Male C57BL⁄6 mice (aged 6–8 weeks) were obtained from the Shanghai SLAC Experimental Animal Centre (Shanghai, China) and maintained in specific pathogen-free conditions at 24°C with a 12 h light/dark cycle and unlimited access to food and water. The animal experiments were approved by the Research Ethics Committee of Renji Hospital (Shanghai, China; no. SYXY (hu) 2011–0121). The animals were cared for in accordance with protocols approved by the Animal Care and Use Committee of Renji Hospital.

A total of 30 mice were randomly divided into three groups (n=10). To induce liver fibrosis, the mice were injected intraperitoneally, twice each week for 8 weeks, with 5 µl/g of 20% CCl₄ (Shanghai Jiahe Biotechnology, Shanghai, China) dissolved in olive oil. Negative control mice were administered with the same volume of olive oil only, for for 8 weeks. Beginning on the day of the CCl₄ injection, the mice in the treatment group were treated intraperitoneally with rapamycin (1.25 mg/kg/day; Gene Operation, Ann Arbor, MI, USA) for the duration of the investigation. In the remaining group of mice, rather than rapamycin, the mice received equivalent volumes of phosphate-buffered saline (PBS) during the course of the experiment. All the mice were sacrificed 72 h following the final CCl₄ injection at 8 weeks, using CO₂ asphyxia.

At the time of sacrifice, blood samples were collected from the mouse eyes (~1 ml) and immediately centrifuged for 10 min. Liver function tests were performed at Renji Hospital on serum samples to evaluate the levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin.

At the time of sacrifice, the liver was dissected from 6–8 mice. The liver sections were fixed in 4% formaldehyde, embedded in paraffin, and sectioned (5 mm). The liver sections were then stained with hematoxylin and eosin (H&E) for histopathological examination. Masson's trichrome staining was used for collagen determination. The content of collagen deposition was quantified using an image analyzer (Image-Pro Plus; Media Cybernetics, Inc., Rockville, MD, USA). Immunohistochemical examinations were performed to detect the expression of α-smooth muscle actin (SMA). In brief, the paraffin sections were deparaffinized and rehydrated. The sections were exposed to fresh 3% hydrogen peroxide for 20 min, and then washed with PBS. Antigens were retrieved in 0.01 M citric acid. The samples were incubated for 30 min at room temperature in 5% normal blocking serum, and incubated with a 1:100 dilution of polyclonal rabbit anti-α-SMA (ab5694) or a 1:100 dilution of monoclonal mouse anti-TGF-β (ab64715; Abcam, Cambridge, MA, USA) overnight at 4°C. The slides were then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:2,000; ab7090; Abcam) or HRP-conjugated goat anti-mouse (1:2,000; ab6789; Abcam) for 60 min at room temperature, and with 3,3′-diaminobenzidine as a substrate. Negative controls were obtained by omitting the primary antibodies. Finally, the sections were counterstained with haematoxylin, and mounted.

Frozen liver tissue (50 mg) was rapidly thawed and homogenized at 4°C in 0.5 ml lysis buffer (Beyotime Institute of Biotechnology) for 30 min. Homogenates were centrifuged at 12,000 × g for 10 min at 4°C to yield supernatants. A bicinchoninic acid protein assay kit was used to detect the protein concentrations. The samples were then heated at 95°C for 5 min. The proteins (30 µg) were separated by 12% SDS-PAGE (Beyotime Institute of Biotechnology, Shanghai, China) and then transferred onto a polyvinylidene difluoride membrane (Beyotime Institute of Biotechnology). The membrane was blocked with 5% non-fat milk for 2 h at room temperature, and incubated with indicated antibodies, including anti-α-SMA (1:500 dilution), monoclonal mouse anti-forkhead/winged helix transcription factor P3 (FoxP3; 1:500 dilution; ab20034; Abcam) and polyclonal rabbit anti-ROR-γt (1:1,000 dilution; ab78007; Abcam) in Tris-buffered saline with 0.05% Tween overnight at 4°C. All antibodies were purchased from Abcam. The membranes were then rinsed and incubated at room temperature with HRP-conjugated secondary antibody (1:10,000 dilution; LI-COR Biosciences, Lincoln, NE, USA). Signals were detected using an Odyssey Infrared image system (LI-COR, Inc., Lincoln, NE, USA).

