Sprague Dawley (SD) rats were anaesthetised with 10% chloral hydrate (3 µl/g). Disc fibre rings were isolated in a sterile manner, and superficial disc fibre rings were harvested and rinsed in 1X phosphate-buffered saline (PBS; Hyclone, Carlsbad, CA, USA) three times. The disc fibre rings were cut into sections (volume, 1 mm³). To isolate the cells, the disc tissues were digested with 0.25% trypsin including 0.02% ethylenediaminetetraacetic acid (Hyclone) for 20 min, followed by another treatment with 0.2% collagenase II (Solarbio Science and Technology Co., Ltd., Beijing, China) for 8 h at 37°C. Following the enzyme digestion, the suspension was filtered through a 70-µm mesh. The filtered cells were harvested, and the digestion solution was refreshed twice. The cells were adjusted to a density of 2×10⁵/ml by a hemocytometre; the cells were then placed in a humidified incubator with 5% CO₂ at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS). The cells were passaged at 80% confluence, which was determined by microscopy.

Third-passage annulus fibrosus cells were plated onto coverslips, rinsed in 1X PBS, fixed in 4% paraformaldehyde for 30 min, and stained with 1% toluidine blue for 30 min at room temperature. The sections were briefly rinsed in absolute ethyl alcohol, dried, transparentised, mounted and observed via microscopy (original magnification, ×200).

One-month-old male or female SD rats (n=50; weight, 100–120 g) were purchased from Slaccas Laboratory Animal Co., Ltd. (Shanghai, China; certification no. SCXX (SH) 2008-0005). The care and use of animals conformed to the regulations of the Guidelines for Laboratory Animal Welfare established by the Ministry of Science and Technology of China (16).

Third-passage annulus fibrosus cells were trypsinised and subcultured into 96-well plates at 5×10³ cells/well. Each group comprised five wells with an untreated group as a control. When the cells were anchored to the plates, various concentrations of PF (2080, 208, 20.8, 2.08, 0.208 and 0.0208 µM; National Institute for Control of Pharmaceutical and Biologic Products, Fujian, China) were incubated for 24, 48 and 72 h. The activity of the annulus fibrosus cells was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

The cells were digested, centrifuged, collected and rinsed in 1X PBS. Twenty microlitres of 0.5% MTT (Solarbio Science and Technology Co., Ltd.) solution was added to each well and incubated at 37°C for 4 h. MTT was discarded and replaced by 150 µl dimethyl sulfoxide (Solarbio Science and Technology Co., Ltd.), and the mixture was blended for 10 min. The optical density (OD) was determined for each group by a microplate reader (Thermo Scientific, Waltham, MA, USA) at 490 nm; the mean values were calculated.

Third-passage annulus fibrosus cells were trypsinised and subcultured into 6-well plates at 3×10⁵ cells/well (final volume, 2 ml), and then incubated in DMEM with 20% FBS (Hyclone). After reaching 80% confluence, the cells were freshly incubated in DMEM with 1% FBS (Hyclone) for 8 h (15) and induced with FasL (R&D Systems, Minneapolis, MN, USA) at final concentrations of 0, 10, 20 and 40 ng/ml for 24 h. The apoptotic rate was measured by flow cytometry using Annexin V-FITC/PI (Invitrogen, Carlsbad, CA, USA).

The cells were digested, centrifuged and rinsed in 1X PBS at 4°C. The rinsed cells were resuspended with binding buffer at a density of 1×10⁶/ml. The cells were stained with 5 µl Annexin V-FITC and 10 µl PI in the dark at room temperature for 15 min. A 400 ml volume of the buffer was added to the resuspending cells; the cells were then sampled by flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA, USA) to analyse the apoptotic fraction of the annulus fibrosus cells.

