Patient tissue samples

Human IVD specimens were collected from 10 patients (5 males and 5 females; age range:15–25 years) with idiopathic scoliosis and 12 patients (7 males and 5 females; age range:21–65 years) with lumbar disc herniation. Informed consent was obtained from each participant and the study protocol was approved by the Medical Ethical Committee of Qilu Hospital of Shandong University (KYll-2021 (ZM) −058).

Cell culture

The rat nucleus pulposus cells (NPCs), rat annulus fibrosus cells(AFCs) and rat adipose-derived stem cells (ADSCs) were extracted from 4-week old rats and cultured in DMEM/F-12 1:1 (Gibico, USA) supplemented with 10% fetal bovine serum (FBS; Gibico) and 1% penicillin/streptomycin (Gibico). MC3T3-E1 cells were purchased from the Chinese Academy of Sciences and maintained in α-MEM (Gibico) supplemented with 10% FBS.

Mechanical stress culture model

A mechanical compression device was used as previously described. Briefly, the cells were seeded on 24 mm glass slides and placed on a compressed support in a closed chamber filled with complete medium. The medium in the closed chamber was placed in a cell incubator to maintain cell viability. Static stress (1 MPa) can induce apoptosis and change structural characteristics, matrix composition, and gene expression.67 Herein, the cells were exposed to mechanical stress of 1 MPa at a frequency of 1 Hz (induced by pneumatic elements; FESTO, Germany). The influence of Ca2+ on cells was eliminated using Ca2+-free DMEM/F-12 (Basalmedia, China) medium. The cells were pretreated with 1 μmol/L GsMTx4 (M10039, Abmole, USA) or 10 μmol/L Fer-1 (HY-100579, MCE, USA) for 24 h, then cultured in Ca2+-free DMEM/F-12 medium and 1 MPa mechanical stress for 24 h. The cells were grouped into four groups as follows: (i) untreated, (ii) 1 MPa (treated with 1 MPa), and (iii) 1 MPa+GsMTx4 (treated with 1 MPa plus GsMTx4, 1 μmol/L, Piezo1 channel inhibitor) (iv) 1 MPa+Fer-1 (treated with 1 MPa plus 10 μmol/L Fer-1, the inhibitor of ferroptosis). The above treatments were conducted in a Ca2+-free medium for 24 h. To examine the time profile after application of 1 MPa mechanical stress, specific indicators were selected at six stages (0, 1, 3, 6, 12, and 24 h).

Chemical stimulation model

To determine the effect of Piezo1 on iron metabolism and ferroptosis of cells, we first established an extracellular high iron environment with 100 μmol/L FAC (F5879, Sigma, USA). Yoda1 reagent (HY-18723, MCE, USA) and GsMTx4 reagent were introduced to the environment with high iron ion concentration. The cells were then grouped as follows: (i) untreated, (ii) FAC (treated with 100 μmol/L FAC), (iii) FAC+Yoda1 (treated with FAC plus 10 μmol/L+Yoda1, Piezo1 channel agonist), (iv) FAC+GsMTx4 (treated with FAC plus 1 μmol/L GsMTx4, Piezo1 channel inhibitor), (v) FAC+Yoda1 (treated with FAC plus 10 μmol/L Yoda1 in Ca2+-free medium). These treatments were conducted in a complete medium for 24 h, except for the fifth group. Since GsMTx4 is a non-specific agonist of Piezo1, we treated cells with Yoda1 and FAC in the presence of GsMTx4. Furthermore, rat Piezo1-siRNA was also used to validate subsequent results. The samples were collected from rats or cells subjected to different treatments and examined.

Measurement of iron content

An Iron Assay Kit (ab83366, Abcam, USA) was used to measure intracellular iron content. Insoluble materials were eliminated through centrifugation at 16 000 x g for 10 min, then the cells were homogenized on ice with iron assay solution. The supernatant was collected and incubated with the assay buffer at 37 °C for 30 min. An iron probe was then added to each sample and incubated in the dark at 37 °C for 60 min. The absorbance was recorded at OD 593 nm using a microplate reader (Bio-Rad, USA).

