SPINE Volume 40, Number 9, pp E515-E524 ©2015, Wolters Kluwer Health, Inc. All rights reserved.
BASIC SCIENCE
SIRT1 Plays a Protective Role in Intervertebral Disc Degeneration in a Puncture-induced Rodent Model Xinlei Xia, MD, PhD, Ji Guo, MD, Feizhou Lu, MD, PhD, and Jianyuan Jiang, MD
Study Design. Experimental animal study of treatment of intervertebral disc (IVD) degeneration. Objective. This report aims to evaluate the in vivo effects of SIRT1 on IVD biology and to explore its potential mechanism. Summary of Background Data. Silent mating type information regulator 2 homolog 1 (SIRT1) has attracted immense attention because of its functions in a variety of aging-related diseases. Despite previous studies indicated that SIRT1 showed a unique expression with degeneration in some in vitro study, there is no in vivo research on the role SIRT1 plays in IVD and its mechanism. Methods. Coccygeal discs were punctured to induce disc degeneration. Sixteen C57BL/6J mice received either Carboxy methocel (Vehicle) or Resveratrol (RES) gavage. Eight SIRT1+/− mice and their SIRT1+/+ littermates were also used in this study. At 2 and 6 weeks after puncture, magnetic resonance images were obtained. The mice were subsequently killed, and the spine was extracted for further evaluation. Results. Coccygeal disc puncture caused IVD degeneration in the mice. A SIRT1 activator, RES, markedly ameliorated this pathological change, as demonstrated by stronger signal intensity in the T2-weighted images, as well as a significantly lower magnetic resonance imaging grade (at 2 wk vs. Vehicle group P < 0.001). Histological analysis also revealed an improvement in the RES group compared with the Vehicle group (P < 0.05). Genetic ablation of 1 allele significantly enhanced the level of damage relative to the wild-type mice. In addition, SIRT1 activation suppressed the
From the Huashan Hospital, Fudan University, Shanghai, People’s Republic of China. Acknowledgment date: August 4, 2014. First revision date: December 4, 2014. Second revision date: January 5, 2015. Acceptance date: January 10, 2015. The manuscript submitted does not contain information about medical device(s)/drug(s). Shanghai Science and Technology Development Funds (grant No: 12ZR1403900) and AOSpine China Research Grant (Project No: AOSCN(R)2013-12) funds were received to support this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Feizhou Lu, MD, PhD, Orthopeadics Department, Huashan Hospital, Fudan University, No. 12 Wulumuqi Mid Rd, Shanghai, 200040, People’s Republic of China. E-mail: [email protected] DOI: 10.1097/BRS.0000000000000817 Spine
expression of p16 and at the same time, promoted proliferating cell nuclear antigen and type II collagen expression in disc cells, whereas genetic ablation of 1 allele SIRT1 exhibited the opposite consequence. Conclusion. The SIRT1 activator RES protects against punctureinduced disc injury whereas SIRT1 deficiency aggravates tissue injury; the protective role of SIRT1 is partly mediated by suppressing p16, which plays a role in elevating the decreased proliferative ability of the senescent nucleus pulposus cells. Key words: intervertebral disc (IVD) degeneration, SIRT1, Resveratrol, annulus puncture, cellular senescence. Level of Evidence: N/A Spine 2015;40:E515–E524
T
he high morbidity of low back pain causes a severe incapacity that increases medical expenses and impacts the workforce, thereby resulting in high socioeconomic costs and human suffering. It is reported that more than 400,000 individuals are unable to work properly because of low back pain in the United States each year.1 Although the exact pathomechanism of low back pain remains poorly understood, intervertebral disc (IVD) degeneration has been thought to be a major cause. A number of diverse etiological factors are thought to serve as primary initiating events that lead to IVD degeneration. These factors include genetic predisposition, smoking, infection, abnormal biomechanical loading, decreased nutrient transport across the vertebral endplates, and cell senescence.2–5 However, the relative importance of each of these factors in IVD degeneration is currently unknown. In addition, the precise pathogenesis of IVD degeneration and its relationship to cellular senescence is unclear as well. Most therapeutic methods for disc diseases continue to concentrate on the treatment of the various pathological conditions that result in disc degeneration, such as surgery, medications, and steroid injections, as opposed to focusing on the prevention or reversal of the degenerative discs. Novel approaches, such as peptide or growth factor injections, gene delivery, and tissue-engineering techniques,6–9 have been proposed to manage the underlying causes of IVD degeneration, but significant limitations exist for these therapies. In addition, many approaches are invasive to the disc, and the ability to transfer results in animal models to human patients remains unclear. www.spinejournal.com
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BASIC SCIENCE Therefore, additional clinical trials are needed to establish the safety and efficacy of these innovative treatments.10 Silent information regulator 2 proteins (sirtuins) are a group of nicotinamide (NAD+)-dependent deacetylases that were initially discovered in yeast.11,12 A human homolog of silent information regulator 2, silent mating-type information regulator 2 homolog 1 (SIRT1), has been reported to play a key role in transcriptional silencing, genome stability, and an extended life span after calorie restriction because of its ability to deacetylate both histone and nonhistone substrates, such as p53, NF-κB, and forkhead box type O-3. Recent studies determined that SIRT1 is crucial in the pathophysiology of metabolic disease, degenerative disorders, cancer, and aging.13 IVD is also a type of degenerative process that can occur quite early even in adolescents.14–16 Several studies have shown that SIRT1 expression decreased significantly with serial cell passage in human and murine cells; a significant positive correlation between the level of SIRT1 and cell proliferation was also found.17 A recent study18 demonstrated that SIRT1 can be expressed in cultured human nucleus pulpous (NP) cells, and the SIRT1 mRNA expression levels significantly decreased with age. Therefore, we hypothesized that SIRT1 may play an important role in IVD degeneration via its regulation of cellular senescence. In the present study, we examined IVD susceptibility to puncture-induced injury under various SIRT1 expression levels.
SIRT1 Protects Against IVD Degeneration • Xia et al
purchased from Sigma (Sigma-Aldrich, St. Louis, MO) was prepared as a solution in carboxymethyl cellulose. The mice were treated with 100 mg RES/kg body weight or carboxymethyl via oral gavage twice per day. The treatment was administered at 1 and 4 weeks after puncture, and each time period lasted for 7 days. Twelve-week-old SIRT1+/+ (WT) and SIRT1+/− (HT) mice were also used in this study (n = 8 per group).
The Annulus Needle Puncture Model
MATERIALS AND METHODS
After an intraperitoneal anesthetic injection of a combination of 10 mg/kg xylazine and 60 mg/kg ketamine hydrochloride, mice were prone on the experiment table, a sagittal small skin incision was performed from Co7 to Co9 to help locate the disc position for needle insertion in the tail. Next, Co7–Co8 coccygeal discs were punctured using a syringe needle following a previously described method.19 The syringe needle was inserted into Co7–Co8 discs along vertical direction and then rotated in the axial direction by 180° and held for 5 seconds, The puncture was made parallel to the endplates through the annulus fibrosus (AF) into the NP using a 31-gauge needle; a locking forceps clamped at 1.5 mm helped control the depth of penetration. The coccygeal segments 8 to 9 were left undisturbed as a contrast segment. In addition, all muscles and soft tissues under the skin were retracted to the same extent, and the coccygeal discs were exposed under aseptic conditions during the operation. The experimenter who did the operation was blinded to mouse grouping.
Mouse Breeding
Magnetic Resonance Imaging Examination
C57BL/6J mice were purchased from the Shanghai Slac Laboratory Animal Co. Ltd (Songjiang, Shanghai, China). The mice were housed in a temperature- and humidity-controlled environment with a 12:12-hour light-dark cycle and were allowed free access to standard rodent chow (Shanghai Slac Laboratory Animal Co. Ltd) and tap water. The use of mice in this study was reviewed and approved by the Laboratory Animal Care Committee of the Shanghai Medical College of Fudan University. All mice used in this study were 12-weekold male mice that weighed approximately 20 to 25 g. Mice with 1 allele of the SIRT1 gene deleted were obtained by crossing a SIRT1floxed/+ mouse with a universal Cre mouse (EIIA-Cre mice in the B6 background). This mouse was further bred with a C57BL/6J mouse to generate heterozygous (HT) SIRT1 knockout mice and wild-type (WT) littermates. Genomic DNA from mouse-tail fragments was periodically sampled for genotyping via polymerase chain reaction (PCR) using the Extract-N-Amp Tissue PCR kit (Sigma-Aldrich, St. Louis, MO). The PCR primer sequences for SIRT1 amplification were as follows: (Forward) 5-TTCACATTGCATGTGTGTGTG-3′ and (Reverse) 5′-TAGCCTGCGTAGTGTTGGTG-3′.
