The present study was approved by the Ethics Committee of Xinqiao Hospital (Chongqing, China). All protocols were subject to the standards set forth in the 8th edition of the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences (Washington, DC, USA).

Rabbit monoclonal anti-rat microtubule-associated protein light chain 3 (LC3)B (cat. no. 3868, 1:1,000 dilution), Beclin-1 (cat. no. 3495, 1:1,000 dilution) and cleaved caspase-3 (cat. no. 9661, 1:1,000 dilution) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse monoclonal anti-rat glyceraldehyde3-phosphate dehydrogenase (GAPDH; cat. no. sc-47724, 1:1,000 dilution) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no.. ZB2301, 1:400 dilution) were purchased from ZSGB-BIO (Beijing, China).

A total of 60 male Sprague-Dawley rats (age, 10-12 weeks; weight, 200-240 g; specific pathogen-free grade) were supplied by the Laboratory Animal Research Center of Daping Hospital (Chongqing, China). Animals were all housed at 20-24°C under a 12-h light/dark cycle, and provided with food and water ad libitum. The caudal IVDs of 10-12-week-old male Sprague-Dawley rats were aseptically excised immediately following sacrifice. The NP tissues were carefully separated from the discs under aseptic conditions and digested in 0.2% type II collagenase (Merck KGaA, Darmstadt, Germany) at 37°C for 2 h to release the NP cells. The cell suspension was passed through a 70-µm cell strainer and centrifuged at 200 × g for 5 min at room temperature. The supernatant was removed and the cells were suspended in DMEM/F12 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.). The NP cells were cultured in 5% CO₂ at 37°C. The medium was replaced every 3 days. When the cells grew to 90% confluence, they were digested using 0.25% trypsin and subcultured in culture flasks. The cells of passages 2-5 were used in the subsequent experiments.

The NP cells were seeded on a 6-well BioFlex™ plate (Flexcell International Corporation, McKeesport, PA, USA) in DMEM/F12 medium containing 10% fetal bovine serum. On reaching 80% confluence, the NP cells were stretched with CMT (20% elongation) at a frequency of 1 Hz for 2, 4, 6, 12, 24 or 48 h using a FX-5000T Flexercell Tension Plus system (Flexcell International Corporation) at 37°C and 5% CO₂. Cells cultured in the same plates under the same conditions were used as the control. Following cyclic tension loading, the morphology of NP cells was observed using a phase contrast microscope (Olympus Corporation, Tokyo, Japan).

Total RNA was isolated from the NP cell using TRIzol reagent (Takara Bio, Inc., Otsu, Japan). The RNA quality and quantity were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc). RNA (1 µg) was reverse transcribed using a PrimeScript RT Reagent kit (Takara Bio). The PrimeScript RT Reagent kit (Takara Bio, Inc.) was used for RT-qPCR according to the manufacturer's instructions. RT-qPCR was performed using a ViiA™ 7 Real-Time PCR system using SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.), according to the manufacturer's instructions. The 20-µl reaction volume was amplified under the following conditions: Initial heat activation for 30 sec at 95°C, followed by 40 cycles of 5 sec at 95°C for template denaturation and 30 sec at 60°C for annealing and extension. GAPDH was used as an internal reference gene. Data were analyzed using the 2^−ΔΔCq method (21). The primers of the genes investigated in the present study are listed in Table I.

Total proteins were extracted from NP cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Inc.). The concentration of protein was quantified using the bicinchoninic acid method (Beyotime Institute of Biotechnology). Protein samples (50 µg) were separated using 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% milk in TBST for 1 h at 37°C to block nonspecific protein binding. The membranes were then incubated at 4°C overnight with primary antibodies, following which the membranes were washed with TBST and incubated at 37°C for 1 h with HRP-conjugated secondary antibodies. Immunolabeling was detected using ECL reagents (Thermo Fisher Scientific, Inc.). The optical density of the bands was measured using Image J software (National Institutes of Health).

Following stretching, the NP cells were isolated with trypsin and fixed in 2% glutaraldehyde at 4°C for 2 days. The cells were then treated with 1% osmium tetroxide (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature, dehydrated in an ethanol gradient and embedded with Epon 812 (Shell Chemical Co., Houston, TX, USA). The fixed cells were then sliced into ultrathin sections (60 nm) and observed under a Tecnai-10 TEM (Philips Healthcare, Amsterdam, The Netherlands).

The apoptotic incidence was detected using the Annexin V-FITC apoptosis detection kit I (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Following stretching, the NP cells were isolated with trypsin and washed twice with PBS. The NP cells were then resuspended in 500 µl binding buffer at a concentration of 1×10⁶ cells/ml. PE Annexin V solution (5 µl) and 7-amino-actinomycin D (5 µl) were added to the cells at room temperature for 15 min in the dark. Finally, the cells were analyzed on a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Following stretching, the NP cells were isolated with trypsin and washed twice with PBS, resuspended in PBS with 0.05 mM monodansylcadaverine (Merck KGaA) at a concentration of 1×10⁶ cells/ml, and incubated at 37°C and 5% CO₂ for 20 min in the dark. Following incubation, the cells were washed three times with PBS. The mean fluorescence intensity (MFI) was analyzed on a flow cytometer (Beckman Coulter, Inc.).

