Data collection and preprocessing

We downloaded the datasets GSE205535, GSE199866, and GSE165722 from the Gene Expression Omnibus database. Database CNP0002664 was downloaded from China National GeneBank DataBase (CNGBdb). These databases contain single cell sequencing results from human normal and degenerated nucleus pulposus tissues. R software (version 4.2.0) and the Seurat package (version 4.0), developed by Satija Lab, were utilized for downstream clustering analysis, identification of differentially expressed genes, differential analysis, and identification of marker genes in single-cell data.

Gene Set Variation Analysis (GSVA)

GSVA is an unsupervised analysis technique that evaluates the enrichment of gene sets on microarrays and transcriptomes, employing a non-parametric approach. The evaluation determines if various metabolic pathways are enriched in samples by transforming the gene expression matrix across samples into a gene set expression matrix across samples. pathways across different conditions or groups. a specific biological state is associated with a pathway [39].

GSVA (version 1.48.2) scoring of Hallmark gene sets, such as ANGIOGENESIS, TNFA_SIGNALING VIA NFKB, KRAS SIGNALLING, HYPOXIA was utilized in this study.

Cell culture and drug intervention

Human normal nucleus pulposus cells (iCell-0028a, iCell Bioscience Inc, China) were utilized, while the cell culture medium was obtained from the iCell primary chondrocyte culture system (PriMed-iCELL-020, iCell Bioscience Inc, China). For the subsequent experiments, the nucleus pulposus cells were cultured in an incubator system set at a temperature of 37°C and a CO2 concentration of 5% after reaching the second passage.

The use of LPS (L2880, Sigma-Aldrich) can simulate the degeneration of nucleus pulposus cells by causing inflammation and cell degeneration. PT2399 selectively inhibits HIF-2α [16].

Slides were used to seed nucleus pulposus cells, which were then cultured in an incubator. After the cell density reached 50% -70%, PBS, LPS, LPS + PT2399, LPS + PT2399 loaded PP20 or PT2399 loaded PP20 were added to different groups. Cells were collected after 48 hours of drug exposure.

Preparation and Characterization of PT2399 Loaded PHBV or PP20

To prepare PHBV-PEG20k (PP20), First, 8.0 g of mPEG-20k (Solarbio, China) was placed in a 50 mL round-bottom flask with a pumping valve, heated at 80 °C while vacuuming for 2 h, followed by nitrogen insufflation until cooled to room temperature. Once cooled, a round-bottom flask was used to transfer 40 mL of anhydrous dichloromethane, which was then dissolved by shaking for future use. The solution was subsequently transferred to a constant-pressure drip funnel and protected with nitrogen for later use. Transfer 100 μL of hexamethylene diisocyanate (HMDI) to a nitrogen-protected round-bottom flask and mix well with 20 mL of dry dichloromethane. mPEG-20k dissolved in dichloromethane was added to the HMDI solution while stirring at 800 rpm, with a flow rate of 1 mL/min. The reaction was carried out in the dark at room temperature for 6 hours to yield mPEG20k-NCO. In a 100 mL round-bottom flask equipped with a pumping valve, 13.3 grams of PHBV from Tianan Biologic Material Ltd. in China were heated at 80 °C under vacuum for 5 hours. After that, nitrogen was introduced until the temperature reached room temperature. Following the cooling process, a round-bottom flask was used to transfer 80 mL of anhydrous dichloromethane. The solution was dissolved by shaking and reserved for future use. Subsequently, the solution was transferred to a constant-pressure drip funnel and slowly added to the mPEG20k-NCO solution at a rate of 5 mL/min. This process took place at room temperature in the absence of light for a duration of 3 days. PHBV-PEG20k was obtained at the conclusion of the reaction by eliminating dichloromethane through rotary evaporation. It was then dissolved in 80 mL of tetrahydrofuran and the refined product was precipitated by gradually adding 1 L of distilled water while stirring. After filtration, the product was washed three times with distilled water and vacuum dried at 50 °C overnight.

Characterization of PP20, which is PHBV-PEG20k, is performed. The Perkin Elmer spectrophotometer was used to record the Fourier transform infrared spectra of PP20 (spectrum 2) via the solid-state KBr pellet technique, covering the 4,000–400 cm−1 range. The polymer's 1H-NMR was measured with a JEOL AL 500 FT-NMR Proton NMR spectrometer using FT-NMR. An internal reference was employed, utilizing tetramethylsilane.