Magnetic resonance (MR) images were obtained at 2 time points: 2 and 6 weeks after the puncture. The effects of the needle puncture and SIRT1 on disc degeneration were evaluated using a Siemens Trio Tim 3.0T MR scanner (Siemens Medical Solutions, Erlangen, Germany) with a quadrature extremity coil receiver at 2 to 6 weeks after puncture. All mice were anesthetized throughout the magnetic resonance imaging (MRI) examination using a combination of 10 mg/kg xylazine and 60 mg/kg ketamine hydrochloride. The animals were laid prone, the tails were straightened in the MR scanner, and the body temperature was maintained at 37°C using circulating heated air. A serial T2-weighted sagittal plane that covered the entire experimental disc area was obtained using the following parameter settings: repetition time, 2200 ms; echo time, 66 ms; field of view, 60 mm × 60 mm; slice thickness, 0.8 mm; and the in-plane resolution, which is 135.47 mm × 135.47 mm. Disc degeneration degree was scored according to a modified Thompson classification on the basis of the changes in the degree and area of signal intensity from grades 1 to 4.20 All image assessments were independently measured by 2 experienced observers in a double-blind fashion, and the data were recorded as the mean of the 2 evaluations.
Grouping
Disc Harvest and Subsequent Examinations
The C57BL/6J mice were randomly divided into 2 groups: 8 mice received Resveratrol (RES), whereas the other 8 mice received only carboxymethyl as a control (Vehicle). RES
At 6 weeks after the puncture, the coccygeal vertebrae were isolated from the killed mice and dissected with the aid of a 5× magnifier; entire IVDs were removed en bloc from the
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BASIC SCIENCE surrounding vertebral bodies by creating an incision along the endplates using a surgical no. 11 blade. The IVDs were fixed for histology or immediately placed in liquid nitrogen and then frozen at −80°C prior to use in real-time reversetranscriptase polymerase chain reaction analysis or western blotting. Among the discs, Co7–Co8 (punched) and Co8– Co9 (sham) sections were used for the histological analysis; RNA was extracted from 2 discs (Co5–Co6 and Co6–Co7), and the other discs were used for western blotting.
Histological Evaluation The discs were fixed in 10% neutral-buffered formalin for 1 week, decalcified in Ethylenediaminetetraacetic acid for 2 weeks, paraffin-embedded, and carefully sectioned to a 5-μm thickness. Midsagittal sections were stained with hematoxylin and eosin. The histological images were analyzed using the Olympus BX51 microscope (Olympus, Center Valley, PA). The extent of disc degeneration was investigated using a semi-quantitative grading scale according to the cellularity and morphology of the AF and NP, as well as the border between the 2 structures applied by Han et al.21 Two independent and blinded observers conducted the histological sections, and the data are presented as the mean of the 2 evaluations.
Immunohistochemical Examination For immunohistochemical examination, the sections were stained with anti-SIRT1, anti–type II collagen, anti–proliferating cell nuclear antigen (PCNA), and antip16INK4a antibodies (Abcam Plc, Cambridge, United Kingdom). The sections embedded in paraffin were deparaffinized and rehydrated and then microwaved in 0.01 mol/L sodium citrate for 15 minutes each. Next, 3% hydrogen peroxide was used to block endogenous peroxidase activity for 10 minutes, and 1% bovine serum albumin was used to block nonspecific binding sites for 30 minutes at room temperature. The sections were then incubated with the primary antibody (rabbit polyclonal IgG anti-SIRT1, amino acids 722-737 of mouse SIRT1, ab12193) overnight at 4°C. After washing, the sections were incubated at room temperature for 40 minutes in a mixture of fluorescent-labeled secondary antibody (Abcam Plc, Cambridge, United Kingdom). The type II collagen, PCNA, and p16INK4a were visualized by the diaminobenzidine method,22 and the sections were counterstained using hematoxylin. Two independent pathologists blinded to the immunohistochemical information performed the analysis of type II collagen, PCNA, and p16INK4a. Ten representative fields analyzed from each section were recorded. All sections were semi-quantitatively analyzed using the Image-Pro Plus (IPP) software, version 6.0, and the integrated optical density was measured on the images for the type II collagen.
Reverse-Transcriptase Polymerase Chain Reaction A 2-step quantitative real-time polymerase chain reaction (RT-q PCR) was performed to evaluate the SIRT1 expression in the HT and WT groups. Total RNA was extracted Spine
SIRT1 Protects Against IVD Degeneration • Xia et al
using TRIzol reagent (Invitrogen, Carlsbad, CA), and RNA was reverse-transcribed using random primers according to the manufacturer’s instructions (Thermo, K1622, Thermo Fisher Scientific Inc. Waltham, MA). Quantitative reversetranscriptase polymerase chain reaction was performed using the SYBR Green PCR Kit (Thermo F-415XL) and the Applied Biosystems 7300 RT-PCR System. Glyceraldehyde phosphate dehydrogenase was used as an endogenous control. The target gene expression levels were determined by the comparative threshold cycle method, normalized to the expression values of glyceraldehyde phosphate dehydrogenase and presented as the fold induction relative to the control. The sequences were as follows: SIRT1: Primer F 5′-TGACGCTGTGGCAGATTG-3′, R 5′-CAAGGCGAGCATAGATACCG-3′; glyceraldehyde phosphate dehydrogenase: Primer F 5′-ATCACTGCCACCCAGAAG-3′, R 5′-TCCACGACGGACACATTG-3′.
Western Blotting The samples were homogenized and centrifuged, and the protein concentration was measured using the Dc Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). The blots were incubated overnight with an anti-SIRT1 antibody (1:5000, mAb, Sigma-Aldrich, St. Louis, MO) at 4°C. A horseradish peroxidase–labeled secondary antibody (1:2500 in 5% milk TBS-T) was added, and the blots were incubated at room temperature for 1 hour. The protein bands on the western blots were visualized using an ECL Plus kit (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions and developed on film. β-Actin was detected using a mouse antiβ-actin polyclonal antibody (Sigma-Aldrich, St. Louis, MO).
Statistical Analyses
All values are expressed as the means ± SE. Statistical analyses were performed using GraphPad Prism 4.00 (GraphPad Software, Inc., San Diego, CA), with paired and unpaired Student t tests for 2 sets of data. P values of less than 0.05 were considered to be statistically significant.
RESULTS SIRT1 Expression Immunofluorescence studies revealed that SIRT1 was expressed in the coccygeal disc (Figure 1). There were no striking differences in the discs of the WT mice and the HT group, even in the AF, the NP, or the cartilage endplate. The deletion of 1 allele of SIRT1 (HT) was associated with reduced SIRT1 mRNA and protein expression in the coccygeal discs relative to the levels in the WT mice, as determined by real-time PCR (gene expression HT 0.1782 ± 0.0245 vs. WT 0.3267 ± 0.0415; P < 0.05) (Figure 2A) and immunoblotting, respectively (SIRT1 protein HT 0.1906 ± 0.0561 vs. WT 0.4009 ± 0.0823; P < 0.05) (Figure 2C). In addition, the mice treated with RES gavage expressed relatively higher SIRT1 level than the mice that received vehicle only (RES 0.4767 ± 0.0174 vs. vehicle 0.3987 ± 0.0413; P < 0.05) (Figure 2C). www.spinejournal.com
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2 groups (WT [sham] 5.125 ± 0.125 vs. HT [sham] 5.250 ± 0.164; P = 0.55). The WT group displayed a loss of disc height, whereas the border between the AF and the NP was clear, but chondrocyte-like cells could be found in the newborn fibrocartilaginous tissue that replaced the NP tissue; however, the loss of 1 allele of SIRT1 significantly aggravated puncture-induced injury. The staining of HT mouse discs demonstrated that fibers were ruptured in the AF, the disc height was severely decreased, and the NP area was completely occupied by fibrocartilaginous tissue with almost no chondrocyte-like cells (Figure 6A). HT mice exhibited significantly higher levels of injury score than the WT controls (HT 10.88 ± 0.398
Figure 1. SIRT1 allocation in disc samples by immunofluorescence. Representative pictures are both from heterozygous SIRT1 knockout mice (HT) and wild-type (WT) littermates (×100). AF indicates annulus fibrosus; WT, wild-type; HT, heterozygous; NP, nucleus pulposus; EP, endplate.
MRI Assessment of the Coccygeal Disc Degeneration MR images obtained 2 weeks after puncture had stronger T2-weighted signal intensities in the RES group than in the vehicle group. Similar results were also observed at 6 weeks. Representative MR images of the punctured discs revealed restoration of the signal intensities and areas of the discs in the RES-treated mice compared with the vehicle-treated mice. The Thompson MRI grade scores, which indicate the degree of disc degeneration, were significantly lower in the REStreated mice than in the Vehicle (2 wk: RES 1.375 ± 0.3536 vs. Vehicle 2.250 ± 0.2673; P < 0.001) (6 wk: RES 1.750 ± 0.3780 vs. Vehicle 2.375 ± 0.2315; P < 0.05, Figures 3A and 4A). In addition, the scores of the HT mouse discs were significantly higher than those of the WT group (2 wk: HT 3.438 ± 0.3204 vs. WT 2.938 ± 0.3204; P < 0.01; 6 wk: HT 3.688 ± 0.3720 vs. WT 2.938 ± 0.4173; P = 0.02, Figures 3B and 4B). Figure 5 shows that the Thompson MRI grade scores both in 2 weeks and 6 weeks indicate that the score of the HT group remained lower than that of the WT littermates, even in 6 weeks.
