Isolation and culture of mouse primary nucleus pulposus cells (MNPCs)
In this study, mice were sacrificed by cervical vertebra dislocation and then soaked in 75% ethyl alcohol for 10 minutes to disinfect the entire body. After the dorsal hair had been shaved, the whole spine was separated from back. The disc tissue was separated under microscope, and cut into pieces and placed in culture dishes. The cells were digested with 0.2% collagenase type II (Gibco, USA) at 37°C for 8 hours. The cells were then cultured in DMEM/F12 (HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin and streptomycin (P1400, Solarbio, China) under standard incubation conditions (37°C, 5% CO2). The culture medium was replaced every 3 days and the cells were passaged when they reached 80–90% confluence. The cells from within five generations were used in all vitro experiments. In subsequent experiments, the control-group and TNF-α (10 ng/ml) group were cultured under standard incubation conditions (37°C, 5% CO2), while ML228 (1µM) group and Oltipraz (10µM) group were cultured under hypoxic conditions (37°C, 1% O2, 5% CO2 and 94% N2).
Western blotting analysis
Total protein was extracted from MNPCs of each group with the precooled RIPA Lysis Buffer (P0013C, Beyotime Biotechnology) containing 1mM PMSF on ice for 30 minutes. The collected liquid was centrifuged at 12000 rpm for 15 min at 4°C and the supernatant was retained. Protein concentration was detected with a BCA protein assay kit (PC0020, Solarbio). Then to destroy the 3-dimensional protein structure, the proteins in loading buffer were heated at 100°C for 10 min. Equal amount of protein from each sample was separated by SDS-PAGE on 8%, 10% or 12% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) for 20 minutes at room temperature, the membranes were incubated with anti-iNOS(1:1000, 18985-1-AP, Proteintech), anti-COX-2(1:1000, 27308-1-AP, Proteintech), anti-Tubulin(1:1000, 10068-1-AP, Proteintech), anti-Aggrecan(1:1000, 13880-1-AP, Proteintech), anti-Col-2(1:1000, 28459-1-AP, Proteintech), anti-ADAMTs-5(1:1000, ab41037, abcam), anti-MMP-13(1:1000, 18165-1-AP, Proteintech), anti-Bcl-2(1:1000, ab196495, Abcam), anti-Bax(1:1000, BM3964, Boster), anti-Caspase-3(1:1000, 19677-1-AP, Proteintech) antibodies at 4°C overnight. The next day, after washing with Tris-buffered saline Tween-20 (TBST), these membranes were incubated with goat anti-rabbit IgG-HRP secondary antibody (1:5000, Jackson ImmunoResearch) at room temperature for 1 h. Bound antibody was visualized using an enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL, USA) and the density of protein bands was quantified using ImageJ software.
Real-time PCR
An RNAfast200 Kit (220011, Fastagen) was used to extract total RNA from the MNPCs of each group according to recommended procedure. Total RNA (1µg) was reverse-transcribed to complementary DNA (cDNA) using HiScript II Q RT SuperMix for qPCR (R222-01, Vazyme). Real-time PCR was carried out with RealStar Fast SYBR qPCR Mix (A301, GenStar). The experiment was repeated three times for each target gene of each group. The nucleotide sequences of the primers are listed in Table. 1. The expression levels of target genes were normalized to Tubulin and was calculated by the 2 − ΔΔCT method
Immunofluorescence staining
The cells were treated as indicated and after 24 h, the cells were fixed with 4% paraformaldehyde for 20 minutes. After permeabilized with 0.2% Triton X-100 for 20 minutes, samples were blocked by BSA at 37℃ for 1 hour. Then, the cells were incubated with anti-iNOS (1:500, 18985-1-AP, Proteintech), anti-COX-2(1:500, 27308-1-AP, Proteintech), MMP-13(1:500, 18165-1-AP, Proteintech) antibodies at 4°C overnight. The next day, the cells were incubated with fluorescently labelled goat anti-rabbit IgG (1:100, Abbkine) for 1 hour at 37℃. The nuclei were stained with DAPI. The images were taken using a fluorescence microscope(ZEISS Vert. A1) and analysed with ImageJ software.
TUNEL staining
To examine the apoptosis of MNPCs in each experimental group, cells were stained with a TMR (red) Tunel Cell Apoptosis Detection Kit (G1502, Servicebio). All the procedures were performed according to the manufacturer’s instructions. The images were captured using a fluorescence microscope (ZEISS Vert. A1).
Flow cytometry
The apoptosis of MNPCs from each group were detected by flow cytometry. Cells were stained with propidium iodide (PI) and Annexin V-FITC for 15 minutes at room temperature in the dark with a FITC Annexin V Apoptosis Detection Kit (E-CK-A211, Elabscience) according to the manufacturer’s instructions. Cell apoptosis was detected with a CytoFLEX S flow cytometer (Beckman Coulter, USA) and the data obtained were analysed with CtyExpert software.
