All the subjects were patients (three males and two females; Age, 59.3±7.2 years) undergoing lumbar spine surgery admitted to Hubei Provincial Hospital of Traditional Chinese Medicine (Wuhan, China) between May 2014 and December 2015. The IDD tissues were harvested under sterile conditions and immediately sent to the laboratory (within 30 min of harvesting). Written informed consent was obtained from each patient. The study was approved by the Ethics Committee of Hubei Provincial Hospital of Traditional Chinese Medicine. Patients with ankylosing spondylitis or diffuse idiopathic skeletal hyperostosis were excluded. Complete Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) medium with serum at 4°C was used as transport medium. The nucleus pulposus tissues were carefully isolated from IDD tissues by a scalpel microscopically under sterile conditions.

3-methyladenine (3-MA), TBHP and the type II collagenases were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The primary antibodies against sequestosome-1 (p62; 1:2,000; cat. no. ab207305), horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090) and GAPDH (1:2,000; cat. no. ab9485; all Abcam, Cambridge, UK). The microtubule-associated proteins 1A/1B light chain 3A (LC-3; 1:2,000; cat. no. 12741), Beclin-1 (1:2,000; cat. no. 3495), Bax (1:2,000; cat. no. 5023) and cleaved caspase 3 (1:2,000; cat. no. 9661) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). DAPI was obtained from Beyotime Institute of Biotechnology (Haimen, China). The cell culture reagents were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Total protein was extracted from nucleus pulposus cells by radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The protein lysates (40 µg per lane) were separated using 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked using 5% non-fat milk in PBS (Thermo Fisher Scientific, Inc.) containing 0.1% Tween-20 (Sigma-Aldrich; Merck KGaA) at room temperature for 2 h. Subsequently, the membrane was incubated for 2 h at 25°C with specific primary anti-human antibodies, followed by incubation for 1 h at 25°C with a goat horseradish peroxidase-conjugated secondary antibody. Membranes were then washed with PBS for 10 min at room temperature, and the protein bands were visualized using an Enhanced Chemiluminescence Western Blotting kit (Pierce; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's protocol. Protein densitometry was performed using ImageJ software (version 1.41; National Institutes of Health, Bethesda, MD, USA). GAPDH was used as a control. The experiment was repeated 3 times.

Chx (Cycloheximide, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; 1 µg/ml) was added into cultured cell medium. Nucleus pulposus cells were cultured for 0, 12, and 24 h. Then cells were collected and Beclin-1 expression was measured using western blotting.

The human nucleus pulposus tissues were carefully isolated from IDD tissues by a scalpel microscopically under sterile condition as previously described (19). Then, they were washed with PBS twice and cut into 1 mm³ fragments. The fragments of nucleus pulposus tissues were digested in 0.25% trypsin solution for 30 min at 37°C, following 3–4 h in 0.2% type II collagenase at 37°C. Tissue debris was removed by passing through a 200 µm filter and then the nucleus pulposus cells were resuspended in Dulbecco's modified Eagle's medium (DMEM)/F12 containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. When the cells grew to 80–90% confluence, they were digested by 0.25% trypsin solution and sub-cultured in culture flasks. The third generation of nucleus pulposus cells was used for all experiments.

The total RNA was extracted from the cells in a 6-well plate using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). The RNA quality and concentration was determined using a Thermo Scientific NanoDrop ND-100 (Wilmington, DE, USA). A total of 1 µg of total RNA was used to synthesize cDNA using a Reverse Transcription System kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The thermocycling conditions were: 37°C for 25 min, incubated at 85°C for 5 sec in 20 µl of reaction volume. For the PCR amplification, a 20 µl reaction volume was used, including 10 µl 2X SYBR Premix Ex Taq mixture (Takara Bio, Inc., Otsu, Japan), 0.2 mmol/l primer, 2 µl 2-fold diluted cDNA and sterile distilled water. The reaction and detection were conducted in a light cycler (Roche Diagnostics GmbH, Mannheim, Germany). The thermocycling conditions were as follows: Denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and elongation at 60°C for 1 min. The cycle threshold (Cq) values were collected and normalized to the level of the housekeeping gene GAPDH according to the 2^−ΔΔCq method (24). The primer sequences were as follows: Collagen type II alpha 1 (Col2α1) forward (F), 5′-ACGCTCAAGTCGCTGAACAA-3′ and reverse (R), 5′-TCAATCCAGTAGTCTCCGCTCT-3′; Aggrecan F, 5′-TCCAAACCAACCCGACAAT-3′ and R, 5′-TCTCATAGCGATCTTTCTTCTGC-3′; a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts-5) F, 5′-CGACAAGAGTCTGGAGGTGAG-3′ and R, 5′-CGTGAGCCACAGTGAAAGC-3′; matrix metalloproteinase-3 (MMP-3) F, 5′-ATGATGAACGATGGACAGATGA-3′ and R, 5′-CATTGGCTGAGTGAAAGAGACC-3′; Beclin-1 F, 5′-TCCGGGCTCCCGAGG-3′ and R, 5′-TTCCTCCTGGGTCTCTCCTG-3′; Bax F, 5′-TCATGGGCTGGACATTGGAC-3′ and R, 5′-GAGACAGGGACATCAGTCGC-3′; GAPDH F, 5′-ATGTTGCAACCGGGAAGGAA-3′ and R, 5′-AGGAAAAGCATCACCCGGAG-3′.

