Adult gender-matched (n=20 each) C57BL rats weighing 200±10.2 g were purchased from Tongji University Laboratories (Shanghai, China) and fed with a commercial diet and water. All animal experiments were performed according to the National Institutes of Health (NIH) guidelines for the ethical care and use of laboratory animals, and the experimental protocol was approved by the Tongji Animal Care and Use Committee of China.

A total of 40 adult numbered rats were sorted into liver fibrosis model and normal control groups. A total of 20 rats in the liver fibrosis group received intraperitoneal injections of 40% CCl₄ and olive oil admixture (0.5 ml/100 mg, Sigma-Aldrich, St. Louis, MO, USA) tert as previously described (4). Rats were sacrificed after 8 weeks of treatment.

Glass slides (75×25 mm²) were obtained from Gibco (Carlsbad, CA, USA). (3-Acryloxypropyl) trichlorosilane was purchased from Gelest, Inc. (Morrisville, PA, USA). Streptavidin-conjugated Alexa 546, AlexaFluor 488 anti-mouse IgG, BMP-7 and Snail were obtained from Sigma-Aldrich. Mouse anti-E-cadherin antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Concentrated phosphate-buffered saline (10X PBS) was purchased from Lonza (China). Minimal essential medium (MEM), sodium pyruvate, non-essential amino acids, fetal bovine serum (FBS), Superscript III, RNaseOut (RNase inhibitor) and dNTPs were purchased from Lonza. The 384-well polypropylene microarray plates were obtained from Genetix (China). Goat anti-rat cross-adsorbed albumin antibody was obtained from Sigma-Aldrich. Formalin was purchased from Fisher Scientific (China). ApopTag Red in situ Apoptosis Detection kit was obtained from Chemicon (China). DAPI stain mounting media were purchased from Vectorshield (China).

Established LEPC cells were obtained from the ATCC collection (LGC Standards-SLU, Barcelona, Spain). Cell lines were maintained in DMEM supplemented with 10% FBS and antibiotics (100 μg/ml ampicillin, 32 μg/ml gentamicin; Sigma-Aldrich). Stable and transient transfections were performed using Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions for the generation of stable clones.

Tissue and cell lysates were prepared and immunoblot analysis was performed as described previously (10). Band intensity was determined using ImageMaster 2D Elite version 4.01 software (Amersham/GE Healthcare, Uppsala, Sweden). For immunoprecipitation, after liver cells were treated with Snail or BMP-7 for 48 h, the cells were lysed in buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% Nonidet P-40 (NP-40)] and centrifuged at 16,000 × g for 15 min to remove debris. Cleared lysates were subjected to immunoprecipitation with antibodies. For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized in 5% Triton X-100 for 5 min and then stained using pAbs. The secondary antibodies used were anti-mouse Alexa Fluor 594 dye conjugate and anti-rabbit Alexa Fluor 488 dye conjugate (Molecular Probes/Life Technologies, Carlsbad, CA, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI Blue; Molecular Probes/Life Technologies). After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY, USA).

Briefly, LEPC cells were transiently transfected with the indicated vectors for 48 h. Lysates were then obtained in immunoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40) containing protease and phosphatase inhibitors (2 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM PMSF, 1 mM Na₃VO₄, 10 mM NaF) and precleared with Sepharose G-beads. Supernatants were subjected to overnight incubation with anti-HA affinity matrix (Roche Diagnostics, Indianapolis, IN, USA) or Sepharose G-beads coated with anti-rat IgG as an immunoprecipitation control. Immunoprecipitates were resolved by PAGE on 7.5–12% SDS gels, transferred to membranes and incubated with the indicated antibodies. The membranes were then developed using ECL reagent following the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Blots were incubated with rat anti-HA (Roche Diagnostics; 1:100) or mouse anti-flag (Sigma-Aldrich; 13:000). The secondary antibodies used were HRP-coupled goat anti-rat (Pierce Biotechnology, Inc., Rockford, IL, USA; 110:000) or sheep anti-mouse (Pierce Biotechnology, Inc.; 11:000). For detection of E-cadherin, α-smooth muscle actin (α-SMA) and Snail expression, western blotting was performed on whole-cell lysates using rat anti-E-cadherin ECCD2 mAb (1:200, produced in our laboratory from the ECCD2 hybridoma, a gift of M. Takeichi, Ricken Center, Japan), mouse anti-α-SMA (1:500, Dako, Carpinteria, CA, USA) or rat anti-Snail (Roche Diagnostics), followed by HRP-coupled secondary antibodies.

RT-PCR analysis of cDNA samples was performed with specific primers designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). The primers used for Snail were 5′-AAGGATCTCCAGGCTCGAAAG-3′ (forward) and 5′-GCTTCGGATGTGCATCTTGA-3′ (reverse) and those used for β-actin were 5′-GCAAAGACCTGTACGCCAACA-3′ (forward) and 5′-TGCATCCTGTCGGCAATG-3′ (reverse). Total RNA was extracted from cultured cells using an RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using 1 μg of RNA with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA) and oligo(dT) primers. Transcript levels were assessed by RT-PCR (ABI 7300; Applied Biosystems) and all experiments were normalized to β-actin.

