HSC-T6 cells were obtained from The Third University Hospital of Sun Yat-Sen University cell bank (Guangzhou, China). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM/F12; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Sijiqing Biological Engineering Materials, Hangzhou, China), 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom GmbH, Berlin, Germany). The cells were maintained at 37°C in 5% CO₂ in a humidified atmosphere. All experiments were performed following 3–5 cell passages. Pirfenidone was purchased from Sigma-Aldrich (St. Louis, MO, USA).

HSC-T6 cells were cultured in six-well plates (10⁵ cells/well) in DMEM/F-12 with 10% FBS. When cells grew to 80% confluence, the media was changed to DMEM/F-12 without penicillin or streptomycin but containing pirfenidone (0, 10 and 100 μM) and growth was continued for 24 h, following which cell morphology was examined under the optical microscope (DP22; Olympus, Tokyo, Japan).

HSC-T6 cells were seeded in 96-well plates (10⁵ cells/well) for 24 h in DMEM/F-12 with 10% FBS. When cells grew to 80% confluence, they were incubated in DMEM/F-12 supplemented with pirfenidone (0, 10 and 100 μM) for 24 h. Following incubation with 100 μl DMEM/F-12 and 10 μl CCK-8 solution (Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at 37°C, the absorbance at 450 nm was measured using a microplate reader (Meterterch Σ960; Sigma-Aldrich).

After a 24-h incubation in DMEM/F-12 with 10% FBS, HSC-T6 cells were incubated with pirfenidone (0, 10 or 100 μM) for an additional 24 h, then trypsinized, washed in phosphate-buffered saline (PBS) and fixed with 70% cold methanol (4°C) overnight. Following fixation, the cells were washed to remove alcohol and resuspended in PBS containing 100 μg/ml RNase A (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) at 37°C for 30 min. The nuclei were stained by incubation in 50 μg/ml propidium iodide (Wako Pure Chemicals Industries, Ltd.) at 4°C for 30 min and analyzed by flow cytometry using a FACS Aria cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). The cell cycle distribution was analyzed using Modifit software (BD Biosciences).

After a 24 h incubation in DMEM/F-12 with 10% FBS, HSC-T6 cells were treated with pirfenidone (0, 10 or 100 μM) for an additional 24 h. Cultured cells were harvested and lysed in mammalian protein extraction reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Supernatants obtained using centrifugation at 12,000 × g for 30 min at 4°C were used as cytoplasmic extracts. Protein concentrations were determined using a bicinchoninic acid assay kit (Shanghai Shenergy Biocolor BioScience and Technology Company, Shanghai, China). Prepared samples were heated to 100°C for 5 min prior to 10% acrylamide SDS-PAGE (Amersham Biosciences, Freiburg, Germany), and an identical quantity of total protein (20 μg) was added to each well. Separated proteins were transferred onto a nitrocellulose membrane (Hybond-ECL; Amersham Biosciences, Freiburg, Germany). Nonspecific binding was blocked by incubation with Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat milk for 2 h prior to an overnight incubation with a 1:1,000 dilution of the mouse monoclonal anti-human HSP-47 antibody (Stress Gen, Victoria, BC, Canada) diluted in TBST at 4°C with constant agitation. Following several washes with TBST, the membranes were incubated with a 1:10,000 dilution of the anti-mouse immunoglobulin G secondary antibody (ZSGB-BIO, Beijing, China) for 1 h. Following several subsequent washes with TBST, blots were developed using chemiluminescence (Phototope-HRP Western Blot Detection system; Cell Signaling Technology, Inc, Danvers, MA, USA) and the signal was captured on X-ray film (Eastman Kodak, Rochester, NY, USA) according to the manufacturer’s instructions. The abundance of HSP-47 was correlated against a 1:10,000 dilution of mouse anti-GAPDH monoclonal antibody (Kang Chen Bio-tech Inc., Shanghai, China) and quantified using densitometry.

At 48 h after treatment with pirfenidone, total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The ThermoScript RT system (Fermentas, Burlington, ON, Canada) was used to conduct the RT reactions. Total RNA (1 μg) was used for RT in a total volume of 20 ml. The following primers (mixed with probes) were purchased from Geneseed Biotech (Guangzhou, China): Collagen type I forward, 5′-CCCTACCCAGCACCTTCAAA-3′ and reverse, 5′-GCACAGGCCCTCAAAAACA-3′; and 18srRNA forward, 5′-CCTGGATACCGCAGCTAGGA-3′ and reverse, 5′-GCGGCGCAATACGAATGCCCC-3′.

PCR amplification was performed in a Gene Amp 2400 thermal cycler (Perkin Elmer Inc., Waltham, MA, USA). PCR amplification was performed with an initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C and extension at 72°C for 32 sec, with a final extension at 72°C for 32 sec. Quantification of signal intensity was confirmed using the IBAS 2.5 Auto Image analysis program (Kontron, Eching, Germany). The fidelity of the RT-PCR fragments was subsequently verified by comparing the size of the amplified products with the expected cDNA bands and the sequencing of the PCR products.

Values are expressed as the mean ± standard deviation. All data were analyzed using a one-way analysis of variance and with the Bonferroni test for replicate measurements. Statistical analyses were conducted using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). A Bonferroni adjusted P<0.05 was considered to indicate a statistically significant difference. For the Kontron IBAS 2.0 automatic image analysis system (Amersham Biosciences), semi-quantitative analysis was applied to scan strips. The stripe area and optical density were analyzed.

Under the optical microscope, the HSC-T6 cells were a characteristic myofibroblastic shape. In the medium containing penicillin and streptomycin, cells were observed to be attached to the culture plate and stretching pseudopodia were identified after 4 h. By the following day, cells were more uniform, with slender pseudopodia. After two days, star-shaped cells were fully-fused. Following exposure to 10–100 μM pirfenidone for 24 h, cells were generally smaller in size and of a more slender shape (Fig. 1). In addition, quantities of the cells were markedly reduced (Fig. 2).

HSC-T6 cells were treated with pirfenidone (0, 10 or 100 μM) for 24 h and cell proliferation was measured using the CCK-8 assay. Pirfenidone significantly reduced the level of cell proliferation in a dose-dependent manner (10 μM pirfenidone vs. control, P=0.005; 100 μM pirfenidone vs. control, P<0.001; 10 μM vs. 100 μM pirfenidone, P=0.0037; Fig. 1).

The cell cycle distribution was determined using flow cytometric analysis of released nuclei. Following treatment with pirfenidone for 24 h, the proportion of cells in the G1 phase was decreased; however the number in the S-phase was increased (Fig. 3).

The effects of pirfenidone on HSP-47 expression were investigated using western blot analysis. The expression of HSP-47 was clearly decreased by pirfenidone treatment in a dose-dependent manner (10 μM pirfenidone vs. control, P= 0.042; 100 μM pirfenidone vs. control, P=0.003; 10 μM vs. 100 μM pirfenidone, P=0.011; Fig. 4A, B).

The mRNA levels of collagen type I were analyzed 24 h after pirfenidone treatment. Of note, collagen type I mRNA levels were significantly decreased by pirfenidone (10 μM pirfenidone vs. control, P=0.079; 100 μM pirfenidone vs. control, P=0.017; 10 μM vs. 100 μM pirfenidone, P=0.044; Fig. 5). Furthermore, quantitative analysis of collagen I transcription normalized against 18S RNA exhibited a similar dose-dependent pattern to that observed with HSP-47 expression (Fig. 4). This indicated that a reduction of collagen type I mRNA resulted from the decreased HSP-47 protein levels caused by pirfenidone treatment.