Polydatin suppresses nucleus pulposus cell senescence, promotes matrix homeostasis and attenuates intervertebral disc degeneration in rats

Abstract

Intervertebral disc degeneration (IVDD) is one of the major causes of low back pain. Polydatin (PD) has been shown to exert multiple pharmacological effects on different diseases; here, we test the therapeutic potential of PD for IVDD. In in-vitro experiments, we confirmed PD is nontoxic to nucleus pulposus cells (NPCs) under the concentration of 400 μmol/L. Furthermore, PD was able to decrease the level of senescence in TNF-α-treated NPCs, as indicated by β-gal staining as well as senescence markers p53 and p16 expression. In the aspect of extracellular matrix (ECM), PD not only reduced metalloproteinase 3 (MMP-3), metalloproteinase 13 (MMP-13) and a disintegrin-like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) expression, but also increased aggrecan and collagen II levels. Mitochondrion is closely related to cellular senescence and ECM homeostasis; mechanistically, we found PD may rescue TNF-α-induced mitochondrial dysfunction, and it may also promote Nrf2 expression and activity. Silencing Nrf2 partly abolished the protective effects of PD on mitochondrial homeostasis, senescence and ECM homeostasis in TNF-α-treated NPCs. Correspondingly, PD ameliorated IVDD in rat model by promoting Nrf2 activity, preserving ECM and inhibiting senescence in nucleus pulposus cells. To sum up, our study suggests that PD exerts protective effects in NPCs against IVDD and reveals the underlying mechanism of PD on Nrf2 activation in NPCs.

Keywords: Nrf2; extracellular matrix; intervertebral disc degeneration; mitochondria; polydatin; senescence.

Figure 1

Effects of PD on the viability of NPCs. A, Chemical structure of PD. B, The cytotoxic effect of PD on NPCs was determined at various concentrations for 24 h using a CCK8 assay. C, The cytotoxic effect of PD on NPCs was determined at various concentrations for 72 h using a CCK8 assay. The values presented are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, n = 5

Figure 2

Polydatin (PD) protects NPCs from senescence induced by TNF‐α. A, Representative micrographs showed the effects of PD on senescence in NPCs treated with 10 ng/mL TNF‐α for 72 h, which was manifested by the SA‐β‐gal staining assay. Scale bar: 100 μm. B, D, The expression of p53 and p16 was detected by Western blotting. NPCs treated with or without preadministration of PD for 2 h were administrated with 50 ng/mL TNF‐α for another 72 h. C, NPCs were treated with PD or TNF‐α as described above and incubated with EdU (10 μmol/L). Then, NPCs were fixed and stained with the antibody for EdU, and nuclei were stained with DAPI. Scale bar: 10 μm. The values presented are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, n = 5

Figure 3

Polydatin (PD) suppresses extracellular matrix degradation induced by TNF‐α in NPCs. NPCs were pretreated with different concentrations of PD before administration with 50 ng/mL TNF‐α for another 72 h. A, B, The expressions of aggrecan were detected by Western blot. C, D, The expression of collagen II (COL II) in NPCs treated as above was detected by immunofluorescence. Scale bar: 50 μm. E‐H, The expression of MMP‐3, MMP‐13 and ADAMTS‐4 was detected by Western blot analysis. Data presented are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs control group; *P < 0.05, **P < 0.01, n = 5

Figure 4

Polydatin (PD) alleviates TNF‐α‐induced mitochondrial dysfunction in NPCs. After 2 h of pretreatment with PD, TNF‐α was added into culture media at the concentration of 50 ng/mL and NPCs were incubated for another 72 h. A, B, MitoSOX assay showed that PD reversed the ROS accumulation in TNF‐α‐treated NPCs. Scale bar: 50 μm. C, Intracellular MDA level was assessed by MDA Assay Kit. D, E, The effect of PD on mitochondrial potential in NPCs treated with TNF‐α using MitoTracker Green. Scale bar: 10 μm. F, Intracellular ATP level was assessed by ATP‐Glo Bioluminometric Cell Viability Assay. The values presented are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, n = 5

Figure 5

Polydatin (PD) promotes nucleus translocation of Nrf2 and activate the Nrf2/HO‐1 signalling pathway in rat NPCs. A, B, The expressions of nucleus Nrf2 in NPCs treated with different concentration of PD for 6 h. C, Representative images of Nrf2 in NPCs with increasing concentration of PD treatment for 6 h. Scale bar: 10 μm. D, The level of Keap1 was detected by Western blot. NPCs were administrated with PD for 6 h. E, The level of HO‐1 was detected by Western blot. NPCs were administrated with PD for 12 h. F, The ratios of Keap1/β‐actin and HO‐1/β‐actin were quantified. Data presented are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, n = 5

Figure 6

Knockdown of Nrf2 compromises the antisenescence, anti‐ECM disorder and antimitochondrial dysfunction properties of PD on NPCs. A‐C, Successful knockdown of Nrf2 suppressed the expression level of HO‐1. D, E, The expression of p53 and p16 in NPCs treated with or without pretreatment of PD for 2 h and another administration of TNF‐α for 72 h. F, G, The levels of ECM‐related proteins such as aggrecan, MMP‐13 and MMP‐3 were detected by Western blotting. H‐J, Representative images for MitoSOX and MitoTracker of PD and TNF‐α‐treated NPCs with or without Nrf2 knockdown. Scale bar: 50 μm. Data presented are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, n = 5

Figure 7

Polydatin (PD) ameliorates disc degeneration development in IVDD rat model in vivo. A, Magnetic resonance image (MRI) of IVDs in rats from different experimental groups. Loss of signal density was found in both IVDD and PD+IVDD group (white arrows). B, Diagrams showed the Pfirrmann grades of IVDs. C, Representative alcian staining of IVDs from different groups at 2 or 4 wk postsurgery. Scale bar: 100 μm. D, Representative images for Nrf2 in IVDs from the IVDD group and the PD+IVDD group. Scale bar: 100 μm. E, Representative images for p16 and p53 in IVDs from the IVDD group and the PD+IVDD group. Scale bar: 100 μm. F, The quantification of Nrf2, p16 and p53 fluorescence intensity of IVDs from the IVDD group and the PD group. Data presented are the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, n = 5

Figure 8

Schematics of mechanism of PD on IVDD. PD treatment suppresses Keap1 and enhances Nrf2 to activate Nrf2/HO‐1 signalling pathway to scavenge TNF‐α‐induced ROS, rescuing senescence and excessive ECM destruction in NPCs