Experimental materials
Establishment of IDD animal models
All animal experiments were performed in accordance with the Guiding Principles of the Care and Use of Animals and approved by the Animal Experimental Ethics Committee of Nanjing University of Chinese Medicine. Male SD rats (250 ± 20 g, n = 60) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., The rats were randomly divided into the sham operation (Sham), model (M), HIF-1α inhibitor (YC-1), and solvent groups (DMSO), with 15 participants each in the Sham and M groups and 10 participants each in the YC-1 and DMSO groups. After 1 week of adaptive feeding, the modeling intervention began, with fibro annular puncture used to prepare the disc degeneration model(22). On the day after the modeling, two rats from each group were randomly selected for magnetic resonance imaging (MRI)examination, and the signal intensity of the annulus fibrosus and nucleus pulposus was observed under T2-weighted MRI. The success of annulus fibrosus puncture was determined by the blurred boundary of annulus fibrosus and the decreased signal intensity of nucleus pulposus. The intervention began 3 days after the modeling was finished. The Sham and M groups were intraperitoneally injected with normal saline (0.1ml/100g), the YC-1 group was intraperitoneally injected with 5%DMSO + 40%PEG300 + 5%Tween80 + 50%saline (YC-1,1mg/kg;0.1ml/100g), and the solvent DMSO group was injected with 5%DMSO + 40%PEG300 + 5%Tween80 + 50%saline; (0.1ml/100g) three times a week for a total of 8 weeks.
Extraction and intervention of NPCs
Extraction and culture of NPCs
Male SD rats (220 ± 20g, n = 10) were sacrificed for cervical dislocation, and disinfected in 75% ethanol for 10 min. The whole spine was separated from the back under aseptic conditions, and the nucleus pulposum tissue from L1-L5 was separated under a microscope and cut into fragments of approximately 1mm3. The tissue was digested with 0.2% type II collagenase (Gibco, USA) at 37°C for 8 h, screened with 200 mesh and centrifuged for cell precipitation. The cells were then cultured in DMEM/F12 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin and streptomycin dual antibody (NCM, China) under the standard incubation conditions (37°C, 5% CO2) (23). Passage was performed when the cells reached 80–90% confluence, and cells from within three generations were used for all in vitro experiments.
NPCs in vitro intervention and grouping
For the in vitro experiments, NPCs were divided into the Lv-NC-H group (NC-H group) and Lv-HIF-1α transfection group (H group). After transfection with the corresponding lentivirus, TNF-α (300-01A, Peprotech, USA) (10ng/ml) (24)was then induced under standard incubation conditions (37°C, 5%CO2) for 24h.
Lentivirus transfection
Corues Biotechnology (Nanjing, Nanjing, China) designed and constructed a lentiviral vector expressing GFP labeled HIF-1α (NM_024359) in the PLv3LLTr-ECMV-ZSGRECn-PGK-Puro-U6 plasmid according to the standard protocol. For viral transduction, NPCs were inoculated and lentivirus infection was achieved after reaching 40–60% confluence with a multiplicity of infection (MOI) of 50. After transfection for 24 h, the culture medium was changed every other day. When the transfected NPCs reached 80–90% confluence, they were used in further experiments to detect the knockdown efficiency and expression level of the HIF-1α gene by immunofluorescence and qPCR.
Experimental method
MRI
On the day after modeling, two rats in each group were randomly selected for MRI examination. At 8 weeks after the intervention, Four rats from each group were randomly selected for MRI examination. The changes and degeneration of IVD signal intensity were observed using T2-weighted MRI, and Pfirrmann grading was performed under the guidance of professional physicians.
Pathological staining
HE staining
Three rats were randomly selected from each group, After administering anesthesia, L5-L6 IVD tissues were taken from marked locations, f fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Subsequently, 4µm paraffin sections were produced from the embedded tissues, These sections were dewaxed, dehydrated with hematoxylin-eosin staining solution, and sealed, and the disc histopathological changes were observed under an optical microscope at 50× and 400× magnification.
Masson’s staining
Paraffin sections were dewaxed in water, immersed in Masson’s stain, differentiated with 1% hydrochloric acid alcohol, rinsed and differentiated with 1% acetic acid, dehydrated with anhydrous ethanol, and examined under a microscope (50×, 400×) after transparent sealing. Images were then collected and analyzed.
Safranin O-fast green staining
Paraffin sections were dewaxed into water, immersed in solid green dye for approximately 10 min, differentiated and rinsed until colorless, and then placed in safranine O dye for 30 seconds for staining. Finally, the sections underwent conventional dehydration and were made transparent before being sealed with neutral gum. Photographs were taken with an optical microscope (50×, 400×) and used for analysis.
