Sirt1 protects against intervertebral disc degeneration induced by 1,25-dihydroxyvitamin D insufficiency in mice by inhibiting the NF-κB inflammatory pathway

Abstract

Background: It has been demonstrated that vitamin D deficiency is associated with an increased risk of patients developing lumbar disc herniation. However, intervertebral disc degeneration caused by active vitamin D deficiency has not been reported. Thus, the purpose of this study was to e investigate the role and mechanism of 1,25-dihydroxyvitamin D (1,25(OH)₂D) insufficiency in promoting intervertebral disc degeneration.

Methods: The phenotypes of intervertebral discs were compared in wild-type mice and mice with heterozygous deletion of 1α-hydroxylase [1α(OH)ase^+/-] at 8 mouths of age using iconography, histology and molecular biology. A mouse model that overexpressed Sirt1 in mesenchymal stem cells on a 1α(OH)ase^+/- background (Sirt1^Tg/1α(OH)ase^+/-) was generated by crossing Prx1-Sirt1 transgenic mice with 1α(OH)ase^+/- mice and comparing their intervertebral disc phenotypes with those of Sirt1^Tg, 1α(OH)ase^+/- and wild-type littermates at 8 months of age. A vitamin D receptor (VDR)-deficient cellular model was generated by knock-down of endogenous VDR using Ad-siVDR transfection into nucleus pulposus cells; VDR-deficient nucleus pulposus cells were then treated with or without resveratrol. The interactions between Sirt1 and acetylated p65, and p65 nuclear localization, were examined using co-immunoprecipitation, Western blots and immunofluorescence staining. VDR-deficient nucleus pulposus cells were also treated with 1,25(OH)₂D₃, or resveratrol or 1,25(OH)₂D₃ plus Ex527 (an inhibitor of Sirt1). Effects on Sirt1 expression, cell proliferation, cell senescence, extracellular matrix protein synthesis and degradation, nuclear factor-κB (NF-κB), and expression of inflammatory molecules, were examined, using immunofluorescence staining, Western blots and real-time RT-PCR.

Results: 1,25(OH)₂D insufficiency accelerated intervertebral disc degeneration by reducing extracellular matrix protein synthesis and enhancing extracellular matrix protein degradation with reduced Sirt1 expression in nucleus pulposus tissues. Overexpression of Sirt1 in MSCs protected against 1,25(OH)₂D deficiency-induced intervertebral disc degeneration by decreasing acetylation and phosphorylation of p65 and inhibiting the NF-κB inflammatory pathway. VDR or resveratrol activated Sirt1 to deacetylate p65 and inhibit its nuclear translocation into nucleus pulposus cells. Knockdown of VDR decreased VDR expression and significantly reduced the proliferation and extracellular matrix protein synthesis of nucleus pulposus cells, significantly increased the senescence of nucleus pulposus cells and significantly downregulated Sirt1 expression, and upregulated matrix metallopeptidase 13 (MMP13), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) expression; the ratios of acetylated and phosphorylated p65/p65 in nucleus pulposus cells were also increased. Treatment of nucleus pulposus cells with VDR reduction using 1,25(OH)₂D₃ or resveratrol partially rescued the degeneration phenotypes, by up-regulating Sirt1 expression and inhibiting NF-κB inflammatory pathway; these effects in nucleus pulposus cells were blocked by inhibition of Sirt1.

Conclusion: Results from this study indicate that the 1,25(OH)₂D/VDR pathway can prevent the degeneration of nucleus pulposus cells by inhibiting the NF-κB inflammatory pathway mediated by Sirt1.The Translational Potential of This Article: This study provides new insights into the use of 1,25(OH)₂D₃ to prevent and treat intervertebral disc degeneration caused by vitamin D deficiency.

Keywords: Intervertebral disc degeneration; NF-κB; Sirt1; Vitamin D; Vitamin D receptor.

