Table 1.
Primer sequences used in the study.
Fig 1.
Pathological changes of livers in CCl4-treated mice.
Pathological changes of livers were analyzed by H&E staining. Arrows represent lesion area.
Fig 2.
Levels of serum ALT and AST and TGF-β1 in mice treated with CCl4.
(A) Levels of ALT and AST were detected in sera of mice treated with CCl4 (■) and control mice treated with olive oil (●). (B) Levels of TGF-β1 were determined by ELISA in sera of mice treated with CCl4 (■) and control mice treated with olive oil (●). *P<0.01, **P<0.001, compared with control group (n = 6).
Fig 3.
Expression of TGF-β1 in livers of mice treated with CCl4.
(A) Levels of TGF-β1 were examined by ELISA in hepatic homogenates of mice with CCl4 (●) and control mice treated with olive oil (○). *P<0.01, compared with control group (n = 6). (B) Expression of TGF-β1 mRNA was examined by RT-PCR in livers of mice treated with CCl4 and control mice treated with olive oil. The graph represents relative levels of TGF-β1 mRNA from triplicate determinations. *P<0.01, compared with olive oil control group.
Fig 4.
Expressions of TβRII and Smads mRNA and protein in livers of mice treated with CCl4.
(A) Expressions of TβRII and signaling molecules Smad2, 3, 4, 6, 7 mRNA were examined by RT-PCR in livers of mice treated with CCl4 and control mice treated with olive oil. The graph represents relative levels of mRNA from triplicate determinations. Olive oil (grey bars), CCl4 (black bars). *P<0.01, compared with olive oil control group. (B) pSmad3 and Smad3 protein levels were analyzed by Western blotting in livers of mice treated with CCl4 and control mice treated with olive oil. The graph represents relative protein levels of pSmad3 and Smad3 from triplicate determinations. *P<0.01, compared with olive oil control group.
Fig 5.
Expression of Smad3 in livers of Smad3-overexpressing mice.
(A) Smad3 mRNA expressions were evaluated by RT-PCR in livers of CCl4-treated Smad3-overexpressing mice (Smad3 + CCl4) and plasmid control mice (PC + CCl4). The graph represents relative levels of Smad3 mRNA from triplicate determinations. *P<0.05, **P<0.01, compared with plasmid control group. (B) pSmad3 protein levels were examined by Western blotting in livers of CCl4-treated Smad3-overexpressing mice (Smad3 + CCl4) and plasmid control mice (PC + CCl4). The graph represents relative protein levels of pSmad3 from triplicate determinations. *P<0.01, compared with plasmid control group. (C) Expression of Smad3 protein was examined by immunohistochemical staining with rabbit anti-mouse Smad3 antibody in livers of Smad3-overexpressing mice (Smad3 + CCl4) and plasmid control mice (PC + CCl4) treated with CCl4 for 1 day. A procedural control staining in the liver was performed using normal rabbit IgG instead of anti-Smad3 antibody (Control). Arrows represent Smad3 positive cells (x100).
Fig 6.
Effects of Smad3 overexpression in vivo on liver injury of CCl4-treated mice.
(A) Levels of ALT and AST were examined by ELISA in sera of CCl4-treated Smad3-overexpressing mice (■) and plasmid control mice (●). *P<0.05, **P<0.01, compared with control group (n = 6). (B) Pathological changes of livers were analyzed by H&E staining in 1 day olive oil-treated control mice (Olive), plasmid control mice (PC + Olive) and Smad3-overexpressing mice (Smad3 + Olive) (x40). (C) Pathological changes of livers were analyzed by H&E staining in CCl4-treated Smad3-overexpressing mice (Smad3 + CCl4) and plasmid control mice (PC + CCl4). Arrows represent lesion area (x40). The graph represents liver injury index. *P<0.05.
Fig 7.
Assay of inflammatory cytokines and inflammatory cells in Smad3-overexpressing mice.
(A) Levels of IL-1β and IL-6 were examined by ELISA in sera of CCl4-treated Smad3-overexpressing mice (■) and plasmid control mice (●). *P<0.05, compared with control group (n = 6). (B) The infiltration of neutrophils and macrophages in livers of plasmid control mice (PC + CCl4) and Smad3-overexpressing mice (Smad3 + CCl4) treated with CCl4 for 3 days were examined by MPO staining and F4/80 immunofluorescence staining. Arrows represent neutrophils (dark blue cells) (x400) and macrophages cells (red cells) (x200). (C) The inflammatory cells were examined in livers of CCl4-treated plasmid control mice (PC + CCl4) and Smad3-overexpressing mice (Smad3 + CCl4) by flow cytometry with anti-CD14/CD11b and anti-Ly-6G/CD11b antibodies. *P<0.05, **P<0.01, compared with control group (n = 6).
Fig 8.
Analysis of hepatocytes apoptosis in Smad3-overexpressing mice.
(A) The apoptosis of hepatocytes was examined by TUNEL staining in livers of CCl4-treated Smad3-overexpressing mice (Smad3 + CCl4) and plasmid control mice (PC + CCl4). Arrows represent apoptotic cells (red cells). The graph represents the apoptotic index of hepatocytes. *P<0.05 and **P<0.01. (B) Levels of pSmad3 and apoptosis-associated proteins Bax, Bcl2, Cyt C and the cleaved caspase 3 (cleaved cap3) were evaluated by Western blotting in livers of non-treated mice (Cont), CCl4-treated plasmid control mice (PC + C) and CCl4-treated Smad3-overexpressing mice (Sm + C). The graph represents levels of relative protein from triplicate determinations.*P<0.01. CCl4-treated Smad3-overexpressing mice (b) vs CCl4-treated plasmid control mice (a).