Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration

Abstract

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert-butyl hydroperoxide (TBHP)-treated nucleus pulposus cells of SD rats in vitro as well as in a puncture-induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen-II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts-5. Additionally, autophagy blockade using 3-MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine-treated rats had an accentuated T2-weighted signal and a diminished histological degenerative grade than vehicle-treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.

Keywords: apoptosis; autophagy; intervertebral disc degeneration; oxidative stress; spermidine.

Figure 1

Spermidine treatment inhibits TBHP‐induced nucleus pulposus cell apoptosis. Nucleus pulposus cells were untreated, or treated with TBHP alone, or treated with spermidine (50 μmol/L) and TBHP, or treated with TBHP (A) Cell Counting Kit‐8 (CCK‐8) results of nucleus pulposus cells treated with different concentrations of spermidine for 24 hours. (B) CCK‐8 results of nucleus pulposus cells treated with different concentrations of TBHP for 4 hours. (C) CCK‐8 results of spermidine pre‐treated nucleus pulposus cells induced by TBHP. (D‐G) the protein expression of c‐caspase3, Bax, Bcl‐2 of the nucleus pulposus cells as treated above. (H) TUNEL assay was performed in nucleus pulposus cells as treated above (original magnification × 200, scale bar: 50 μm). The data in the figures represent the averages ± SD. *P < .05, **P < .01 vs the control group. ^## P < .01 vs TBHP group, n = 3 per group

Figure 2

Spermidine regulates the expression of anabolic‐ and catabolic‐related proteins. Nucleus pulposus cells were untreated (DMEM 10%FBS), or treated with TBHP alone, or treated with spermidine (50 μmol/L) and TBHP, or treated with TBHP (A‐D) Protein content of Collagen‐II, Aggrecan, Sox‐9 of nucleus pulposus cells as treated above. (E‐H) Protein content of MMP9, MMP13, Adamts‐5 of nucleus pulposus cells as treated above. (I‐J)The representative Collagen‐II and MMP13 were detected by the immunofluorescence combined with DAPI staining for nuclei (Collagen‐II: original magnification ×400, scale bar: 25 μm, MMP13: ×400, scale bar: 25 μm). The data in the figures represent the averages ± SD. ***P < .001 vs the control group. ^## P < .01, ^### P < .001 vs TBHP group, n = 3 per group

Figure 3

Regulation of autophagy in the nucleus pulposus cells cotreated with TBHP and spermidine. Nucleus pulposus cells were untreated, or treated with TBHP alone, or treated with spermidine (50 μmol/L) and TBHP, or treated with TBHP and spermidine. (A‐E) Protein content of Lc3, P62, Atg‐7, Beclin1 of nucleus pulposus cells as treated above. (F) Immunofluorescence of LC3 protein in nucleus pulposus cells. (Green signal represents LC3, scale bar: 25 μm). (G) Autophagosomes were detected by transmission electron microscopy (×25 000) in nucleus pulposus cells. (Black arrow: autophagosome). *P < .05, **P < .01 vs the control group. ^## P < .01, ^### P < .001 vs TBHP group, n = 3 per group

Figure 4

The protective effect of spermidine against apoptosis is related to the stimulation of autophagy. Nucleus pulposus cells were untreated, or treated with TBHP alone, or treated with spermidine (50 μmol/L) and TBHP, or treated with TBHP and spermidine (50 μmol/L) combined with 3‐MA (10 mmol/L). (A‐C) The protein expression of LC3, P62 in the nucleus pulposus cells as treated above. (D‐G)The protein expression of C‐casepase3, Bax, Bcl‐2 in the nucleus pulposus cells as treated above. (H‐J) The protein expression of Collagen‐II and aggrecan in the nucleus pulposus cells as treated above. The data in the figures represent the averages ± SD. Significant differences between the treatment and control groups are indicated as **P < .01, ***P < .001, n = 3 per group

Figure 5

Spermidine treatment ameliorates rat IDD in vivo. (A‐B) Representative safranin O‐fast green and haematoxylin, and haematoxylin and eosin staining of disc samples from different experimental groups at 4 and 8 weeks post‐surgery. Scale bars are 200 μm (40×) and 50 μm (200×), respectively. (C) T2‐weighted MRI of a rat tail with a needle‐punctured disc at 4 and 8 weeks post‐surgery. (D) The histological grades evaluated at week 4 and week 8 in three groups. Samples from 48 rats (sixteen in each group) were used for imageological and histopathologic analysis. (E) The Pfirrmann MRI grade scores in three groups at week 4 and week 8. Significant differences between the treatment and control groups are indicated as **P < .01, *P < .05, n = 6 per group