2.1 Human NP tissue collection and classification

The magnetic resonance imaging (MRI) data of human NP tissues were classified according to the Pfirrmann grading method.²⁵ Discs categorized as Pfirrmann Grade I or II was considered as early IVDD group, and Pfirrmann Grade III, IV, or V was classified as late IVDD group. Early NP tissue was collected from patients diagnosed with idiopathic scoliosis and lumbar fractures (5 females and 3 males; age 19.4 ± 4.88; Grade I [n = 3] and Grade II [n = 5]), and late NP tissue was collected from patients diagnosed with lumbar disc herniation or lumbar spondylolisthesis undergoing disc fusion surgery (14 females and 10 males; age 43 ± 6.42 years; Grade III [n = 8], Grade IV [n = 8] and Grade V [n = 8]). Pfirrmann Grades I–V tissue samples were randomly selected three for immunohistochemistry (IHC) analysis. The remaining NP tissue samples were frozen in liquid nitrogen and stored for western blotting analysis. All patients provided written informed consent, and all protocols were approved by the Ethics Committee of the Second Hospital of Lanzhou University (2023A-588).

2.2 Culture of rat nucleus pulposus cells

The extraction method for Sprague–Dawley rat NP cells was based on our previous research.²⁶ Extracted NP cells were cultured in flasks containing Dulbecco's Modified Eagle Medium (DMEM)/F12 medium containing 10% fetal bovine serum (FBS) in an incubator at 37°C, 5% CO₂. When the cells reached approximately 90% confluency, NP cells were dissociated using 0.25% trypsin and subcultured. Third-generation NP cells were used for the subsequent experiments in this study.

2.3 Lentiviral transfection

DNMT3a short hairpin RNA (shRNA) and scrambled shRNA were purchased from Gemma (Shanghai, China). Transfection with DNMT3a shRNA inhibited DNMT3a expression Lentiviruses containing DNMT3a shRNA or negative control lentivirus (shRNA-NC) were added to NP cells at a multiplicity of infection (MOI) of 100. NP cells were cultured in a 37°C, 5% CO₂ incubator. After 24 h, the medium was changed, and the cells were cultured for 3 days and transferred for subsequent experiments. Transfection efficiency was determined using western blotting and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The sequences were as follows:

Dnmt3a-Rat-2182-1:5′-TCGCTCCGCTGAAGGAATATT-3′.

Dnmt3a-Rat-764-2:5′-GAATCCTTACAAGGAAGTTTA-3′.

Dnmt3a-Rat-1292-3:5′-CCAGATGTTCTTCGCCAATAA-3′.

2.4 Western blotting assay of protein expressions

Human and rat NP tissues were lysed in a lysis buffer containing protease inhibitors for 30 min. The cell lysate was centrifuged at 12,000 rpm, 4°C for 15 min. Protein detection in the supernatant was performed using a Bradford Protein Assay Kit. Equal-sized protein samples were separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% bovine serum albumin for 2 h at room temperature, then incubated overnight at 4°C with primary antibodies against the following proteins: DNMT3a (anti-rabbit, 1:1000), PPARγ (anti-rabbit, 1:1000), Caspase-3 (anti-rabbit, 1:1000), Bcl-2 (anti-rabbit, 1:1000), Bax (anti-rabbit, 1:1000), Collagen II (anti-rabbit, 1:1000), Aggrecan (anti-rabbit, 1:1000), Adamts-4 (anti-rabbit, 1:1000), MMP-3 (anti-rabbit, 1:1000), p-P65 (anti-rabbit, 1:1000), P65 (anti-rabbit, 1:1000), p-IKBα (anti-rabbit, 1:1000), IKBα (anti-rabbit, 1:1000) and β-actin (anti-mouse, 1:1000). Subsequently, the membrane was washed thrice for 10 min each and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5000; Beyotime) or horseradish peroxidase-conjugated goat anti-mouse antibody (1:5000; Beyotime) at room temperature for 2 h. ImageJ software (https://imagej.nih.gov/ij/) was used to analyse the western blot data.

