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. 2018 Jan 19;9(2):56.
doi: 10.1038/s41419-017-0085-5.

Sirt6 Overexpression Suppresses Senescence and Apoptosis of Nucleus Pulposus Cells by Inducing Autophagy in a Model of Intervertebral Disc Degeneration

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Free PMC article

Sirt6 Overexpression Suppresses Senescence and Apoptosis of Nucleus Pulposus Cells by Inducing Autophagy in a Model of Intervertebral Disc Degeneration

Jian Chen et al. Cell Death Dis. .
Free PMC article

Abstract

Treatment of intervertebral disc degeneration (IDD) seeks to prevent senescence and death of nucleus pulposus (NP) cells. Previous studies have shown that sirt6 exerts potent anti-senescent and anti-apoptotic effects in models of age-related degenerative disease. However, it is not known whether sirt6 protects against IDD. Here, we explored whether sirt6 influenced IDD. The sirt6 level was reduced in senescent human NP cells. Sirt6 overexpression protected against apoptosis and both replicative and stress-induced premature senescence. Sirt6 also activated NP cell autophagy both in vivo and in vitro. 3-methyladenine (3-MA) and chloroquine (CQ)-mediated inhibition of autophagy partially reversed the anti-senescent and anti-apoptotic effects of sirt6, which regulated the expression of degeneration-associated proteins. In vivo, sirt6 overexpression attenuated IDD. Together, the data showed that sirt6 attenuated cell senescence, and reduced apoptosis, by triggering autophagy that ultimately ameliorated IDD. Thus, sirt6 may be a novel therapeutic target for IDD treatment.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. Sirt6 level declines in senescent NP cells both in vivo and in vitro
a Immunofluorescence of sirt6 in aging (16 months) group and young (3 months) group (scale bar: 200 μm). b Immunofluorescence of sirt6 in NP cells that were isolated from aging and young rats (scale bar: 50 μm). c, d Representative western blots and quantification data of sirt6 in NP cells of each group. e SA-β-gal staining assay was performed in rat NP cells as treated above (scale bar: 50 μm). fh Representative western blots and quantification data of p16 and sirt6 in NP cells of each group. Columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, n = 5
Fig. 2
Fig. 2. Sirt6 overexpression attenuated replicative senescence of rat NP cells
We passaged NP cells to the 15th generations to detect sirt6 and p16. a SA-β-gal staining assay was performed in NP cells of each group (scale bar: 50 μm). bd Representative western blots and quantification data of p16 and sirt6 in human NP cells of each group; columns represent mean ± SD. Significant differences between the treatment and sham groups are indicated as **P < 0.01, ***P < 0.001, n = 5
Fig. 3
Fig. 3. Sirt6 overexpression suppressed IL-1β-induced premature senescence and apoptosis of human NP cells
ae Representative western blots and quantification data of p16, cleaved caspase3, Bax, Bcl-2 in NP cells of each group as treated above; columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, n = 5
Fig. 4
Fig. 4. Sirt6 overexpression activated human NP cell autophagy
a Transmission electron microscopy showed the autophagosomes (black arrow: autophagosome) in NP cells after lentivirus transfection. bf Representative western blots and quantification data of p-mTOR, mTOR, LC3, P62, and Beclin-1 protein in NP cells of each group; columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as **P < 0.01, *P < 0.05, n = 5. g Double immunofluorescence of sirt6 (red) and LC3-II (green) in NP cells treated by Lenti-sirt6 (scale bar: 10 μm)
Fig. 5
Fig. 5. Inhibition of autophagy attenuated the anti-apoptotic effect of sirt6
a, b TUNEL assay was performed to assess the apoptosis in NP cells of each group as treated above (scale bar: 50 μm). cf Representative western blots and quantification data of cleaved caspase3, Bax, Bcl-2 in NP cells of each group as treated above. g Caspase3 activity in NP cells of each group. Columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, n = 5
Fig. 6
Fig. 6. Sirt6 inhibits stress-induced premature senescence via autophagy
a SA-β-gal staining assay was performed in NP cells of each group as treated above (scale bar: 50 μm). be Representative western blots and quantification data of p-p53, p53, p21, and p16 in NP cells of each group as treated above; columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, n = 5
Fig. 7
Fig. 7. Sirt6 regulated the expression levels of degeneration-associated proteins via autophagy of human NP cells
af PCR assay of collegan-II, aggrecan, MMP-3, MMP-13, ADAMT4, and ADAMT5 in NP cells of each group as treated above; columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, n = 5. g, h Immunofluorescence of collegan-II and MMP-3 in NP cells of each group as treated above (scale bar: 50 μm)
Fig. 8
Fig. 8. Sirt6 overexpression ameliorated puncture-induced IDD in vivo
a, b T2-weighted MRI and relative Pfirrmann MRI grade scores of a rat tail with a needle-punctured disc at 8 weeks in each group (white arrows); columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, ***P < 0.001, n = 5. c HE staining of each group. d, e Immunohistochemical staining of cleaved-caspase3 and LC3-II expression in the disc samples of each group (scale bar: 200 μm)

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