All animal experiments were approved by the Ethics Committee on Animal Experiments of Fudan University (Shanghai, China). A total of 2 male Sprague-Dawley rats (age, ~12 weeks; weight, 400 g) were used in the present study and were supplied by the Shanghai Public Health Center (Shanghai, China). Animals were housed with free access to food and water under a 12-h light/dark cycle, with a constant temperature (20–23°C) and humidity (55±5%). Rats were euthanized by cervical dislocation following anesthesia with pelltobarbitalum natricum (1.5%) by intraperitoneal injection (50 mg/kg; Shanghai Shangxiao New Asia Pharmaceutical Co., Ltd., Shanghai, China; http://www.xinyapharm.com). Lumbar spines were obtained within 1 h of sacrifice, and the discs were carefully dissected under a microscope using aseptic conditions to obtain the NP. Tissues were sequentially treated with 0.25% trypsin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 2 h followed by 0.02% collagenase (Sigma-Aldrich; Merck KGaA) at 37°C for 24 h, then washed with phosphate-buffered saline (PBS). Subsequently, the cells were released from the matrix by centrifugation at 200 × g for 5 min at room temperature, seeded into 6-well plates (2×10⁴ cells/well) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml of penicillin (Beyotime Institute of Biotechnology, Nantong, China) and 100 µg/ml of streptomycin (Beyotime Institute of Biotechnology) under 5% CO₂ in a humidified incubator at 37°C. Primary cells were maintained in a high-density monolayer culture for 2 weeks. The cells were then trypsinized again and subcultured into 6-well plates; these cells were used in the subsequent experiments as secondary cells after reaching 80% confluence.

A DNA template and oligonucleotides corresponding to the caveolin-1 gene (Gene ID: NM_001753; www.ncbi.nlm.nih.gov/nuccore/NM_001753) were targeted. The oligonucleotide sequences (Shanghai R&S Biotechnology Co., Ltd., Shanghai, China) were designed and synthesized as follows: Caveolin-1 small interfering (sh)RNA, forward, 5′-GACGUGGUCAAGAUUGACUTT-3′ and reverse, 5′-AGUCAAUCUUGACCACGUCTT-3′. A control shRNA unassociated with these gene sequences was used as the negative control (NC): Caveolin-1 shRNA-NC, forward, 5′-CUGUGAUCCACUCUUUGAATT-3′ and reverse, 5′-UUCAAAGAGUGGAUCACAGTT-3′. The combined sequences of the enhanced green fluorescent protein (GFP) gene and the caveolin-1 shRNA were cloned into the AscI and PmeI sites of the pLenti6.3-MCS vector (Shanghai R&S Biotechnology Co., Ltd., Shanghai, China) containing a cytomegalovirus-driven GFP reporter (25). All of the constructed plasmids (1 µg/µl; 5 µg) were confirmed by sequencing analysis, as previously described (26); prior to transfection into 293T cells (iCell Bioscience, Inc., Shanghai, China) using ViraPower lentiviral Packaging Mix (5 µg, Invitrogen; Thermo Fisher Scientific, Inc.) at 40% confluence (5×10 ⁵ cells/well) in 10 cm plate. Supernatants containing lentiviruses were harvested at 96 h following transduction. Subsequent purification was performed using ultracentrifugation at 1610 × g and 4°C for 10 min. The isolated lentiviruses were stored at −80°C until use within 2 weeks; the lentivirus titre was 1.5×10⁶.

Secondary cells were transferred to 6-well plates at a density of 5×10⁵ cells/well in DMEM with 10% FBS without antibiotics the day prior to transduction procedures. Once cells had reached 80% confluency, cells were transfected with the aforementioned recombinant experimental or control lentivirus at a multiplicity of infection of 50 using polybrene (5 µg/ml; Sidansai Biotechnology Co., Ltd., Shanghai, China) for 24 h at 37°C. All cells were then refreshed with DMEM containing 10% FBS without antibiotics and cultured in DMEM with 10% FBS for 48 h. The transduction efficiency was determined by fluorescence microscopy, which was the average rate of green fluorescent cells in five fields (magnification, ×100).