Intracellular cytokine staining was performed, as previously described (18). To investigate the frequency of Tregs, lymphocytes from the spleen and liver were prepared. To obtain single-cell suspensions from the spleen and liver, tissue was dissected into small pieces, ground and filtered through stainless steel meshes. Splenocytes were isolated from the cell suspensions by gradient centrifugation at 1,400 × g for 25 min at 4°C with Lymphoprep (Dakewei Biotechnology, Shenzhen, China). Following surface staining with anti-CD4-fluorescein isothiocyanate (FITC; 11-0041; eBioscience, San Diego, CA, USA) and anti-CD25-peridinin-chlorophyll-protein (45-0251; eBioscience), the cells were fixed, permeabilized and stained with anti-FoxP3-phycoerythrin (PE; 12-5773; eBioscience). For Th17 cell detection, the lymphocytes from the spleen and liver were stimulated for 5 h with phorbol-12-myristate-13-acetate (50 ng/ml), ionomycin (1 µg/ml) and 5 µg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) in RPMI-1640 (GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS; GE Healthcare Life Sciences). The cells were harvested and stained with anti-CD4-FITC. The cells were fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) and stained intracellularly with anti-IL-17A-PE (12-7177; eBioscience). Cells stained with IgG isotype control were used as controls. All antibodies were purchased from eBioscience. Data were obtained using a FACSCalibur flow cytometer and analyzed using CXP analysis software, version 2.1 (Beckman Coulter, Inc., Brea, CA, USA).

To obtain single-cell suspensions from the spleen, tissue was dissected into small pieces, ground and filtered through stainless steel meshes. Splenocytes were isolated from the cell suspensions by gradient centrifugation at 1,400 × g for 25 min at 4°C with Lymphoprep (Dakewei Biotechnology). The splenocytes were separated into CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells using a CD4⁺CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's protocol. These cells were cultured in RPMI-1640 medium supplemented with 10% FBS and penicillin/streptomycin. All cells were cultured at 37°C in a humidified 5% CO₂ incubator.

The splenocytes from the rapamycin- or PBS-treated mice were separated into CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells, as described above. Subsequently, 2×10⁵ CD4⁺CD25⁻ T cells (responder cells) with titrated CD4⁺CD25⁺ T cells (effector cells) were suspended in 200 µl RPMI-1640, seeded into 96-well plates, and stimulated with a combination of soluble anti-CD3 (1 µl/ml; 557306; BD Pharmingen) and soluble anti-CD28 (1 µl/ml; 553295; BD Pharmingen). In co-culture experiments, purified CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells were simultaneously added into the wells, and cultured at 37°C for 72 h. Following an 18 h pulse with [³H] thymidine (1 µCi/well; Shanghai Institute of Nuclear Research, Shanghai, China), proliferation was analyzed using a scintillation counter (Beckman LE 5000CE, Beckman Coulter, Inc.).

Mouse HSCs were isolated from the livers of the C57BL⁄6, as described previously (19). In brief, collagenase perfusion in situ and density gradient centrifugation were used for isolating the HSCs. The liver was perfused with collagenase at 37°C at a flow rate of 18, until the hepatic parenchyma beneath the capsule appeared liquefied. This was then subjected to gradient centrifugation using OptiPrep (Sigma-Aldrich), at 2,000 × g for 20 min at 4°C. The isolated HSCs were cultured in RPMI-1640 (GE Healthcare Life Sciences) containing 10% FBS and penicillin/streptomycin. The HSCs were plated at 3×10⁵ cells/well into 6-well plates, and were cultured for 6 days, following which they were harvested for subsequent use. Cell viability was >90%, determined using a trypan blue exclusion assay. The purity of the HSCs was >92%, as determined by the typical appearance of the lipid droplets under a light microscope (IX50; Olympus Corporation, Tokyo, Japan) (20). All cells were cultured in a humidified incubator with 5% CO₂ at 37°C.

A co-culture experiment was performed using the HSCs and splenic Tregs isolated from the rapamycin- and PBS-treated mice. The HSCs were plated at a density of 1×10⁵ cells/well in a 24-well plate for 24 h, following which Treg cells (1×10⁵ or 2×10⁵ cells/well) were added to the culture system. The cells were co-cultured for 3 days and the expression levels of α-SMA were detected using immunofluorescence staining and western blot analysis.

The cells were fixed in 4% formaldehyde and permeabilized in methanol. Following blocking, the cells were incubated overnight with rabbit anti-mouse α-SMA monoclonal antibody (1:100 dilution; Abcam) followed by CY3-conjugated goat anti-rabbit antibody (1:400 dilution; 112-165-143, Jackson ImmunoResearch, West Grove, PA, USA) at room temperature. The nuclei were stained with DAPI. The expression of α-SMA was observed under a fluorescent microscope (Nikon Eclipse Ti-S, Nikon Corporation, Tokyo, Japan) and fluorescence intensity was analyzed using Image-Pro Plus software, version 6.0.

