The human stellate cell line LX-2 was obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China) and maintained in RPMI 1640 culture medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen Life Technologies) and 1% penicillin/streptomycin (Invitrogen Life Technologies) in 5% CO₂ at 37°C. Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of resveratrol in dimethylsulfoxide (DMSO; Aladdin Reagents Co., Ltd., Shanghai, China) at a concentration of 100 mg/ml was prepared.

LX-2 cells were seeded at a density of 5×10³ cells per well in 96-well plates. After 24 h, various concentrations of resveratrol were added to the wells (0, 3.125, 6.25, 12.5, 25.0, 50.0, 75.0, 100 and 125 µg/ml) and the plates were incubated for 72 h. After treatment, MTT (Sigma-Aldrich) was added to each well at a final concentration of 0.5 mg/ml. Plates were incubated at 37°C for an additional 4 h. After incubation, the supernatant was removed and the cells were lysed in 150 µl DMSO. Absorbance of the blue formazan derivative was measured at 570 nm using a microplate reader (VICTOR X Multilabel; PerkinElmer, Waltham, MA, USA). All measurements were performed in triplicate and all experiments were repeated three times.

Cells were seeded in 96-well plates at a density of 5×10³ cells per well. After 24 h, the cells were incubated for 48 h with resveratrol (0, 10, 20 and 50 µg/ml). Cells were then fixed at room temperature (RT) in 4% paraformaldehyde (Aladdin Reagents Co., Ltd.) for 20 min, permeabilized at RT in 0.1% Triton-X 100 (Sigma-Aldrich) in 0.01 M phosphate-buffered saline (PBS; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 10 min and then blocked at RT in 5% horse serum in PBS for 20 min. After blocking, the cells were incubated at 4°C overnight with a primary antibody against α-SMA (1:400, rabbit polyclonal; cat no. ab5694; Abcam, Cambridge, MA, USA). After the overnight incubation, cells were washed three times with PBS for 10 min each prior to incubation for 30 min at RT with Alexa 488-conjugated secondary antibody (1:800; goat anti-rabbit; Sangon Biotech Co. Ltd, Shanghai, China). Cells were then washed with PBS and the nuclei were counterstained with DAPI (Invitrogen Life Technologies) in PBS for 10 min at RT. Immunofluorescently labelled cells were observed and images were captured under a fluorescence microscope (BX71; Olympus, Tokyo, Japan) equipped with a DP70 digital camera (Olympus). All measurements were performed in triplicate and all experiments were repeated three times.

Cells were seeded in 6-cm dishes at a density of 5×10⁵ cells per well. After 24 h, the cells were incubated with resveratrol (0, 10, 20 and 50 µg/ml) for 48 h. For fluorescence detection, a single-cell suspension was prepared by treatment with 0.25% trypsin (Invitrogen Life Technologies). Single cells were fixed in ice-cold methanol (Aladdin Reagents Co., Ltd.) for 30 min and then washed three times in PBS. For antibody staining, ~0.2×10⁶ cells were incubated with α-SMA primary antibody at 4°C for 1 h in independent reactions. Afterwards, cells were washed three times with PBS buffer, followed by incubation at 4°C for 30 min in the dark with AlexaFluor 488-labeled rabbit-specific secondary antibody (Invitrogen Life Technologies, Inc.). Subsequently, cells were washed and re-suspended in 0.2 ml sheath fluid. Flow-cytometric analysis was performed using a BD FACSCalibur fluorescence-assisted cell sorting machine (BD Biosciences, Franklin Lakes, NJ, USA) using FlowJo software 7.6 (FlowJo LLC, Ashland, OR, Canada). All measurements were performed in triplicate and all experiments were repeated three times.