Graphical abstract

Figure 1

1,25(OH)₂D₃ deficiency accelerated intervertebral disc degeneration (A) Representative radiographs and (B) images of micro-CT-scanned sagittal plane and coronal plane of L1/2 segment from 8-month-old wild-type (WT) and 1α(OH)ase^+/− mice (C) Disc height of L1/2 in wild-type (WT), 1α(OH)ase^+/− mice. Representative micrographs of decalcified paraffin-embedded sections through the intervertebral disc from 8-month-old WT, and 1α(OH)ase ​± ​mice were stained with (D) hematoxylin and eosin (HE) (E) Safranin O-Fast Green (S–O) and (H) immunohistochemically for Sirt1 (F) Intervertebral disc volume of L1/2 in WT and 1α(OH)ase^+/− mice (G) Real-time RT–PCR of tissue extracts of intervertebral discs for expression of Col-2, aggrecan and MMP13. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to WT mice (I) The percentages of Sirt1 positive area (J) Western blots of intervertebral disc extracts were carried out for the expression of Col-2, aggrecan and MMP13 (K) Protein levels relative to β-actin were assessed by densitometric analysis, and expressed relative to WT mice. Values are mean ​± ​S.E.M. of 5 determinations per group. The differences between the two groups were analyzed by using the Student's t-test. ∗: P ​< ​0.05 ∗∗: P ​< ​0.01, ∗∗∗: P ​< ​0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

Overexpression of Sirt1 in NP tissues prevents 1,25(OH)₂D₃ deficiency-induced intervertebral disc degeneration (A) Representative radiographs and (B) images of micro-CT-scanned sagittal plane and coronal plane of L1/2 segment from 8-month-old WT, Sirt1^Tg, 1α(OH)ase^+/− and Sirt1^Tg/1α(OH)ase^+/− mice (C) Disc height of L1/2 in WT, Sirt1^Tg, 1α(OH)ase^+/− and Sirt1^Tg/1α(OH)ase^+/− mice. Representative micrographs of decalcified paraffin-embedded sections through the intervertebral disc from 8-month-old mice were stained with (D) hematoxylin and eosin (HE) (E) Safranin O-Fast Green (S–O) and immunohistochemically for (H) Sirt1 (J) Col-2 (L) aggrecan and (N) MMP13 (F) Intervertebral disc volume of L1/2 in above 4 genotype mice (G) Real-time RT–PCR of tissue extracts of intervertebral discs for expression of Prx-1, Sirt1, Col-2, aggrecan, MMP3 and MMP13. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to WT mice. The percentages of (I) Sirt1 positive area (K) Col-2 positive area (M) aggrecan positive area and (O) MMP13 positive area (P) Western blots of intervertebral disc extracts were carried out for expression of Prx-1, Sirt1, Col-2, aggrecan, MMP3 and MMP13 (Q) Protein levels relative to β-actin were assessed by densitometric analysis. Values are mean ​± ​S.E.M. of 5 determinations per group. A comparison between multiple groups was assessed by using one-way ANOVA test followed by Post-Hoc Test (Least Significant Difference). ∗: P ​< ​0.05 ∗∗: P ​< ​0.01, ∗∗∗: P ​< ​0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 3

Sirt1 prevents 1,25(OH)₂D₃ deficiency-induced intervertebral disc degeneration by inhibiting inflammation mediated by the NF-κB pathway. Representative micrographs of decalcified paraffin-embedded sections through the intervertebral discs from 8-month-old WT, Sirt1^Tg, 1α(OH)ase^+/− and Sirt1^Tg/1α(OH)ase^+/− mice were stained with double immunofluorescence for (A) p16 and p65, and immunohistochemically for (C) TNF-α and (E) IL-1β. The percentages of (B) p16 and p65 single positive cells and p16/p65 double positive cells (D) TNF-α positive area and (F) IL-1β positive area (G) Western blots of intervertebral disc extracts were carried out for expression of Ace-p65, p-p65, p65, TNF-α, and IL-1β (H) Protein levels relative to β-actin were assessed by densitometric analysis. Protein levels of (I) Ace-p65 and (J) p-p65 relative to p65 were assessed by densitometric analysis (K) Real-time RT–PCR of tissue extracts of intervertebral discs for expression of p65, TNF-α, and IL-1β. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to WT mice. Values are mean ​± ​S.E.M. of 5 determinations per group. A comparison between multiple groups was assessed by using one-way ANOVA test followed by Post-Hoc Test (Least Significant Difference). ∗: P ​< ​0.05 ∗∗: P ​< ​0.01, ∗∗∗: P ​< ​0.001.

