Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathway-mediated inhibition of extracellular matrix and autophagy

Abstract

Liver fibrosis is a dynamic reversible pathological process in the development of chronic liver disease to cirrhosis. However, the current treatments are not administered for a long term due to their various side effects. Autophagy is initiated to decompose damaged or excess organelles, which had been found to alter the progression of liver fibrosis. In this article, we hypothesized that fucoidan from Fucus vesiculosus may attenuate liver fibrosis in mice by inhibition of the extracellular matrix and autophagy in carbon tetrachloride- and bile duct ligation-induced animal models of liver fibrosis. The results were determined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. Fucoidan from F. vesiculosus could inhibit the activation of hepatic stellate cells and the formation of extracellular matrix and autophagosomes, and its effect may be associated with the downregulation of transforming growth factor beta 1/Smads pathways. Fucoidan, as an autophagy and transforming growth factor beta 1 inhibitor, could be a promising potential therapeutic agent for liver fibrosis.

Keywords: bile duct ligation; hepatic stellate cells; liver cirrhosis.

Figure 1

Effect of fucoidan on liver cytolysis. Notes: (A) Fucoidan decreased the level of ALT, AST and hydroxyproline content in a dose-dependent manner. Data are expressed as mean ± SD (n=8, *P<0.05 for CCl₄ [BDL] versus control, ^#P<0.05 for CCl₄ [BDL] + fucoidan versus CCl₄ [BDL], ^{^}P<0.05 for CCl₄ [BDL] + fucoidan [25 mg/kg] versus CCl₄ [BDL] + fucoidan [10 mg/kg], +P<0.05 for CCl₄ [BDL] + fucoidan [50 mg/kg] versus CCl₄ [BDL] + fucoidan [25 mg/kg]). (B and C) Fucoidan ameliorated pathological change, showed by H&E and Masson’s trichrome (original magnification: ×200). Abbreviations: AST, aspartate aminotransferase; SD, standard deviation; CCl₄, carbon tetrachloride; BDL, bile duct ligation; H&E, hematoxylin and eosin; ALT, alanine aminotransferase.

Figure 2

Effect of fucoidan on ECM in liver fibrosis. Notes: (A) Fucoidan decreased the level of HA and LN. (B) The analysis of Western blotting showed that fucoidan obviously changed the expression of collagen I, α-SMA, MMP-9, and TIMP1. (C) The mRNA levels of collagen 1α1, α-SMA were significantly downregulated by fucoidan. Data are expressed as mean ± SD (n=8, *P<0.05 for CCl₄ [BDL] versus control, ^#P<0.05 for CCl₄ [BDL] + fucoidan versus CCl₄ [BDL], ^{^}P<0.05 for CCl₄ [BDL] + fucoidan [25 mg/kg] versus CCl₄ [BDL] + fucoidan [10 mg/kg], +P<0.05 for CCl₄ [BDL] + fucoidan [50 mg/kg] versus CCl₄ [BDL] + fucoidan [25 mg/kg]). (D) The areas of positive cells of collagen I and α-SMA were diminished by fucoidan as shown by immunohistochemistry staining (original magnification: ×200). Abbreviations: ECM, extracellular matrix; HA, hyaluronic acid; LN, laminin; α-SMA, α-smooth muscle actin; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; mRNA, messenger ribonucleic acid; SD, standard deviation; CCl₄, carbon tetrachloride; BDL, bile duct ligation.