2.5 qRT-PCR

Total RNA was extracted from rat NP cells using AG RNAex Pro Reagent (Accurate Biotechnology, Ltd., China), and the absorbance ratios at 260/280 nm and 260/230 nm were determined using a UV–Vis spectrophotometer (Thermo, USA) to assess the purity and concentration of total RNA in each sample. Reverse transcription of mRNA into cDNA was conducted using Evo M-MLV RT Kit. Subsequently, qRT-PCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology Ltd., China). The primers used were as follows: Rat DNMT3a, forward 5’-AAGGACCCTGCGGTGATCT-3,’ reverse 5′-TTGGGTAATAGCTCTGAGGCG-3′; GAPDH, forward 5′-GGAAGCTTGTCATCAATGGAAATC-3′; reverse 5′-TGATGACCCTTTTGGCTCCC-3′. Three replicate wells were set up per reaction to quantify the gene expression of interest using the 2^−ΔΔCt method.

2.6 Methylation-specific PCR (MSP)

The CpG island of the rat PPARγ promoter was analysed using MetPrimer software (http://www.urogene.org/methprimer/), and MSP primers were designed. Genomic DNA was extracted from NP cells according to the instructions provided with the EasyPure® Genomic DNA Kit (Beijing, China). DNA was modified with bisulfite using a DNA Bisulfite Conversion Kit (TIANGEN, Beijing, China). PCR products were analysed on agarose gels for density using ImageJ software. Methylated and unmethylated PPARγ primer sequences were as follows: Methylated primers: forward 5′-CGTGGGATTTTTGTGGC-3′, Reverse 5′-CACGAAACCCGGTAACT-3′; Unmethylated primers: Forward 5′-GAATGTGGGATTTTTGTGTT-3′, Reverse 5′-CCCACAAAACCCACATAACTC-3.

2.7 Immunofluorescence staining

Rat NP cells were transferred to flat-bottomed 24-well plates at a specified density (5 × 10³ cells/well). After treatment, cells were fixed with 4% paraformaldehyde, followed by permeability with 0.5% tritonX-100 for 30 min, blocked with 5% BSA, and incubated overnight at 4°C with corresponding antibodies. After washing, cells were incubated with DyLight 649 or DyLight 488 (Abbkine) anti-rabbit secondary antibody, which was diluted at 1:300, incubated at 37°C for 1 h, and then photographed using an inverted fluorescence microscope (Olympus).

2.8 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining

DNA damage level was detected using TUNEL staining. NP cells were seeded into 24-well plates, fixed with fresh 4% paraformaldehyde for 30 min, and incubated with 3% H₂O₂ and 0.5% Triton X-100 for 30 min. Finally, according to the manufacturer's instructions, the cells and the TUNEL reaction mixture were incubated at 37°C for 1 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min in the dark. After washing the cells thrice with PBS at room temperature in the dark, images of the cells were obtained using a fluorescence microscope (Olympus, Tokyo, Japan). Apoptotic cells showed dense fluorescent particles in the nucleus.

2.9 Cell viability assay

Cell Counting Kit-8 (CCK-8; Yeasen Biotechnology) was used to measure NP cell viability. NP cells were seeded in 96-well plates containing 100 μL of whole medium (containing 10% FBS) and incubated for 24 h in a 37°C 5% CO₂ incubator. PPARγ inhibitor (T0070907) (Medchem Express, Shanghai, China) was added to 5, 10, 15, 25, 50 and 100 μM complete medium culture for 24 h; on the next day, 10 μL of CCK-8 solution per well was added and incubated at 37°C for 2 h. Finally, the 450 nm optical density (OD450) was measured using a microplate analyser (Bio-Rad, Hercules, CA, USA). Similarly, another batch of cells was used to detect cell viability of the PPARγ activator (GW1929) (Medchem Express, shanghai, China) and NF-κB pathway activator (diprovocim) (Medchem Express, shanghai, China). The percentage cell viability was calculated as $\left(1-\left[=\text{mean}\mathrm{OD}\text{in the drug group}/\text{mean}\mathrm{OD}\text{in the control group}\right]\right)\times 100\%$.