FerroOrange Fe2+ staining

Intracellular Fe2+ was detected by using an FerroOrange Kit (F374, Dojindo, Japan). According to the instructions, 10 μmol/L FerroOrange was introduced to the cell cultures and incubated for 60 min at 37 °C with 5% CO2. Images were taken under a fluorescence microscope (Olympus, Japan).

Perl’s Prussian blue stain

Prussian Blue Iron Stain Kit (G1428, Solarbio, China) was used to evaluate the iron concentration in tissue slices. Human NP sections were deparaffinized and rehydrated. Next, slides containing tissue sections were stained with iron staining solutions following the manufacturer’s instructions. The final staining directly correlates with nonchelated iron in the human NP tissue.

Transmission electron microscopy

Isolated rat NPCs were fixed for 1 h using Electron Microscope Fixative Solution (G1102, Servicebio, China), and then they were re-fixed for 2 h at room temperature with 1% osmic acid in 0.1 mol/L phosphate buffered saline (pH 7.4). The samples were then embedded in epoxy resin and dehydrated using a series of ethanol concentrations. Finally, a transmission electron microscope (HT7700, Tokyo, Japan) was used to view the sections.

JC-1 assay

The mitochondrial membrane potential was measured using the JC-1 test kit (C2003S, Beyotime, China) following the manufacturer’s instructions. NPCs were stained with a JC-1 staining solution at 37 °C for 20 min after exposure to different stimulations for 24 h. A fluorescence microscope (Olympus, Japan) was used to capture fluorescent images. The ratio of red to green fluorescence was calculated as an indicator of alterations in the potential of the mitochondrial membrane.

Following the manufacturer’s instructions, the JC-1 test kit (C2003S, Beyotime, China) was used to determine the mitochondrial membrane potential. After being exposed to various stimuli for 24 h, NPCs were stained with a JC-1 staining solution at 37 °C for 20 min. Fluorescent pictures were taken using an Olympus fluorescence microscope (Japan). The ratio of red to green fluorescence was estimated as an indicator of alterations in the mitochondrial membrane’s potential.

MitoTracker assay

MitoTracker Red CMXRos (C1035, Beyotime, China) was applied in line with the instructions as directed to detecte biologically active mitochondria. As shown, rat NPCs were activated. Cells were treated with MitoTracker Red CMXROS working fluid for 30 min at 37 °C in complete darkness after 24 h. A fluorescence microscope was used to capture fluorescence from the cells.

Detection of lipid peroxidation level

MDA was measured using a Lipid Peroxidation MDA Assay Kit (S0131M, Beyotime, China). Cells were treated in accordance with the kit’s instructions. The reaction solution was added onto the 96-well plate and its absorbance was measured using a microplate reader (Bi-Rad, USA) at 532 nm. C11-BODIPY 581/591 (GC40165, Glpbio, USA) is used to detect lipid peroxidation in living cells. A fluorescent microscope (Olympus, Japan) was used to capture the images after addition of C11-BODIPY 581/591 (10 mol/L) directly into various groups and incubated for 30 min at 37 °C. For flow cytometry analysis, cells were digested and resuspended in 400 µL of serum-free medium with C11-BODIPY C11 (10 μmol/L). After that, NPCs were incubated for 30 min and the nuclei were stained using Hoechst 33342 (C1022, Beyotime, China) for 5 min. The samples were subsequently detected using a flow cytometer (CytoFLEX LX, Beckman Colter) and data were collected from the FL1 channel.

Cell transfection

The rat Tfrc-siRNA, rat Piezo1-siRNA and their negative control (NC) were sourced from GenePharma (Shanghai, China). The Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, USA) was used for cell transfection following the manufacturer’s instructions. The cells were harvested after 48 h of transfection for further experiments. The sequences are listed in Table S1.