Figure 2

Effect of fucoidan on ECM in liver fibrosis. Notes: (A) Fucoidan decreased the level of HA and LN. (B) The analysis of Western blotting showed that fucoidan obviously changed the expression of collagen I, α-SMA, MMP-9, and TIMP1. (C) The mRNA levels of collagen 1α1, α-SMA were significantly downregulated by fucoidan. Data are expressed as mean ± SD (n=8, *P<0.05 for CCl₄ [BDL] versus control, ^#P<0.05 for CCl₄ [BDL] + fucoidan versus CCl₄ [BDL], ^{^}P<0.05 for CCl₄ [BDL] + fucoidan [25 mg/kg] versus CCl₄ [BDL] + fucoidan [10 mg/kg], +P<0.05 for CCl₄ [BDL] + fucoidan [50 mg/kg] versus CCl₄ [BDL] + fucoidan [25 mg/kg]). (D) The areas of positive cells of collagen I and α-SMA were diminished by fucoidan as shown by immunohistochemistry staining (original magnification: ×200). Abbreviations: ECM, extracellular matrix; HA, hyaluronic acid; LN, laminin; α-SMA, α-smooth muscle actin; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; mRNA, messenger ribonucleic acid; SD, standard deviation; CCl₄, carbon tetrachloride; BDL, bile duct ligation.

Figure 3

Effect of fucoidan on the process of autophagy in liver fibrosis. Notes: (A) The mRNA levels of Beclin-1 and LC3-11 were decreased by fucoidan in a dose-dependent manner. Data are expressed as mean ± SD (n=8, *P,0.05 for CC [BDL] versus control, ^#P<0.05 for CCl₄ [BDL] + fucoidan versus CCl₄ [BDL], ^{^}P<0.05 for CCl₄ [BDL] + fucoidan [25 mg/kg] versus CCl₄ [BDL] + fucoidan [10 mg/kg], +P<0.05 for CCl₄ [BDL] + fucoidan [50 mg/kg] versus CCl₄ [BDL] + fucoidan [25 mg/kg]). (B) The analysis of Western blotting showed that fucoidan obviously changed the expression of Beclin-1, LC3, and P62. (C) The areas of positive cells of Beclin-1 and LC3 were diminished by fucoidan as shown by immunohistochemistry staining (original magnification: 200×). (D) The amount of autophagosome significantly decreased as shown by TEM (original magnification: ×20,000). The red arrows indicate autophagosomes detected in liver tissue. Abbreviations: mRNA, messenger ribonucleic acid; SD, standard deviation; BDL, bile duct ligation; CCl₄, carbon tetrachloride; TEM, transmission electron microscopy.

Figure 4

Effect of fucoidan on the TGF-β1/Smads pathways in liver fibrosis. Notes: (A) The serum and mRNA levels of TGF-β1 were decreased by fucoidan in a dose-dependent manner. Data are expressed as mean ± SD (n=8, *P<0.05 for CCl₄ [BDL] versus control, ^#P<0.05 for CCl₄ [BDL] + fucoidan versus CCl₄ [BDL] + fucoidan [25 mg/kg] versus CCl₄ [BDL] + fucoidan [10 mg/kg], ⁺P<0.05 for CCl₄ [BDL] + fucoidan [50 mg/kg] versus CCl₄ [BDL] + fucoidan [25 mg/kg]). (B) The analysis of Western blotting showed that fucoidan obviously reduced the expression of TGF-β1, p-Smad2, and p-Smad3. (C) The areas of positive cells of TGF-β1, p-Smad2, and p-Smad3 were diminished by fucoidan as shown by immunohistochemistry staining (original magnification: ×200). Abbreviations: TGF-β1, transforming growth factor beta 1; mRNA, messenger ribonucleic acid; SD, standard deviation; CCl₄, carbon tetrachloride; BDL, bile duct ligation.

Figure 5

Mechanism of fucoidan action. Notes: Fucoidan inhibited HSCs activation that could secrete TGF-β1, thus inhibiting the downstream TGF-β1/Smads pathway. Smad2/3 cannot be transferred from pulp to the nucleus to combine with specific DNA sequences to promote Beclin-1 transcription so that fucoidan hindered the formation of autophagosomes. The autophagy associated with cracked organelles was then activated to induce cell death. Abbreviations: HSCs, hepatic stellate cells; TGF-β1, transforming growth factor beta 1; DNA, deoxyribose nucleic acid; CCl₄, carbon tetrachloride; BDL, bile duct ligation; MFBs, myofibroblasts; ECM, extracellular matrix; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitor of matrix metalloproteinases; α-SMA, α-smooth muscle actin; SEC, sinusoidal endothelial cells. CRE, c-AMP response element.