Metformin protects against apoptosis and senescence in nucleus pulposus cells and ameliorates disc degeneration in vivo

Abstract

Intervertebral disc degeneration (IDD) is a complicated process that involves both cellular apoptosis and senescence. Metformin has been reported to stimulate autophagy, whereas autophagy is shown to protect against apoptosis and senescence. Therefore, we hypothesize that metformin may have therapeutic effect on IDD through autophagy stimulation. The effect of metformin on IDD was investigated both in vitro and in vivo. Our study showed that metformin attenuated cellular apoptosis and senescence induced by tert-butyl hydroperoxide in nucleus pulposus cells. Autophagy, as well as its upstream regulator AMPK, was activated by metformin in nucleus pulposus cells in a dose- and time-dependent manner. Inhibition of autophagy by 3-MA partially abolished the protective effect of metformin against nucleus pulposus cells' apoptosis and senescence, indicating that autophagy was involved in the protective effect of metformin on IDD. In addition, metformin was shown to promote the expression of anabolic genes such as Col2a1 and Acan expression while inhibiting the expression of catabolic genes such as Mmp3 and Adamts5 in nucleus pulposus cells. In vivo study illustrated that metformin treatment could ameliorate IDD in a puncture-induced rat model. Thus, our study showed that metformin could protect nucleus pulposus cells against apoptosis and senescence via autophagy stimulation and ameliorate disc degeneration in vivo, revealing its potential to be a therapeutic agent for IDD.

Figure 1

Metformin treatment inhibits TBHP-induced nucleus pulposus cell apoptosis and senescence. (a) Cell Counting Kit-8 (CCK-8) results of nucleus pulposus cells treated with different concentrations of metformin for 24 h. (b) CCK-8 results of nucleus pulposus cells treated with different concentrations of TBHP for 4 h. (c) CCK-8 results of metformin-pretreated nucleus pulposus cells induced by TBHP. (d) Nucleus pulposus cells were pretreated with metformin and then TBHP and imaged by phase-contrast microscopy (original magnification × 100, scale bar: 50 μm). (e–g) Protein content of cleaved caspase3, p16INK4a of nucleus pulposus cells treated with TBHP and TBHP plus metformin. (h) The mRNA expression of Col2a1 of nucleus pulposus cells treated with TBHP and TBHP plus metformin was measured by real-time PCR. The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01,*P<0.05, n=3

Figure 2

Metformin treatment induces autophagy in the nucleus pulposus cells. The nucleus pulposus cells were incubated with 0, 10, 50, 100 or 200 μM metformin for 24 h or 100 μM metformin for 0, 6, 12, 24 or 48 h. (a–h) Protein content of p-AMPK, AMPK, LC3, Beclin-1 and p62 of nucleus pulposus cells as treated above. (i) Autophagosomes and autophagolysosomes were detected by transmission electron microscopy (× 25000) in nucleus pulposus cells. (Black arrow: autophagosome; black triangle: autophagolysosome). The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01,*P<0.05, n=3

Figure 3

Inhibition of AMPK by siRNA significantly attenuates metformin-induced autophagy in nucleus pulposus cells. Cells were transfected with negative control siRNA (con-siRNA) or AMPK-siRNA before receiving metformin(100 μM). (a–c) The protein expression of p-AMPK, AMPK, LC3, Beclin-1 and p62 in the nucleus pulposus cells as treated above. (d, e)The representative LC3-positive autophagic vesicles were detected by immunofluorescence staining combined with DAPI staining for nuclei (scale bar: 25 μm). The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01, *P<0.05, n=3.

Figure 4

TBHP and 3-MA regulate autophagy in the nucleus pulposus cells. Nucleus pulposus cells were untreated (DMEM 10%FBS), or treated with TBHP alone, or treated with metformin (100 μM) and TBHP, or treated with TBHP and metformin (100 μM) combined with 3-MA (10 mM). (a,b)The protein expression of LC3, Beclin-1 and p62 in the nucleus pulposus cells as treated above. (c,d) Double immunofluorescence of LC3 protein and cleaved-caspase3 protein in nucleus pulposus cells. (Green signal represents LC3, red signal represents cleaved-caspase3, scale bar: 25 μm). The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01, *P<0.05, n=3

Figure 5

Metformin treatment reduces nucleus pulposus cell apoptosis under oxidative stress. (a–b) TUNEL assay was performed in nucleus pulposus cells as treated above (original magnification × 200, scale bar: 50 μm). (c–f)The protein expression of cleaved-caspase3, Bax and Bcl-2 in nucleus pulposus cells treated as above. The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01, *P<0.05, n=3

Figure 6

Metformin treatment alleviates nucleus pulposus cells senescence under oxidative stress. (a–b) SA-β-gal staining assay was performed in nucleus pulposus cells as treated above (original magnification × 200, scale bar: 20 μm). (c–d) The protein expression of p-p53, P53, p21WAF1 and p16INKa in nucleus pulposus cells treated as above. The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01, *P<0.05, n=3

Figure 7

Effects of metformin on degeneration-related genes and protein expression in nucleus pulposus cells induced by oxidative stress. (a–d)The mRNA expression of Col2a1, Aggrecan, Adamts-5 and Mmp-3 were measured by real-time PCR. Gene expression was normalized by individual GAPDH expression, and expressed as mean of fold-change mean±S.D. compared with control level. (e–f) The representative collagen-II and Mmp3 were detected by the immunofluorescence combined with DAPI staining for nuclei (collagen-II: original magnification × 200, scale bar: 50 μm, Mmp3: × 400, scale bar: 25 μm). (g–j)The expression of Col2a1, Aggrecan, Adamts-5 and Mmp-3 were measured by ELISA. The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01, *P<0.05, ^#P>0.05, n=3

Figure 8

Metformin treatment amiliorates rat IDD in vivo. (a) T2-weighted MRI of a rat tail with a needle-punctured disc at 8 and 16 weeks post surgery (white arrows). (b) The Pfirrmann MRI grade scores in three groups at week 8 and week 16. (c) Representative S-O staining of disc samples from different experimental groups at 8 and 16 weeks post surgery (original magnification × 40, scale bar: 100 μm). (d) The histological grades evaluated at week 8 and week 16 in three groups. (e–g) Immunohistochemical staining of LC3-II and cleaved-caspase3 expression in the disc samples (original magnification × 100, scale bar: 50 μm).The data in the figures represent the averages±S.D. Significant differences between the treatment and control groups are indicated as **P<0.01, *P<0.05, n=6