Figure 1.
AA treatment attenuates CCl4-induced liver damage and functional impairment in a dosage-dependent manner in rats.
A. Histology (H&E). B. Liver function. Data represent mean ± SEM for groups of 8 animals. **p<0.01, *** p<0.001 versus normal control; #p<0.05, ##p<0.01, ###p<0.001 versus CCl4-induced disease control. Magnification: ×100.
Figure 2.
Immunohistochemistry detects that AA treatment attenuates CCl4-induced liver fibrosis in a dosage-dependent manner in rats.
A. Collagen I expression. B. Collagen III expression. Data represent mean ± SEM for groups of 8 animals. **p<0.01, *** p<0.001 versus normal control; ##p<0.01, ###p<0.001 versus CCl4-induced disease control. Magnification: ×100.
Figure 3.
Immunohistochemistry detects that AA treatment attenuates CCl4-induced activation of HSC in a dosage-dependent manner in rats.
Activation of HSC was determined by a-SMA+ myofibroblast transition. A. Representative picture from a control rat treated with 20% peanut oil. B. Representative picture from a rat treated with CCl4. C–E. Representative pictures from CCl4-treated rats received AA in a dosage-dependent manner. F. Quantitation of a-SMA+ cells. Data represent mean ± SEM for groups of 8 animals. *** p<0.001 versus normal control; ##p<0.01, ###p<0.001 versus CCl4-induced disease rats. Magnification: ×200.
Figure 4.
Real-time PCR and Western blot analysis show that AA treatment blocks CCl4-induced liver fibrosis in a dosage-dependent manner in rats.
A. Real-time PCR for a-SMA, collagen type I and III mRNA expression. B. Western blot analysis for a-SMA, collagen type I and III protein expression. Data represent mean ± SEM for groups of 8 animals. *p<0.05, **p<0.01, *** p<0.001 versus normal control; #p<0.05, ###p<0.001 versus CCl4-induced disease control.
Figure 5.
TGF-beta1 induces collagen I and a-SMA expression by HSC-T6 cells in vitro.
A. Real-time PCR show that TGF-beta1 (1 ng/ml) induces collagen I and a-SMA mRNA expression in a time and dosage-dependent manner. B. Western blot analysis for a time (TGF-beta1 1 ng/ml) and dosage (24 h)-dependent effects of TGF-beta1 on collagen I expression. Data represent mean ± SEM for at least 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus medium control.
Figure 6.
AA inhibits TGF-beta1-induced collagen I and a-SMA expression by HSC-T6 cells in a dosage-dependent manner in vitro.
A. Dose-dependent effects of AA on cytotoxicity of HSC-T6 cells by MTT and LDH release assays. B and C. Real-time PCR and Western blot analysis for a dosage-dependent inhibitory effect of AA on collagen type I and a-SMA expression. Data represent mean ± SEM for at least 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus medium control; #p<0.05, ##p<0.01, ###p<0.001 versus TGF-beta1-treated, isotype control antibody-treated (CTL Ab), or DMSO-treated cells.
Figure 7.
AA treatment upregulates hepatic Smad7, but blocks expression of TGF-beta1 and CTGF and activation of Smad2/3 in CCl4-induced liver disease in rats.
A. Real-time PCR analysis of TGF-beta1, CTGF, and Smad7 mRNA expression. B. Western blot analysis for levels of phospho-Smad2/3 and Smad7 protein expression. C. Immunohistochemistry for nuclear location of phospho-Smad2/3. Data represent mean ± SEM for groups of 8 animals. *p<0.05, **p<0.01, ***p<0.001 versus normal control; #p<0.05, ##p<0.01, ###p<0.001 versus CCl4-induced disease control. Magnification: ×200 (C).
Figure 8.
AA blocks TGF-beta1-induced phosphorylation of Smad2/3, a-SMA, and collagen matrix production by HSC-T6 cells in vitro.
Western blot analysis detects that HSC-T6 cells pretreated with AA (20 uM) for overnight blocks TGF-beta1 (1 ng/ml)-induced Smad2/3 phosphorylation at 30 mins (A) and upregulation of α-SMA and collagen I expression at 24 h (B,C). Data represent mean ± SEM for at least 3 independent experiments. **p<0.01, ***p<0.001 versus medium control; #p<0.05, ##p<0.01 versus TGF-beta1-treated or DMSO-treated cells.
Figure 9.
AA induces Smad7 expression by HSC-T6 cells in a time and dosage-dependent manner in vitro.
A. Real-time PCR. B. Western blots. Results show that addition of AA induces Smad7 mRNA and protein expression in a time (at a dose of 20 uM) and dosage (3 h for mRNA and 24 h for protein)-dependent manner. Data represent mean ± SEM for at least 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus medium control.
Figure 10.
Knockdown of Smad7 from HSC-T6 cells prevents the inhibitory effect of AA on TGF-beta1-induced hepatic fibrosis in vitro.
A. Real-time PCR shows reduction of Smad7 mRNA by siRNA technique. B. Western blot analysis detects that knockdown of Smad7 from HSC-T6 cells results in a loss of AA (20 uM)-induced inhibition of TGF-beta1 (1 ng/ml)-mediated collagen I and a-SMA expression at 24 h. Data represent mean ± SEM for at least 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus control; ###p<0.001 versus TGF-beta1, DMSO, and control vector (P-super)-treated cells.