Bmi deficiency causes oxidative stress and intervertebral disc degeneration which can be alleviated by antioxidant treatment

Abstract

The transcriptional repressor Bmi-1 is involved in cell-cycle regulation and cell senescence, the deficiency of which has been shown to cause oxidative stress. This study investigated whether Bmi-1 deficiency plays a role in promoting disc degeneration and the effect of treatment with antioxidant N-acetylcysteine (NAC) on intervertebral disc degeneration. Bmi-1^-/- mice were treated with the antioxidant NAC, supplied in drinking water (Bmi-1^-/- +NAC). For in vitro experiments, mouse intervertebral discs were cultured under low oxygen tension and serum-limiting conditions in the presence of tumour necrosis factor α and interleukin 1β in order to mimic degenerative insult. Disc metabolism parameters in these in vitro and in vivo studies were evaluated by histopathological, immunohistochemical and molecular methods. Bmi-1^-/- mice showed lower collagen Ⅱ and aggrecan levels and higher collagen Ⅹ levels than wild-type and Bmi-1^-/- +NAC mice. Bmi-1^-/- mice showed significantly lower superoxide dismutase (SOD)-1, SOD-2, glutathione peroxidase (GPX)-1 and GPX-3 levels than their wild-type littermates and Bmi-1^-/- + NAC mice. Relative to Bmi-1^-/- mice, the control and Bmi-1^-/- +NAC mice showed significantly lower p16, p21, and p53 levels. These results demonstrate that Bmi-1 plays an important role in attenuating intervertebral disc degeneration in mice by inhibiting oxidative stress and cell apoptosis.

Keywords: Bmi-1; N-acetylcysteine; cell apoptosis; intervertebral disc degeneration; oxidative stress.

FIGURE 1

Alcian blue staining and collagen X and matrix metalloproteinase 3 (MMP3) immunostaining in mouse intervertebral discs cultured under the control or degeneration condition in vitro. A, Mouse intervertebral discs were removed under sterile conditions and cultured in 6‐well plate. B, The intervertebral disc structure and the expression of aggrecan in intervertebral disc were determined by HE‐Alcian blue composite staining. C, D, The expression of collagen X and MMP3 in intervertebral disc of mice was determined by immunohistochemistry. The positive areas of aggrecan, collagen X and MMP3 were analysed. (*P < .05)

FIGURE 2

NAC treatment alleviates intervertebral disc degeneration caused by loss of Bmi‐1. A, Representative micrographs stained immunohistochemically for collagen II and collagen X. B, The percentage of areas with positive collagen II and collagen X staining. C, Western blots of intervertebral disc extracts showing levels of collagen II and collagen X. D, mRNA levels of collagen II and collagen X as determined by real‐time RT–PCR. Values are means ± SE of determinations in 6 mice of each group. * P < .05; ** P < .01; *** P < .001

FIGURE 3

The loss of Bmi‐1 aggravates oxidative stress of intervertebral disc degeneration in mice, which can be alleviated by NAC treatment. A, Representative micrographs stained immunohistochemically for SOD‐1. B, The percentage of SOD‐1‐stained area. C, Western blots of intervertebral disc extracts showing levels of SOD‐1 and SOD‐2. D, mRNA levels of SOD‐1, SOD‐2, GPX1 and GPX3 as determined by real‐time RT–PCR. Values are means ± SE of determinations in 6 mice of each group. * P < .05; ** P < .01; *** P < .001

FIGURE 4

The loss of Bmi‐1 aggravates nucleus pulposus cell senescence in intervertebral disc degeneration in mice. A, Representative micrographs stained immunohistochemically for p16. B, The percentage of p16‐stained area. C, Western blots of intervertebral disc extracts showing p16, p21 and p53. D, mRNA levels of p16, p21 and p53 as determined by real‐time RT–PCR. Values are means ± SE of determinations in 6 mice of each group. *P < .05; **P < .01; ***P < .001

FIGURE 5

NAC alleviates nucleus pulposus cell degeneration induced by IL‐1β and TNF‐α. After one and two weeks of culturing, intervertebral discs were stained by haematoxylin‐eosin staining (A). The expression of collagen II, collagen X and Bmi‐1 was determined by Western blotting (B) and/or real‐time RT‐PCR (C). Values are means ± SE of determinations in 6 mice of each group. *P < .05; **P < .01; ***P < .001

FIGURE 6

Proposed model depicting the mechanism of Bmi‐1 in regulating oxidative stress in IDD