Hypoxia-Inducible Factor-1α Protects Against Intervertebral Disc Degeneration Through Antagonizing Mitochondrial Oxidative Stress

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 min to disinfect the entire body. After the dorsal hair had been shaved, the whole spine was separated from the back. The disc tissue was separated under a microscope, 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 h. The cells were then cultured in DMEM/F12 (HyClone, USA) supplemented with 10% foetal 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 (HY-P7058, MCE, USA) (10 ng/ml) group were cultured under standard incubation conditions (37 °C, 5% CO2), while the ML228 (HY-12754, MCE, USA) (1 μM) group and Oltipraz (HY-12519, MCE, USA) (10 μM) 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 1 mM PMSF on ice for 30 min. The collected liquid was centrifuged at 12,000 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 the loading buffer were heated at 100 °C for 10 min. An 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 being blocked with QuickBlockTM Blocking Buffer (P0252, Beyotime Biotechnology) for 20 min at room temperature, the membranes were incubated with anti-iNOS(1:1000, 18,985–1-AP, Proteintech), anti-COX-2(1:1000, 27,308–1-AP, Proteintech), anti-Tubulin (1:1000, 10,068–1-AP, Proteintech), anti-Aggrecan (1:1000, 13,880–1-AP, Proteintech), anti-Col-2 (1:1000, 28,459–1-AP, Proteintech), anti-ADAMTs-5 (1:1000, ab41037, Abcam), anti-MMP-13 (1:1000, 18,165–1-AP, Proteintech), anti-Bcl-2 (1:1000, ab196495, Abcam), anti-Bax (1:1000, BM3964, Boster) and anti-Caspase-3 (1:1000, 19,677–1-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 the ImageJ software.

Real-Time PCR

An RNAfast200 Kit (220011, Fastagen) was used to extract the total RNA from the MNPCs of each group according to the recommended procedure. Total RNA (1 µg) 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 were calculated by the 2 − ΔΔCT method.

Table 1 Primers Used for Quantitative Real-Time PCRImmunofluorescence Staining

The cells were treated as indicated, and after 24 h, the cells were fixed with 4% paraformaldehyde for 20 min. After being permeabilized with 0.2% Triton X-100 for 20 min, the samples were blocked by BSA at 37 ℃ for 1 h. Then, the cells were incubated with anti-iNOS (1:500, 18985–1-AP, Proteintech), anti-COX-2 (1:500, 27308–1-AP, Proteintech) and MMP-13 (1:500, 18,165–1-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 h at 37 ℃. The nuclei were stained with DAPI. The images were taken using a fluorescence microscope (ZEISS Vert. A1) and analysed with the 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 was detected by flow cytometry. Cells were stained with propidium iodide (PI) and Annexin V-FITC for 15 min 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 the 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 min in the dark. Then, the cells were washed with a 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’s cells were stained with the JC-1 staining solution at 37 °C for 20 min to protect them 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 the mitochondrial membrane potential of each group following the instructions of the Mito-Tracker Red CMXRos (C1049B, Beyotime Biotechnology). The cells were incubated with the culture medium containing 20 nM Mito-Tracker Red CMXRos for 30 min at 37 ℃ in the dark. Then, the images were captured using a fluorescence microscope (ZEISS Vert. A1) after changing the fresh culture medium.

X-Ray and Magnetic Resonance Imaging (MRI)

The rabbits in each group were performed an X-ray before execution. 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; echo time: 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 sagittal plane. The MRI grade of NPs was 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 the nucleus pulposus with a H&E Staining Kit (EE0012, Sparkjade), and the histological grading of these samples was evaluated in accordance with the grading scale based on the morphology of AF and the cellularity of NP. Safranin O staining was performed to detect the 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. Twelve New Zealand white rabbits (female), ranging from 2.5 to 3.0 kg in body weight (Jinfeng Experimental Animal Co. Ltd., Jinan, China), were used in this study. Rabbits were housed in separate cages under standard conditions with a light–dark cycle (12 h-12 h) and dry-bulb room temperature at 22–24 °C and provided ad libitum access to tap water and food pellets daily. Rabbits were anaesthetized by an intravenous injection of 10% chloral hydrate (3 mL/kg). Rabbits were then placed into a left lateral position, and the vertical line for outward 1/3 of the connecting line between the anterior superior spine and navel or the outer margin of the transverse process was chosen as the incision. The external oblique muscle was outwardly separated from the beginning of its fascia to find the outer margin of the longissimus muscle, the deep fascia was cut open to locate the reference transverse process, the transverse abdominis was separated to expose the psoas major, and the psoas major was retracted toward the abdomen and the position was leaned 20° toward the back to expose the vertebral body. The lumbar spine’s lowest levels (L6–L7) should be avoided to eliminate possible influences of the lumbosacral junction. After the nucleus pulposus was removed, HIF-1α recombinant protein (11977-H07E, Sino Biological) was injected into the target intervertebral disc in the HIF group, and Oltipraz (HY-12519, MCE, USA) was injected into the target intervertebral disc in the HIF inhibitor group. PBS was injected into the target intervertebral disc in the PBS group. The sham group only underwent surgery, but no substance was injected into the intervertebral disc. MRI examinations were performed at 3, 6, 11 and 14 weeks postoperatively, and X-ray examinations were performed before execution. After euthanasia, disc specimens were obtained, and histological analysis was performed.

Statistical Analysis

Analysis of data was 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 ± SEM”. Statistical significance was indicated when two-sided P < 0.05.

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