To establish the apoptosis model of nucleus pulposus cells, different concentrations of TBHP (50, 100, 200, 300 and 500 µM) were added into the culture medium of nucleus pulposus cells for 24 h. Cells were pretreated with different concentrations of β-ecdysterone (10, 50,100 and 200 µM) for 12 h prior to the addition of TBHP (100 µM) to investigate its effect on cell apoptosis. To examine the role of autophagy in β-ecdysterone-mediated cell protection, nucleus pulposus cells were pretreated with 10 µM 3-MA, an autophagy inhibitor, for 1 h prior to β-ecdysterone administration. All experiments were performed in triplicate. Small interfering (si)-Beclin-1 transfection (5′-CGGAGAGGAGCCATTTATTGAA-3′; 50 nM) was performed using Lipofectamine 2000 siRNA transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The efficiency was confirmed 48 h after transfection, using RT-qPCR.

Cell viability was assayed with the Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's protocol. In brief, the nucleus pulposus cells were plated in 96-well plates (5,000 cells/cm²) and incubated in DMEM with 10% FBS at 37°C for 24 h. Then, the cells were treated with TBHP, β-ecdysterone and 3-MA, as aforementioned. Following treatment, the cells were washed with PBS, and then 100 µl DMEM containing 10 µl CCK-8 solution was added to each well, and the plate was incubated for an additional 1 h. The absorbance of the wells was then measured at 450 nm using a microplate reader.

Nucleus pulposus cells were pretreated with β-ecdysterone for 12 h prior to TBHP stimulation for 24 h. Culture supernatants were collected and stored at −20°C until analysis. The concentration of Col2a1 (cat. no. DY7589-05), Aggrecan (cat. no. DY1220), Adamts-5 (DY2198-05) and MMP-3 (cat. no. DMP300) in cell culture supernatants was measured using ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.

Experiments were performed at least three times. The results are presented as mean ± standard deviation. Statistical analyses were performed using SPSS statistical software program v.18.0 (SPSS, Inc., Chicago, IL, USA). Data were analyzed by Student's t-test or one-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of β-ecdysterone on nucleus pulposus cells, the cytotoxic effect of β-ecdysterone was first evaluated. Nucleus pulposus cells from patients with lumbar vertebral fracture were treated with β-ecdysterone at different concentrations (0, 10, 50, 100, 200 and 500 µM) or for different times (0, 6, 12, 18, 24 and 48 h). As indicated in Fig. 1A and B, β-ecdysterone was not cytotoxic to nucleus pulposus cells for 24 h or at concentrations of ≤100 µM. However, the cell viability was significantly downregulated following TBHP treatment in a dose-dependent manner (Fig. 1C). Then, nucleus pulposus cells were treated with β-ecdysterone and TBHP, and it was identified that β-ecdysterone markedly protected nucleus pulposus cells from TBHP-induced cell apoptosis (Fig. 1D and E). The expression of B cell lymphoma 2-associated X protein (Bax) and cleaved caspase 3 were also measured by RT-qPCR and western blot analysis. The results indicated that pretreatment with β-ecdysterone decreased the mRNA level of Bax and the protein level of cleaved caspase 3 induced by TBHP in nucleus pulposus cells (Fig. 1F and G). Taken together, β-ecdysterone exhibited a marked protective effect against TBHP-induced apoptosis in nucleus pulposus cells.

