Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice

Abstract

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16^INK4a, p19^ARF, and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration.

Fig. 1. Senolytic drugs Dasatinib and Quercetin alleviate age-dependent intervertebral disc degeneration and decrease abundance of senescence markers.

a Schematic showing study design: peritoneal injections were administered once every week to mice starting at 6 (6 M), 14 (14 M), or 18 (18 M) months of age and ending at 23 (23 M) months of age. Vehicle (1:1 PBS/DMSO), D + Q (5 mg/kg Dasatinib plus 50 mg/kg Quercetin). b Histology of the 14–23 M cohort showed preservation of tissue architecture and cell morphology in the NP, AF, and CEP of D + Q-treated mice. 5X and 20X images of 14–23 M show NP, AF, and CEP compartment morphologies. c, d Modified Thompson Grading averages and grade distributions showed lower average scores of degeneration in mice that were treated from 6 M and 14 M. Mice in 18–23 M D + Q cohort showed comparable scores the Veh group. e Level-by-level analysis of L3-6 in the lumbar spine evidenced decreases in degeneration grades in the NP compartment at L5-6 and in the AF compartment at L3-4 and L5-6. f–j Staining and abundance of key markers of senescence p16^INK4a, p19^ARF and p21, and cell cycle: Ki67 in disc compartments. Two-tailed t-test or Mann–Whitney test were used as appropriate for comparing differences between Veh and D + Q groups. 6–23 M Veh (n = 13), D + Q (n = 15); 14–23 M Veh (n = 8), D + Q (n = 7); 18–23 M Veh (n = 11), D + Q (n = 9). Data are represented as mean ± SEM. Scale bar b (disc) = 200 µm; Scale bar b (NP, AF, and CEP) = 50 µm; Scale bar f–f’, g–g’, i–i’, and j–j’ = 200 µm. NP: nucleus pulposus; AF: annulus fibrosus; EP: endplate; GP: growth plate, VB: vertebral bone. Source data are provided as a Source Data file.

Fig. 2. D + Q treatment prevents age-dependent SASP progression and promotes NP cell phenotype maintenance and disc cell viability.

a–c”’ Immunohistological staining and staining area quantifications of SASP markers IL-1, IL-6, and MMP13 in the disc compartments of 1-year-old healthy Ctr (1 y Ctr), 14–23 M Veh-, and 14–23 M D + Q-treated mice. d–e”’ Quantitative immunostaining of NP cell phenotypic markers CA3 and GLUT1. f–f”’ Cell death assessment in the disc compartments by TUNEL staining. Two-tailed ANOVA or Kruskal–Wallis tests were used as appropriate; n = 6 mice/group, 2–3 levels per mouse. Data are represented as mean ± SEM. Scale bar = 200 µm. Source data are provided as a Source Data file.

Fig. 3. D + Q treatment preserved healthy extracellular matrix composition with a decrease in Aggrecan degradation during aging.

a–f”’ Staining and abundance in the disc compartment of essential extracellular matrix proteins in 1 y Ctr, 14–23 M Veh, and 14–23 M D + Q groups: Collagen I, Collagen II, COMP, Collagen X, Chondroitin sulfate, and ARGXX neoepitope. Two-tailed ANOVA or Kruskal–Wallis tests were used as appropriate; n = 6 mice/group, 2–3 levels per mouse were analyzed. Data are represented as mean ± SEM. Scale bar a–f” = 200 µm. Source data are provided as a Source Data file.

Fig. 4. D + Q treatment results in lower incidence of NP fibrosis.

a Bright field and chemical map of mean second-derivative peaks for collagen (1338 cm⁻¹). Red: high relative absorbance; yellow: intermediate relative absorbance; blue: low relative absorbance. b Quantification of mean second-derivative peaks for collagen (1338 cm⁻¹). c Quantification of the percentage of presence/absence of collagen fibers within the NP compartment. d 1-year Ctr, Veh and D + Q lumbar discs showing collagen fiber organization under bright field and polarized light (scale bar = 100 μm). e Quantification of collagen fiber thickness distribution and proportion in the AF. f Denatured collagen, measured by a collagen hybridization peptide (CHP) biding assay (scale bar = 200 µm). Two-tailed ANOVA or Kruskal–Wallis tests were used as appropriate; n = 6 mice/group, 2–3 levels per mouse were analyzed. Data are represented as mean ± SEM. Source data are provided as a Source Data file.

