The collection of human NP samples was approved by the Ethics Committee of Shanghai East Hospital, Tongji University School of Medicine. All experiments were conducted in accordance with the Helsinki Declaration. All patients and the next of kin were fully legally competent and consented to the use of lumbar NP tissues for research. Written informed consent from the patients or the next of kin was obtained. None of the patients belonged to a vulnerable population, and all patients or next of kin freely provided written informed consent. The privacy rights of the patients or next of kin were always protected. The patients or the next of kin provided written informed consent to publish case details in this manuscript.
Extraction, culture and identification of NPCs
NP samples in percutaneous endoscopic lumbar discectomy (PELD) surgery were collected, 0.25% trypsin was added, and the samples were placed in a 37° incubator for digestion for half an hour. After centrifugation at 1500 RPM/min, the supernatant was removed, 0.1% type II collagenase was added (diluted with 10% FBS), and the cells were collected in an incubator at 37 °C overnight. Cultured cells were validated by immunofluorescence staining with aggrecan and collagen-II antibodies (Cell Signaling), which are NP cell-specific markers.
Immunofluorescence staining in vitro
Twenty thousand living cells were loaded onto each coverslip and incubated overnight. PFA (4%) was fixed at room temperature for 15 min. Then, 1 mL of blocking solution (containing 10% horse serum, 1% BSA and 0.1% Triton X-100 DPBS) was added and blocked for 1 hour. The blocking solution was discarded, and diluted primary antibodies (including Aggrecan, Collagen-II, GSK-3β, Caspase-3/7, and 8-OHDG) were added for incubation. After rinsing, the coverslips were placed in the dark and incubated in florescent-labeled goat-anti-rabbit IgG for 1 hour. Then, Prolong Gold antifade reagent with DAPI was added to the coverslips. All coverslips were randomly photographed, and at least 200 cells were counted (Leica DMI 6000B).
NP cells were stimulated with different concentrations of TBHP (Sigma–Aldrich) for 6 h, 12 h and 24 h. The cells were collected and treated with the pre-prepared 1× Annexin V Binding Solution at a final concentration of 5×105 cells/mL. Annexin V-APC (5 ml) and PI (5 ml) were added, and NPCs were cultured at room temperature in the dark for 15 min. Apoptosis was detected by flow cytometry within 1 h. Similarly, NPCs were treated with 175 µM TBHP for 12 h and then precipitated and resuspended in 37° preheated TMRM staining fluid for 30 min. The 488 nm laser excitation and 570±10 nm emission filter were used to detect flow data and further analyzed by FlowJo software (7.6.1).
TBHP cytotoxicity assay (CCK8 method）
NPCs in logarithmic growth phase were made into a cell suspension. NPCs were loaded in a 96-well plate and cultured at 37 °C in a 5% CO2 incubator. NPCs were treated with 100 µl of TBHP at different concentrations for 12 h. Ten microliters of CCK8 solution was added to a 96-well plate, and the absorbance at 450 nm was measured.
TMRM is a commonly used fluorescent dye that depends on mitochondrial membrane potential, and MTG is a fluorescent dye that is independent of mitochondrial membrane potential. Both of them can stain the mitochondria of living cells. When NPCs grew to 80% confluence, they were treated with 175 µM TBHP for 12 h, and then TMRM and MTG staining working solutions were added for incubation. After rinsing, DAPI (5 µg/ml) was used to stain nuclei. Photographs were taken with a confocal microscope.
The attached NP cells were trypsinized and lysed using RIPA (Bio-Rad) to extract the total proteins. Samples were denatured and loaded into sodium dodecyl sulfate-polyacrylamide gels to run for 1 hour at 100 V. The proteins were transferred to a polyvinylidene difluoride membrane (Bio–Rad). After blocking with 5% milk solution, the membranes were incubated in primary antibody solution overnight at 4 °C. The membranes were incubated with the appropriate diluted HRP-conjugated secondary antibody (1/2000 in 1× TBST) for 1 hour at room temperature. ECL chemical luminescent agents were prepared and exposed and developed with a chemiluminescence imager.
GSK-3β knockdown in NPCs
The lentivirus stock was diluted with fresh medium containing 5 μg/mL polycoagulant. The dilution containing negative control (NC) was added to the control group, and the dilution containing lentivirus was added to the treatment group. The culture medium containing lentivirus was replaced with normal culture medium 16 h after infection. Western blot and qRT–PCR were used to detect the effectiveness of lentivirus infection.
Extraction of RNA and quantitative reverse transcription-polymerase chain reaction (qRT–PCR)
Total RNA of NPCs was extracted and purified using a QIAGEN mini kit. After that, one microgram of RNA was reverse transcribed, and complementary DNA was subjected to PCR. GAPDH was used as the normalizing gene. The primers used were as follows:
GSK-3β Forward CCCAGAACCACCTCCTTTGC
GSK-3β Reverse TGCCACCACTGTTGTCACCTT
GAPDH Forward GTCTCCTCTGACTTCAACAGCG
GAPDH Reverse ACCACCCTGTTGCTGTAGCCAA
The mRNA expression level of GSK-3β was measured and quantified using an ABI Prism 7000 sequence detection system.
The cells were collected with a scraper, and the supernatant was assembled after centrifugation. Two hundred microliters was taken for the input sample, 400 µl for the IgG sample, and 400 µl for the co-IP sample. Input samples were added to 5× SDS–PAGE protein loading buffer and placed at -20 °C for later use. The IgG and co-IP samples were separately added with a primary antibody for immunoprecipitation (dilution ratio 1:50). Then, 50 µl of pretreated magnetic beads was added, mixed and incubated at 4° for 3 h. Sixty microliters of 5×SDS–PAGE protein loading buffer was added. The IP samples were boiled in boiling water for 10 mins. After cooling, the protein samples were loaded for verification using western blotting.
All data are expressed as the mean±SD. Statistical analyses were performed by using SPSS (version 23.0, SPSS Inc., Chicago, IL, USA). Statistical analysis was performed using one-way analysis of variance and independent samples t-test. Post hoc pairwise comparisons were performed using Dunnett's T3 or Student-Newman–Keuls (S-N-K) test. A value of P<0.05 was considered statistically significant.