At room temperature, paraffin tissue sections of lumbar intervertebral disc tissue were soaked in xylene for 5 min and replaced with new xylene for another 5 min to remove the paraffin completely. Next, following being immersed into 100% ethanol for 5 min and then into new 100% ethanol for another 5 min, the sections were soaked in gradient ethanol (90, 80, 70%) sequentially. Subsequently, 20 µg/ml Protease K was added into the sections for incubation for 20 min. The apoptosis of the lumbar intervertebral disc tissue was evaluated using a TUNEL kit (cat.no C1086, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. 50 µL TUNEL solution was added to sections for incubation for 60 min at 37℃. The apoptosis was observed under a fluorescence microscope.

Immunohistochemistry (IHC)

The paraffin-embedded lumbar intervertebral disc tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval by high-pressure boiling in citrate buffer (pH 6.0) for 2 min. To block non-specific binding, sections were blocked with goat serum (Sigma-Aldrich, St. Louis, Missouri, USA), then incubated at 4 °C overnight with anti-PDK4 antibody (1:250, Proteintech, Rosemont, Illinois, USA). After staining with diaminobenzidine for 2 min and counterstaining with hematoxylin, the sections were observed under a microscope.

ELISA assay

The collected blood from rats was centrifuged at 3500 r/min at 4℃ for 20 min to obtain the supernatant. The supernatant of the cultured NPC cells was collected and centrifuged at 3000 r/min to obtain the supernatant. Following the manufacturer’s guidance of ELISA kit (IL-18, cat.no P1555; IL-1β, cat.no PI303; TNF-α, cat.no PT516, Beyotime Biotechnology, Shanghai, China), the levels of IL-18, IL-1β, and TNF-α were evaluated by the detection of the absorbance at 450 nm with a microplate reader (Thermo Fisher Scientific, Pittsburgh, PA, USA).

RNA-sequencing

The rat intervertebral disc tissues from the control and IVDD were collected, separately. RNA extraction was performed using MasterPure™ Complete DNA and RNA Purification Kit MasterPure™ (MC85200, Epicentre, Madison, WI, Madison), followed by the determination of RNA concentration and purity by Nanodrop2000. RNA integrity number (RIN) was assessed by agarose gel electrophoresis and RIN was determined by Agilent 2100. Raw RNA sequencing required quality control (QC) on the raw reads to evaluate the suitability of the sequencing for subsequent analysis. RNA sequencing was performed on Illumina HiSeq using Next-Generation Sequencing (NGS).

NPC culture and transfection

Rat nucleus pulposus cells (NPC, Procell Life Science&Technology Co., Ltd, Wuhan, China) were cultured in DMEM/F12 medium at 37℃ with 5% CO2, containing 10% fetal bovine serum, 1% streptomycin, and 1% penicillin (Hyclone, USA). NPCs were transfected with siPDK4 or its negative control siNC using lipo2000 (Invitrogen) according to the manufacturer’s guidance. After 4–6 h, NPCs were treated with 10 µg/L IL-1β for 24 h for further experiments.

CCK-8 assay

The NPC proliferation of each group was detected by CCK-8 kit at 0 h, 24 h, 48 h, and 72 h (Abcam, England). The cells (200 µg/ml) were seeded into 96-well plates at 37℃ with 5% CO2 for 24 h, 48 h, or 72 h. Then, 10 µL CCK-8 solution was added for incubation with cells for 2 h and the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).

Determination of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)

The MDA and GSH levels, and SOD activity for NPC were measured using Malondialdehyde (MDA) colorimetric assay kit (cat.no.E-BC-K028-M, Elabscience, Wuhan, China), reduced glutathione (GSH) colorimetric assay kit (cat.no.E-BC-K030-M) and total superoxide Dismutase (T-SOD) activity assay kit (WST-1 Method, cat.no.E-BC-K020-M) according to the manufacturer’s guidance.

Iron ion concentration determination

The iron determination in the NPC was determined using an Iron determination kit(colorimetry) (cat.no #K390-100BioVision.Inc) and the absorbance was measured at 593 nm using a microplate reader (Thermo Fisher Scientific).

RNA extraction and quantitative real-time RT-PCR

Total RNAs from NPCs were extracted using TRIZOL reagent, 5 µL of which was diluted 20 times with RNase-free Water. After the concentrations and purity of RNA were evaluated using an ultraviolet spectrophotometer, the RNA was reversed into complementary DNA (cDNA), which then was used to perform Real-Time fluorescence quantitative PCR in ABI7500 quantitative PCR instrument (Applied Biosystems, USA). The PCR conditions contained pre-denaturation for 5 min at 95℃ and 40 cycles of denaturation for 10 s at 95℃, annealing for 30 s at 60℃. The relative RNA levels were evaluated by 2−ΔΔCt method The sequence of primers (TaKaRa, Japan) used in this study is shown in Table 1.

Table 1 The primer sequencesWestern blotting assay

After transfection for 48 h, NPCs were collected and lysed with RIPA Lysis solution (Beyotime technology, Shanghai, China). Then, the proteins were separated by the technology of SDS-PAGE and transferred into the PVDF membrane. Next, the membrane was blocked with 5%-TBST nonfat milk for 2 h at room temperature and incubated with the primary antibodies (anti-FTH1, cat.no ab183781, the dilution of 1:1000; anti-GPX4, cat.no ab125066, the dilution of 1:5000. anti-β-actin, cat.no ab6276, the dilution of 1:5000, abcam, England) (anti-ACLS4, cat.no PAB25681, the dilution of 1:1000, Amylet Scientific) overnight at 4℃. Then, the membrane was washed with TBST and incubated with secondary antibodies (horseradish peroxidase-conjugated Goat Anti-Rabbit IgG, the dilution of 1;5000, CWBIO, Beijing, China) for 2 h at room temperature. After the visualization of protein bands using enhanced chemiluminescence (cat.no 34095, Thermo Fisher Scientific), the protein levels were analyzed using the ChemiDoc Touch Imaging System (Bio-Rad). β-actin was used as an internal reference.

Statistical analysis

The experimental data were shown as mean ± standard deviation (SD) and analyzed by GraphPad Prism 8.0 statistical software. Comparison between two groups was performed by t-test and comparison among multiple groups was carried out by One-Way ANOVA analysis of variance, followed by Tukey’s hoc test. P < 0.05 was considered as statistically significant.