A total of 32 healthy male Sprague-Dawley rats (age, 6–8 weeks; weight, 250–300 g) were provided by the Experimental Animal Center of Tongji Medical College (Wuhan, China). All the rats were housed under habitual conditions at 21±2°C, under a 12-h light/dark cycle and with free access to food and standard laboratory chow. At all times the animals were bred in accordance with the Principles and Guidelines for the Care and Use of Laboratory Animals of Tongji Medical College, recommended by the National Academy of Sciences and published by the National Institute of Health (NIH publication 85–23 revised 1996). The methodology was approved by the Ethics Committee of Tongji Medical College.

The rats were randomly divided into four groups (n=8 per group): i) Sham group, Sham-operated; ii) BDL+NS group, animals underwent BDL and were treated with normal saline (NS; 0.02 mg/kg/day); iii) BDL+Sim 0.02; and iv) BDL+Sim 0.2 groups underwent BDL and were treated with 0.02 or 0.2 mg/kg/day Sim (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), respectively.

Prior to surgery the animals were anesthetized using cotton soaked with 1.5 ml sulfuric ether (Guidechem, Hangzhou, China). Under sterile conditions, an upper abdominal midline incision was performed. Muscle and peritoneal structure were then dissected to expose the common bile duct, which was carefully isolated and double-ligated with a 5-0 silk close to the liver hilus immediately below the bifurcation and cut between the ligatures (BDL group). The control animals underwent a Sham operation in which the common bile duct was not ligated but exposed, and the abdominal incision was then closed using single sutures (13).

In all of the groups the route of administration was intraperitoneal via a single daily injection at 9:00 am for seven days. This route was selected because it appeared to lead to more stable levels of the drug in equilibrium. In total, 24 h after the last injection each animal was weighed, anesthetized with sulfuric ether and sacrificed by exsanguination (abdominal aorta puncture). Blood and tissues were then collected and processed as described below.

Blood samples were collected with 10 IU/ml sodium heparin (GL Biochem, Shanghai, China) as an anticoagulant, and were then centrifuged at 2,000 × g for 5 min at 4°C to obtain plasma. The plasma was then used to measure the total bilirubin (TB), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as parameters that indicate hepatic function. These biochemical analyses were then performed using an autoanalyzer (7600–020; Hitachi Ltd., Tokyo, Japan).

Liver specimens were homogenized in lysis buffer (P0013; Beyotime Institute of Biotechnology, Haimen, China), and the total protein concentration was determined and equalized. The 20 µg samples were then boiled in Laemmli buffer (P0015A; Beyotime Institute of Biotechnology) and analyzed by western blotting as previously described (14). Equal amounts of proteins were separated by 12% SDS-PAGE (P0012A; Beyotime Institute of Biotechnology) with 100 V for 1 h and transferred onto polyvinylidene difluoride membranes (Merck Millipore). The membranes were blocked with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) for 2 h and then incubated with primary antibodies at 4°C overnight. Primary antibodies were used at a dilution of 1:800 and horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG (sc-2005) and goat anti-rabblit IgG (sc-2004; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a dilution of 1:5,000. Moreover, detection was performed using Supersignal West Pico chemiluminescent substrate kit (Pierce Protein Biology; Thermo Fisher Scientific, Inc., Rockford, IL, USA) and X-ray film, and was converted to digital images. Densitometric quantification was performed using the Quantity One version 4.62 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). In all instances, the membranes were stripped and reported using an antibody specific against α-tubulin, and densitometric values were corrected to α-tubulin expression. Furthermore, the commercial antibodies used were: TGF-β1 (ab92486) from Abcam (Cambridge, MA, USA), Smad2 (12584) and Smad3 (9523) from Cell Signaling Technology, Inc. (Danvers, MA, USA) and α-tubulin (sc-8035) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc.).