Third-passage annulus fibrosus cells were trypsinised and subcultured into 6-well plates at 3×10⁵ cells/well (final volume, 2 ml), and then incubated in DMEM with 20% FBS. After reaching 80% confluence, the cells were replaced by DMEM with 1% FBS for 8 h and induced with FasL at final concentrations of 20 ng/ml; the cells were then treated with PF at final concentrations of 208, 20.8 and 2.08 µM for 24 h.

Total proteins were isolated from the cells; protein concentrations were tested by the bicinchoninic acid assay method (Sangon Biotech Co., Ltd., Shanghai, China). Samples of 40 µg total protein were separated by gel electrophoresis using 12% SDS gel; the samples were then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was incubated with 1X Tris-buffered saline with 0.1% Tween-20 (TBST) and 5% dehydrated skimmed milk to block non-specific protein binding for 2 h; the proteins were incubated with rabbit anti-Fas (Cell Signaling Technology, Inc., Beverly, MA, USA).

Subsequently, 1:1,000 each of anti-caspase-3, anti-GAPDH, and anti-β-actin antibodies (all from Cell Signaling Technology, Inc.) were incubated overnight at 4°C; anti-rabbit secondary antibodies (Cell Signaling Technology, Inc.) were incubated for 1 h at room temperature. The membranes were rinsed in TBST three times for 5 min, labelled with enhanced chemiluminescence substrates, and exposed to X-ray. Protein bands were analysed using a gel imaging analysis system (Bio-Rad, Hercules, CA, USA) and normalised to β-actin in the sample.

Data are expressed as the mean ± standard deviation. The groups were compared by analysis of variance; P<0.05 was considered to indicate a statistically significant difference.

The primary digestion annulus fibrosus cells were small and round; strong refraction was observed in the medium (Fig. 1A). On the first day of culture, the annulus fibrosus cells adhered to the culture flask and extended pseudopod-like projections; their nuclei were mainly round or oval. On the fourth day, the cells were mainly polygonal and in grouped distributions. The cells became confluent and arranged regularly on the eighth day (Fig. 1B). Second-, third- and fourth-passage cells grew rapidly; the passaged cells adhered to the culture flask within 12 h. The period of subculture was 7 days; third-passage cells were mainly polygonal and all equal in size (Fig. 1C). The annulus fibrosus cells were detected by toluidine blue staining for characterisation. The third-passage annulus fibrosus cells were purple and exhibited metachromatic granules. The nuclei were mainly round or oval and dark blue (Fig. 1D).

The OD values were significantly higher in the cells treated with 208, 20.8 and 2.08 µM PF after 24 h than those in untreated cells (P<0.01). At 48 and 72 h, the OD values of the cells treated with 208, 20.8 or 2.08 µM PF were also significantly higher than those of the untreated cells (P<0.01; Fig. 2).

Flow cytometry of annexin V-FITC/PI-stained cells was used to analyse the inhibitory effects of PF treatment on FasL-induced apoptosis in annulus fibrosus cells. The percentages of early and late apoptotic cells following treatment with 208, 20.8 or 2.08 µM PF were significantly lower than those in untreated cells (P<0.01). The percentages of early and late apoptotic cells following treatment with 208 µM PF were lower than those of cells treated with 2.08 µM PF (P>0.05). The percentage of late apoptotic cells was lower following treatment with 208 µM PF than when treated with 20.8 µM PF (P>0.05); no significant differences were observed in the percentages of early apoptotic cells (P>0.05; Fig. 3).

Following treatment with 208, 20.8 and 2.08 µM PF, the annulus fibrosus cells demonstrated significantly lower levels of caspase-3 protein than untreated cells (P<0.01); the protein levels in FasL-induced cells were higher than those in normal cells (P<0.05). Fas levels following 208, 20.8 and 2.08 µM PF treatment were significantly lower than those in untreated cells (P<0.05); FasL-induced annulus fibrosus cells exhibited higher Fas levels compared with normal fibrosus cells (P<0.05; Fig. 4).