To assess the water-loving properties of the scaffolds, we employed the sessile drop technique. This method entails using a contact angle analyzer (Surface Electro Optics, Phoenix 150, Korea) to measure the angle at which liquid droplets make contact with the surface of the scaffold. Each scaffold sample was gently covered with around 2 µL of distilled water, and the contact angles were documented. The immobile droplet technique is a commonly employed method for evaluating surface wetness, as it offers a numerical assessment of the relationship between the fluid and the surface. The degree of hydrophilicity or hydrophobicity of the scaffold surface can be determined by measuring the contact angle. A surface is more hydrophilic when the contact angle is smaller, and conversely, it is less hydrophilic when the contact angle is larger. Using a contact angle analyzer, like the Surface Electro Optics Phoenix 150, guarantees precise and uniform measurements. This method provides a reliable and efficient means of evaluating the hydrophilicity of scaffold surfaces, which is crucial for assessing their suitability for various biomedical applications.

The PT2399-loaded PHBV or PP20 was prepared using the double emulsion (W1/O/W2) technique. To summarize, 4.2 μg PT2399 was added gradually to 10 g of PHBV dissolved in chloroform using a homogenizer operating at 20000 rpm (SilentCrusher M, Heidolph, Germany) in order to create the initial emulsion (W1/O) whti a drug concentration of 1μmmol/L. This primary emulsion was then added to an aqueous phase (W2) containing PVA as an emulsifier and homogenized again. The resultant mixture was agitated until the liquid evaporated entirely and subsequently spun at a speed of 15000 revolutions per minute for a duration of 30 minutes using a centrifuge (MPW-350R, Poland). Afterwards, PHBV or PP20 loaded with PT2399 were rinsed three times using distilled water and then subjected to freeze-drying at a temperature of -50 ̊℃ for a duration of 24 hours in preparation for subsequent experiments.

In vitro drug release behavior

The initial step involved creating a standard curve for PT2399 by utilizing PBS (pH = 7.4) as the solvent at the wavelength corresponding to the highest absorption peak (278 nm). PT2399-loaded PHBV, PT2399-loaded PP20 (100 mg) were each dissolved in PBS and placed in a dialysis bag. Next, the dialysis pouch was positioned inside a beaker filled with the identical pH PBS buffer. It was then subjected to horizontal vibrations in a temperature-controlled shaker (37 °C ± 0.5 °C) at a frequency of 50 vibrations per minute. At specific time intervals, the 5 ml of released liquid was substituted with equal quantities of new PBS. The solution's absorbance was measured at a wavelength of 278 nm, and the rate of drug release was calculated as follows.

$$}(\mathrm)=\frac_V+__^_}}_}\times 100$$

The symbol R represents the total percentage of drug release, while n represents the number of samples taken. Indicates the amount of the medication in the nth release, measured in grams per liter. The release liquid's total volume is represented by V. The ith release is represented by the mass concentration (g/l) of the drug, while the volume of the release liquid at the ith sampling is denoted as Vi. MD refers to the mass of drug loaded (g).

Detection of alterations in gene expression in nucleus pulposus cells following intervention with LPS and PT2399 loaded PP20 (RT-qPCR)

For a duration of 48 hours, human typical nucleus pulposus cells were exposed to LPS at a concentration of 10 µg/ml [40], PT2399 at a concentration of 1 ummol/L, and PT2399 loaded PP20 at a concentration of 1 ummol/L [41]. The expression levels of HIF-1α, CITED2, HIF-2α, CA9, PPP1R15A, VEGFA, and EGLN3 genes in nucleus pulposus cells from various experimental groups were detected using RT-qPCR. The verification of target differential genes in human normal and degenerated nucleus pulposus cell populations was done by analyzing the changes in target gene expression levels after each experimental group treatment. Additionally, the effects of drugs on HIF-2α and other indicators were also confirmed. The levels of mRNA were measured using the 2-ΔΔCt method, with GAPDH serving as the endogenous controls. The primer sequences were as follows: HIF-1α-F 5'-GCACAGTTACAGTATTCCAGCAGAC-3'; HIF-1α-R 5'-TTCATCAGTGGTGGCAGTGGTAG-3'. CITED2-F:5'-TCCTTGGTGATAGAAATGGGTTTGG-3'; CITED2-R:5'-CTCTGCTGGGCTGCTGTTTG-3’. HIF-2α-F:5'-TGATGTGGAAACGGATGAAGAACC-3'; HIF-2α-R:5'-TGGCAGCGGCAGATGTCTC-3'. VEGFA-F:5'-GGAGGAGGAAGAAGAGAAGGAAGAG-3'; VEGFA-R:5'-GCGGCTGGAGCACTGTCTG-3'. PPP1R15A-F:5'-ACAGAGGAAGAGGAAGATGAGGAAG-3'; PPP1R15A-R:5'-TGTAGCAGGAGTGGAAGAGGAAG-3'. CA9-F:5'-TGGCTGCTGGTGACATCCTAG-3'; CA9-R:5'-CTTCTGTGCTGCCTTCTCATCTG-3'. EGLN3-F:5'-TGGAGTACATCGTGCCCTGTC-3'. The sequences are as follows: EGLN3-R 5'-GCAGCGACCATCACCGTTG-3'; GAPDH-F 5'-GTCTCCTCTGACTTCAACAGCG-3'; and GAPDH-R 5'-ACCACCCTGTTGCTGTAGCCAA-3'.