Data are presented as the mean ± standard error of the mean. Statistical analyses were performed using SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA). Data were analyzed using Student's t-test or one way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The present study aimed to assess the antifibrogenic effects of rapamycin via the expansion of Tregs and inhibition of Th17 cells in CCl₄-induced hepatic fibrosis. To examine the effect of rapamycin on liver function, total bilirubin and serum aminotransferases were detected. The liver function tests showed that rapamycin significantly reduced the concentrations of total bilirubin and aminotransferases in the CCl₄-induced liver fibrosis model, compared with the PBS-treated control group (Table I).

The liver fibrosis model was established by chronic CCl₄ injection, and the degree of hepatic fibrosis was detected using H&E and Masson's trichrome staining (Fig. 1A). Following CCl₄ administration, the liver tissues of the mice treated with PBS exhibited a distorted architecture, with extensive fibrosis combined with the development of micronodules throughout the liver parenchyma shown in the H&E staining. Liver injury was attenuated in the rapamycin-treated mice. The deposition of collagen fibers as an indicator of liver fibrosis was determined using Masson's trichrome staining. The pathological progression in liver fibrosis was attenuated by rapamycin, with fewer and smaller fibrotic nodules observed. As a marker of HSC activation, α-SMA is one of the sensitive indices of the rate of fibrogenesis (21). As shown in Fig. 1B, the expression of α-SMA was significantly elevated in the mice with CCl₄-induced liver fibrosis, and was reduced following rapamycin administration. As a key factor of fibrogenesis, TGF-β in the rapamycin-treated mice showed reduced expression, compared with that in the PBS-treated mice.

To analyze the immunoregulatory effect of rapamycin on Th17 cells in CCl₄-induced liver fibrosis, the percentages of Th17 cells in the mouse spleen and liver were measured using flow cytometry (Fig. 2A). As shown in Fig. 2B, the cells from the PBS-treated mice showed markedly higher percentages of Th17 cells, compared with the negative control group (oil without CCl₄). Following rapamycin administration, the percentages of Th17 cells were significantly lower, compared with those in the PBS-treated mice, but were higher, compared with the negative control group (Fig. 2B). In addition, as a crucial transcription factor of Th17 cell differentiation, the expression of ROR-γt in the rapamycin-treated mice was decreased, compared with that in the PBS-treated mice (Fig. 2C). The relative protein levels are shown in Fig. 2D.

The present study subsequently investigated whether the effect of rapamycin was associated with modulation of Treg cell function in CCl₄-induced hepatic fibrosis. The percentages of Tregs in the spleen and liver were analyzed using flow cytometry (Fig. 3A). The frequencies of Tregs in the spleen and liver from the rapamycin-treated mice were significantly higher, compared with those of the PBS-treated mice (Fig. 3B). The hepatic protein expression of FoxP3 was then examined using western blot analysis. Consistent with the results of the flow cytometric analysis, the expression of FoxP3 in the liver from the rapamycin-treated mice was markedly enhanced, compared with that from the PBS-treated mice (Fig. 3C). The relative protein levels are shown in Fig. 3D.

Furthermore, to clarify the effect of the mTOR inhibitor on the suppressive efficacy of Tregs, splenic CD4⁺CD25⁻ responder T cells from the PBS-treated mice were stimulated with anti-CD3/CD28 monoclonal antibody and co-cultured with CD4⁺CD25⁺ Tregs from the rapamycin- or PBS-treated mice. As shown in Fig. 3E, the suppressive activities of the Tregs in the splenic tissues of the rapamycin-treated mice were markedly higher, compared with those of the PBS-treated mice.

It is known that regulatory immune cells, including CD4⁺CD25⁺ T cells, are important in the pathogenesis of liver fibrosis and the activation of HSCs (9,11). To examine the effect of the Treg cell expansion by rapamycin on HSCs, HSCs were isolated from the mouse liver, and then cultured with splenic Tregs from the rapamycin- or PBS-treated mice. Immunofluorescence staining and western blot analysis were performed to evaluate changes in the expression of α-SMA. As shown in Fig. 4A–C, immunofluorescence staining showed that the expression of α-SMA was significantly reduced following exposure to the Tregs from the rapamycin-treated mice, compared with those from the PBS-treated mice. Fig. 4D shows the fluorescence intensity of each group, which shows the trends described above. The results of the western blot analysis showed that, compared with the control group, the expression of α-SMA was reduced following exposure to the Tregs, however, the difference was not significant. The protein expression of α-SMA was also markedly decreased following exposure to Tregs from the rapamycin-treated mice, compared with the PBS-treated mice (Fig. 4E). The expression levels of α-SMA are shown in Fig. 4F. These data showed that the expansion of the Treg cell population by rapamycin increased the capacity to inhibit the activation of HSCs.