Ruxolitinib Delays Nucleus Pulposus Cell Senescence in Rat Intervertebral Discs

Abstract

Background: Intervertebral disc degeneration involves aging and senescence of nucleus pulposus cells (NPCs), and JAK/STAT signaling may contribute to this process. The aim of this study was to investigate the therapeutic effect of the JAK2 inhibitor, ruxolitinib, on NPC senescence.

Methods: Control (third passage), Senescence (sixth passage), JAK inhibitor (ruxolitinib-treated), siRNA-NC (control siRNA-treated), and siRNA-JAK2 (JAK2-targeting siRNA-treated) groups of rat NPCs were established. Cell senescence ratios were determined by β-galactosidase staining and Edu staining was conducted to assess cell proliferation. Cell cycle and apoptosis were analyzed by flow cytometry and Aggrecan and Col II expression detected by immunofluorescence staining. Levels of IL-1β, IL-6, TNF-α, MMP-3, and MMP-13 were detected by ELISA, and p16, p21, p53, p-p53, JAK2, STAT3, p-JAK2, p-STAT3, ADAMTS4, and ADAMTS5 levels were examined by western blot.

Results: More cell senescence was detected by β-galactosidase staining in the Senescence group than in the Control group, while cell proliferation was lower, apoptosis ratio higher, and the percentage of NPCs in G0/G1 phase higher. Levels of senescence-related proteins, including p16, p21, p53, and p-p53, were higher in the Senescence group than the Control group, as were those of IL-1β, IL-6, TNF-α, MMP-3, MMP-13, ADAMTS4, and ADAMTS5. Further, Aggrecan and Col II levels were lower in the Senescence group, while those of JAK2 and STAT3 (JAK2/STAT3 signaling pathway) were higher. Ruxolitinib reversed the changes described above to varying degrees, and the results were supported by those of experiments involving targeted silencing of JAK2.

Conclusions: NPC senescence is characterized by low cell proliferation, a high apoptosis ratio, cell cycle arrest, and generation of senescence-associated secretory phenotypes. NPC senescence can be delayed by inhibiting JAK2/STAT3 signaling using ruxolitinib.

Keywords: JAK2/STAT3 signaling pathway; nucleus pulposus cells; ruxolitinib; senescence.

FIGURE 1

Detection of NPC senescence rates by β‐galactosidase staining. (A) Representative images of β‐galactosidase staining; senescent NPCs are stained dark blue as SA‐β‐gal accumulates in lysosomes (scale bar: 50 μm). (B) Calculation of NPC senescence ratio = (number of senescent cells per unit field of view/total number of cells) × 100%. Data are presented as mean ± standard deviation from three independent experiments; *p < 0.05 versus control group, #p < 0.05 between two groups.

FIGURE 2

Edu staining to detect NPC proliferation capacity. (A) Representative images of EdU staining of NPCs; green fluorescence indicates replication‐capable DNA; living cells were stained with DAPI. Scale bar: 20 μm. (B) Statistical analysis of cell proliferation ratio data. Cell proliferation ratio = (number of green fluorescently labeled cells per unit field of view/total number of cells) × 100%. Data are presented as mean ± standard deviation from three independent experiments; *p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 3

Measurement of the cell cycle by flow cytometry. (A) The fluorescent dye, PI, binds to DNA, and is used to detect the DNA content in each period according to differences in fluorescence intensity, to analyze the proportion of cells in each phase, 5 × 10⁵ events were collected during flow cytometry measurements. (B) Histogram of cell cycle distribution in each group. (C) Proportions of cells in cell cycle phases compared among groups. Data are presented as mean ± standard deviation from three independent experiments; *p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 4

Ruxolitinib reduces senescence‐associated protein expression in NPCs. (A) Senescence‐related proteins (p16, p21, p53, and p‐p53) were analyzed by western blotting. (B) Data are presented as mean ± standard deviation from three independent experiments. (C) Phosphorylated/total ratio for p53; *p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 5

Ruxolitinib reduces NPC apoptosis. Measurement of apoptosis by flow cytometry. (A) Apoptotic NPCs were stained with Annexin VPE/7‐AAD double‐fluorescence and analyzed by flow cytometry, 5 × 10⁵ events were collected during flow cytometry measurements. (B) The apoptosis rate was analyzed by counting 7‐AAD mono‐stained, Annexin V mono‐stained, and double‐stained cells. Data are presented as mean ± standard deviation from three independent experiments;*p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 6

Ruxolitinib reduces the SASP in NPCs. ELISA quantification of SASP‐associated proteins: (A) IL‐1β, (B) IL‐6, (C) TNF‐α, (D) MMP‐3, (E) MMP‐13. Data are presented as mean ± standard deviation from three independent experiments; *p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 7

Ruxolitinib promoted anabolism in nucleus pulposus cells (NPCs). (A) Senescence‐related proteins (ADAMTS4 and ADAMTS5) were analyzed by western blotting. (B) Relative expression levels of ADAMTS4 and ADAMTS5. Data are presented as mean ± standard deviation from three independent experiments; *p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 8

Ruxolitinib inhibits catabolism in nucleus pulposus cells (NPCs). (A) Representative images showing immunofluorescence staining for Aggrecan and Collagen II (Aggrecan: Alexa Fluor 488‐labeled secondary antibody; Collagen II: Alexa Fluor 594‐labeled secondary antibody). Scale bar: 20 μm. (B) Fluorescence intensity of Aggrecan. (C) Fluorescence intensity of Col II. Data are presented as mean ± standard deviation from three independent experiments; *p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 9

Ruxolitinib inhibited JAK2/STAT3 signaling in nucleus pulposus cells (NPCs). (A) Expression of JAK2/STAT3 signaling pathway‐related proteins analyzed by western blotting. (B) Relative expression levels of JAK2, STAT3, p‐JAK2, and p‐STAT3. Data are presented as mean ± standard deviation from three independent experiments. (C) Phosphorylated/total ratio for STAT3; *p < 0.05 versus control group, #p < 0.05 between groups.

FIGURE 10

Determination of transfection conditions based on fluorescence intensity. An siRNA transfection concentration of 0.1 μM and transfection time of 48 h were selected for subsequent experiments. Scale bar: 50 μm.

FIGURE 11

(A) Expression of JAK2/STAT3 signaling pathway. (B) β‐galactosidase staining. (C) Edu staining. (D) Cell cycle. (E) Expression of senescence‐related proteins. (F) Cell apoptosis. (G) ELISA. (H) Expression of ADAMTS. (I) Immunofluorescence staining. Data are shown as mean ± SD, n = 3; *p < 0.05 versus si‐NC group.