A previous study indicated that β-ecdysterone inhibited cell apoptosis by induction of autophagy in osteoblasts (20). To determine whether β-ecdysterone has a role in autophagy in nucleus pulposus cells, cells were treated with different concentrations of β-ecdysterone or for different time intervals in the present study. LC3-II/LC3-I, beclin-1 and p62 were recognized as indicators of autophagy formation. Their levels in nucleus pulposus cells treated with β-ecdysterone were assessed by western blot analysis, and it was identified that the protein levels of LC3-II and beclin-1 were significantly increased in a dose-dependent manner, while the expression level of p62 was decreased following treatment (Fig. 2A-C). Their expression levels were then determined at indicative time points following β-ecdysterone treatment (100 µM). As demonstrated, the expression levels of LC3-II and Beclin-1 were increased, while the expression of p62 was significantly downregulated (Fig. 2D-F). Collectively, β-ecdysterone is an effective stimulus for autophagy in nucleus pulposus cells.

Previous evidence indicates that autophagy protects cells from apoptosis, including in nucleus pulposus cells (25). To determine whether β-ecdysterone-induced autophagy is responsible for the protection against TBHP-mediated apoptosis, autophagy was inhibited with 3-MA. The administration of 3-MA abolished the autophagy induced by β-ecdysterone in nucleus pulposus cells (Fig. 3A). As demonstrated, the protein levels of LC3-II and beclin-1 were significantly decreased while the expression of p62 was increased following 3-MA treatment (Fig. 3A-C). These experiments indicated that β-ecdysterone may effectively induce autophagy flux in TBHP-treated nucleus pulposus cells, which may be completely reversed by 3-MA treatment. To additionally determine whether autophagy was involved in β-ecdysterone-mediated protection against apoptosis in nucleus pulposus cells, the cell viability and the expression level of cleaved caspase 3 were examined. As indicated, β-ecdysterone increased the proportion of cell viability and inhibited the protein level of cleaved caspase 3 in TBHP-treated cells, while addition of 3-MA completely reversed this trend (Fig. 3D and E).

Subsequently, the present study aimed to determine the molecular mechanism by which β-ecdysterone regulated autophagy in nucleus pulposus cells. To examine whether β-ecdysterone may promote the expression of Beclin-1; cells were treated with β-ecdysterone at different concentrations and it was identified that the Beclin-1 mRNA protein levels were markedly upregulated (Fig. 4A and B). Similarly, administration with β-ecdysterone for different time intervals also promoted the mRNA and protein levels of Beclin-1 (Fig. 4C and D). In addition, it was identified that β-ecdysterone significantly improved the stability of Beclin-1 in nucleus pulposus cells. As demonstrated in Fig. 4E, following the addition of Chx, the degradation of Beclin-1 in β-ecdysterone-treated cells was slower.

To additionally investigate the role of Beclin-1 in the process of β-ecdysterone-induced autophagy, nucleus pulposus cells were transfected with Beclin-1-siRNA (Fig. 5A). By western blot analysis, it was demonstrated that depletion of Beclin-1 protein levels by siRNA abolished the increased autophagy in nucleus pulposus cells (Fig. 5B-D). Then, the effect of Beclin-1 knockdown on the protective effect of β-ecdysterone was also evaluated; it was identified that β-ecdysterone improved the cell activity and inhibited the expression of cleaved caspase 3 in nucleus pulposus cells while Beclin-1 knockdown abolished this effects (Fig. 5E and F), which indicated that β-ecdysterone-mediated autophagy and protective function against apoptosis relied on upregulation of Beclin-1.

Decrease in ECM proteins including aggrecan, collagen II and other factors often results in IDD (26,27). To additionally explore the degeneration of nucleus pulposus cells, the expression levels of major ECM synthesis genes (Col2a1 and aggrecan) and ECM degrading genes (MMP-3 and Adamts5) in nucleus pulposus cells were examined. As demonstrated, the mRNA levels of Col2a1 and aggrecan were downregulated following TBHP treatment, while TBHP upregulated the mRNA levels of MMP-3 and Adamts5 in nucleus pulposus cells (Fig. 6A). Notably, β-ecdysterone reversed the effects of TBHP on the mRNA levels of Col2a1, aggrecan, MMP-3 and Adamts5 via activation of autophagy (Fig. 6A), which was validated by the addition of 3-MA. Furthermore, the ELISA results also exhibited a similar trend. β-ecdysterone promoted the protein levels of collagen-II and aggrecan but inhibited that of MMP-3 and Adamts-5 (Fig. 6B). Finally, nucleus pulposus cells were isolated from patients with IDD, and it was identified that β-ecdysterone upregulated the protein levels of collagen-II and aggrecan but inhibited that of MMP-3 and adamts-5 by ELISA (Fig. 6C). In addition, β-ecdysterone also increased the autophagy of nucleus pulposus cells from patients with IDD and promoted their survival by inhibiting apoptosis (Fig. 6D-F).