To establish the cellular senescence model, secondary cells were treated with 70% tert-butyl hydroperoxide (t-BHP; Sigma-Aldrich; Merck KGaA) for 6 h at 37°C, and the sublethal final concentration was observed to be 100 µmol/l. Cells treated with t-BHP were infected with either the caveolin-1 short hairpin (sh)RNA vector or the NC vector, which, were termed NP-LV and NP-LV-NC, respectively. An additional untransfected group treated with t-BHP was used as the control group, which, was termed NP-CTR. Untransfected cells treated under normal conditions without t-BHP treatment formed the normal control group, termed NP-NOM. All four treatment groups were incubated under DMEM with 10% FBS for 72 h at 37°C; the subsequent experiments were performed using cells at different time points throughout this 72 h period.

SA-β-gal staining was performed using a SA-β-gal staining kit (Cell Signaling Technology, Inc., Danvers, MA, USA) according to the manufacturer's instructions. Briefly, cells were washed twice with PBS and fixed with 3% formaldehyde for 15 min at 37°C. Cells were then incubated overnight at 37°C with the kit staining solution. Cells were photographed under reflected light using a digital high-fidelity fluorescence microscope (VH-8000; Keyence Corporation, Osaka, Japan). A total of 5 fields, that were distributed throughout the well, were counted (magnification, ×200), and the average rate of positive staining was recorded.

The proliferation of NP cells exposed to t-BHP treatment was assessed by cell counting kit-8 (CCK-8; Beyotime Institute of Biotechnology) at 0, 24, 48 and 72 h. Briefly, cells were replated at 1×10⁴ cells/well in a 96-well plate, and were then incubated with 10 µl CCK-8 solution at 37°C for 2 h. The mixtures were analyzed with an Epoch Multi-Volume Spectrophotometer System (BioTek Instruments, Inc., Winooski, VT, USA) at an absorbance of 490 nm. Proliferative activities were each calculated as the change in absorbance at 490 nm.

RT-qPCR was performed to detect caveolin-1 mRNA in the different treatment groups at 24, 36 and 72 h; GAPDH was used as an internal standard control. Briefly, total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Single-strand cDNA templates were prepared from 1 µg total RNA using the RT-for-PCR kit (Clontech Laboratories, Inc., Mountainview, CA, USA.). Specific cDNAs were then amplified by PCR using the following primers: Caveolin-1, forward, 5′-AAGGAGATCGACCTG-3′ and reverse, 5′-GGAATAGACACGGCTG-3′; GAPDH, forward, 5′-CCCCAATGTATCCGTTGTG-3′ and reverse, 5′-CTCAGTGTAGCCCAGGATGC-3′. PCR amplification from cDNA was performed using the Takara TP800 Thermal Cycler Dice (Takara Bio, Inc., Otsu, Japan) with a final volume of 20 µl [2X SYBR Green Mix 10 µl (Invitrogen; Thermo Fisher Scientific, Inc.), 1 µl primer mix, 1 µl template DNA and 8 µl diethylpyrocarbonate water]. The thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 59°C for 20 sec and elongation at 72°C for 20 sec, and then a final extension at 72°C for 10 min. PCR products were subjected to amplification curve analysis and quantified using SYBR Green (Invitrogen; Thermo Fisher Scientific, Inc.). The data were normalized to GAPDH and quantified using the 2^−ΔΔCq method (27,28).

The protein expression levels of caveolin-1, p53, p21 and p16 were detected by western blot analysis following 72 h of cell treatment. Total protein was extracted with SDS-PAGE Sample Loading Buffer 5X (cat. no. P0015, Beyotime Institute of Biotechnology). The total protein concentration was determined by a bicinchoninic acid assay (Sigma-Aldrich; Merck KGaA). Protein (20 µg/lane) extracts were separated by 8–12% SDS-PAGE and were then transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween (TBST) for 1 h at 37°C, and incubated overnight at 4°C in TBST with the following primary antibodies: Mouse monoclonal anti-caveolin-1 (dilution 1:1,000; cat. no. AF0087; Beyotime Institute of Biotechnology), mouse monoclonal anti-p53 (dilution 1:1,000; cat. no. AF0255; Beyotime Institute of Biotechnology), mouse monoclonal anti-p21 (dilution 1:500; cat. no. AP021; Beyotime Institute of Biotechnology), polyclonal rabbit anti-p16 (dilution 1:1,000; cat. no. SAB4500072; Sigma-Aldrich; Merck KGaA) and mouse monoclonal anti-β-actin (dilution 1:2,000; cat. no. AF0003; Beyotime Institute of Biotechnology). The membranes were then further incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:5,000; cat. no. A0208; Beyotime Institute of Biotechnology) for 1 h at room temperature, prior to treatment with Electrochemiluminescence Plus (Tanon Science and Technology Co., Ltd., Shanghai, China), according to the manufacturer's protocol.