RT-PCR assay

Total RNA was extracted using the TRIzol Reagent (Invitrogen, USA) and reversed using the Evo M-MLV RT Mix Kit (AG11728, Accurate Biology, China). To accomplish RT-qPCR, a SYBR Green PCR master mix (AG11701, Accurate Biology, China) was used. The relative mRNA expression levels of the target genes were determined using the 2−ΔΔCT method with GAPDH serving as the endogenous reference. Table S2 shows a complete list of all the primers utilized.

Western blotting

Proteins were extracted by RIPA (P0013B, Beyotime, China) with PMSF (AR1192, Boster, China). BCA Protein Assay Kit (P0011, Beyotime, China) were used to quantify proteins. The standard western blotting was performed with primary antibodies against: NRF2 (1:1 000, 16396-1-AP, Proteintech, USA); ACSL4 (1:1 000, 22401-1-AP, Proteintech); FSP1 (1:1 000, 20886-1-AP, Proteintech); GPX4 (1:1 000, ab125066, Abcam, USA); TFRC (1:3 000, ab269513 Abcam); FPN (1:1 000, 26601-1-AP, Proteintech), DMT1 (1:1 000, YN3198, Immunoway, USA); FTH1 (1:1 000, ab183781, Abcam); MFN1 (1:3 000, ab221661, Abcam); MFN2 (1:3 000, ab205236, Abcam); DRP1 (1:3 000, ab184247, Abcam); OPA1 (1:3 000, ab157457, Abcam); Hepcidin (1:1 000, DF6492, Immunoway, USA); GAPDH (1:3 000, 10494-1-AP, Proteintech). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Abcam (1:5 000, Abcam).

Animals

All animal experiments in this study were performed in accordance with the International Guiding Principles for Animal Research and were approved by the Laboratory Animal Center of Shandong University. Col2a1-CreERT mice were established by and purchased from Cyagen (USA). Gpx4flox/+ mice were created by Cyagen (USA) through ES genome engineering. GPX4flox/+ mice were mated with Gpx4flox/+ mice to generate Gpx4flox/flox mice. Gpx4flox/flox mice were mated with Col2a1-CreERT mice to generate Col2a1-CreERT, Gpx4flox/+ mice. Col2a1-CreERT Gpx4flox/+ mice were mated with Col2a1-CreERT Gpx4flox/+ mice to generate Col2a1-CreERT Gpx4flox/flox mice. Male mice with the Col2a1-CreERT and Gpx4flox/flox genes were used in experiments. Ten-week-old Col2a1-CreERT Gpx4flox/flox mice were intraperitoneally injected with tamoxifen (1 mg/d for 5 d) (HY-13757A, MCE, USA) to obtain Gpx4 conditional knockout (Gpx4-CKO) mice. Piezo1flox/+ mice were created by Cyagen (USA) through ES genome engineering. Piezo1 conditional knockout (Piezo1-cKO) mice were produced by the same way. Col2a1-CreERT Gpx44flox/+ mice were mated with Col2a1-CreERT Piezo1flox/+ mice to generate Col2a1-CreERT Piezo1flox/flox/Gpx4flox/flox mice. Male mice with the Col2a1-CreERT and Piezo1flox/flox/Gpx4flox/flox genes were used in experiments. Ten-week-old Col2a1-CreERT Piezo1flox/flox/Gpx4flox/flox mice were intraperitoneally injected with tamoxifen (1 mg/d*5d) (HY-13757A, MCE, USA) to obtain Piezo1/GPX4 conditional knockout (Piezo1/Gpx4-cKO) mice. Col2a1-CreERT GPX4+/+Piezo1+/+ littermates were assigned to the wild-type (WT) group. Three-month-old Sprague-Dawley (SD) rats were purchased from the Animal Center of Shandong University. All of the animals were housed under controlled identical specific pathogen-free (SPF) standard environmental conditions (23 ± 2 °C, 12 h light/dark cycle) with free access to food and allowed to move freely. WT and Gpx4-cKO mice were fed with water supplemented with Se-Met (2 mg/L, HY-114245, MCE, USA) and maintained on the diet for 8 weeks around establishment of IVD needle puncture model.