Histological Assessment Hematoxylin and eosin staining of nonpunctured normal discs revealed clear demarcations between the NP and the AF as well as abundant extracellular matrix (ECM) in both WT mice and HT mice. NP cells consisted mainly of large, vacuolated notochordal cells and chondrocyte-like cells. In punctured discs from the RES group, HE staining showed that the IVD structure was nearly maintained; NP was filled with a gelatinous matrix and the border between AF and NP was basically clear. However, the NP area decreased to some extent and became irregular in sections from mice treated with Vehicle. In addition, serpentine-patterned fibers in the AF were easily detected. The injury score in 2 sham operation groups was low, and there is no significant difference between E518
Figure 2. SIRT1 expression in coccygeal discs. A, SIRT1 mRNA expression in the coccygeal discs was examined by quantitative RT-PCR. The samples were extracted from Co5–Co6 and Co6–Co7 discs of each mouse in groups HT and WT (n = 8). B, Representative bands from western blotting for SIRT1 in the coccygeal discs of all mice in the 4 groups. C, Quantitative data of SIRT1 protein level in 4 groups (n = 8/ group, densitometry). WT indicates wild-type; HT, heterozygous.
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SIRT1 Protects Against IVD Degeneration • Xia et al
Figure 8A shows the expression of p16INK4a. Positive cells could be detected in all specimens, but this staining was least frequently located in the nonpunctured discs. p16INK4a expression was significantly less in the RES-treated group than in the Vehicle-treated group (RES 6.5 ± 1.003 vs Vehicle 11.4 ± 0.9911; P < 0.01) (Figure 8B). The expression level in HT group was higher than that in WT group (HT 16.1 ± 1.400 vs WT 10.1 ± 1.100; P < 0.001) (Figure 8B). Immunohistochemical analysis of PCNA expression revealed that positive cells were barely present in the discs of the HT mice, whereas only several PCNA-positive cells appeared in the WT and vehicle-treated mice (Figure 9A). A higher number of positive cells were identified in the segments of the RES-treated mice than in the Vehicle-treated group (RES 6.1 ± 0.5677 vs Vehicle 4.1 ± 0.3786; P < 0.01) (Figure 9B). Almost no PCNA-positive cells could be detected in the nonpunctured discs (Figure 9B).
DISCUSSION
SIRT1 is an NAD+-dependent deacetylase with a wide range of substrates, such as p53, NF-κB, FOXO transcription factors, and PGC-1α, and has been reported to mediate
Figure 3. MRI evaluation at 2 weeks. A, Four representative T2-weighted magnetic resonance images of each group (the original in-plane resolution is 135.47 mm × 135.47 mm). Blue arrows indicate that Co7–Co8 discs received needle puncture and yellow arrows show that Co8–Co9 discs received sham operation. B, Quantitative data of MRI grade based on modified Thompson classification20 in 4 groups (n = 8/group). HT indicates heterozygous; WT, wild-type; MRI, magnetic resonance imaging.
vs. WT 7.250 ± 0.313; P < 0.001). In contrast, the SIRT1 activator RES but not the Vehicle significantly reduced injury in mice after puncture (injury score RES 5.75 ± 0.313 vs. Vehicle 7.375 ± 0.498; P < 0.05) (Figure 6B).
Immunohistochemical Assessment The representative images for type II collagen staining are shown in Figure 7A. Sham segments revealed a substantially stronger stain than the punctured segments in all 4 groups. The staining in the WT mice after needle puncture was stronger than that in the HT mice (HT 1805 ± 122.9 vs WT 2748 ± 112.2; P < 0.001) (Figure 7B), whereas the degree of staining was nearly the same between the nonpunctured WT and HT mice. In addition, the integrated optical density score of type II collagen was higher in the RES-treated segments than in the Vehicle-treated segments (RES 3627 ± 160.3 vs Vehicle 2667 ± 176.9; P < 0.001) (Figure 7B). Besides, the 2 sham operation groups showed much higher scores (WT [sham] 4925 ± 122.2 vs HT [sham] 4852 ± 140.6; P = 0.70) (Figure 7B). Spine
Figure 4. MRI evaluation at 6 weeks. A, Four representative T2-weighted magnetic resonance images of each group (the original in-plane resolution is 135.47 mm × 135.47 mm). B, Quantitative data of MRI grades in 4 groups (n = 8/group). HT indicates heterozygous; WT, wild-type; MRI, magnetic resonance imaging. www.spinejournal.com
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SIRT1 Protects Against IVD Degeneration • Xia et al
the MRI T2-weighted signal intensity is one of the most usual and practical parameters to assess the degree of disc degeneration.41,42 Different levels of decrease in the T2-weighted signal images indicate different grades of disc degeneration because of the loss of water content in NP.43,44 Our results showed a loss of signal intensity and area from slight to severe on the T2-weighted midsagittal images in the punctured discs from different groups. The T2-weighted signal intensity of the RES group demonstrated a minimal decrease, whereas the HT mice showed the most severe degeneration. The histological
Figure 5. MRI evaluation of punctured discs at 2 weeks and 6 weeks. The Thompson MRI grade scores in 4 groups at 2 weeks and 6 weeks. MRI indicates magnetic resonance imaging; WT, wild-type; HT, heterozygous.
numerous physiologic events, including transcriptional silencing, cell survival, and an extension of the life span after caloric restriction.23–26 William Giblin et al thought that sirtuins not only extend the life span but also extend the health span.27 In mammals, interventions that slow the aging process concomitantly retard the onset and pace of different types of degenerative and metabolic diseases.28 SIRT1 has been demonstrated to play a crucial part in many pathophysiological processes, especially age-related diseases, such as neurodegenerative disorders, chronic obstructive pulmonary disease, different types of cancer, and osteoarthritis, because of its involvement of the stress responses, DNA repair, and inflammation.29–35 IVD degeneration is also a type of degenerative disease that is closely related to aging, but, to date, very little attention has been paid to whether SIRT1 slows IVD degeneration. In the present study, the HT mice and their WT littermates were used to enable genetic and environmental variables to be controlled. Total body SIRT1 knockout mice (SIRT1−/−) were not used in the present study because they are significantly smaller than their WT littermates and cannot survive for more than 1 month postnatal,36 whereas HT SIRT1 knockout mice offer more homogeneity. In addition, a mouse model of human disc degeneration was chosen as mouse discs have been confirmed to be one of the most similar to human discs both proportionally and geometrically among all animal models tested.37 The annulus puncture model is a practical modeling method. Previous studies have shown that needle puncture can induce clear biochemical and molecular changes.38,39 First, we found that SIRT1 is expressed in the coccygeal disc, and the deletion of 1 allele of SIRT1 resulted in significantly lower mRNA and protein expression, whereas RES helped increase the expression of SIRT1 protein. As a natural activator of SIRT1, RES has been proven to have the ability to stimulate the synthesis of the ECM in degenerative human NP cells via upregulation of the expression of SIRT1 at mRNA and protein levels.40 In addition, we evaluated the degree of murine disc degeneration using both MRI and histological analyses. The change in E520
Figure 6. Histological evaluation of coccygeal discs. A, Representative section HE staining pictures of each group, including sham discs (×40). B, Quantitative data of histological grades in all 4 operation groups and 2 sham groups (n = 8/group). WT indicates wild-type; HT, heterozygous.
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SIRT1 Protects Against IVD Degeneration • Xia et al
Figure 8. p16INK4a staining in coccygeal discs. A, Four representative p16INK4a immunohistochemical staining pictures from each group (×400). B, Semi-quantitative data of p16-positive cells in all 4 groups that were quantified by counting p16-positive cells in 10 random highpower fields (n = 10*8). NP indicates nucleus pulposus; AF, annulus fibrosus; WT, wild-type; HT, heterozygous. The arrows indicate p16INK4a positive cells in specimen.
Figure 7. Expression of type II collagen in coccygeal discs. A, Representative type II collagen immunohistochemical images from each group including sham discs (×100). B, Quantitative data of type II collagen integrated optical density scores in 4 operation groups and 2 sham groups (n = 8/group). NP indicates nucleus pulposus; EP, endplate; WT, wild-type; HT, heterozygous; AF, annulus fibrosus.
assessments were consistent with the imaging findings, and the morphological changes in the NP area were most outstanding. The NP was filled with a gelatinous substance in the disc treated with RES, whereas the NP in HT mouse discs was occupied by a fiber-like tissue with few chondrocyte-like cells. Therefore, these 2 different valuation methodologies indicated that SIRT1 activation reduces puncture-induced injury, whereas the loss of 1 allele of SIRT1 enhances IVD degeneration after puncture. Besides, MR images were used to give a continuous monitoring of disc degeneration. We compared the Thompson grade of disc degeneration for each group at Spine
different time points; only HT group showed further deterioration at the sixth week when compared with 2 weeks. This phenomenon indicates that decreased SIRT1 level leads to not only more serious disc degeneration but also to impaired recovering capability. A wide range of research initially demonstrated the function of SIRT1 in articular chondrocytes. Matsuzaki et al34 verified that the disruption of SIRT1 in chondrocytes caused an accelerated progression of osteoarthritis under mechanical stress and during aging in mice. Dvir-Ginzberg et al35 demonstrated that the expression of some cartilage-specific genes, such as aggrecan, collagen type 2, and collagen type 9, were in proportion to the SIRT1 protein expression levels. These reports suggest that SIRT1 plays a vital role in maintaining the stability of articular cartilage cells. NP cells in the IVDs originate from the hyaline cartilage endplate44,45 and share some similarity with chondrocytes in articular cartilage. The NP cell environment is characterized by low nutrition levels and oxygen tension and is www.spinejournal.com
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Figure 9. PCNA-positive nucleus pulposus cells in coccygeal discs. A, Representative PCNA immunohistochemical staining pictures from RES group and Vehicle group (×400). B, Semi-quantitative data of PCNA-positive cells that were quantified by counting PCNA-positive cells in 10 random high-power fields (n = 10*8). NP indicates nucleus pulposus; PCNA, proliferating cell nuclear antigen; WT, wild-type; HT, heterozygous. The arrows indicate p16INK4a positive cells in specimen.
