Hif 1α

The cells of the nucleus pulposus in every group were rinsed twice using PBS, then treated with 1% phenylmethanesulfonyl fluoride (PMSF) in RIPA lysis buffer (R0010, Solarbio) for a duration of 10 minutes. After that, they were subjected to centrifugation at 15,000×g for 10 minutes at a temperature of 4°C. The BCA protein assay kit (P0010, Beyotime) was used to measure protein concentrations. After that, the proteins were separated using 12.5% SDS-PAGE and then transferred to PVDF membranes (TM-PVDF-R-45, LABSELECT). Following a 90-minute incubation at room temperature with Western Blocking Solution (P0023B, Beyotime), the membranes were then subjected to overnight incubation at 4 °C with the primary antibodies HIF-1α (BF0593, Affinity, USA), CITED2 (DF2455, Affinity, USA), HIF-2α (DF2928, Affinity, USA), VEGFA (AF5131, Affinity, USA), and GAPDH (AF7021, Affinity, USA). Next, the membranes were exposed to secondary antibodies (LF102, EpiZyme, MA) for a duration of 1 hour at ambient temperature. Proteins of interest were identified following the established procedure. Blots were visualized using BeyoECL Plus (P0018FM, Beyotime, China). The quantification of the density of each band was performed using Image J version 1.8.0.

Polyvinyl alcohol, (NH₄)₂HPO₄ (99%), Ca(NO₃)₂•4H₂O (99%), Li3PO4 (99%), CoCl₂ (99%), NH3•H2O (25–28%), and Tris–HCl were purchased from Sinopharm Chemical Reagent Co., Ltd. Simulated body fluid (SBF) was purchased from Leagene (China). Minimum Eagle's medium (MEM), Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12), and fetal bovine serum (FBS) were purchased from HyClone (USA). Endothelial cell medium was obtained from ScienCell (USA). The NBT/BCIP staining kit and detection kit for ALP activity and calcium content were purchased from Beyotime Company (China). The CCK-8 assay was obtained from Dojindo (Japan). MTT, phalloidin, and 4',6-diamidino-2-phenylindole (DAPI) were purchased from AAT Bioquest (USA). Primary antibodies against ALP, BMP-2, RUNX2, OCN, HIF-1α, VEGF, GAPDH, and HRP-conjugated secondary antibodies were obtained from Affinity (China).

The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Co., Ltd. P0027) was used to extract nuclear and plasma proteins from the tissues and cells. All lysates contained protease inhibitors. The quantified proteins were separated by 10% SDS-PAGE. After transferring the protein to the PVDF membrane and blocking with 5% BSA, the PVDF membrane was blocked at room temperature with the following primary antibodies: HIF-1α (Affinity, Changzhou, China), TGFΒ1 (Affinity, Changzhou, China), SAMD3 (Affinity, Changzhou, China), p-SMAD3 (Affinity, Changzhou, China) E-cadherin (Affinity, Changzhou, China), Vimentin (Affinity, Changzhou, China), Lamin B (Affinity, Changzhou, China) and GAPDH (Affinity, Changzhou, China). GAPDH and Lamin B were used as loading controls. After 4 h, the excess primary antibody was removed, and the PVDF membrane was incubated with HRP-labelled secondary antibody at room temperature for 2 h. Protein intensity was detected with an Image Lab instrument (Bio-Rad, USA). Each experiment was performed in triplicate.

Whole-cell lysates were prepared using RIPA containing protease and phosphatase inhibitors (Beyotime, Shanghai, China). The protein was quantified with BCA kit (Solarbio, Beijing, China), separated on a 10% SDS-polyacrylamide gel electrophoresis and transferred onto 0.45 μm PVDF membranes (Invitrogen, Carlsbad, USA). Membranes were blocked using 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight After washed three times by TBST for 10 min intervals, membranes were incubated with diluted secondary antibodies for 1 h at room temperature. The expression of proteins was assessed using an ECL kit (Tanon, Shanghai, China), and quantified by the image J software. The expression of β-actin did not change significantly either under hypoxia or after drug treatments, which was used as an internal parameter in our experiment accordingly.
The primary antibodies used were antibodies against α-ENaC (1:2000, Invitrogen, Carlsbad, USA), γ-ENaC (1:2000, Abcam, Cambridge, USA), β-actin (1:2000, Proteintech, Chicago, USA), HIF-1α (1:1000, Affinity Biosciences, Cincinnati, USA), ERK1/2, p-ERK1/2 (1:1000, Cell Signaling, Mass, USA), inhibitor κBα (IκBα), p-IκBα, p65, and p-p65 (1:1000, Abmart, Shanghai, China). The secondary antibodies used were goat anti-rabbit or goat anti-mouse antibodies (1:5000, ZSGB-bio, Beijing, China), respectively.

Healthy adult male Sprague–Dawley rats, aged 6–9 weeks and weighing 200–250 g, were purchased from the Experimental Animal Center of Wenzhou Medical University (WMU) and kept in separate cages under controlled temperature (24°C ± 0.5°C) and humidity (45–50%) conditions, with good ventilation and ad libitum access to water and standard rodent chow. Animal experiments were performed in accordance with the Guidelines for Ethical Review of Laboratory Animals and 3R principles, and were approved by the Ethics Committee for Experimental Animals of Wenzhou Medical University (ID number: WYDW 2017-0509). All experimental procedures were performed using standard methods. Superoxide dismutase (SOD) and malondialdehyde (MDA) detection kits were obtained from Jiangsu Jiancheng Technology Co., Ltd. (Nanjing, Jiangsu, China). Antibodies to IL-1β, IL-18, CD34, VEGF, and HIF-1α were purchased from Affinity Biosciences (Cincinnati, OH, United States). Rivastigmine (≥98% purity) was purchased from Novartis (Basel, Switzerland).