A total of 50 healthy male Wistar rats (age, 6–8 weeks; weight, 180–200 g) were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). The rats were housed in a temperature- and humidity-controlled facility under a 12 h light-dark cycle, and were given ad libitum access to a standard laboratory diet and tap water. The experimental procedures and ethical requirements of the present study conformed to the Laboratory Animal Management Regulations of the People's Republic of China. All experimental procedures were approved by the Animal Experimental Committee of Beijing Friendship Hospital affiliated with Capital Medical University (Beijing, China).

The 50 rats were randomly divided into the normal control group (n=6) and the liver fibrosis model group (n=44). The liver fibrosis model group received intraperitoneal injections of 0.15 ml/100 g 50% CCl₄ in olive oil (5:5, v/v) twice weekly for eight weeks. Rats in the normal control group received intraperitoneal injections of the same volume of physiological saline over the same period. Reversal of liver fibrosis was investigated for 4 weeks after the final intraperitoneal injection. Rats were sacrificed at 2, 4, 6, 8, 10 and 12 weeks. Seven rats were sacrificed at each time point. Two rats succumbed without investigator intervention during week 12. All rats from the normal group were sacrificed during week 8.

All rats were euthanized under anesthesia using 1% sodium pentobarbital (50 mg/kg, i.p). Blood samples were collected for liver function tests. In addition, liver specimens were collected, divided into three parts and subjected to the following analyses: i) mRNA extraction using TRIzol for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses; ii) protein expression analysis of fibrotic indices using western blotting; and iii) histological and immunohistochemical analyses.

The HSC-T6 immortalized rat cell line (Tumor Cell Bank of Chinese Academy of Medical Sciences, Beijing, China) was cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 µg/ml streptomycin (Beijing Suolaibao Biotechnology Co., Ltd., Beijing, China). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. All experiments were carried out 24 h after the cells were seeded.

Cells were divided into two groups: The control group and the transforming growth factor (TGF)-β group. Recombinant human TGF-β (Sigma -Aldrich; Merck Millipore, Darmstadt, Germany) was used, since it is a major fibrogenic mediator in the liver (13,14). TGF-β was administered to the cultured cells at 0, 1, 2, 5, 10 and 20 ng/ml for 0, 1, 3, 6, 12, 24 and 48 h. Cells were exposed to starvation conditions using FBS-free medium for 24 h prior to treatment with recombinant human TGF-β.

Cells were plated at a density of 3×10⁵ cells/well in six-well plates, and were allowed to adhere overnight. The cells were then cultured in serum-free medium for an additional 24 h at 37°C. Subsequently, cells were rinsed in phosphate-buffered saline (3×10 min), and were fixed in 4% paraformaldehyde (40 min, 37°C) and 3% H₂O₂ (30 min, 37°C). Following fixation, cells were blocked with 1% FBS (40 min, 37°C) and were incubated overnight at 4°C with rabbit anti-mouse elastin primary antibody (FN; 1:100; sc-9068; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Goat anti-rabbit fluorescein isothiocyanate probes (1:200; bs-0295G-FITC; BIOSS, Beijing, China) were used as secondary antibodies at 37°C for 1 h. The cell nuclei were stained using 4′,6-diamidino-2-phenylindole (Santa Cruz Biotechnology, Inc.). Immunofluorescence was analyzed using a fluorescence microscope. Cells incubated with normal rabbit serum instead of the primary antibody served as a negative control.

Blood samples were centrifuged at 1,200 × g for 5 min at 4°C to separate serum from the blood. Serum aspartate transaminase (AST) and alanine transaminase (ALT) (15) activities were determined by the Clinical Laboratory (Beijing Friendship Hospital, Beijing, China).