dependent upon diffusion across the AF and cartilaginous endplates.46–50 Therefore, NP cells are under a stressful environment of nutrient reduction during the process of IVD degeneration. In some in vitro studies, SIRT1 has been verified to affect both the ECM metabolism and the proliferation. It also seems to function in homeostasis during the process of human disc degeneration.18 Our study suggested that SIRT1 activation increased the expression of type II collagen, whereas the loss of 1 allele of SIRT1 showed the opposite effect; these findings indicated that SIRT1 helps maintain the normal components of the ECM in vivo and may have a similar effect during the process of IVD degeneration as during osteoarthritis. A few reports have revealed that the population of the senescent disc cells increases with advancing disc degeneration.51,52 In addition, senescent cells can persist and acquire altered functions, and this characteristic alters the tissue microenvironments in ways that can promote aging phenotypes.53,54 Significant accumulation of p16 expression has been widely observed in a variety of aging tissues in animal models and humans, thus indicating that p16 plays an important role in aging and can also serve as a potent aging biomarker. As a result, increased p16 expression identified in the punctured disc compared with the nonpunctured segments revealed that the expression of p16 was increased in the degenerative discs. Furthermore, SIRT1 activation downregulated the expression E522
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of p16 in disc cells, whereas the mice with 1 allele of SIRT1 presented more p16 expression in their injured discs, which indicates that SIRT1 functioned in retarding disc degeneration through p16 pathway by preventing cellular senescence. As we know, p16 often correlates with both replicative senescence and oncogene-induced senescence because it plays an important role in cell cycle regulation by decelerating cells’ progression from G1 phase to S phase. So, we also used PCNA as a marker for detecting cell proliferation in our experiment. Our results demonstrated that higher level of SIRT1 is related to increased cell proliferation. Above all, SIRT1 may lead to the suppression of p53 through deacetylation, thus resulting in the inactivation of p16, which plays a role in the proliferative ability of the senescent NP cells. Therefore, the lack of proliferation in senescent NP cells is a significant factor in disc degeneration, and SIRT1 activation could help retard the process of disc degeneration. Kwon39 demonstrated that disc injection of RES has anabolic effects on degenerated discs induced by puncture in rabbits. Our research used oral gavage as the administration route to avoid secondary injury. These results revealed that SIRT1 had a protective effect on disc degeneration and RES gavage could be an effective route for SIRT1 activation. However, there are some limitations in the present research. In this study, RES was administered at 100 mg/kg body weight twice per day according to published data; however, we have not performed a pharmacokinetic study. Further study should be conducted to determine the proper dosage. The present study provides valuable information regarding the protective role of SIRT1 in IVD degeneration. We also explored the potential mechanisms of this function; however, additional studies are needed to clarify and confirm this unique mechanism. Although biological treatment approaches to disc repair remain in the early stages, advances in our understanding of disc biochemistry and the role of genetic inheritance have provided an initiation point for the development of new concepts in the diagnosis, therapy, and prevention of disc degeneration.10,54
CONCLUSION In summary, the present data identify SIRT1 as a protective mediator in puncture-induced IVD degeneration. The SIRT1 activator RES protects against IVD injury by promoting regeneration, whereas the SIRT1 deficiency worsens IVD degeneration after needle puncture. These studies suggest that SIRT1 plays a preventive role against IVD degeneration by inducing cell proliferation, and these results may contribute to new therapeutic approaches for disc degeneration.
➢ Key Points Genetic ablation of 1 allele (SIRT1+/−) in mice enhanced the level of disc degeneration compared with the wild-type (SIRT1+/+) mice through magnetic resonance imaging evaluation and histological evaluation.
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BASIC SCIENCE Administration of Resveratrol, as SIRT1 activator, protected against puncture-induced disc degeneration. Increased expression of SIRT1 leads to the suppression of p16, which plays a role in elevating the decreased proliferative ability of the senescent nucleus pulpous cells.
Acknowledgments The authors thank Dr. Chuanming Hao, Department of Nephrology, Huashan Hospital, for providing heterozygous SIRT1 knockout mice.
References
1. Praemer A, Furner S, Rice DP. Musculoskeletal Conditions in the United States. Rosemont, IL: American Academy of Orthopaedic Surgeons; 1999. 2. An HS, Masuda K, Inoue N. Intervertebral disc degeneration: biological and biomechanical factors. J Orthop Sci 2006;11:541–52. 3. Seki S, Kawaguchi Y, Chiba K, et al. A functional SNP in CILP, encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease. Nat Genet 2005;37:607–12. 4. Kim KW, Ha KY, Lee JS, et al. The apoptotic effects of oxidative stress and antiapoptotic effects of caspase inhibitors on rat notochordal cells. Spine 2007;32:2443–8. 5. Poveda L, Hottiger M, Boos N, et al. Peroxynitrite induces gene expression in intervertebral disc cells. Spine 2009;34:1127–33. 6. Chujo T, An HS, Akeda K, et al. Effects of growth differentiation factor-5 on the intervertebral disc—in vitro bovine study and in vivo rabbit disc degeneration model study. Spine (Phila Pa 1976) 2006;31:2909–17. 7. Kandel R, Roberts S, Urban JP. Tissue engineering and the intervertebral disc: the challenges. Eur Spine J 2008;17(suppl 4):480–91. 8. Kwon YJ, Lee JW, Moon EJ, et al. Anabolic effects of Peniel 2000, a peptide that regulates TGF-β1 signaling on intervertebral disc degeneration. Spine (Phila Pa 1976) 2013;38:E49–58. 9. Nishida K, Kang JD, Gilbertson LG, et al. Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene. Spine (Phila Pa 1976) 1999;24:2419–25. 10. Bae WC, Masuda K. Emerging technologies for molecular therapy for intervertebral disk degeneration. Orthop Clin North Am 2011;42:585–601. 11. Blander G, Guarente L. The Sir2 family of protein deacetylases. Annu Rev Biochem 2004;73:417–35. 12. Rahman S, Islam R. Mammalian Sirt1: insights on its biological functions. Cell Commun Signal 2011;9:11. 13. Carafa V, Nebbioso A, Altucci L. Sirtuins and disease: the road ahead. Front Pharmacol 2012;3:4. 14. Boos N, Weissbach S, Rohrbach H, et al. Classification of agerelated changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine (Phila Pa 1976) 2002;27:2631–44. 15. Erkintalo MO, Salminen JJ, Alanen AM, et al. Development of degenerative changes in the lumbar intervertebral disk: results of a prospective MR imaging study in adolescents with and without low-back pain. Radiology 1995;196;529–33. 16. Salminen JJ, Erkintalo MO, Pentti J, et al. Recurrent low back pain and early disc degeneration in the young. Spine (Phila Pa 1976) 1999;24:1316–21. 17. Banks AS, Kon N, Knight C, et al. SirT1 gain of function increases energy efficiency and prevents diabetes in mice. Cell Metab 2008;8:333–41. 18. Zhang Z, Kakutani K, Maeno K, et al. Expression of silent mating type information regulator 2 homolog 1 and its role in human intervertebral disc cell homeostasis. Arthritis Res Ther 2011;13:R200.
