This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. All experiments and procedures were reviewed and approved by the institutional review board of Emory University (IRB #00099028) approval. Human degenerated NP and AF tissues (Grade IV or V on Pfirrman grade; 64.6±8.5 y old) were obtained as the surgical waste from donors with disc herniation, with each donor providing written informed consent. Healthy NP and AF cells (23.0±3.7 y old) were gifted by Professor Lisbet Haglund from McGill University (Tissue Biobank #2019–4896). The donor information was listed in Table 1. NP and AF cells were extracted as previously described (Zhang et al., 2024). Isolated cells were expanded in growth media containing low glucose DMEM, 10% FBS, 1 X antibiotic-antimycotic (Thermo Fisher Scientific, 15240062), and 50 µg/ml L-Ascorbic acid 2-phosphate (Sigma, A8960). Cells were grown at 37 °C under 5% CO₂ and 20% O₂. To minimize the variability between experiments that may be caused by changes in cell behavior with each passage, cells of passage 3 were used for each experiment.

To synchronize cells and enhance the sensitivity of cells to PDGF stimulation, degenerated NP (n=5/each group) and AF cells (n=6/each group) were serum-deprived in low glucose media containing 0.2% FBS for 1 d. Cells were then treated with recombinant human PDGF-AB (40 ng/ml; PeproTech, 10770584) or -BB (20 ng/ml; PeproTech, 10771918) for 5 d.

Total RNA was isolated with TRIzol (Thermo Fisher Scientific, 15596018) and purified using miRNeasy kit (Qiagen, 217084). Genomic DNA contamination was eliminated through on-column DNase digestion. The concentration and quality of RNA were determined using Nanodrop and RNA integrity was assessed by Agilent 2200 Bioanalyzer. RNA samples (NP: n=5, AF: n=6) with RIN >7 were shipped in dry ice to Novogene for RNA sequencing, which was carried out using NovaSeq 6000 (Novogene Corporation Inc). Each sample represents a unique biological replicate, and no technical replicates were performed for RNA sequencing. Libraries were constructed from 0.2 μg RNA using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina). Quality control was performed for the distribution of sequencing error rate and GC content and data filtering was performed to remove the low-quality reads and reads containing adapters. The resulting sequences were uploaded into the NCBI Sequence Read Archive (SRA) database (PRJNA1150962). The clean reads were mapped to the human reference genome sequence using DNASTAR.

Unnormalized raw counts exported from DNASTAR were used for pairwise gene expression comparison between untreated and PDGF-AB/BB samples. Differential expression genes (DEGs) were identified using the DESeq2 package in R and DEGs with a fold change greater than 1.5 and adjusted p value less than 0.05 were considered statistically significant. Heatmap and volcano plots and principal component analysis were performed to visualize the DEGs between untreated and PDGF-AB/BB samples.

To identify the biological process that DEGs were enriched, Gene ontology (GO) analysis was performed using upregulated and downregulated DEGs separately in R. The significantly enriched GO terms were visualized by dot plot analysis. GeneRatio in the X-axis was calculated by the ratio of gene counts enriched in a particular GO term to all the gene counts annotated in the GO term.

Gene Set Enrichment Analysis (GSEA) was performed on all the genes to determine whether a predefined set of genes is significantly enriched in untreated or PDGF-BB samples. GSEA was conducted based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database using the clusterProfiler package in R (Wu et al., 2021) and a gene set was considered significantly enriched when the false discovery rate (FDR) was less than 0.05. Enrichment score (ES) was calculated to reflect the degree to which a gene set is overrepresented in our dataset, which was ranked based on fold change. Normalized enrichment score (NES) is the ES normalized to the gene set size.

The interactions among DEGs were performed using the Search Tool for the Retrieval of Interacting Genes (STRING) database (v12.0) with an interaction score greater than 0.4. Discrete proteins were removed, and the node files were exported and visualized in Cytoscape (v3.10.0). To identify essential nodes in the interaction network, the betweenness centrality among the nodes was calculated using the CytoNCA plugin (v2.1.6) in Cytospace (Tang et al., 2015) and the top 10 hub nodes were identified using the CyoHubba plugin (v0.1) (Chin et al., 2014).