Male C57BL/6 mice (weight, 20–25 g; age, 8–12 weeks; n=25) were obtained from the Animal Division of Fudan University, Shanghai Medical College (Shanghai, China). The mice were maintained at 24°C on a 12-h light/dark cycle and had access to rodent chow and water ad libitum. All experimental procedures were approved by the Ethics Committee for Animal Care of Fudan University (Shanghai, China). Mice were randomly divided into five groups, including a normal group, a resveratrol (50 mg/kg) treatment group, a CCl₄ (Aladdin Reagents Co., Ltd.) treatment group, a combined resveratrol (20 mg/kg) plus CCl₄ treatment group and a combined resveratrol (50 mg/kg) plus CCl₄ treatment group. Resveratrol was dissolved in 2% DMSO and saline prior to administration. To induce liver fibrosis, mice were intraperitoneally injected with 0.3 ml/kg CCl₄ (mixed 1:1 with vegetable oil) twice a week for four weeks and then once a week for the following four weeks. In the combined resveratrol plus CCl₄ treatment groups, mice were given an intragastric administration of resveratrol (20 or 50 mg/kg) everyday, and were also intraperitoneally injected with CCl₄ three times per week, for a total co-administration time of eight weeks. Resveratrol dosages were selected according to guidelines established by previous studies (18,23). Mice were sacrificed at eight weeks and blood samples were collected for serum biochemistry. The liver was dissected, weighed, frozen in liquid nitrogen and stored at −80°C until analysis.

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and TNF-α were determined using ELISAs according the manufacturer's instructions. The ELISA kit for ALT, AST and TNF-α was obtained from Shanghai Kemin Bioscience Ltd. (Shanghai, China). All measurements were performed in triplicate and all experiments were repeated three times.

Cells and tissues were homogenized in a commercial lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). For isolation of cytoplasmic and nuclear fractions, all tissue samples were processed using a Cytoplasmic/Nuclear Extraction kit (Fermentas, Thermo Scientific, Pittsburg, PA, USA) according to the manufacturer's instructions. Protein concentrations were quantified using a bicinchoninic acid protein assay kit (Sangon Biotech Co., Ltd.) with bovine serum albumin (Wuhan Boster Biological Technology, Ltd.) as the standard. Protein was denatured by heating at 100°C for 5 min and the cellular debris was removed by centrifuging at 12,000 × g for 10 min. Equal amounts of protein (30 µg) were loaded and subjected to 10% SDS-PAGE followed by electotransfer onto a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST; Sigma-Aldrich) and 5% (w/v) non-fat dry milk (Wuhan Boster Biological Technology, Ltd.) for 1 h. After blocking, the membranes were incubated overnight at 4°C with primary antibodies against α-SMA (1:400; rabbit polyclonal; cat. no. ab5694; Abcam), Collagen I (1:800; mouse monoclonal; cat. no. ab6308; Abcam), NF-κB (1:200; rabbit polyclonal; cat. no. ab7972; Abcam); IκB-α (1:500; mouse monoclonal; cat. no. sc-1643; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), pIκB-α (1:500; mouse monoclonal; cat. no. sc-101713; Santa Cruz Biotechnology, Inc.), GAPDH (1:10,000; rabbit monoclonal; cat. no. ab181603; Abcam), p65 (1:500; rabbit polyclonal; cat. no. ab7970; Abcam), α-tubulin (1:500; rabbit polyclonal; cat. no. ab126165; Abcam), β-tubulin (1:1,000; rabbit monoclonal; cat. no. ab179513; Abcam). After washing with TBST, the membranes were incubated with constant agitation with horseradish peroxidase-conjugated secondary antibodies (Sangon Biotech Co., Ltd.) at a dilution of 1:2,000 at RT for 1 h. The membranes were visualized using an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA) following the manufacturer's instructions. Chemiluminescent signals were captured digitally using a chemiluminescence imaging system (Shanghai Clinx Science Instruments, Shanghai, China). The intensity of each band was quantified using ImagePro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). All measurements were performed in triplicate and all experiments were repeated three times.