Analysis of western blot with a rating

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.

Immunofluorescence stain

Prior to immunofluorescence, cultured nucleus pulposus cells were treated with 4% PFA and fixed for a duration of 20 minutes. After treating the cells with 0.5% Triton X-100 for 20 minutes to make them permeable, they were then subjected to a 10-minute blocking step at room temperature using 5% BSA. After incubating overnight with primary antibodies targeting VEGFA (AF5131, Affinity, USA) at a temperature of 4 ° C, the samples were then exposed to secondary antibodies (LF102, EpiZyme, MA) for a duration of 1 hour at ambient temperature.BY-1002) to mount the slides and prevent fading of the DAPI stain. P0131) for installation. Acquisition of images was performed with a confocal microscope, specifically the Zeiss LSM 880.

Rats were subjected to experiments, followed by histological staining and grading using a scoring system

Prior to commencing animal experiments, this study received approval from the Ethics Committee of the Affiliated Hospital at Guangdong Medical University.

This study utilized 36 Sprague–Dawley (SD, YC-SD004) rats. The 36 rats used in the experiment were divided into four groups randomly: Acupuncture-induced pathological degeneration group (APD), Acupuncture with PT2399 injection (APD+PT2399), Acupuncture with PT2399-loaded PHBV injection (APD+PT2399-loaded PHBV), and Acupuncture with PT2399-loaded PP20 injection (APD+PT2399-loaded PP20). Each experimental group consisted of nine rats subjected to different experimental methods. A 1 milliliter syringe (HSZSQ-1ML-01, KERONG, CHINA) having a 0.45 mm diameter was employed for administering a dose of PT2399, equivalent to 10 nanomoles per kilogram, into the middle disc of every rat. Each rat was kept in separate enclosures, provided with unrestricted access to water and food, and their well-being was monitored on a daily basis. The Ethics Committee of Affiliated Hospital of Guangdong Medical University approved all animal experiments.

Tissue staining was performed on the intervertebral discs and nearby tissues at the surgical site of two groups of rats at 4 (n = 3), 6 (n = 3), and 8 (n = 3) weeks after degeneration or drug injection following puncture.

The tissues were fixed in a solution containing 10% neutral-buffered formalin and 10% cetylpyridinium chloride. Following decalcification using Cal-Ex II Fixative/Decalcifier (Fisher Scientific, Pittsburgh, PA, USA), the specimens were then immersed in paraffin and sliced into sections measuring 6 m in thickness.

Hematoxylin and eosin (H&E) were used to stain the cellular components of the sections, while safranin-O was used to stain the proteoglycans. After deparaffinization and rehydration, the sections were stained using the H&E staining kit (Solarbio, Beijing, China), safranin-O cartilage staining kit (Solarbio), and alcian blue staining kit (Solarbio) as per the provided instructions. The pictures were taken with a Leica light microscope from Wetzlar, Germany, at magnifications of 50× and 40×.

Assessing the level of deterioration involved employing a revised grading system (Supplementary Table 1), which drew inspiration from both rabbit and human grading systems. The grading scores for AF and NP varied between 1 and 4. To assess the extent of degeneration, the scores of AF and NP were combined.

Reagent

LPS (L2880, Sigma-Aldrich), mPEG-20k (Solarbio, China), PHBV (Tianan Biologic Material Ltd, in China), PT2399 (HY-108697, MCE, USA), PBS (HY-K3005, MCE, USA), RIPA lysis buffer (R0010, Solarbio), BCA protein assay kit (P0010, Beyotime), PVDF membranes (TM-PVDF-R-45, LABSELECT), Western Blocking Solution (P0023B, Beyotime), BeyoECL Plus (P0018FM, Beyotime, China), Cal-Ex II Fixative/Decalcifier (Fisher Scientific, Pittsburgh, PA, USA), H&E/safranin-O cartilage/alcian blue staining kit (Solarbio, Beijing, China). The RT-qPCR primer sequences ( Sangon, China), The WB and Immunofluorescence primary antibodies (Affinity, USA), The WB and Immunofluorescence secondary antibodies (LF102, EpiZyme, MA).

Statistical analysis

All statistical analyses and figures in the online database were generated using R software (version 4.2.0). All data in the experiments were analyzed and histograms were generated using GraphPad Prism software (version 8.0).