To induce cellular senescence, cells were seeded in a six-well plate at a density of 5×10⁴ cells/ml and cultured in low glucose DMEM supplemented with 10% FBS, 1 X antibiotic-antimycotic (Thermofisher Scientific, 15240062), and 50 µg/ml L-Ascorbic acid 2-phosphate (Sigma, A8960). After 48 hr of culture, growth media was removed, and cells were washed with 1 X PBS once. To minimize the interference of culture media with the irradiation process, 500 μl PBS was added before irradiation. Healthy NP and AF cells (n=4/each group) were exposed to a single dose of 5 Gy, 10 Gy, or 15 Gy of X-ray in a CellRad benchtop X-ray irradiator (Precision X-Ray) at a rate of 2.6 Gy/min. The irradiation doses and rate were selected based on prior studies (Ungvari et al., 2013; Zhong et al., 2024) and optimized to effectively induce senescence-associated changes in human IVD cells without triggering excessive cell death, which is often observed at higher irradiation doses. After irradiation, cells were immediately incubated with growth media. Cells exposed to 10 Gy of irradiation showed the most pronounced senescent phenotype on day 10 and were used for further experiments. For treatment experiments, cells were exposed to 10 Gy of X-ray and treated with PDGF-AB/BB (20 ng/ml) immediately after irradiation for 10 d. The culture of media was changed every other day.

SA-β-Gal staining was performed according to the manufacturer’s protocol (Cell Signaling Technology, #9860). In brief, cells cultured in six-well plates were washed in PBS and fixed with 1 X fixative solution for 10 min. 1 ml of β-Gal staining solution (containing 1 mg/ml X-Gal) was added and the cells were incubated at 37 °C in the absence of CO₂ overnight. Cells were washed in PBS and observed under a bright-field microscope (ECHO Revolve). Technical triplicates were performed for each sample.

Cells cultured on glass coverslips were washed in 1 X PBS and fixed in 4% (v/v) formaldehyde for 10 min. After washing in 1 X PBS, cells were premetallized in PBS with 0.1% Triton X-100 for 15 min. Non-specific binding sites were blocked in 0.1% Tween-20/PBS with 1% bovine serum albumin for 30 min, followed by incubating with primary antibodies against P21 (1:500, Cell Signaling Technology, #2947), P16 (1:500, Cell Signaling Technology, #80772), or Lamin B1 (1:500, Cell Signaling Technology, #68591 S). After overnight incubation at 4 °C, the cells were washed and incubated with the corresponding host-specific secondary antibodies goat anti-mouse Alex Fluor 488 (1:1000, #ab150113), or goat anti-rabbit Alex Fluro 647 (1:1000, #ab150079) for 1 hr. Cells were counterstained and mounted with mounting medium with DAPI (Abcam, #ab104139) and visualized using an Olympus BX63 automated fluorescence microscope. Technical triplicates were performed for each sample.

Total RNA was extracted from cells collected in TRIzol reagent (Thermo Fisher Scientific, #15596018) and RNA quality was measured using a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific, #ND-2000). 1 μg of RNA was reverse transcribed using Verso cDNA synthesis kit (Thermo Fisher Scientific, #AB-1453B). Quantification of mRNA expression was determined by SYBR-based real-time PCR using PowerUp SYBR Green Master Mix (Applied Biosystems, #A25742). PCR primer sequences were listed in Table 2. Values were normalized to ACTB. mRNA levels were presented as fold change compared to untreated cells according to the 2^-ΔΔCT method. Each qPCR assay was performed in technical triplicates for each sample.

Whole-cell protein extraction and immunoblotting analysis were performed as previously described (Zhang et al., 2024). Briefly, cells were lysed using RIPA lysis buffer with 1 X protease and phosphatase inhibitor (Thermo Fisher Scientific, #PI78440). Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, #23225) and 20 μg of cell lysate from each sample was used to determine the protein expression of P21 (1:2000, Cell Signaling Technology, #2947), NF-kB (1:2000, Cell Signaling Technology, #8242 S), and PDGFRα (1:1000, Cell Signaling Technology, #3164 S). All proteins were normalized to β-actin (1:2000, Cell Signaling Technology, #5125 S). Technical triplicates were performed for each sample.