Fig. 5. D + Q treatment promoted transcriptomic modulation of cell death, response to cytokines, regulation of RNA polymerase II, and the ERK1/ERK2 cascade in AF and NP compartments.

a Transcriptomic profiles of AF (n = 5) and NP (n = 5) tissues from 14–23 M Veh mice clustered distinctly with principal component analysis. b Volcano Plot, showing up- and downregulated DEGs from the 23 M AF vs. NP comparison used for GO Process enrichment analysis, p-value ≤ 0.05. c Schematic summarizing the DEGs between the AF and NP at 23 M related to cell cycle regulation. d Volcano Plot, showing up- and downregulated DEGs from the D + Q AF vs. Veh AF comparison, p ≤ 0.05. e Representative GO processes of upregulated genes from D + Q AF vs. Veh AF. f Representative GO processes of downregulated genes from D + Q AF vs. Veh AF. g Volcano Plot, showing up- and downregulated DEGs from the D + Q NP vs. Veh NP comparison, p-value ≤ 0.05. h Representative GO processes of downregulated genes from D + Q NP vs. Veh NP. i Venn Diagram of common DEGs from D + Q AF vs. Veh AF and D + Q NP vs. Veh NP, p-value ≤ 0.05. j Representative GO processes of common DEGs from panel I. k Representative DEGs from selected GO processes from panel j. p-values were calculated using eBayes ANOVA Method (unadjusted). GO analysis was performed using the PANTHER Overrepresentation Test with GO Ontology database annotations and a binomial statistical test with FDR ≤ 0.05. Source data are provided as a Source Data file.

Fig. 6. D + Q modulates aging-associated DEGs related to negative regulation of apoptotic process, regulation of kinase activity and regulation of cell differentiation pathways.

a, b PCA and hierarchical clustering of transcriptomic signature from 23 M vs. 6 M BL6 NP of deposited data in GSE134955, p < 0.05. c Venn diagram from intersection analysis of aging and D + Q modulated DEGs and the representative GO processes associated with the 166 common DEGs. d Representative common DEGs from selected GO processes from panel C. e Schematic of common DEGs showing directionality of change in each experiment study (Aging vs. D + Q). f Representative DEGs with opposite modulation between aging and D + Q selected from panel e. p-values were calculated using eBayes ANOVA Method (unadjusted). GO analysis was performed using the PANTHER Overrepresentation Test with GO Ontology database annotations and a binomial statistical test with FDR ≤ 0.05. Source data are provided as a Source Data file.

Fig. 7. D + Q treatment prevented the age-associated systemic increase in proinflammatory molecules, cytokines, and Th17-related proteins.

Multiplex analysis of the main (a–a’) proinflammatory molecules, (b–b’) cytokines, and (c–c’) Th17-related proteins in plasma of young (6–12 M, n = 7), old (23 M, n = 8), Veh-14–23 M (n = 8), and D + Q-14–23M (n = 7) groups. Two-tailed t-test or Mann–Whitney test were used as appropriate. Data are represented as mean ± SEM. Source data are provided as a Source Data file.

Fig. 8. Long-term D + Q treatment was well tolerated by mice.

a Survival curve from the 14–23 M Veh and. D + Q cohorts. b Weight progression in males from 14–23 M Veh (n = 4) and D + Q (n = 4) cohorts. c Weight progression in females from 14–23 M Veh (n = 4) and D + Q (n = 3) cohorts. d Disc height index comparison between 14 and 23 M Veh and D + Q groups. e Representative lumbar spine μCT pictures and bone properties:BV/TV, trabecular thickness, and cortical bone thickness of Veh (n = 4) and D + Q (n = 4) males from the 14–23 M cohort, L4-L6/mouse. f Representative lumbar spine μCT pictures and bone parameter analysis of BV/TV, trabecular thickness, and cortical bone thickness of Veh (n = 4) and D + Q (n = 3) females from the 14–23 M cohort, L4-L6/mouse. Scale bar e, f = 500 µm. t-test or Mann–Whitney test was used as appropriate. g Representative hindlimb histology from the 14–23 M cohort as assessed by Safranin-O/Fast Green/Hematoxylin. Imaged from the lateral side of the joint. Anatomical and histological features of the joint are denoted: Femur (F), Tibia (T), meniscus (M), tidemark (TM with diamond arrow), Saf-O loss (dotted arrow), and vertical clefts of cartilage loss (solid arrow). OARSI grading (0–6) summed across 4 quadrants of joint from 14–23 M Veh (n = 7) and D + Q (n = 8) cohorts. Males denoted by circles, females by triangles. Scale bar e, f = 500 µm, G = 20 µm. Two-tailed t-test or Mann–Whitney test was used as appropriate. Data are represented as mean ± SEM. Source data are provided as a Source Data file.

Fig. 9. Senolytics ameliorate age-related disc degeneration.

a Schematic summarizing the overall beneficial effects of D + Q contributing to alleviation of intervertebral disc degeneration during aging.