2.10 Animal experiments

Adult male Sprague–Dawley rats (200–250 g) were purchased from Lanzhou Veterinary Research Institute in Gansu Province. The rats were placed at an indoor temperature of 23 ± 2°C, were alternately reared under light and dark conditions for 12 h, and were fed regular supplementation with feed and purified water. A classic rat fibre ring-needled IVDD model was used as previously described.²⁷ All experimental rats were anaesthetised using pentobarbital sodium (40 mg/kg, intraperitoneal injection). They were pierced with a 21G needle at Co6-7, CO7-8, and CO8-9, rotated 180°, and held for 30 s. Subsequently, we administered a 2 μL PBS injection into the NP of Co6-7, 2 μL of shRNA-NC into the NP of Co7-8, and 2 μL of shRNA-DNMT3a into the NP of Co8-9. The rats were returned to their cages and fed continuously.

2.11 X-ray and MRI scan

All rats' tails underwent X-ray and MRI 8 weeks after surgery. The rats remained in a horizontal supine position with the tail extending straight on the mammography device (GE). X-ray irradiation was performed at a distance of 66 cm between the collimator and film, penetration voltage of 35 kV and exposure intensity of 63 mA. The sagittal T2-weighted MRI image was acquired using a 3.0 T clinical magnet with a fast-spin echo sequence with a time-to-repetition of 5400 ms, a time-to-echo of 920 ms, a 320 (h) × 256 (v) matrix, a field-of-view of 260°, and four excitations. The section thickness was 2 mm, and the gap was 0 mm. After the X-ray and MRI examinations were completed, the rats were euthanized, and images were acquired and analysed.

2.12 Haematoxylinand eosin (HE) staining, toluidine blue staining and safranine O staining

Fresh 4% paraformaldehyde was used to fix human NP tissue and rat NP tissue, which was embedded in paraffin and then sectioned into 5 μm sections for use. HE, toluidine blue and Safranine O stainings were performed according to the manufacturer's instructions. After staining, the seal was fixed with gel and photographed using an EVOS microscope (EVOS-FL Cell imaging System, Thermo Fisher Scientific).

2.13 IHC staining

Fresh 4% paraformaldehyde was used to fix human NP tissue and rat NP tissue, which was embedded in paraffin and then sectioned into 5 μm sections for use. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 min and then incubated with 5% bovine serum albumin (BSA) and 1% Tween-20 in phosphate-buffered saline (PBS) for 30 min to block non-specific antigens. The tissue sections were incubated overnight with anti-PPARγ, DNMT3a, Collagen II, MMP-3 and Caspase-3 antibodies at 4°C. Subsequently, tissue sections were incubated with the corresponding enzyme-labelled secondary antibody (Proteintech, 1:5000) and counterstained with haematoxylin. Photographic records were generated using a panoramic histiocyte scanner and analysed using the ImageJ software.

2.14 Statistical analysis

Data were analysed using GraphPad Prism 9 software, and all experiments were repeated at least thrice. We used t-tests and one-way or two-way analysis of variance (anova) to compare data from different groups. The Kruskal–Wallis test was used to analyse differences between groups. The values of p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001) were considered statistically significant.

3.1 Expression of DNMT3a and PPARγ in human NP tissues

Early and late human NP tissue samples were collected, and the degree of NP tissue degeneration was assessed based on the patient's T2 MRI images (Figure 1A). Haematoxylin and eosin (HE) and toluidine blue staining showed a change in the characteristics of NP tissues during the progression of IVDD. In the early IVDD group, the collagen fibres in NP tissues were neatly arranged, and the ECM staining of NP cells was uniform. In the late IVDD group, the collagen fibres in the NP tissue were highly disordered, and the NP cells were clustered in large quantities, showing large vacuolar cells (Figure 1B). Additionally, western blotting analysis showed increased DNMT3a expression and decreased PPARγ expression in late IVDD group, compared with early IVDD group (Figure 1C–E). Further, the expression levels of DNMT3a and PPARγ were measured in human NP tissues by immunohistochemical analysis. Compared to early NP tissues, DNMT3a expression in late NP tissues was increased and PPARγ expression was decreased (Figure 1F–H). These results suggest that DNMT3a level increase, and PPARγ level gradually decrease during the IVDD progression.

