Overloaded axial stress activates the Wnt/β-Catenin pathway in nucleus pulposus cells of adult degenerative scoliosis combined with intervertebral disc degeneration

Abstract

Background: Intervertebral disc degeneration (IVDD) is the initiating factor of adult degenerative scoliosis (ADS), and ADS further accelerates IVDD, creating a vicious cycle. Nevertheless, the role of the Wnt/β-Catenin pathway in ADS combined with IVDD (ADS-IVDD) remains a mystery. Accordingly, this study was proposed to investigate the effect of axial stress on the Wnt/β-Catenin pathway in nucleus pulposus cells (NPCs) isolated from DS-IVDD patients.

Methods: Normal NPCs (N-NPC) were purchased and the NPCs of young (25-30 years; Y-NPC) and old (65-70 years; O-NPC) from ADS-IVDD patients were primary cultured. After treatment of NPC with overloaded axial pressure, CCK-8 and Annexin V-FITC kits were applied for detecting proliferation and apoptosis of N-NPC, Y-NPC and O-NPC, and western blotting was performed to assess the expression of Wnt 3a, β-Catenin, NPC markers and apoptosis markers (Bax, Bcl2 and Caspase 3).

Results: N-NPC, Y-NPC and O-NPC were mainly oval, polygonal and spindle-shaped with pseudopods, and the cell morphology tended to be flattened with age. N-NPC, Y-NPC and O-NPC were capable of synthesizing proteoglycans and expressing the NPC markers (Collagen II and Aggrecan). Notably, the expression of Wnt 3a, β-Catenin, Collagen II and Aggrecan was reduced in N-NPC, Y-NPC and O-NPC in that order. After overload axial stress treatment, cell viability of N-NPC and Y-NPC was significantly reduced, and the percentage of apoptosis and expression of Wnt 3a and β-Catenin were significantly increased.

Conclusions: Overloaded axial pressure activates the Wnt/β-Catenin pathway to suppress proliferation and facilitate apoptosis of NPC in ADS-IVDD patients.

Keywords: Axial stress; Degenerative scoliosis; Intervertebral disc degeneration; Nucleus pulposus cells; Wnt/β-Catenin pathway.

Fig. 1

Identification of N-NPC, Y-NPC and O-NPC. A: Representative cell images of N-NPC, Y-NPC and O-NPC (P3). Original magnification: 100 ×. B: The ability of N-NPC, Y-NPC and O-NPC (P3) to synthesize proteoglycans was identified by toluidine blue staining. Scale bar: 200 or 50 μm. C: Representative gel blots of NPC markers Collagen II and Aggrecan. D: Statistical analysis of Collagen II and Aggrecan grayscale values. Compared to the N-NPC group, ^*P < 0.05; compared to the Y-NPC group, ^#P < 0.05

Fig. 2

Changes in Wnt/β-Catenin pathway activity in different NPCs. A: The cell viability of N-NPC, Y-NPC and O-NPC (P3) was detected by CCK-8. Representative cell images display the cell states of N-NPC, Y-NPC and O-NPC at 72 h. Original magnification: 100 ×. B: Western blotting was performed to detect the expression of Wnt 3a and β-Catenin in N-NPC, Y-NPC and O-NPC (P3). C: Statistical analysis of changes in Wnt 3a and β-Catenin expression in each group. Compared to the N-NPC group, ^*P < 0.05; compared to the Y-NPC group, ^#P < 0.05

Fig. 3

Effect of axial stress on proliferation and apoptosis of N-NPC and Y-NPC. A: Cell viability of N-NPC and Y-NPC (P3) at 72 h before and after axial stress treatment. B-C: The expression of apoptosis-related proteins Bax, Bcl 2 and Caspase in N-NPC and Y-NPC (P3) was detected by Western blotting. D: Effects of axial stress on apoptosis of N-NPC and Y-NPC (P3) detected by flow cytometry. Compared to the N-NPC group, ^*P < 0.05; compared to the Y-NPC group, ^#P < 0.05; compared to the N-NPC + F group, ^&P < 0.05

Fig. 4

Effect of axial stress on Wnt/β-Catenin pathway activity in N-NPC and Y-NPC. A: Representative gel blots of Wnt 3a and β-Catenin proteins in N-NPC and Y-NPC (P3). B: Statistical analysis of Wnt 3a and β-Catenin protein expression in each group. C: Effect of axial stress on Wnt 3a and β-Catenin mRNA expression in N-NPC and Y-NPC (P3) examined by RT-qPCR. Compared to the N-NPC group, ^*P < 0.05; compared to the Y-NPC group, ^#P < 0.05; compared to the N-NPC + F group, ^&P < 0.05