One-way analysis of variance (ANOVA) testing was performed to assess the effects of treatment on NP and AF cells with subsequent Dunnett post hoc testing. To analyze the effects of ionizing radiation on the expression of senescence markers in cells, a t-test was utilized. Data are presented as mean ± standard deviations. There were no samples were excluded from the analysis. The statistical analyses were performed using GraphPad Prism 9. A p-value < 0.05 was considered statistically significant.

Bulk RNA sequencing was performed to investigate the global changes in gene expression of degenerated AF and NP cells in response to rhPDGF-AB/BB treatment. Principal component analysis (PCA) on all detected genes revealed distinct clusters between untreated and rhPDGF-AB/BB-treated samples, while clusters of rhPDGF-AB and rhPDGF-BB-treated samples were overlapping (Figure 1—figure supplement 1). As shown in volcano plots, differential expression analysis (|FC|>1.5 and FDR <0.05) revealed 880 downregulated and 740 upregulated genes in AF cells treated with rhPDGF-AB and 996 downregulated and 821 upregulated genes induced by rhPDGF-BB treatment after 5 d of exposure (Figure 1A). In contrast, NP cells had 563 downregulated and 423 upregulated genes in rhPDGF-AB-treated group and 409 downregulated and 404 upregulated genes in rhPDGF-BB-treated group. The top differentially expressed genes (DEGs) were annotated according to FDR and most of these genes exhibited similarity between treatment groups in both AF and NP cells (Figure 1A and B). Furthermore, a heat map visualizing the top 50 DEGs by fold change revealed variability among donors while demonstrating an overall consistent response to rhPDGF-AB/BB treatment across the samples (Figure 1C and D). Tissue-specific changes were observed in AF and NP cells when treated with rhPDGF-AB/BB. For example, SLC26A4 (Pendrin) and CA2, regulators of the process of transporting and producing bicarbonate, are involved in the homeostatic control of pH in various types of cells (Vince and Reithmeier, 1998; Silagi et al., 2018) and their expression was upregulated in NP cells when treated with rhPDGF-AB/BB (Figure 1D). MAMDC2 and COL21A1, involved in extracellular matrix organization (Chou and Li, 2002; Mavillard et al., 2023), were also upregulated in NP cells, while WISP2 (Ruiz-Fernández et al., 2022) and RARRES2 (Liu-Chittenden et al., 2017), modulators of the induction of inflammatory mediators and immune response, were downregulated after treatment. In contrast, in addition to the commonly upregulated CA2, MDMC2 and RARRES2, AF cells showed alterations in genes involved in mechanical stress, pain sensation, and angiogenesis, such as CSRP3 (Rashid et al., 2015), PI16 (Singhmar et al., 2020), EFEMP1 (Song et al., 2011), and SLC26A4-AS1 (Li et al., 2021; Figure 1C). Notably, PRELP, potentially associated with Hutchinson-Gilford progeria (Lewis, 2003), was also downregulated in rhPDGF-AB/BB-treated AF cells (Figure 1A).

To investigate the enriched biological processes (BP) induced by rhPDGF-AB/BB treatment in AF cells, gene ontology (GO) analysis was performed using upregulated and downregulated DEGs separately. As expected, the biological processes between rhPDGF-AB and -BB-treated samples were mostly overlapping (Figure 2A and B). In AF cells, upregulated DEGs were significantly enriched in biological processes including regulation of cell cycle and chromosome segregation. Furthermore, AF cells demonstrated upregulated pathways including mesenchymal cell differentiation and response to oxygen levels and downregulated pathways including response to ROS and oxidative stress (Figure 2A). Moreover, the treatment downregulated groups of genes related to neurogenesis and response to mechanical stimulus (Figure 2B). In line with the results of GO analysis, GSEA analysis revealed an overrepresentation of cell cycle pathway in rhPDGF-AB/BB-treated cells (Figure 2C). Additionally, the ErbB signaling pathway, proteasome pathway, and cytosolic DNA-sensing pathway were enriched, suggesting that PDGF-AB/BB treatment promoted cell growth, protein turnover, and maintenance of cellular homeostasis. The leading-edge genes that contributed most significantly to these pathways include CCNB1, CCND1, AREG, PSMB9, TREX1, and POLR3A, as shown in Figure 2D. Protein-protein interaction (PPI) network was employed to further investigate the potential connectivity of DEGs between untreated and treated groups using the STRING and Cytoscape softwares (Figure 2E). PPI analysis revealed that the hub genes in rhPDGF-AB-treated samples were associated with inflammation (CCN2, CSF3, OASL, and SOCS1), macrophage function (CSF1R), neurogenesis (NGF, APOE), and angiogenesis (PTGS2). In rhPDGF-BB-treated AF cells, PXDNL, a member of the peroxidase enzyme family and participating in scavenging ROS interacted with IL-18, an activator of NF-kB signaling. Additionally, CD36, involved in angiogenesis, was among the top 10 hub genes. Taken together, these findings suggest that PDGF treatment promoted cell mitogenesis while inhibiting oxidative stress, inflammation, neurogenesis, and response to mechanical stimulus.