Total RNA was extracted from frozen liver samples or cultured cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RT was performed using random primers, Moloney Murine Leukemia Virus Reverse Transcriptase and RNase inhibitor (Fermentas; Thermo Fisher Scientific, Inc.). The expression levels of all transcripts were normalized to the expression of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in the same sample. PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and an ABI 7500 platform (Applied Biosystems; Thermo Fisher Scientific, Inc.). RNase-free DNase (Promega Corporation, Madison, WI, USA) was used to eliminate the contamination of genomic DNA. The first cDNA strand was synthesized by the RT reaction in a reaction mixture of 20 µl containing 4 µl MgCl₂, 3 µl 10X RT buffer, 2 µl dNTP mixture, 0.5 µl recombinant RNase inhibitor, 0.6 µl AMV reverse transcriptase, 1 µl oligo dT-Adaptor primer and 5 µg RNA sample. The RT reaction product was then used for the amplification of cDNA with the SYBR Master Mix reaction system (total, 10 µl) including 1 µl upstream primers, 1 µl downstream primers, 1 µl cDNA and 7 µl diethylpyrocarbonate-treated water. PCR was conducted as follows: Initial denaturation at 95°C for 10 min, 40 cycles at 95°C for 15 sec and 64°C for 30 sec, followed by 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and a final extension step at 60°C for 15 sec. Data were analyzed according to the 2^−ΔΔCq method, as described by Livak and Schmittgen (16). The following primer pairs were used: FN, forward 5′-CCAGCCTACGGATGACTC-3′, reverse 5′-AATGACCACTGCCAAAGC-3′; and GAPDH, forward 5′-GGCACAGTCAAGGCTGAGAATG-3′, and reverse 5′-ATGGTGGTGAAGACGCCAGTA-3′ [Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China].

Liver tissues and cultured HSCs were homogenized in radioimmunoprecipitation assay buffer and were centrifuged as previously described (17). The supernatants were considered whole-cell lysates. Total protein concentration was determined using a Bicinchoninc Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (~80 µg) were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes by electroblotting. Subsequently, the membranes were incubated for 3 h with 5% non-fat dry milk at 37°C, followed by an overnight incubation at 4°C with mouse anti-FN polyclonal antibody (1:600; sc-8422; Santa Cruz Biotechnology, Inc.), rabbit anti-TGF-β antibody (1:800; ab92486; Abcam, Cambridge, UK), rabbit anti-α-smooth muscle actin (α-SMA) antibody (1:800; ab5694; Abcam) and mouse anti-β-actin monoclonal antibody (1:800; ab8226; Abcam). Following the overnight incubation, membranes were incubated for 1 h at 37°C with peroxidase-conjugated goat anti-mouse secondary antibodies (1:6,000 in Tris-buffered saline containing 1% Tween; sc-2005; Santa Cruz Biotechnology, Inc.). Enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA) was used to detect immunoreactive bands. Finally, densitometric analysis of the bands was performed using Image Lab software, version 4.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Liver samples were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 3 µm. The tissue sections were then stained with hematoxylin and eosin (H&E) or Masson's trichrome, and were examined for changes in liver pathology. The histopathological scores for fibrosis were evaluated according to previously published criteria (18), as follows: 0, Normal liver; 1, increased collagen matrix accumulation without septa formation (small stellate expansions of the portal fields); 2, formation of incomplete septa from the portal tract to the central vein (septa that do not interconnect with each other); 3, complete but thin septa interconnecting with each other to divide the parenchyma into separate fragments; and 4, similar to grade 3, except for the presence of thick septa (complete cirrhosis).

Immunohistochemical analysis of paraffin-embedded sections was performed using a microwave-based antigen retrieval method, as described previously (19), and antibodies against FN were used (mouse polyclonal; 1:100; sc-8422; Santa Cruz Biotechnology, Inc.). In brief, histological sections were heated for 60 min at 60°C, deparaffinized with xylene and rinsed in a reducing gradient of alcohol. Subsequently, antigens were retrieved with citrate buffer in a microwave oven. Subsequent to treatment with hydrogen peroxide at 37°C for 30 min to block endogenous peroxidase activity, the sections were incubated with the primary antibody overnight at 4°C and then incubated with the secondary antibody (PV-9002; OriGene Technologies, Inc., Beijing, China) for 30 min at room temperature. Finally stained sections were developed using diaminobenzidine and were counterstained with hematoxylin and a BX43 optical microscope (Olympus Corporation, Tokyo, Japan) was used for analysis. FN expression was analyzed using a quantitative image analysis system (Image-Pro Plus 6.0; Media Cybernetics Inc., Rockville, MD, USA). Briefly, 10 fields were randomly selected from each section and positive signals within the section were highlighted, measured and expressed as a percentage of the positive areas within the entirety of the examined liver tissue.