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19. Yang F, Leung VY, Luk KD, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. J Pathol 2009;218:113–21. 20. Masuda K, Aota Y, Muehleman C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976) 2005;30:5–14. 21. Han B, Zhu K, Li FC, et al. A simple disc degeneration model induced by percutaneous needle puncture in the rat tail. Spine 2008;33:1925–34. 22. Zhang H, La Marca F, Hollister SJ, et al. Developing consistently reproducible intervertebral disc degeneration at rat caudal spine by using needle puncture. J Neurosurg Spine 2009;10:522–30. 23. Bordone L, Guarente L. Calorie restriction, SIRT1 and metabolism: understanding longevity. Nat Rev Mol Cell Biol 2005;6:298–305. 24. Cohen HY, Miller C, Bitterman KJ, et al. Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science 2004;305:390–2. 25. Dali-Youcef N, Lagouge M, Froelich S, et al. Sirtuins: the ‘magnificent seven’, function, metabolism and longevity. Ann Med 2007;39:335–45. 26. Guarente L. Sirtuins in aging and disease. Cold Spring Harbor Symp Quant Biol 2007;72:483–8. 27. Giblin W, Skinner ME, Lombard DB. Sirtuins: guardians of mammalian healthspan. Trends Genet 2014;30:271–86. 28. Lombard DB, Miller RA. Aging, disease, and longevity in mice. Annu Rev Gerontol Geriatr 2014;34:93–138. 29. Gagarina V, Gabay O, Dvir-Ginzberg M, et al. SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin-like growth factor receptor pathway. Arthritis Rheum 2010;62:1383–92. 30. Guarente L, Franklin H. Epstein lecture: sirtuins, aging, and medicine. N Engl J Med 2011;364:2235–44. 31. Kim S, Bi X, Czarny-Ratajczak M, et al. Telomere maintenance genes SIRT1 and XRCC6 impact age-related decline in telomere length but only SIRT1 is associated with human longevity. Biogerontology 2012;13:119–31. 32. Satoh A, Stein L, Imai S. The role of mammalian sirtuins in the regulation of metabolism, aging, and longevity. Handb Exp Pharmacol 2011;206:125–62. 33. Haigis MC, Sinclair DA. Mammalian sirtuins: biological insights and disease relevance. Annu Rev Pathol 2010;5:253–95. 34. Matsuzaki T, Matsushita T, Takayama K, et al. Disruption of Sirt1 in chondrocytes causes accelerated progression of osteoarthritis under mechanical stress and during ageing in mice. Ann Rheum Dis 2014;73:1397–1404. 35. Dvir-Ginzberg M, Gagarina V, Lee EJ, et al. Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase. J Biol Chem 2008;283: 36300–10. 36. McBurney MW, Yang X, Jardine K, et al. The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis. Mol Cell Biol 2003;23:38–54. 37. O’Connell GD, Vresilovic EJ, Elliott DM. Comparison of animals used in disc research to human lumbar disc geometry. Spine 2007;32:328–33. 38. Ohta R, Tanaka N, Nakanishi K, et al. Heme oxygenase-1 modulates degeneration of the intervertebral disc after puncture in Bach 1 deficient mice. Eur Spine J 2012;21:1748–57. 39. Kwon YJ. Resveratrol has anabolic effects on disc degeneration in a rabbit model. J Korean Med Sci 2013;28:939–45. 40. Shen JL, Hu ZM, Zhong XM, et al. Resveratrol stimulates extracellular matrix synthesis in degenerative nucleus pulposus cells via upregulation of SIRT1. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2012;28:1146–50. 41. Crevensten G, Walsh AJ, Ananthakrishnan D, et al. Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs. Ann Biomed Eng 2004;32:430–4. 42. Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen www.spinejournal.com
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to the degenerated intervertebral disc. Biomaterials 2006;27:335– 45. Roberts N, Gratin C, Whitehouse GH. MRI analysis of lumbar intervertebral disc height in young and older populations. J Magn Reson Imaging 1997;7:880–6. Schiebler ML, Camerino VJ, Fallon MD, et al. In vivo and ex vivo magnetic resonance imaging evaluation of early disc degeneration with histopathologic correlation. Spine (Phila Pa 1976) 1991;16:635–40. Ishii T, Tsuji H, Sano A, et al. Histochemical and ultrastructural observations on brown degeneration of human intervertebral disc. J Orthop Res 1991;9:78–90. Holm S, Maroudas A, Urban JP, et al. Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res 1981;8:101–19. Diamant B, Karlsson J, Nachemson A. Correlation between lactate levels and pH in discs of patients with lumbar rhizopathies. Experientia 1968;24:1195–6.
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48. Nachemson A. Intradiscal measurements of pH in patients with lumbar rhizopathies. Acta Orthop Scand 1969;40: 23–42. 49. Zhao CQ, Wang LM, Jiang LS, et al. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev 2007;6:247–61. 50. Takayama K, Ishida K, Matsushita T, et al. SIRT1 regulation of apoptosis of human chondrocytes. Arthritis Rheum 2009;60:2731–40. 51. Campisi J. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 2005;120:513–22. 52. Krtolica A, Campisi J. Cancer and aging: a model for the cancer promoting effects of the aging stroma. Int J Biochem Cell Biol 2002;34:1401–14. 53. Rodier F, Campisi J, Bhaumik D. Two faces of p53: aging and tumor suppression. Nucleic Acids Res 2007;35:7475–84. 54. Masuda K. Biological repair of the degenerated intervertebral disc by the injection of growth factors. Eur Spine J 2008;17(suppl 4):441–51.
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BASIC SCIENCE
SIRT1 Plays a Protective Role in Intervertebral Disc Degeneration in a Puncture-induced Rodent Model Xinlei Xia, MD, PhD, Ji Guo, MD, Feizhou Lu, MD, PhD, and Jianyuan Jiang, MD
Study Design. Experimental animal study of treatment of intervertebral disc (IVD) degeneration. Objective. This report aims to evaluate the in vivo effects of SIRT1 on IVD biology and to explore its potential mechanism. Summary of Background Data. Silent mating type information regulator 2 homolog 1 (SIRT1) has attracted immense attention because of its functions in a variety of aging-related diseases. Despite previous studies indicated that SIRT1 showed a unique expression with degeneration in some in vitro study, there is no in vivo research on the role SIRT1 plays in IVD and its mechanism. Methods. Coccygeal discs were punctured to induce disc degeneration. Sixteen C57BL/6J mice received either Carboxy methocel (Vehicle) or Resveratrol (RES) gavage. Eight SIRT1+/− mice and their SIRT1+/+ littermates were also used in this study. At 2 and 6 weeks after puncture, magnetic resonance images were obtained. The mice were subsequently killed, and the spine was extracted for further evaluation. Results. Coccygeal disc puncture caused IVD degeneration in the mice. A SIRT1 activator, RES, markedly ameliorated this pathological change, as demonstrated by stronger signal intensity in the T2-weighted images, as well as a significantly lower magnetic resonance imaging grade (at 2 wk vs. Vehicle group P < 0.001). Histological analysis also revealed an improvement in the RES group compared with the Vehicle group (P < 0.05). Genetic ablation of 1 allele significantly enhanced the level of damage relative to the wild-type mice. In addition, SIRT1 activation suppressed the
From the Huashan Hospital, Fudan University, Shanghai, People’s Republic of China. Acknowledgment date: August 4, 2014. First revision date: December 4, 2014. Second revision date: January 5, 2015. Acceptance date: January 10, 2015. The manuscript submitted does not contain information about medical device(s)/drug(s). Shanghai Science and Technology Development Funds (grant No: 12ZR1403900) and AOSpine China Research Grant (Project No: AOSCN(R)2013-12) funds were received to support this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Feizhou Lu, MD, PhD, Orthopeadics Department, Huashan Hospital, Fudan University, No. 12 Wulumuqi Mid Rd, Shanghai, 200040, People’s Republic of China. E-mail: [email protected] DOI: 10.1097/BRS.0000000000000817 Spine
expression of p16 and at the same time, promoted proliferating cell nuclear antigen and type II collagen expression in disc cells, whereas genetic ablation of 1 allele SIRT1 exhibited the opposite consequence. Conclusion. The SIRT1 activator RES protects against punctureinduced disc injury whereas SIRT1 deficiency aggravates tissue injury; the protective role of SIRT1 is partly mediated by suppressing p16, which plays a role in elevating the decreased proliferative ability of the senescent nucleus pulposus cells. Key words: intervertebral disc (IVD) degeneration, SIRT1, Resveratrol, annulus puncture, cellular senescence. Level of Evidence: N/A Spine 2015;40:E515–E524
T
he high morbidity of low back pain causes a severe incapacity that increases medical expenses and impacts the workforce, thereby resulting in high socioeconomic costs and human suffering. It is reported that more than 400,000 individuals are unable to work properly because of low back pain in the United States each year.1 Although the exact pathomechanism of low back pain remains poorly understood, intervertebral disc (IVD) degeneration has been thought to be a major cause. A number of diverse etiological factors are thought to serve as primary initiating events that lead to IVD degeneration. These factors include genetic predisposition, smoking, infection, abnormal biomechanical loading, decreased nutrient transport across the vertebral endplates, and cell senescence.2–5 However, the relative importance of each of these factors in IVD degeneration is currently unknown. In addition, the precise pathogenesis of IVD degeneration and its relationship to cellular senescence is unclear as well. Most therapeutic methods for disc diseases continue to concentrate on the treatment of the various pathological conditions that result in disc degeneration, such as surgery, medications, and steroid injections, as opposed to focusing on the prevention or reversal of the degenerative discs. Novel approaches, such as peptide or growth factor injections, gene delivery, and tissue-engineering techniques,6–9 have been proposed to manage the underlying causes of IVD degeneration, but significant limitations exist for these therapies. In addition, many approaches are invasive to the disc, and the ability to transfer results in animal models to human patients remains unclear. www.spinejournal.com
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BASIC SCIENCE Therefore, additional clinical trials are needed to establish the safety and efficacy of these innovative treatments.10 Silent information regulator 2 proteins (sirtuins) are a group of nicotinamide (NAD+)-dependent deacetylases that were initially discovered in yeast.11,12 A human homolog of silent information regulator 2, silent mating-type information regulator 2 homolog 1 (SIRT1), has been reported to play a key role in transcriptional silencing, genome stability, and an extended life span after calorie restriction because of its ability to deacetylate both histone and nonhistone substrates, such as p53, NF-κB, and forkhead box type O-3. Recent studies determined that SIRT1 is crucial in the pathophysiology of metabolic disease, degenerative disorders, cancer, and aging.13 IVD is also a type of degenerative process that can occur quite early even in adolescents.14–16 Several studies have shown that SIRT1 expression decreased significantly with serial cell passage in human and murine cells; a significant positive correlation between the level of SIRT1 and cell proliferation was also found.17 A recent study18 demonstrated that SIRT1 can be expressed in cultured human nucleus pulpous (NP) cells, and the SIRT1 mRNA expression levels significantly decreased with age. Therefore, we hypothesized that SIRT1 may play an important role in IVD degeneration via its regulation of cellular senescence. In the present study, we examined IVD susceptibility to puncture-induced injury under various SIRT1 expression levels.