GO analysis revealed similar yet distinct responses to rhPDGF-AB/BB treatment between NP and AF cells. In NP cells, rhPDGF-AB and -BB treatment upregulated groups of genes related to cartilage development, cell-matrix adhesion, Wnt signaling pathway, and response to decreased oxygen levels, which were all important to maintaining NP phenotype (Figure 3A). Downregulated DEGs were enriched in GO terms associated with fatty acid metabolism, cellular respiration, mitochondrial function, and reactive oxygen species production (Figure 3B), suggesting alterations in cellular metabolism and oxidative stress. Gene set enrichment analysis (GSEA) on the entire transcriptomics also revealed that Wnt signaling pathway and neuroactive ligand-receptor interaction were upregulated, while the pathways related to oxidative phosphorylation and ribosome were downregulated in rhPDGF-BB-treated NP cells (Figure 3C). The core enrichment genes associated with these pathways were visualized in a chord diagram (Figure 3D). Specifically, the leading-edge DEGs enriched in Wnt signaling pathway include WNT7B, WNT5A, NKD1, and SFRP1 and oxidative phosphorylation pathway showed enrichment in NDUFB7, NDUFA11, COX5B, and CYC1.

To better understand the regulatory mechanisms of PDGF-AB/BB in degenerated NP cells, DEGs between untreated and treated groups were used to construct PPI networks. The network of the top 10 hub nodes based on betweenness was demonstrated in Figure 3E and it revealed the strong correlation between PDGFRA (PDGF receptor alpha) and IL6, MDM2, MDM2, CCND1, PIK3R1, NOP53, FN1, and IDH2. The enrichment of hub gene IDH2, which plays a role in generating NADPH within mitochondria, further validated the alterations in oxidative phosphorylation pathway and subsequent ROS production, as indicated by GSEA and GO analysis in rhPDGF-AB/BB-treated NP cells. MDM2 (a negative regulator of P53) and predicted NOP53 (a regulator of P53 binding activity) suggested that PDGF-BB interfered with P53 signaling pathway. The interaction between these genes and inflammatory cytokine IL6 indicated a potential involvement of PDGF-AB/BB in cellular senescence. The gene expression of top hub genes PDGFRA and IL6 was further verified in both NP (Figure 3F) and AF (Figure 3G) cells by qPCR. PDGF-AB/BB increased the gene expression of PDGFRA in only NP cells and reduced the IL6 gene expression in both types of cells. Interestingly, the protein expression of PDGFRA was decreased in treated NP and AF cells compared to untreated groups (Figure 3—figure supplement 1). The discrepancy in changes between gene and protein expression levels reflects the complexity in the dynamic regulation of PDGFRA (Figure 3—figure supplement 1). Taken together, NP and AF cells responded differently to PDGF-AB/BB treatment, but both cell types showed a positive regulation of cell cycle and inhibited inflammation, response to ROS, oxidative stress, and mitochondrial dysfunction, indicating a role of PDGF-AB/BB in IVD cell senescence.