Analyses were performed using SPSS software (SPSS version 17.0; SPSS Inc., Chicago, IL, USA), and the differences between multiple groups were evaluated using one-way analysis of variance and the least significant difference test was used for multiple comparisons. Results are presented as the mean ± standard deviation and experiments were repeated 6 times. P<0.05 was considered to indicate a statistically significant difference.

In the liver samples of the control group, normal lobular architecture was observed with hepatic cords radiating from the central veins, and the absence of regenerated collagen fibers, as detected by H&E and Masson's trichrome staining. Conversely, liver samples from the experimental group (2–12 weeks) exhibited severe fibrosis. Morphological alterations in the experimental group included thick fibrotic septa, fatty degeneration, enlarged hepatocytes with nodular arrangement, focal inflammatory changes, thickened vessel walls and early capillarization (Fig. 1).

The hepatic lobule structure persisted beyond 4 weeks; however, in the CCl₄ treatment group the appearance of the hepatic lobules became disorganized, and were characterized by fibrous septa, severe fatty degeneration, hydropic degeneration and focal necrosis. After 6 and 8 weeks of CCl₄ treatment, pseudolobules, and inflammatory cell infiltration into the portal areas and fibrous septa were observed. At 12 weeks (or 4 weeks after the final CCl₄ injection), fibrous septa began to attenuate or exhibit discontinuous shapes, with some of the fibrous septa becoming barely visible (Fig. 1).

Treatment with CCl₄ (2–8 weeks) resulted in a significant elevation in AST and ALT levels. However, at 10 weeks (2 weeks after the final CCl₄ injection), AST and ALT levels were markedly lower compared with those measured at 8 weeks (Fig. 2).

α-SMA expression in HSCs reflects myofibroblast-like cell activation, has been directly linked to experimental liver fibrogenesis, and is indirectly associated with fibrosis in chronic human liver disease (20).

The expression levels of α-SMA were increased in the CCl₄ treatment group compared with in the control group. However, when CCl₄ treatment ceased, protein expression returned to baseline levels (Fig. 3A). In addition, the expression levels of TGF-β were elevated in the CCl₄ treatment group (2–8 weeks), and this increase corresponded with the degree of liver fibrosis (Fig. 3B).

Markedly increased FN staining was detected in hepatocytes, sinusoidal areas, extracellular spaces, and portal and central areas in the experimental group tissues compared with in the control tissues, as determined by immunohistochemical staining of FN in liver sections. FN expression increased with increasing fibrosis, and peaked during week 8. However, after week 8 and the discontinuation of CCl₄ treatment, FN staining gradually decreased (Fig. 4A). The criterion used to assess FN positivity was yellow or brown staining in the cell membrane or mesenchyme.

The protein expression levels of FN in CCl₄-injured liver tissues gradually increased during the course of liver fibrogenesis. As detected by western blotting, FN expression reached its maximal point during week 8, after which expression gradually decreased (Fig. 4B). The mRNA expression levels of FN rapidly increased to a maximum level during the first 2 weeks. After the first 2 weeks, expression gradually fell (Fig. 4C).

Immunofluorescence assays demonstrated that FN was primarily located in the cytoplasm of HSC-T6 cells (Fig. 5A). TGF-β stimulation upregulated the mRNA and protein expression levels of FN in HSC-T6 cells in a dose- and time-dependent manner (Fig. 5B–E).