SIRT1 Protects Against IVD Degeneration • Xia et al
purchased from Sigma (Sigma-Aldrich, St. Louis, MO) was prepared as a solution in carboxymethyl cellulose. The mice were treated with 100 mg RES/kg body weight or carboxymethyl via oral gavage twice per day. The treatment was administered at 1 and 4 weeks after puncture, and each time period lasted for 7 days. Twelve-week-old SIRT1+/+ (WT) and SIRT1+/− (HT) mice were also used in this study (n = 8 per group).
The Annulus Needle Puncture Model
MATERIALS AND METHODS
After an intraperitoneal anesthetic injection of a combination of 10 mg/kg xylazine and 60 mg/kg ketamine hydrochloride, mice were prone on the experiment table, a sagittal small skin incision was performed from Co7 to Co9 to help locate the disc position for needle insertion in the tail. Next, Co7–Co8 coccygeal discs were punctured using a syringe needle following a previously described method.19 The syringe needle was inserted into Co7–Co8 discs along vertical direction and then rotated in the axial direction by 180° and held for 5 seconds, The puncture was made parallel to the endplates through the annulus fibrosus (AF) into the NP using a 31-gauge needle; a locking forceps clamped at 1.5 mm helped control the depth of penetration. The coccygeal segments 8 to 9 were left undisturbed as a contrast segment. In addition, all muscles and soft tissues under the skin were retracted to the same extent, and the coccygeal discs were exposed under aseptic conditions during the operation. The experimenter who did the operation was blinded to mouse grouping.
Mouse Breeding
Magnetic Resonance Imaging Examination
C57BL/6J mice were purchased from the Shanghai Slac Laboratory Animal Co. Ltd (Songjiang, Shanghai, China). The mice were housed in a temperature- and humidity-controlled environment with a 12:12-hour light-dark cycle and were allowed free access to standard rodent chow (Shanghai Slac Laboratory Animal Co. Ltd) and tap water. The use of mice in this study was reviewed and approved by the Laboratory Animal Care Committee of the Shanghai Medical College of Fudan University. All mice used in this study were 12-weekold male mice that weighed approximately 20 to 25 g. Mice with 1 allele of the SIRT1 gene deleted were obtained by crossing a SIRT1floxed/+ mouse with a universal Cre mouse (EIIA-Cre mice in the B6 background). This mouse was further bred with a C57BL/6J mouse to generate heterozygous (HT) SIRT1 knockout mice and wild-type (WT) littermates. Genomic DNA from mouse-tail fragments was periodically sampled for genotyping via polymerase chain reaction (PCR) using the Extract-N-Amp Tissue PCR kit (Sigma-Aldrich, St. Louis, MO). The PCR primer sequences for SIRT1 amplification were as follows: (Forward) 5-TTCACATTGCATGTGTGTGTG-3′ and (Reverse) 5′-TAGCCTGCGTAGTGTTGGTG-3′.
Magnetic resonance (MR) images were obtained at 2 time points: 2 and 6 weeks after the puncture. The effects of the needle puncture and SIRT1 on disc degeneration were evaluated using a Siemens Trio Tim 3.0T MR scanner (Siemens Medical Solutions, Erlangen, Germany) with a quadrature extremity coil receiver at 2 to 6 weeks after puncture. All mice were anesthetized throughout the magnetic resonance imaging (MRI) examination using a combination of 10 mg/kg xylazine and 60 mg/kg ketamine hydrochloride. The animals were laid prone, the tails were straightened in the MR scanner, and the body temperature was maintained at 37°C using circulating heated air. A serial T2-weighted sagittal plane that covered the entire experimental disc area was obtained using the following parameter settings: repetition time, 2200 ms; echo time, 66 ms; field of view, 60 mm × 60 mm; slice thickness, 0.8 mm; and the in-plane resolution, which is 135.47 mm × 135.47 mm. Disc degeneration degree was scored according to a modified Thompson classification on the basis of the changes in the degree and area of signal intensity from grades 1 to 4.20 All image assessments were independently measured by 2 experienced observers in a double-blind fashion, and the data were recorded as the mean of the 2 evaluations.
Grouping
Disc Harvest and Subsequent Examinations
The C57BL/6J mice were randomly divided into 2 groups: 8 mice received Resveratrol (RES), whereas the other 8 mice received only carboxymethyl as a control (Vehicle). RES
At 6 weeks after the puncture, the coccygeal vertebrae were isolated from the killed mice and dissected with the aid of a 5× magnifier; entire IVDs were removed en bloc from the
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BASIC SCIENCE surrounding vertebral bodies by creating an incision along the endplates using a surgical no. 11 blade. The IVDs were fixed for histology or immediately placed in liquid nitrogen and then frozen at −80°C prior to use in real-time reversetranscriptase polymerase chain reaction analysis or western blotting. Among the discs, Co7–Co8 (punched) and Co8– Co9 (sham) sections were used for the histological analysis; RNA was extracted from 2 discs (Co5–Co6 and Co6–Co7), and the other discs were used for western blotting.
Histological Evaluation The discs were fixed in 10% neutral-buffered formalin for 1 week, decalcified in Ethylenediaminetetraacetic acid for 2 weeks, paraffin-embedded, and carefully sectioned to a 5-μm thickness. Midsagittal sections were stained with hematoxylin and eosin. The histological images were analyzed using the Olympus BX51 microscope (Olympus, Center Valley, PA). The extent of disc degeneration was investigated using a semi-quantitative grading scale according to the cellularity and morphology of the AF and NP, as well as the border between the 2 structures applied by Han et al.21 Two independent and blinded observers conducted the histological sections, and the data are presented as the mean of the 2 evaluations.
Immunohistochemical Examination For immunohistochemical examination, the sections were stained with anti-SIRT1, anti–type II collagen, anti–proliferating cell nuclear antigen (PCNA), and antip16INK4a antibodies (Abcam Plc, Cambridge, United Kingdom). The sections embedded in paraffin were deparaffinized and rehydrated and then microwaved in 0.01 mol/L sodium citrate for 15 minutes each. Next, 3% hydrogen peroxide was used to block endogenous peroxidase activity for 10 minutes, and 1% bovine serum albumin was used to block nonspecific binding sites for 30 minutes at room temperature. The sections were then incubated with the primary antibody (rabbit polyclonal IgG anti-SIRT1, amino acids 722-737 of mouse SIRT1, ab12193) overnight at 4°C. After washing, the sections were incubated at room temperature for 40 minutes in a mixture of fluorescent-labeled secondary antibody (Abcam Plc, Cambridge, United Kingdom). The type II collagen, PCNA, and p16INK4a were visualized by the diaminobenzidine method,22 and the sections were counterstained using hematoxylin. Two independent pathologists blinded to the immunohistochemical information performed the analysis of type II collagen, PCNA, and p16INK4a. Ten representative fields analyzed from each section were recorded. All sections were semi-quantitatively analyzed using the Image-Pro Plus (IPP) software, version 6.0, and the integrated optical density was measured on the images for the type II collagen.
Reverse-Transcriptase Polymerase Chain Reaction A 2-step quantitative real-time polymerase chain reaction (RT-q PCR) was performed to evaluate the SIRT1 expression in the HT and WT groups. Total RNA was extracted Spine
SIRT1 Protects Against IVD Degeneration • Xia et al
using TRIzol reagent (Invitrogen, Carlsbad, CA), and RNA was reverse-transcribed using random primers according to the manufacturer’s instructions (Thermo, K1622, Thermo Fisher Scientific Inc. Waltham, MA). Quantitative reversetranscriptase polymerase chain reaction was performed using the SYBR Green PCR Kit (Thermo F-415XL) and the Applied Biosystems 7300 RT-PCR System. Glyceraldehyde phosphate dehydrogenase was used as an endogenous control. The target gene expression levels were determined by the comparative threshold cycle method, normalized to the expression values of glyceraldehyde phosphate dehydrogenase and presented as the fold induction relative to the control. The sequences were as follows: SIRT1: Primer F 5′-TGACGCTGTGGCAGATTG-3′, R 5′-CAAGGCGAGCATAGATACCG-3′; glyceraldehyde phosphate dehydrogenase: Primer F 5′-ATCACTGCCACCCAGAAG-3′, R 5′-TCCACGACGGACACATTG-3′.