To test our hypothesis that PDGF-AB/BB provides protection against the progression of cellular senescence in the IVD, we first created a cellular senescence model using X-ray radiation. The healthy NP cells were exposed to 5, 10, and 15 Gy of irradiation and cultured for 7–10 d to develop a senescent phenotype. SA-β-gal staining was performed to detect senescent cells. We found that non-irradiated, proliferating NP cells showed elongated, spindle-shaped morphology and faint SA-β-gal staining at day 7 (Figure 4A). In contrast, cells exposed to 5 and 10 Gy of irradiation developed characteristics of a senescent cell phenotype — enlarged cell size, low cell number, and a higher percentage of SA-β-gal positive cells. However, cells exposed to 15 Gy of radiation underwent significant cell death, showing negligible SA-β-gal staining. As a result, we excluded the 15 Gy dosage group in subsequent studies. To quantify cell growth, the MTT assay was performed, which revealed reduced cell proliferation under 5 and 10 Gy of irradiation compared to the non-irradiated group (Figure 4B). Furthermore, we examined the expression of senescence-related markers — Lamin B1, P21, and P16. Lamin B1 is a structural component of the nucleus, and its loss is a senescence-associated biomarker (Freund et al., 2012). Non-irradiated cells displayed nuclear Lamin B1 expression, while its expression was lost in healthy NP cells exposed to 5 and 10 Gy of irradiation for 7 d (Figure 4C). Meanwhile, nuclear localization of P21 (Figure 4C) and P16 (Figure 4D) was observed in irradiated cells, with little immunopositivity in non-irradiated cells at day 7. However, the transcripts of P21 and P16 were not significantly decreased until day 10 (Figure 4E and F). Caspase 3 (CASP3) gene expression was reduced at both timepoints, suggesting that the irradiated cells were resistant to apoptosis. Furthermore, the gene expression of PDGFRA was significantly decreased in cells under 10 Gy of irradiation at day 10. Taken together, when exposed to irradiation for 10 d, healthy human NP cells developed a senescent phenotype, accompanied by decreased PDGFRA expression.

To investigate the protective effects of PDGF-AB/BB against cell senescence in the IVD, healthy human NP and AF cells were exposed to 10 Gy of irradiation, followed by immediate treatment with 20 ng/ml rhPDGF-AB/BB for 10 d. We first examined the cell cycle progression using DAPI-stained cells and found that rhPDGF-BB significantly increased the percentage of cell population in the S phase in irradiated NP (Figure 5A and B) and AF (Figure 5C and D) cells at day 10. In rhPDGF-AB-treated cells, the percentage of cell population in the S phase was increased in AF cells, but not significantly in NP cells. These findings suggested that PDGF-AB/BB rescued IVD cells from cell cycle arrest in irradiation-induced senescent cells. Additionally, SA-β-gal staining demonstrated an increase in the number of senescent cells after irradiation at day 10, whereas a decrease was examined in rhPDGF-AB/BB-treated NP and AF cells (Figure 5E).

At day 10, we analyzed the expression of PDGFRA and key senescence regulators of cell cycle inhibitors P21 and P16, CASP3, and SASP mediator IL6. The treatment of rhPDGF-BB to irradiated NP cells significantly increased the gene expression of PDGFRA and decreased the gene expression of P21, P16, and IL6, while playing no effect in the gene expression of CASP3 (Figure 6A). Comparably, the treatment of rhPDGF-AB to irradiated NP cells significantly decreased the gene expression of P21 and IL6 and played no effect on the gene expression of PDGFRA, P16, and CASP3 (Figure 6A). In AF cells, the results show that the treatment of rhPDGF-BB and rhPDGF-AB reduced the gene expression of P21 to those of NP cells (Figure 6B). However, the treatment of either rhPDGF-BB or rhPDGF-AB had no effect on the gene expression of other genes examined (Figure 6B). The lack of change in the gene expression of PDGFRA from the treatment of either rhPDGF-BB or rhPDGF-AB suggested that PDGF-BB reduced senescent phenotype through other receptors in AF cells.

Furthermore, compared to irradiation group, the protein levels of P21 and NF-κB, a major regulator stimulating SASP, were inhibited in rhPDGF-BB-treated NP cells (Figure 6C). In irradiated AF cells, the protein expression of P21 was significantly reduced by rhPDGF-BB treatment. The expression of NF-kB showed a decreasing trend in rhPDGF-BB-treated cells. rhPDGF-AB had no effects on the protein expression of P21 and NF-kB. Overall, PDGF-BB mitigated the progression of senescent phenotype in both human NP and AF cells.