Liver tissues were fixed in 10% formalin overnight and embedded in paraffin (327204; Sigma Aldrich, Merck Millipore). Immunohistochemical analysis of the liver tissue was performed as described previously (15), and TGF-β1 expression was detected. Polylysine was smeared on the slides, 3.5-µm paraffin sections were cut and a pressure cooker (WQC50A1P; Midea, Foshan, China) was used to prepare the hot citric acid antigen for primary TGF-β1 antibody repair (sc-130348; 1:100; Santa Cruz Biotechnology, Inc.). It was then incubated for 1 h with a biotin-conjugated secondary antibody, goat anti-mouse IgG-B (sc-2039; 1:800; Santa Cruz Biotechnology, Inc.), at room temperature for 30 min, 3,3′-diaminobenzidine (DAB) colored for 5–10 min and stained with hematoxylin and eosin for 1–3 min. An Olympus Corporation (Tokyo, Japan) BX51 microscope was used to photograph the slides. Phosphate-buffered saline (PBS) instead of the primary antibody was used as a negative control. Using a Leica Qwin V3 image analysis system (Leica Microsystems GmbH, Wetzlar, Germany), immunohistochemical analysis of the positive products of the area in each section was selected at a magnification of ×200 of the three non-overlapping representations. The percentage of each sample was then calculated and the positive area (positive area/measurement area) was determined. The scoring criteria used based on a semiquantitative approach, in which the percentage of TGF-β1-positive cells (0–100%) was determined and multiplied by the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong).

Total RNA from liver was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) reagent according to the manufacturer's instructions and treated with RNase-free DNaseI (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. In total, 2 µg total RNA from liver was reverse transcribed into cDNA using the StrataScript first-strand synthesis system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA). TGF-β1 (sense: 5′-AGGGCTTTCGCTTCAGTGCT-3′ and anti-sense: 5′-CCATGAGGAGCAGGAAGGGT-3′); Smad3 (sense: 5′-TGAACACCAAGTGCATTACCA-3′ and anti-sense: 5′-TGACTGGCTGTAGGTCCAAGT-3′), and Smad7 (sense: 5′-CGGAATTCGCCACCATGTTCAGGACCAAACGATC-3′ and anti-sense: 5′-CGGGATCCACTACCGGCTGTTGAAGATG-3′) were amplified using SYBR Green PCR master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and iCycler real-time PCR detection system (Bio-Rad Laboratories, Inc.) for 40 cycles. Relative levels were then calculated using the iCycler software and a standard equation (Applied Biosystems; Thermo Fisher Scientific, Inc.) and normalized against glyceraldehyde 3-phosphate dehydrogenase (sense: 5′-CCTGGTATGACAATGAATATG-3′ and anti-sense: 5′-TCTCTTGCTCTCAGTATCC-3′).

Hepatocyte apoptosis was quantified using the TUNEL assay. All liver tissue specimens were fixed in freshly prepared 3.5% paraformaldehyde (158127; Sigma-Aldrich; Merck Millipore) and sucrose (V900116 Sigma-Aldrich; Merck Millipore). Tissues were then embedded in Thermo Shandon Cryomatrix (Thermo Fisher Scientific, Inc.), sectioned (10 µm) using a Shandon Cryotome (M16512), and then stored at 4°C. After treating the specimen with PBS, the sections were processed following the instructions of an In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN, USA). After rinsing the specimens twice with PBS, the tissue peroxidase activity was visualized using DAB. TUNEL-positive nuclei were counted in five high-powered fields and expressed as a percentage of the total nuclei counted (16). Moreover, two investigators (one of which was blinded) scored the sections, and the mean of the two scores is reported.