Figure 4

VDR or resveratrol activates Sirt1 to deacetylate p65 and inhibit its activity in nucleus pulposus cells. The knockdown efficiency of VDR by adenovirus vectors transfection with siVDR was detected by (A) real-time RT-PCR for the mRNA level (B) Western blots and (C) immunofluorescence staining for VDR protein expression level. NP cells isolated from intervertebral discs of mice were cultured in blank medium (Control), Ad-siVDR medium and Ad-siVDR with resveratrol (RES) medium (D) Whole cell lysates (WCL) of NP cells were treated with anti-Sirt1 antibody and hybridized with anti-p65 or acetylated p65 antibody. The proteins were then analyzed by performing Western blotting (E) Ace-p65 relative to p65 were assessed by densitometric analysis (F) Whole cell lysates (WCL) and nuclear lysates were blotted with anti-Sirt1 or anti-p65 antibody (G) Representative micrographs of immunofluorescence staining for p65 (green), DAPI (blue) and merge. Values are mean ​± ​S.E.M. of 5 determinations per group. The differences between the two groups were analyzed by using the Student's t-test. A comparison between multiple groups was assessed by using one-way ANOVA test followed by Post-Hoc Test (Least Significant Difference). ∗∗: P ​< ​0.01, ∗∗∗: P ​< ​0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 5

1,25(OH)2D3/VDR mediated via Sirt1 stimulates the proliferation and inhibits senescence of nucleus pulposus cells. NP cells isolated from intervertebral discs of mice were cultured in the absence (Control) or presence of transfected with Ad-NC or Ad-siVDR or Ad-siVDR treated with 10⁻⁸ ​M 1,25(OH)₂D₃ (VD) or Ad-siVDR treated with 10⁻⁸ ​M 1,25(OH)₂D₃ and 10 ​μM Sirt1 inhibitor Ex527 or Ad-siVDR treated with 100 ​μM resveratrol (Res). Representative micrographs of immunofluorescence staining for (A) Sirt1 (green) (C) EdU (red) (E) p16 (green), DAPI (blue) and merge. The percentages of (B) Sirt1 (D) EdU and (F) p16 positive cells (G) Representative micrographs of cells from SA-β-gal cytochemical and p16 immunocytochemical double staining (H) The percentage of p16, SA-β-gal single positive cells and p16 and SA-β-gal double positive cells (I) Whole cell lysates were blotted with anti-Sirt1 antibody (J) Protein levels relative to β-actin were assessed by densitometric analysis (K) Real-time RT–PCR of NP cell extracts for expression of Sirt1. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to control. Values are mean ​± ​S.E.M. of 5 determinations per group. A comparison between multiple groups was assessed by using one-way ANOVA test followed by Post-Hoc Test (Least Significant Difference). ∗: P ​< ​0.05 ∗∗: P ​< ​0.01, ∗∗∗: P ​< ​0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 6

1,25(OH)2D3/VDR mediated via Sirt1 inhibits nucleus pulposus cell degeneration. NP cells isolated from intervertebral discs of mice were cultured and treated as Fig. 5. Representative micrographs of immunofluorescence staining for (A) aggrecan (green) (C) MMP13 (green), DAPI (blue) and merge. The percentages of (B) aggrecan and (D) MMP13 intensity to area (E) Whole cell lysates were blotted with anti-Sirt1 or anti-aggrecan or anti-MMP13 antibody (F) Protein levels relative to β-actin were assessed by densitometric analysis (G) Real-time RT–PCR of NP cell extracts for expression of Sirt1, aggrecan and MMP13. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to control. Values are mean ​± ​S.E.M. of 5 determinations per group. A comparison between multiple groups was assessed by using one-way ANOVA test followed by Post-Hoc Test (Least Significant Difference). ∗: P ​< ​0.05 ∗∗: P ​< ​0.01, ∗∗∗: P ​< ​0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 7

1,25(OH)2D3/VDR mediated via Sirt1 inhibits NF-κB inflammatory pathway in nucleus pulposus cells. NP cells isolated from intervertebral discs of mice were cultured and treated as Fig. 5. Representative micrographs of immunofluorescence staining for (A) TNF-α (green) (C) IL-1β (green), DAPI (blue) and merge (B) The percentages of (B) TNF-α and (D) IL-1β intensity to area (E) Whole cell lysates were blotted with anti-Ace-p65, anti-p-p65, anti-p65, anti-TNF-α or anti-IL-1β antibody. Protein levels of (F) Ace-p65 and (G) p-p65 relative to p65 were assessed by densitometric analysis (H) Protein levels relative to β-actin were assessed by densitometric analysis (I) Real-time RT–PCR of NP cell extracts for expression of p65, TNF-α and IL-1β. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio relative to GAPDH, and expressed relative to control group. Values are mean ​± ​S.E.M. of 5 determinations per group. A comparison between multiple groups was assessed by using one-way ANOVA test followed by Post-Hoc Test (Least Significant Difference). ∗: P ​< ​0.05 ∗∗: P ​< ​0.01, ∗∗∗: P ​< ​0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)