3.2 IL-1β treatment causes increased expression of DNMT3a and decreased expression of PPARγ in rat NP cells

Inflammation is considered an important contributing factor to IVDD progression.²⁸ IL-1β can significantly increase the expression of apoptosis-associated proteins (Caspase-3, Bax, and Bcl-2) in IVD cells in vitro, promoting apoptosis and ECM degradation in NP cells.^{29, 30} Therefore, we evaluated the changes in PPARγ and DNMT3a in IL-1β-treated NP cells. Western blot analysis revealed that IL-1β treatment increased the levels of DNMT3a expression while reducing PPARγ levels in NP cells in a dose-dependent manner (Figure 2A–C) and time-dependent manner (Figure 2D–F). Therefore, the subsequent experiment selected IL-1β concentration of 10 ng/mL for 24 h. Additionally, immunofluorescence analysis confirmed that the DNMT3a expression increased and the PPARγ expression decreased in IL-1β-treated NP cells (Figure 2G,H). These results are consistent with the findings in human tissue studies that DNMT3a was up-regulated, and PPARγ was down-regulated during IL-1β-induced NP cell degeneration.

3.3 DNMT3a inhibition attenuates IL-1β-induced apoptosis and ECM degradation

To investigate whether silencing DNMT3a inhibits the apoptosis of IL-1β-induced rat NP cells in vitro, we infected lentiviruses with scrambled shRNA (shRNA-NC) and DNMT3a shRNA (shRNA-DNMT3a) and assessed the silencing efficiency using qRT-PCR and western blotting (Supporting Information S1). Western blotting results showed that DNMT3a inhibition significantly reversed the IL-1β-induced increase in DNMT3a expression (Figure 3A,B). Apoptosis is often accompanied by the upregulation of Caspase-3 and Bax expression and the downregulation of Bcl-2. As shown in Figure 3A,C–E, IL-1β promoted the expression of Bax and Caspase-3 pro-apoptotic proteins and reduced the expression level of anti-apoptotic protein Bcl-2 compared to the control group (p < 0.05). Additionally, the shRNA-DNMT3a group, compared with the IL-1β and shRNA-NC groups, experienced significantly reduced pro-apoptotic proteins (Bax and Caspase-3) expression and increased anti-apoptotic proteins (Bcl-2) expression, indicating that the silencing of DNMT3a can inhibit the apoptosis of IL-1β-induced NP cells (Figure 3A,C–E). Moreover, TUNEL staining showed that shRNA-DNMT3a significantly reduced the IL-1β-induced NP cells apoptosis ratio (Figure 3F,G).

Furthermore, we evaluated whether inhibition of DNMT3a inhibits IL-1β-induced ECM degradation in rat NP cells. The balance between ECM synthesis and degradation contributes to the structural stability of intervertebral discs, which is a key indicator of NP cell function. We examined the expression of matrix decomposition enzymes (MMP-3 and Adamts-4), collagen II, and aggrecan in NP cells to assess the balance between ECM synthesis and degradation in NP cells. As shown in Figure 3H–L, IL-1β promoted MMP-3 and Adamts-4 expression levels and decreased collagen II and aggrecan expression levels compared with the control group (p < 0.05). Additionally, shRNA-DNMT3a increased collagen II and aggrecan expression levels and decreased MMP-3 and Adamts-4 expression levels compared to the IL-1β and shRNA-NC groups (Figure 3H–L). Additionally, immunofluorescence staining showed that shRNA-DNMT3a significantly increased collagen II expression levels and decreased MMP-3 expression levels (Figure 3M–O). These results suggest that DNMT3a silencing inhibits NP apoptosis and ECM degradation.