Immunohistochemistry
IVD tissue from rats in each group were collected, and paraffin-embedded sections were prepared, and dehydrated, Endogenous catalase was blocked using 3% hydrogen peroxide, and the sections were then closed with 3% BSA at room temperature for 1 hour, Rabbit antirat primary antibodies (HIF-1α, 1:200, GB114936, Servicebio; Bnip3, 1:1000, GB111204, Servicebio; Beclin-1, 1:200, GB11228, Servicebio) were incubated overnight at 4℃. After washing with PBS, HRP was added to label goat anti-rabbit secondary antibody (Servicebio, 1:200, GB23303), and DAB was used for color development. Restaining was then performed with hematoxylin, Hydrochloric acid ethanol differentiation was performed, and the sections were then dehydrated and sealed. The expression of HIF-1α, Bnip3 and Beclin-1 positive cells in the IVD tissue sections was observed under the microscope. The hematoxylin-stained nuclei were blue, and the positive expression of DAB was brown-yellow. Three fields (400×) were randomly selected in each section under the microscope and the percentage of positive cells was counted using ImageJ.
ELISA
The rats were anesthetized with isoflurane, blood was collected through the abdominal aorta, and serum was then separated by centrifugation at 4℃ and 3500r·min− 1 for 15min. The expression levels of serum TNF-α and VEGF in rats were detected using the ELISA kit (AF-01587R1; AF-10645R1, Aifang Biotechnology Co., Hunan, China) was used to detect the expression levels of serum TNF-α and VEGF in rats. NPCs cells were cultured in 6-well plates and transfected with Lv-shHIF1α. They were then induced with TNF-α (10ng/ml) and divided into the NC-H group and H group. After 24 hours, the supernatant of the cell culture was extracted to detect the expression level of VEGF in NPCs.
2.5 Transmission electron microscopy
The L4-L5 IVDs were removed from three rats, and the nucleus pulposus was removed, cut into approximately 1mm3 pieces and placed in glutaraldehyde at a temperature of 4℃ and fixed for a duration of 2 h. Then, the pieces were washed with phosphate buffer, fixed again using a solution containing 3% glutaraldehyde and 1% osmic acid for a period of 5 h, and embedded in epoxy resin. The embedded tissue was sliced into ultra-thin sections measuring 50nm and then stained with 2% uranium acetate and 0.2% lead citrate. Finally, transmission electron microscopy at a magnification of 10000× was employed to analyze the resulting images. NPCs cells were cultured in 6-well plates for the cell samples, transfected with Lv-shHIF-1α and induced with TNF-α (10ng/ml). The cells were divided into the NC-H group and H group. After 24 h, the cells were extracted and precipitated using the same procedure as before.
2.6 Western blot
The total protein of NPCs and tissues was extracted with RIPA lysate and quantified according to the instructions of the BCA kit (NCM, Nanjing, China). SDS-PAGE sample loading buffer was added and heated for 10 min. Protein samples were subjected to SDS-P AGE electrophoresis, transferred to a PVDF membrane, and blocked with 5% milk powder at room temperature for 2 h. The PVDF membrane was then incubated with the following primary antibodies(VEGF,1:1000,Abcam; HIF1α,1:1000,Abcam; P62,1:1000,Abcam; LC3-II,1:2000,Abcam; Bnip3,1:1000,CST; Beclin-1,1:1000,CST; beta-actin,1:5000, Proteintech); Then the membrane was incubated with an enzyme-labeled goat anti-rabbit secondary antibody( goat anti-rabbit, 1:10000, Abcam, ab205718) at room temperature for 2 h. Finally, the PVDF membranes were incubated with an ECL chemiluminescence solution (Yeasen, Shanghai, China), and the protein bands were detected using a gel imaging system (Tanon, Shanghai, China). Grayscale values were measured using ImageJ software.
RT-qPCR
Total RNA was isolated from NPCs and IVD tissues using a Fast Pure Cell/Tissue Total RNA Isolation Kit (Vazyme, Nanjing, China).The isolated RNA was then reverse-transcribed into cDNA using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).RT-qPCR was performed using Ace Q Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China).A PCR automatic serialization analyzer (ABI Quant Studio 3, USA) was set to pre-denaturation at 95℃ for 10 min, pre-denaturation at 95℃ for 15 s, annealing at 60℃ for 60 s, and extension for 40 cycles. Ct values of each target gene were obtained with GAPDH as the internal reference gene, a Semi-quantitative calculation and analysis were carried out using the 2^−△△Ct method. The primers are shown in Table 1.
Immunofluorescence
After dewaxing the paraffin sections paraffin sections in water and repairing the antigen, the fixed solution was left at room temperature for 10 min, The sections were washed in washing solution was used to wash three times, followed by sealing the solution at room temperature for 1 h, and VEGF, HIF-1α and P62/LC3-II rabbit monoclonal antibodies were then added (1:500; 1:500; 1µg/ml; 1µg/ml, Abcam) and incubated overnight at 4℃. Subsequently, fluorescently labeled goat anti-rabbit IgG was added at room temperature for 1 h, anti-fluorescence quencher was added, and the sample was observed and photographed under a fluorescence microscope
2.9 Statistical methods
Experimental data are expressed as the mean ± SD. SPSS 20.0 software was used to compare the differences among the groups. GraphPad 8.0 was used to draw relevant data graphs. Independent-samples t-test was used for, two groups of samples, and significant differences between two or more groups were analyzed using one-way analysis of variance or Tukey’s test. Additionally, P < 0.05 was considered statistically significant.