Western Blotting The samples were homogenized and centrifuged, and the protein concentration was measured using the Dc Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). The blots were incubated overnight with an anti-SIRT1 antibody (1:5000, mAb, Sigma-Aldrich, St. Louis, MO) at 4°C. A horseradish peroxidase–labeled secondary antibody (1:2500 in 5% milk TBS-T) was added, and the blots were incubated at room temperature for 1 hour. The protein bands on the western blots were visualized using an ECL Plus kit (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions and developed on film. β-Actin was detected using a mouse antiβ-actin polyclonal antibody (Sigma-Aldrich, St. Louis, MO).
Statistical Analyses
All values are expressed as the means ± SE. Statistical analyses were performed using GraphPad Prism 4.00 (GraphPad Software, Inc., San Diego, CA), with paired and unpaired Student t tests for 2 sets of data. P values of less than 0.05 were considered to be statistically significant.
RESULTS SIRT1 Expression Immunofluorescence studies revealed that SIRT1 was expressed in the coccygeal disc (Figure 1). There were no striking differences in the discs of the WT mice and the HT group, even in the AF, the NP, or the cartilage endplate. The deletion of 1 allele of SIRT1 (HT) was associated with reduced SIRT1 mRNA and protein expression in the coccygeal discs relative to the levels in the WT mice, as determined by real-time PCR (gene expression HT 0.1782 ± 0.0245 vs. WT 0.3267 ± 0.0415; P < 0.05) (Figure 2A) and immunoblotting, respectively (SIRT1 protein HT 0.1906 ± 0.0561 vs. WT 0.4009 ± 0.0823; P < 0.05) (Figure 2C). In addition, the mice treated with RES gavage expressed relatively higher SIRT1 level than the mice that received vehicle only (RES 0.4767 ± 0.0174 vs. vehicle 0.3987 ± 0.0413; P < 0.05) (Figure 2C). www.spinejournal.com
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2 groups (WT [sham] 5.125 ± 0.125 vs. HT [sham] 5.250 ± 0.164; P = 0.55). The WT group displayed a loss of disc height, whereas the border between the AF and the NP was clear, but chondrocyte-like cells could be found in the newborn fibrocartilaginous tissue that replaced the NP tissue; however, the loss of 1 allele of SIRT1 significantly aggravated puncture-induced injury. The staining of HT mouse discs demonstrated that fibers were ruptured in the AF, the disc height was severely decreased, and the NP area was completely occupied by fibrocartilaginous tissue with almost no chondrocyte-like cells (Figure 6A). HT mice exhibited significantly higher levels of injury score than the WT controls (HT 10.88 ± 0.398
Figure 1. SIRT1 allocation in disc samples by immunofluorescence. Representative pictures are both from heterozygous SIRT1 knockout mice (HT) and wild-type (WT) littermates (×100). AF indicates annulus fibrosus; WT, wild-type; HT, heterozygous; NP, nucleus pulposus; EP, endplate.
MRI Assessment of the Coccygeal Disc Degeneration MR images obtained 2 weeks after puncture had stronger T2-weighted signal intensities in the RES group than in the vehicle group. Similar results were also observed at 6 weeks. Representative MR images of the punctured discs revealed restoration of the signal intensities and areas of the discs in the RES-treated mice compared with the vehicle-treated mice. The Thompson MRI grade scores, which indicate the degree of disc degeneration, were significantly lower in the REStreated mice than in the Vehicle (2 wk: RES 1.375 ± 0.3536 vs. Vehicle 2.250 ± 0.2673; P < 0.001) (6 wk: RES 1.750 ± 0.3780 vs. Vehicle 2.375 ± 0.2315; P < 0.05, Figures 3A and 4A). In addition, the scores of the HT mouse discs were significantly higher than those of the WT group (2 wk: HT 3.438 ± 0.3204 vs. WT 2.938 ± 0.3204; P < 0.01; 6 wk: HT 3.688 ± 0.3720 vs. WT 2.938 ± 0.4173; P = 0.02, Figures 3B and 4B). Figure 5 shows that the Thompson MRI grade scores both in 2 weeks and 6 weeks indicate that the score of the HT group remained lower than that of the WT littermates, even in 6 weeks.
Histological Assessment Hematoxylin and eosin staining of nonpunctured normal discs revealed clear demarcations between the NP and the AF as well as abundant extracellular matrix (ECM) in both WT mice and HT mice. NP cells consisted mainly of large, vacuolated notochordal cells and chondrocyte-like cells. In punctured discs from the RES group, HE staining showed that the IVD structure was nearly maintained; NP was filled with a gelatinous matrix and the border between AF and NP was basically clear. However, the NP area decreased to some extent and became irregular in sections from mice treated with Vehicle. In addition, serpentine-patterned fibers in the AF were easily detected. The injury score in 2 sham operation groups was low, and there is no significant difference between E518
Figure 2. SIRT1 expression in coccygeal discs. A, SIRT1 mRNA expression in the coccygeal discs was examined by quantitative RT-PCR. The samples were extracted from Co5–Co6 and Co6–Co7 discs of each mouse in groups HT and WT (n = 8). B, Representative bands from western blotting for SIRT1 in the coccygeal discs of all mice in the 4 groups. C, Quantitative data of SIRT1 protein level in 4 groups (n = 8/ group, densitometry). WT indicates wild-type; HT, heterozygous.
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Figure 8A shows the expression of p16INK4a. Positive cells could be detected in all specimens, but this staining was least frequently located in the nonpunctured discs. p16INK4a expression was significantly less in the RES-treated group than in the Vehicle-treated group (RES 6.5 ± 1.003 vs Vehicle 11.4 ± 0.9911; P < 0.01) (Figure 8B). The expression level in HT group was higher than that in WT group (HT 16.1 ± 1.400 vs WT 10.1 ± 1.100; P < 0.001) (Figure 8B). Immunohistochemical analysis of PCNA expression revealed that positive cells were barely present in the discs of the HT mice, whereas only several PCNA-positive cells appeared in the WT and vehicle-treated mice (Figure 9A). A higher number of positive cells were identified in the segments of the RES-treated mice than in the Vehicle-treated group (RES 6.1 ± 0.5677 vs Vehicle 4.1 ± 0.3786; P < 0.01) (Figure 9B). Almost no PCNA-positive cells could be detected in the nonpunctured discs (Figure 9B).
DISCUSSION
SIRT1 is an NAD+-dependent deacetylase with a wide range of substrates, such as p53, NF-κB, FOXO transcription factors, and PGC-1α, and has been reported to mediate
Figure 3. MRI evaluation at 2 weeks. A, Four representative T2-weighted magnetic resonance images of each group (the original in-plane resolution is 135.47 mm × 135.47 mm). Blue arrows indicate that Co7–Co8 discs received needle puncture and yellow arrows show that Co8–Co9 discs received sham operation. B, Quantitative data of MRI grade based on modified Thompson classification20 in 4 groups (n = 8/group). HT indicates heterozygous; WT, wild-type; MRI, magnetic resonance imaging.
vs. WT 7.250 ± 0.313; P < 0.001). In contrast, the SIRT1 activator RES but not the Vehicle significantly reduced injury in mice after puncture (injury score RES 5.75 ± 0.313 vs. Vehicle 7.375 ± 0.498; P < 0.05) (Figure 6B).
Immunohistochemical Assessment The representative images for type II collagen staining are shown in Figure 7A. Sham segments revealed a substantially stronger stain than the punctured segments in all 4 groups. The staining in the WT mice after needle puncture was stronger than that in the HT mice (HT 1805 ± 122.9 vs WT 2748 ± 112.2; P < 0.001) (Figure 7B), whereas the degree of staining was nearly the same between the nonpunctured WT and HT mice. In addition, the integrated optical density score of type II collagen was higher in the RES-treated segments than in the Vehicle-treated segments (RES 3627 ± 160.3 vs Vehicle 2667 ± 176.9; P < 0.001) (Figure 7B). Besides, the 2 sham operation groups showed much higher scores (WT [sham] 4925 ± 122.2 vs HT [sham] 4852 ± 140.6; P = 0.70) (Figure 7B). Spine
Figure 4. MRI evaluation at 6 weeks. A, Four representative T2-weighted magnetic resonance images of each group (the original in-plane resolution is 135.47 mm × 135.47 mm). B, Quantitative data of MRI grades in 4 groups (n = 8/group). HT indicates heterozygous; WT, wild-type; MRI, magnetic resonance imaging. www.spinejournal.com
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SIRT1 Protects Against IVD Degeneration • Xia et al
the MRI T2-weighted signal intensity is one of the most usual and practical parameters to assess the degree of disc degeneration.41,42 Different levels of decrease in the T2-weighted signal images indicate different grades of disc degeneration because of the loss of water content in NP.43,44 Our results showed a loss of signal intensity and area from slight to severe on the T2-weighted midsagittal images in the punctured discs from different groups. The T2-weighted signal intensity of the RES group demonstrated a minimal decrease, whereas the HT mice showed the most severe degeneration. The histological
Figure 5. MRI evaluation of punctured discs at 2 weeks and 6 weeks. The Thompson MRI grade scores in 4 groups at 2 weeks and 6 weeks. MRI indicates magnetic resonance imaging; WT, wild-type; HT, heterozygous.