PCNA immunostaining was performed in order to examine the hepatocyte proliferation with the mouse monoclonal antibody against PCNA (clone-PC 10; 1:100; Dako Denmark A/S, Glostrup, Denmark). Briefly, remnant liver tissue specimens were fixed in 10% buffered formalin, embedded in paraffin and then cut into 5-µm sections. The deparaffinized sections were then treated by microwave heating thrice in PBS for 5 min each, then washed three times with PBS for 5 min each. Following blocking with endogenous peroxidase, the specimens were washed three times with PBS for 5 min each. The sections were then incubated with the antibody against PCNA overnight at 4°C. After washing several times with PBS, biotin-labeled secondary antibody (sc-57636; 1:2,000; Santa Cruz Biotechnology) was added for 1 h at room temperature, and after washing several times with PBS the tissue peroxidase activity was visualized using DAB (17). The PCNA labeling index was then determined as the number of PCNA-positive cells among 1,000 counted cells at high power (magnification, ×400).

Statistical comparison was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). Student's t-test, Welch's t-test or Mann-Whitney U test were adapted for the evaluation of the significance of the differences between groups. P<0.05 was used to indicate a statistically significant difference. Results are expressed as the mean ± standard error.

The urine color of the rats evidently deepened 24 h-post operation. After 48 h, the tails of these rats began to turn yellow. Following prolonged bile duct obstruction, there was progression in jaundice, and the appetite and mental states gradually deteriorated.

Sections from hepatic tissue from the four experimental groups were analyzed after hematoxylin and eosin staining (Fig 1), however, no histopathological differences were observed between the control group (Fig. 1A) and animals receiving Sim (Fig. 1D). By contrast, BDL caused substantial hepatocellular injury, as indicated by a >7.2-fold increase in liver enzymes. However, Sim treatment significantly reduced BDL-induced liver damage (Fig. 1B and C; P<0.01 vs. BDL+NS group). Moreover, the BDL+NS group significantly elevated the serum TB by >17.6-fold vs. the Sham group, suggesting that an obstructive jaundice model was successfully established (Fig. 1E). Bilirubin levels in rats treated with Sim following BDL were not different from those in NS-treated animals, indicating that the degree of obstructive jaundice was similar in all of the experimental groups (Fig. 1E). Moreover, administration of 0.02 mg/kg/day Sim significantly suppressed the release of ALT and AST from the liver by 61.02 and 58.01%, respectively, compared with the NS-treated rats. The treatment with 0.2 mg/kg/day Sim decreased BDL-induced ALT and AST levels by 69.14 and 65.18%, respectively (Fig. 1F and G; P<0.01 vs. BDL+NS group).

TGF-β1 concentrations in the liver were significantly elevated in the livers of BDL animals compared with those in Sham-operated animals (P<0.01) (Fig. 2A, B and E). Sim administration markedly lowered obstructive jaundice-induced elevation of hepatic concentration of TGF-β1 (P<0.01) (Fig. 2E). Moreover, RT-qPCR and western blot analyses revealed that TGF-β1 was less activated in the liver tissues obtained from Sim-treated rats compared to those from NS-treated rats (Fig. 2F and G). These results indicate that Sim administration blunted the TGF-β1 signaling pathway that is known to contribute to the aggression of obstructive jaundice-induced liver injury.

TUNEL immunohistochemistry stained liver sections taken from Sham, BDL+NS, BDL+ Sim 0.02 and BDL+ Sim 0.2 are shown in Fig. 3A-D. Immunohistochemistry for TUNEL demonstrated a significant increase in TUNEL-positive hepatocytes after BDL compared to the Sham group (P<0.01) (Fig. 3A and B). Furthermore, treatment of BDL animals with Sim resulted in a significant reduction in the percentage of hepatocyte apoptosis compared to BDL rats treated with saline (P<0.01) (Fig. 3B and D). Also, in the Sham animals no significant change on apoptosis was noted (Fig. 3A).

PCNA immunohistochemistry stained liver sections were collected following seven days of BDL. Sham, BDL+NS, BDL+Sim 0.02 and BDL+Sim 0.2 are depicted in Fig. 4A-D. Immunohistochemical staining for PCNA demonstrated a significant increase in DNA synthesis in the BDL+NS group, compared to the Sham group (Fig. 4B). Moreover, treatment of BDL rats with Sim increased DNA synthesis in BDL animals to 27% compared to the controls (Fig. 4C and D).