Following stretching, the NP cells were isolated with trypsin and washed twice with PBS. The cells were then resuspended using serum-free DMEM/F12 medium with 25 µM 2',7'-dichlorodi-hydrofluorescein diacetate (Merck KGaA) at a concentration of 1×10⁶ cells/ml, and incubated at 37°C and 5% CO₂ for 20 min. Following incubation, the cells were washed three times with serum-free DMEM/F12 medium. The MFI was analyzed on a flow cytometer (Beckman Coulter, Inc.).

Data are presented as the mean ± standard deviation. All experiments were repeated at least three times. For comparisons between two independent groups, Student's t-test was used. For comparisons among three or more groups, one-way ANOVA and least significant difference multiple comparisons were used. The RT-qPCR data were analyzed using Kruskal-Wallis nonparametric analysis and Mann-Whitney U post hoc tests. Data were analyzed and displayed using SPSS version 22.0 (International Business Machines Corp., Armonk, NY, USA) and GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Following cyclic tension loading, the morphology of NP cells was observed using a phase contrast microscope. The NP cells that adhered to the outer part of the membrane became clearly spindle-shaped and exhibited a circular arrangement perpendicular to the axial tension (Fig. 1). Only a few cells were detached from the membrane surface.

Following 20% CMT at a frequency of 1 Hz for 6, 12, 24 and 48 h, the apoptotic rate of the NPs was detected by flow cytometry with Annexin V and 7AAD double-labeling. The results demonstrated a time-dependent increase in the apoptotic rate of rat NP cells subjected to CMT (Fig. 2A), and significant differences in the apoptotic rate were observed after 24 h (Fig. 2B). Consistent with the results of flow cytometry, the western blot results showed that the levels of caspase-3 and cleaved caspase-3 were also increased in the NP cells subjected to CMT (Fig. 2C-E).

Examining the ultrastructure of cells by TEM is the gold standard for the detection of autophagy (14). The ultrastructure of the NP cells was observed under TEM. The autophagosomes, manifested as double-membrane vacuolar structures containing organelles and parts of cytoplasm, were observed in NP cells. The number of autophagosomes in the CMT groups was higher than the number in the static control groups (Fig. 3). MDC staining and a flow cytometry quantitative assay were used to quantify the expression of autophagy. The increase in MFI in each CMT group was measured. There was a time-dependent decrease in the MFI of NP cells subjected to 20% CMT for 6, 12, 24 and 48 h (Fig. 4A). The mRNA expression levels of LC3 and Beclin-1 showed a similar trend (Fig. 4B and C). The protein expression of LC3 II and Beclin-1 was also increased significantly in the NP cells subjected to CMT for 6 h (Fig. 4D-F), and exhibited a time-dependent decrease after 6 h. These results indicated the upregulation of autophagy in NP cells subjected to CMT. Furthermore, the protein expression of LC3 II was measured at 2, 4 and 6 h to determine when autophagy was the most marked. The results showed that autophagy increased within 6 h and reached a maximum level between 4 and 6 h (Fig. S1).

The NP cells were administered with rapamycin (1 µM) or 3-MA (100 nM) and placed in an incubator for 1 h, following which the NP cells were loaded with 20% CMT for 24 h. The results of MDC staining and quantitative fluorescence measured by flow cytometry indicated that the activities of autophagy in cells following CMT loading were significantly reduced by 3-MA (P<0.05; Fig. 5A). The difference in autophagic activities between the CMT and the combined CMT and rapamycin was not significant (P=0.39). The mRNA expression levels of LC3 and Beclin-1 were decreased by 3-MA in NP cells subjected to 20% CMT for 24 h, but did not differ significantly following combined CMT and rapamycin treatment (Fig. 5B). Consistent with the results of the RT-qPCR analysis, the western blot analysis results confirmed the downregulation of LC3 and Beclin-1 by 3-MA at the protein level (Fig. 5C and D).

Our previous study indicated that CMT had minimal effect on the production of ROS in NP cells (22). In the present study, the ROS levels of NP cells pretreated with 3-MA or rapamycin following CMT were measured at 24 h. The results showed that the level of ROS in NP cells was increased significantly in the combined CMT and 3-MA group (P<0.05), with no significant differences observed among other groups (Fig. 6). In order to further illustrate the effect of autophagy on ROS production, ROS levels were measured at 6 h when autophagy was the highest (Fig. S2). The results showed that the level of ROS in NP cells increased significantly with 3-MA treatment at 6 h (P<0.05).

The NP cells were loaded with 20% CMT for 24 h following incubation with 1 µM rapamycin or 100 nM 3-MA for 1 h. The apoptotic rates of NP cells were then measured (Fig. 7A). Compared with the CMT group, the apoptotic rate of NP cells was significantly increased in the combined CMT and 3-MA group (P<0.05), with no clear differences observed in the combined CMT and rapamycin group (Fig. 7B). Changes in the protein expression of caspase-3 and cleaved caspase-3 were similar (Fig. 7C-E). According to the results, autophagy was highest following application of CMT for 6 h. To verify the inhibitory effect of autophagy on apoptosis, the apoptotic rates and expression of cleaved caspase-3 were measured in NP cells treated with 3-MA and CMT at 6 h (Fig. S3). The apoptotic rate of NP cells and the protein expression of cleaved caspase-3 were significantly increased in the combined CMT and 3-MA group at 6 h (P<0.05).