Reactive oxygen species assay
To detect intracellular reactive oxygen species (ROS), we used an ROS assay kit (S0033, Beyotime Biotechnology). All the procedures were performed according to the manufacturer’s instructions. Briefly, after washing twice with sterile PBS, cells were stained with 10 µM DCFDA at 37°C for 20 minutes in the dark. Then, the cells were washed with basal culture medium three times. The images were captured using a fluorescence microscope (ZEISS Vert. A1).
JC-1 assay
The mitochondrial membrane potential changes of MNPCs after treatment were detected with a JC-1 assay kit(C2006, Beyotime). Based on the manufacturer’s instructions, each group’cells were stained with the JC-1 staining solution at 37°C for 20 minutes protect from light. Then the cells were washed twice with JC-1 staining buffer and the images were observed and captured using a fluorescence microscope (ZEISS Vert. A1).
MitoTracker assay
MitoTracker staining was performed to visualize the mitochondria and detect mitochondrial membrane potential of each group flowing the instructions of the Mito-Tracker Red CMXRos (C1049B, Beyotime Biotechnology). The cells were incubated with the culture medium containing 20nM Mito-Tracker Red CMXRos for 30 minutes at 37℃ in the dark. Then the images were captured using a fluorescence microscope(ZEISS Vert. A1) after changing fresh culture medium.
X-Ray and Magnetic Resonance Imaging (MRI)
The rabbits in each group were performed X-ray at 14 weeks after the initial puncture. Radiographs were captured at a collimator-to-film distance of 66 cm, an exposure of 63 mAs, and a penetration power of 35 kv. MRI was performed for each group at 3, 6, 11 and 14 weeks, and T2-weighted images (repetition time: 3000 ms; echotime: 80 ms; field of view: 200 mm2; slice thickness: 1.4 mm) were obtained by MRI using a 1.5 T system (GE) in the coronal plane. The MRI grade of NPs were evaluated as previously reported.
Histological staining
The rabbits were sacrificed at 14 weeks after indicated surgery and the IVD tissues were collected and fixed in 4% paraformaldehyde for 2 days. After decalcification in 10% EDTA (pH 7.2–7.4), the samples were processed, embedded in paraffin and cut into 5-µm sections using a microtome. H&E staining was performed to evaluate the morphological changes of nucleus pulposus with a H&E staining Kit (EE0012, Sparkjade) and histological grading of these samples was valuated in accordance with the grading scale based on the morphology of AF and the cellularity of NP. Safranin O staining was performed to detect changes in proteoglycans with a Safranin O staining kit (G1371, Solarbio) according to the manufacturer’s recommended procedure. Masson staining was performed to confirm collagen loss of these samples with a Masson's Trichrome Stain Kit (G1340, Solarbio) according to the manufacturer’s recommended procedure. The images were captured by a microscope (Leica DMI3000B).
Surgery Procedure
The protocol of this study was approved by the Institutional Animal Care and Use Committee. Twenty-four New Zealand white rabbits (female), ranging from 2.9 to 3.4 kg in body weight (SLC, Hamamatsu, Japan), were used in this study. Rabbits were housed in separate cages under standard conditions with a light-dark cycle (12h–12h) and dry-bulb room temperature at 22–24°C and provided ad libitum access to tap water and food pellets daily. Rabbits were anesthetized by an intramuscular injection of ketamine hydrochloride (25mg/kg; Ketalar®; Daiichi Sankyo, Tokyo, Japan) mixed with xylazine (5mg/kg; Selactar®, Bayer, Tokyo, Japan). Lateral plain radiographs were obtained to determine baseline IVD height values before the treatment. Under single fluoroscopy (C-arm image intensifier, Philips Med System), rabbits were then placed into a lateral prone position, and a nonionic, isotonic contrast agent (CA) (Iotrolan 240, Bayer Global, Leverkusen, Germany) or MIA (305-53-3, Sigma-Aldrich, St. Louis, MO, USA) was injected into the rabbit IVDs from the posterolateral to the center percutaneously using micro syringes with a 31G needle. L1–L2 discs received 10 µl of CA only, L2–L3 discs received MIA 0.01 mg (in 10 µl CA), L3–L4 MIA 0.1 mg (in 10 µl CA), and L4–L5 MIA 1.0 mg (in 10 µl CA). L5–L6 discs were used as non-injection (NI) controls. The use of the lumbar spine lowest levels (L6–L7) was avoided to eliminate possible influences of the lumbosacral junction. Six rabbits were euthanized at 3, 6, 11, and 14 weeks post-injection, and the spinal columns (L1 to L6 vertebra) were harvested. At each time point, three spinal columns were processed for micro-computed tomography (CT) and another three for magnetic resonance imaging (MRI), followed by histological analysis.
Statistical analysis
Analysis of data were performed with GraphPad Prism (GraphPad Software Inc., USA). Comparisons of various groups were performed using analysis of variance (ANOVA) with Tukey’s post hoc test. Data were presented as “mean ± SD”. Statistical significance was indicated when two-sided P < 0.05.