Values are expressed as the mean ± standard deviation. Data were analyzed using SPSS 20 (IBM, Armonk, NY, USA). Comparisons between groups were performed using analysis of variance with Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

The cytotoxicity of resveratrol in LX-2 cells was determined using an MTT assay. Resveratrol was not cytotoxic below a concentration of 20 µg/ml. The IC₅₀-value of resveratrol on LX-2 cells was 51.8 µg/ml (Fig. 1A). Therefore, the non-cytotoxic concentrations of 10 and 20 µg/ml were selected as the experimental conditions of all subsequent experiments.

The effects of resveratrol on α-SMA expression in LX-2 cells was determined using immunofluorescence and flow cytometric analyses. Immunofluorescent microscopic observation revealed that resveratrol (20 µg/ml) markedly decreased α-SMA expression (Fig. 1B). Similarly, flow cytometric analysis demonstrated that resveratrol treatment decreased the expression of α-SMA in LX-2 cells (Fig. 1C). The percentage of α-SMA-positive cells was 65.1, 31.0 and 15.1% when cells were treated with 0, 10 and 20 µg/ml resveratrol, respectively. These results demonstrated that resveratrol decreased α-SMA expression in LX-2 cells.

To investigate the potential anti-liver fibrosis activity of resveratrol in vivo, the present study used a CCl₄-induced mouse model of liver fibrosis. Serum levels of ALT and AST were determined in animals treated with or without CCl₄ and resveratrol. As shown in Fig. 2A, CCl₄-induced mice had significantly higher levels of ALT and AST when compared with those in the untreated control mice. Resveratrol treatment decreased the levels of ALT and AST in a dose-dependent manner in the CCl₄-induced mice; however, in mice that were not treated with CCl₄, resveratrol treatment had no effect on ALT or AST expression.

Serum levels of the inflammatory factor TNF-α as well as other markers of liver fibrosis were also detected. IThe serum levels of TNF-α were significantly decreased by resveratrol treatment in CCl₄-induced mice when compared with those in the untreated mice (Fig. 2B). The effects of resveratrol treatment on the expression of the liver fibrosis markers α-SMA and collagen-I were also examined. As shown in Fig. 2C and D, the expression of α-SMA and collagen-I was markedly increased in the CCl₄-induced mice, while resve-ratrol treatment significantly inhibited the CCl₄-induced increase of α-SMA and collagen-I in a dose-dependent manner. Treatment with 50 µg/ml resveratrol decreased the expression levels of α-SMA and collagen-I to almost basal levels of the normal control.

As it has been reported that resveratrol prevents liver fibrosis by inhibiting the activity of NF-κB (19), the effects of resveratrol on NF-κB activity were investigated in the mouse model of liver fibrosis as well as in LX-2 cells. As NF-κB is activated through phosphorylation of IκB and the translocation of p65 from the cytoplasm to the nucleus (24), the levels of IκB, pIκB and p65 were assessed. As shown in Fig. 3A and B, the expression of IκB-α was markedly decreased in CCl₄-induced mice; however, resveratrol treatment partially rescued the expression of IκB-α. Furthermore, NF-κB was markedly increased by CCl₄ stimulation, while resveratrol treatment reduced NF-κB to levels below those of the control group. In a further experiment, LX-2 cells were induced with CCl₄. While resveratrol or CCl₄ treatment had no significant effects on the expression of IκB-α, the levels of pIκB-α were markedly increased in CCl₄-induced cells, which was attenuated by resveratrol treatment (Fig. 3C and D). Furthermore, the expression of NF-κB in CCl₄-induced LX-2 cells was assessed (Fig. 3E and F). The levels of the NF-κB p65 sub-unit were markedly increased in the nuclei of CCl₄-induced cells, which was significantly inhibited by resveratrol treatment. All of these results suggested that resveratrol inhibits the activation of NF-κB during liver fibrosis.

The effects of resveratrol on Akt were also examined in vitro. As shown in Fig. 4A and B, neither CCl₄-induced nor resveratrol-treated cells showed a change in total Akt expression compared with that in the control group. However, Akt phosphorylation was markedly increased in CCl₄-induced cells, which was attenuated by resveratrol treatment. These results indicated that resveratrol inhibited the activation of Akt during liver fibrosis.