, with values expressed as the mean ± SD fold increase over normal control group (n=6). *P < 0.01 for Cur/CCl₄ vs. PBS/CCl₄.