All measurements were carried out using the same instrument under the same experimental conditions and were independently performed at least three times to ensure consistency. Statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Significant differences were analyzed with a one-way analysis of variance. Bonferroni was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effects of t-BHP-induced oxidative stress on NP cells, the percentage of SA-β-gal-positive cells and the cellular proliferative rate were assessed. Premature senescence of NP cells was investigated following treatment with t-BHP and culture for 72 h. As is shown in Fig. 1A, the percentage of SA-β-gal-positive cells was significantly higher in the NP-CTR group when compared with the NP-NOM group at 72 h (34.25±1.45% vs. 5.60±0.68%; P<0.05). In addition, the proliferative rate of NP-CTR cells gradually decreased with increasing time when compared with NP-NOM cells, and at 72 h the proliferative activity decreased to nearly half that of the NP-NOM control group (0.82±0.15% vs. 1.60±0.21%; P<0.05; Fig. 1B). Notably, caveolin-1 expression at the mRNA and protein level was markedly increased following treatment with t-BHP which, is in agreement with the increase observed in the percentage of SA-β-gal-positive cells and the reduction in cellular proliferative ability (Fig. 1C-E).

Lentiviral shRNA was successfully constructed and confirmed by sequencing analysis; the transduction rate of the lentivirus was ~90% at 48 h (Fig. 2A). Caveolin-1 expression was assessed by RT-qPCR and western blot analysis following treatment with t-BHP. As shown in Fig. 2B, the mRNA levels of caveolin-1 decreased in the NP-LV group at 24, 36 and 48 h when compared with the NP-CTR group; however, no marked difference was detected when comparing the NP-CTR and NP-LV-NC groups. Similarly, the caveolin-1 protein level also decreased in the NP-LV group at 72 h and was markedly decreased when compared with the NP-CTR group (Fig. 2C). These results demonstrated that caveolin-1 gene expression was successfully knocked down by lentivirus-mediated RNA interference.

To determine whether caveolin-1 mediated oxidative stress-induced cellular senescence, the present study quantitatively assessed the percentage of SA-β-gal-positive cells and the proliferative rate following silencing of caveolin-1 expression. As shown in Fig. 3, when caveolin-1 was knocked down in the NP-LV group, the increase in SA-β-gal-positive cells at 72 h was not as marked when compared with the NP-CTR group (20.56±1.28% vs. 34.25±1.45%; P<0.05; Fig. 3A). At 72 h, more proliferative cells were observed in the NP-LV group than in the NP-CTR group when caveolin-1 expression decreased (1.22±0.20% vs. 0.82±0.15%; P<0.05; Fig. 3B).

Cellular senescence primarily occurs via two signaling pathways: The p16 pathway and the p53/p21 pathway (16). In the present study, the expression of the p53 protein increased under oxidative stress in the NP-CTR and NP-LV-NC groups when compared with NP-NOM (Fig. 4). The levels of the p21 protein, a pro-senescence modulator of p53 activity, were also increased under t-BHP-induced oxidative stress in the two groups (29). In addition, inhibition of caveolin-1 expression markedly decreased p53 and p21 protein expression levels in the NP-LV group. Notably, the decrease in p53 and p21 protein expression were associated with the decrease in SA-β-gal-positive cells and the recovery of cellular proliferative ability. By contrast, there was no marked difference in p16 protein levels among the three groups when compared with NP-NOM.