3.4 DNMT3a inhibits PPARγ expression by modifying PPARγ promoter methylation

The UCSC Genomic Explorer database (http://genome.ucsc.edu) was used to predict the PPARγ promoter CpG island region. As shown in Figure 4A, the PPARγ promoter in rats has a typical CpG island located in the 1312/1418 region. To further clarify the epigenetic mechanism of PPARγ inhibition due to abnormal DNA methylation, we used shRNA-NC or shRNA-DNMT3a to transfect NP cells and detected the DNA methylation status of PPARγ promoters in rat NP cells (1312/1418 sites) using MSP analysis. The MSP results showed that the methylation degree of the PPARγ promoter was significantly increased after IL-1β treatment in rat NP, whereas the activity of DNMT3a was inhibited after shRNA-DNMT3a treatment compared with the IL-1β and shRNA-NC groups, resulting in a significant decrease in the degree of methylation of the PPARγ promoter (Figure 4B,C). Moreover, western blotting results showed that the shRNA-DNMT3a group experienced significantly increased PPARγ expression levels compared with the IL-1β and shRNA-NC groups (Figure 4D,E). Furthermore, immunofluorescence and quantitative analyses showed that shRNA-DNMT3a significantly increased PPARγ expression levels compared to the IL-1β and shRNA-NC groups (Figure 4F,G). The above results show that DNMT3a inhibits the expression of PPARγ by modifying PPARγ promoter methylation.

3.5 DNMT3a inhibits PPARγ to promote apoptosis and ECM degradation

To determine whether DNMT3a exerts anti-apoptotic effects via PPARγ, we pretreated NP cells with a specific inhibitor of PPARγ (T0070907). First, we detected the toxic effects of T0070907 on rat NP cells, and the results showed that no toxicity existed in NP cells below 10 μM (Figure 5A). Western blotting and immunofluorescence results showed that T0070907 inhibited PPARγ expression (Figure 5B,C,G,H). In addition, compared with the shRNA-NC group, the shRNA-DNMT3a group experienced significantly reduced IL-1 β-induced Bax and Caspase-3 expression levels and increased PPARγ and Bcl-2 expression levels. However, pretreatment with T0070907 reversed the increase in PPARγ owing to shRNA-DNMT3a treatment and increased Bax and Caspase-3 expression levels while reducing the expression of Bcl-2, suggesting that inhibition of PPARγ expression eliminated the anti-apoptotic effect of shRNA-DNMT3a (p < 0.05) (Figure 5B,D–F). Similarly, the TUNEL staining results showed that DNMT3a inhibition significantly reduced the apoptosis rate, whereas T0070907 significantly reversed the anti-apoptotic effect of shRNA-DNMT3a (Figure 5G,I). These results indicate that DNMT3a inhibition regulates NP apoptosis through PPARγ.

In addition, to determine whether DNMT3a participates in anti-ECM degradation via PPARγ, we pretreated NP cells by adding T0070907 to the medium. Western blotting showed that the shRNA-DNMT3a group experienced significantly reduced IL-1β-induced Adamts-4 and MMP-3 expression levels and increased collagen II and aggrecan expression levels compared with the shRNA-NC group. However, T0070907 eliminated the anti-ECM degradation of shRNA-DNMT3a by inhibiting PPARγ expression (Figure 6A–E). Similarly, immunofluorescence results showed that DNMT3a inhibition significantly increased collagen II and decreased MMP-3 expression. The inhibitory effect of shRNA-DNMT3a was reversed in the presence of T0070907 (Figure 6F–H). Thus, DNMT3a inhibition regulates the degradation of ECM in NP cells via PPARγ.