The cells were treated with 10 μM MG132 for 6 h, 24 h after transfection. The treated cells were then harvested with PBS containing 10 mM N-ethylmaleimide (NEM) and 1 mM dithiothreitol (DTT). The cells were washed with PBS, centrifuged and subjected to one freeze-thaw cycle. Cell pellets were then resuspended in 200 μl buffer 1 [10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% NP-40, 5 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM NEM, 1 mM DTT, 5 mM NaF, 1 mM Na₃VO₄ and protease inhibitor cocktail] and sonicated in a water bath (Bioruptor; Diagenode, Denville, NJ, USA). Next, 500 μl buffer 2 (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.5% NP-40, 5 mM EDTA, 1 mM EGTA, 10 mM NEM, 1 mM DTT, 5 mM NaF, 1 mM Na₃VO₄ and protease inhibitor cocktail) was added and the extracts were subjected to a 30-min rotation at 4°C. The extracts were then centrifuged. We added 2 μg of anti-Flag M2 antibody and protein A/G beads to the supernatant, which was then incubated for 2 h. The beads were then washed three times, resuspended in loading buffer and boiled. Immunoblotting was performed as described above.

A total of 15 adult numbered rats were randomly sorted into 3 groups: i) normal control group: 5 rats received intraperitoneal injections of olive oil (0.5 ml/100 mg) twice per week; ii) liver fibrosis model group: 5 rats received intraperitoneal injections of 40% CCl₄ and olive oil admixture (0.5 ml/100 mg); iii) BMP-7-treated group: 5 rats received intraperitoneal injections of 40% CCl₄ and olive oil admixture (0.5 ml/100 mg) twice per week and BMP-7 (300 μg/kg) at same time. Primary rat hepatocytes (1×10⁶/dish) were cultured for 48 h in F12 medium containing 10% fetal bovine serum and 2 μg/ml insulin until they had adhered, which was marked as ‘−’; Primary rat hepatocytes (1×10⁶/dish) were cultured for 48 h in F12 medium containing 10% fetal bovine serum and 2 μg/ml insulin until they had adhered. To induce EMT, the media were replaced with F12 media supplemented with 0.5% fetal bovine serum and 200 mg/μl insulin containing Snail for 96 h, which was marked as ‘+’.

All results shown in the bar graphs are expressed as the fold ratio relative to untreated or control cells. Statistical analysis was performed using SPSS version 17 statistical software (SPSS Inc., Chicago, IL, USA). Student’s t-test was used when comparing two groups. One-way ANOVA was used when comparing multiple groups, followed by Tukey’s post-hoc test. P<0.001 was considered to indicate a statistically significant result.

None of the animals died during the study period. Body weight gain was lower in the Snail-treated compared to the control rats (data not shown). A total of 40 adult numbered rats were randomly sorted into 4 groups: i) normal control group: 5 rats received intraperitoneal injections of olive oil (0.5 ml/100 mg) twice every week; ii) liver fibrosis model group: 10 rats received intraperitoneal injections of 40% CCl₄ and olive oil admixture (0.5 ml/100 mg) tert as previously described (4); iii) Snail-treated group: 10 rats received intraperitoneal injections of 40% CCl₄ and olive oil admixture (0.5 ml/100 mg) twice every week and Snail (500 μg/kg) at the same time; iv) BMP-7-treated group, 10 rats received intraperitoneal injections of 40% CCl₄ and olive oil admixture (0.5 ml/100 mg) twice every week and BMP-7 (300 μg/kg) at the same time. The rats were sacrificed after 8 weeks of treatment.

The present study demonstrated that treatment with Snail induced EMT and increased liver injury during CCl₄-induced liver fibrosis in rats. This was accompanied by increased expression of liver fibrosis mesenchymal markers, including α-SMA, but inhibition of E-cadherin (P<0.001; Figs. 1–5). The ratio of α-SMA/GAPDH in liver fibrosis model group was higher than that in control group, with 200 mg/μl insulin containing Snail-treated for 96 h, ratio of α-SMA increased significantly in liver fibrosis model group than control group. But this was reverse in ratio of E-cadherin/GAPDH (P<0.001; Fig. 5).

The present study demonstrated that rats treated with Snail had increased hepatic fibrosis in CCl₄-induced liver injury. This was accompanied by increased expression of hepatic fibrosis mesenchymal markers, including α-SMA, but repression of E-cadherin (Figs. 1–4).

We demonstrated that CCl₄-induced fibrosis is reversed in rats treated with BMP-7. Significantly more BMP-7 and less Snail mRNA were expressed in the hepatic fibrosis model group than in the controls (P<0.001; Figs. 1–4). This was accompanied by reduced expression of hepatic fibrosis mesenchymal markers, including α-SMA, but increased expression of E-cadherin (Fig. 1). Thus, a strategy that specifically increased BMP-7 in myofibroblasts from cirrhotic livers tended to reverse the myofibroblastic phenotype and caused the cells to acquire a more quiescent and epithelial phenotype.