Genotyping

Tail clippings from 4-week-old mice were taken. According to the manufacturer’s instructions, mouse tail DNA was extracted using a One Step Mouse Genotyping Kit (PD101-01, Vazyme, China). To make agarose gels, agar (1.5 g), 100 mL of 2 × Tris-acetate-EDTA buffer (TAE), and 6 μL of Gel Red were mixed and heated. Agarose gel electrophoresis was used to separate the amplified DNA. Amersham Imager 680 (GE, USA) was used to capture the images. Table S3 lists the primers used for amplification (Gpx4flox, Piezo1flox and Col2a1-CreERT).

IVDD model establishment

To establish an IVDD model in vivo, caudal needle puncture injuries were performed in 12 weeks old WT mice (n = 10), Piezo1-cKO mice (n = 10), Gpx4-cKO mice (n = 10) and Piezo1/Gpx4-cKO animals (n = 5). The surgeries were carried out under general anesthesia (2% isoflurane in oxygen) and sterile conditions. After confirming the location of the IVD with a microscope, needle punctures were made to a depth of 50% of the dorsal-ventral width with 26 G syringe needles. Mice were thoroughly observed to verify that there were no surgical complications and were permitted free movement in their cages with access to water and food.

Magnetic resonance imaging (MRI)

The mice underwent MRI scanning 6 weeks following the initial puncture to assess structural differences and signal intensity changes in sagittal T2-weighted images of IVDs. 3.0 T MRI scanners (GE Signa HDX, USA) were used for the disc imaging evaluation. Mice were restrained in a supine position with their tails straightened. Spin echo repetition time was 2 275 ms; echo time was 80 ms; number of excitations was 8; field of view was 5 cm; slice thickness was 1.5 mm; and there was no phase wrap. T2 intensities and MRI indices (the area of NP multiplied by the average signal intensity) were calculated using the procedures reported in a prior study.

Micro-CT

The scanning protocol included an isometric resolution of 15 μm, as well as X-ray energy settings of 70 kV and 200 A. A Quantum GX2 scanner (PerkinElmer, USA) was used to measure the microstructure of the vertebrae. Before histological processing, samples were fixed in paraformaldehyde and micro-CT was performed. Each group’s scanned pictures were analyzed at the same threshold to enable for 3-dimensional structural reconstruction of each sample. The degenerative score was calculated using the procedures outlined in a previous study.68

HE and Safranine O staining

Safranine O staining was performed to detect the changes in proteoglycans with HE staining kit (C0105, Beyotime, China) and Safranine O staining kit (G1371, SolarBio, China) according to the manufacturer’s recommended procedure.

Immunohistochemistry

The IVD tissues were decalcified, embedded in paraffin, and cut into 5 m slices after being fixed in 4% paraformaldehyde. The paraffin slices were antigen-repaired with citric acid (pH 6.0), blocked with goat serum, and dewaxed with xylene and gradient ethanol. The sections were then treated at 4 °C for an overnight period with primary antibodies to ACSL4 (1:200, Proteintech, USA), Collagen II (1:200, Novus, USA), Aggrecan (1:500, Servicebio, China), and Piezo1 (1:200, Affinity, USA). The sections were exposed to goat anti-rabbit IgG-HRP secondary antibody for 1 h at room temperature the next day. The DAB Substrate kit (ZLI-9018, ZSGB) was used for detection, and the samples were then counterstained with 1% hematoxylin. Using a microscope (Leica DMI3000B, Germany), pictures were recorded. ImageJ quantified the areas that were positive.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.4.0 (GraphPad Software, USA). Two-tailed unpaired t-tests were used to analyze the two groups. Multiple comparisons were analyzed using the one-way analysis of variance. All results are expressed as the mean ± SEM. P < 0.05 was considered statistically significant. n ≥ 3 for all samples. Each experiment was repeated independently three times.