3.6 DNMT3a regulates the activity of the PPARγ/NF-κB axis

NF-κB pathway plays a crucial role in the pathological processes of IVDD. PPARγ can regulate the activity of the NF-κB pathway and participate in the ECM degradation of chondrocytes, anabolism, apoptosis and inflammation, and other pathological processes in osteoarthritis. However, whether PPARγ can participate in IVDD development by regulating the NF-κB pathway activity remains unclear. To further explore the underlying molecular mechanism of PPARγ regulating apoptosis and ECM degradation in NP cells, we observed that DNMT3a inhibition significantly inhibited the activation of the NF-κB pathway induced by IL-1β compared with the IL-1β and shRNA-NC groups (Figure 7A–C), indicating that the NF-κB pathway is involved in apoptosis and ECM degradation of NP cells. In addition, when T0070907 was pretreated, the inhibitory effect of DNMT3a inhibition on the NF-κB pathway declined compared to that of shRNA-DNMT3a (Figure 7D–F), indicating that DNMT3a regulates the activity of the PPARγ/NF-κB axis.

3.7 PPARγ regulates apoptosis and ECM degradation by inhibiting the NF-κB pathway

We further verified whether PPARγ regulates apoptosis and ECM degradation in NP cells via the NF-κB pathway. First, we examined the effects of PPARγ activator (GW1929) and NF-κB pathway activator (diprovocim) on NP cell viability and observed that 10 μM GW1929 and 20 μM diprovocim were not toxic to NP cells (Supporting Information S2). Therefore, we chose GW1929 at a concentration of 10 μM and diprovocim at 20 μM for the relevant experimental studies. Figure 8A–C show that GW1929 pretreatment inhibits-induced activation of the NF-κB pathway induced by IL-1β. In addition, western blotting showed that GW1929 significantly reduced the Bax and Caspase-3 expression levels and increased the Bcl-2 expression levels compared to IL-1β group, while the anti-apoptotic effect of GW1929 was debilitated by activating the NF-κB pathway, indicating that PPARγ regulates the apoptosis of NP cells through the NF-κB pathway (Figure 8D–K). Similarly, compared with the IL-1β, GW1929 significantly reduced the Adamts-4 and MMP-3 expression levels and increased Collagen II and Aggrecan expression levels, while the anti-ECM degradation of GW1929 was weakened by activating the NF-κB pathway using diprovocim (Figure 8D–K). These results suggest that PPARγ regulated ECM degradation in NP cells through the NF-κB pathway.

3.8 DNMT3a inhibition ameliorates puncture-induced IVDD in vivo

To detect the protective effects of DNMT3a inhibition in rat intervertebral discs, we used a classical acupuncture-induced IVDD model. The degree of IVDD in the rats was assessed using radiography, MRI and Pfirrmann grading. We mapped the stages of male Co5–6, Co6–7, Co7-8, and Co8-9 from each SD rat to the Sham, IVDD, IVDD+shRNA-NC and IVDD+shRNA-DNMT3a groups. Radiographic and MRI T2 images of the rat coccygeal vertebrae were acquired 8 weeks after surgery, and disc height measurements and Pfirrmann grades were performed (Figure 9A–C). The HE staining and saffranine O staining were performed on NP tissues of rats in different groups, and histological scores were performed (Figure 9D,E). This indicated that DNMT3a inhibition significantly improved the development of IVDD. In addition, further immunohistochemical analysis showed that the level of DNMT3a in the IVDD+shRNA-DNMT3a group was significantly lower than that in the IVDD and IVDD+shRNA-NC groups, indicating that shRNA-DNMT3a could significantly reduce the expression of DNMT3a in the NP tissues of IVDD rats. More importantly, compared with the IVDD and IVDD+shRNA-NC groups, the expressions of PPARγ and collagen II increased, while the expression levels of MMP-3 and Caspase-3 decreased in the IVDD+shRNA-DNMT3a group (Figure 9F–K). This indicated that inhibition of DNMT3a could significantly inhibit ECM degradation and apoptosis. In summary, DNMT3a inhibition can increase the anabolism of ECM and decrease the apoptosis of NP cells, thus improving the ameliorate puncture-induced IVDD in vivo.