numerous physiologic events, including transcriptional silencing, cell survival, and an extension of the life span after caloric restriction.23–26 William Giblin et al thought that sirtuins not only extend the life span but also extend the health span.27 In mammals, interventions that slow the aging process concomitantly retard the onset and pace of different types of degenerative and metabolic diseases.28 SIRT1 has been demonstrated to play a crucial part in many pathophysiological processes, especially age-related diseases, such as neurodegenerative disorders, chronic obstructive pulmonary disease, different types of cancer, and osteoarthritis, because of its involvement of the stress responses, DNA repair, and inflammation.29–35 IVD degeneration is also a type of degenerative disease that is closely related to aging, but, to date, very little attention has been paid to whether SIRT1 slows IVD degeneration. In the present study, the HT mice and their WT littermates were used to enable genetic and environmental variables to be controlled. Total body SIRT1 knockout mice (SIRT1−/−) were not used in the present study because they are significantly smaller than their WT littermates and cannot survive for more than 1 month postnatal,36 whereas HT SIRT1 knockout mice offer more homogeneity. In addition, a mouse model of human disc degeneration was chosen as mouse discs have been confirmed to be one of the most similar to human discs both proportionally and geometrically among all animal models tested.37 The annulus puncture model is a practical modeling method. Previous studies have shown that needle puncture can induce clear biochemical and molecular changes.38,39 First, we found that SIRT1 is expressed in the coccygeal disc, and the deletion of 1 allele of SIRT1 resulted in significantly lower mRNA and protein expression, whereas RES helped increase the expression of SIRT1 protein. As a natural activator of SIRT1, RES has been proven to have the ability to stimulate the synthesis of the ECM in degenerative human NP cells via upregulation of the expression of SIRT1 at mRNA and protein levels.40 In addition, we evaluated the degree of murine disc degeneration using both MRI and histological analyses. The change in E520
Figure 6. Histological evaluation of coccygeal discs. A, Representative section HE staining pictures of each group, including sham discs (×40). B, Quantitative data of histological grades in all 4 operation groups and 2 sham groups (n = 8/group). WT indicates wild-type; HT, heterozygous.
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SIRT1 Protects Against IVD Degeneration • Xia et al
Figure 8. p16INK4a staining in coccygeal discs. A, Four representative p16INK4a immunohistochemical staining pictures from each group (×400). B, Semi-quantitative data of p16-positive cells in all 4 groups that were quantified by counting p16-positive cells in 10 random highpower fields (n = 10*8). NP indicates nucleus pulposus; AF, annulus fibrosus; WT, wild-type; HT, heterozygous. The arrows indicate p16INK4a positive cells in specimen.
Figure 7. Expression of type II collagen in coccygeal discs. A, Representative type II collagen immunohistochemical images from each group including sham discs (×100). B, Quantitative data of type II collagen integrated optical density scores in 4 operation groups and 2 sham groups (n = 8/group). NP indicates nucleus pulposus; EP, endplate; WT, wild-type; HT, heterozygous; AF, annulus fibrosus.
assessments were consistent with the imaging findings, and the morphological changes in the NP area were most outstanding. The NP was filled with a gelatinous substance in the disc treated with RES, whereas the NP in HT mouse discs was occupied by a fiber-like tissue with few chondrocyte-like cells. Therefore, these 2 different valuation methodologies indicated that SIRT1 activation reduces puncture-induced injury, whereas the loss of 1 allele of SIRT1 enhances IVD degeneration after puncture. Besides, MR images were used to give a continuous monitoring of disc degeneration. We compared the Thompson grade of disc degeneration for each group at Spine
different time points; only HT group showed further deterioration at the sixth week when compared with 2 weeks. This phenomenon indicates that decreased SIRT1 level leads to not only more serious disc degeneration but also to impaired recovering capability. A wide range of research initially demonstrated the function of SIRT1 in articular chondrocytes. Matsuzaki et al34 verified that the disruption of SIRT1 in chondrocytes caused an accelerated progression of osteoarthritis under mechanical stress and during aging in mice. Dvir-Ginzberg et al35 demonstrated that the expression of some cartilage-specific genes, such as aggrecan, collagen type 2, and collagen type 9, were in proportion to the SIRT1 protein expression levels. These reports suggest that SIRT1 plays a vital role in maintaining the stability of articular cartilage cells. NP cells in the IVDs originate from the hyaline cartilage endplate44,45 and share some similarity with chondrocytes in articular cartilage. The NP cell environment is characterized by low nutrition levels and oxygen tension and is www.spinejournal.com
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Figure 9. PCNA-positive nucleus pulposus cells in coccygeal discs. A, Representative PCNA immunohistochemical staining pictures from RES group and Vehicle group (×400). B, Semi-quantitative data of PCNA-positive cells that were quantified by counting PCNA-positive cells in 10 random high-power fields (n = 10*8). NP indicates nucleus pulposus; PCNA, proliferating cell nuclear antigen; WT, wild-type; HT, heterozygous. The arrows indicate p16INK4a positive cells in specimen.
dependent upon diffusion across the AF and cartilaginous endplates.46–50 Therefore, NP cells are under a stressful environment of nutrient reduction during the process of IVD degeneration. In some in vitro studies, SIRT1 has been verified to affect both the ECM metabolism and the proliferation. It also seems to function in homeostasis during the process of human disc degeneration.18 Our study suggested that SIRT1 activation increased the expression of type II collagen, whereas the loss of 1 allele of SIRT1 showed the opposite effect; these findings indicated that SIRT1 helps maintain the normal components of the ECM in vivo and may have a similar effect during the process of IVD degeneration as during osteoarthritis. A few reports have revealed that the population of the senescent disc cells increases with advancing disc degeneration.51,52 In addition, senescent cells can persist and acquire altered functions, and this characteristic alters the tissue microenvironments in ways that can promote aging phenotypes.53,54 Significant accumulation of p16 expression has been widely observed in a variety of aging tissues in animal models and humans, thus indicating that p16 plays an important role in aging and can also serve as a potent aging biomarker. As a result, increased p16 expression identified in the punctured disc compared with the nonpunctured segments revealed that the expression of p16 was increased in the degenerative discs. Furthermore, SIRT1 activation downregulated the expression E522
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of p16 in disc cells, whereas the mice with 1 allele of SIRT1 presented more p16 expression in their injured discs, which indicates that SIRT1 functioned in retarding disc degeneration through p16 pathway by preventing cellular senescence. As we know, p16 often correlates with both replicative senescence and oncogene-induced senescence because it plays an important role in cell cycle regulation by decelerating cells’ progression from G1 phase to S phase. So, we also used PCNA as a marker for detecting cell proliferation in our experiment. Our results demonstrated that higher level of SIRT1 is related to increased cell proliferation. Above all, SIRT1 may lead to the suppression of p53 through deacetylation, thus resulting in the inactivation of p16, which plays a role in the proliferative ability of the senescent NP cells. Therefore, the lack of proliferation in senescent NP cells is a significant factor in disc degeneration, and SIRT1 activation could help retard the process of disc degeneration. Kwon39 demonstrated that disc injection of RES has anabolic effects on degenerated discs induced by puncture in rabbits. Our research used oral gavage as the administration route to avoid secondary injury. These results revealed that SIRT1 had a protective effect on disc degeneration and RES gavage could be an effective route for SIRT1 activation. However, there are some limitations in the present research. In this study, RES was administered at 100 mg/kg body weight twice per day according to published data; however, we have not performed a pharmacokinetic study. Further study should be conducted to determine the proper dosage. The present study provides valuable information regarding the protective role of SIRT1 in IVD degeneration. We also explored the potential mechanisms of this function; however, additional studies are needed to clarify and confirm this unique mechanism. Although biological treatment approaches to disc repair remain in the early stages, advances in our understanding of disc biochemistry and the role of genetic inheritance have provided an initiation point for the development of new concepts in the diagnosis, therapy, and prevention of disc degeneration.10,54
CONCLUSION In summary, the present data identify SIRT1 as a protective mediator in puncture-induced IVD degeneration. The SIRT1 activator RES protects against IVD injury by promoting regeneration, whereas the SIRT1 deficiency worsens IVD degeneration after needle puncture. These studies suggest that SIRT1 plays a preventive role against IVD degeneration by inducing cell proliferation, and these results may contribute to new therapeutic approaches for disc degeneration.
➢ Key Points Genetic ablation of 1 allele (SIRT1+/−) in mice enhanced the level of disc degeneration compared with the wild-type (SIRT1+/+) mice through magnetic resonance imaging evaluation and histological evaluation.
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BASIC SCIENCE Administration of Resveratrol, as SIRT1 activator, protected against puncture-induced disc degeneration. Increased expression of SIRT1 leads to the suppression of p16, which plays a role in elevating the decreased proliferative ability of the senescent nucleus pulpous cells.
Acknowledgments The authors thank Dr. Chuanming Hao, Department of Nephrology, Huashan Hospital, for providing heterozygous SIRT1 knockout mice.
References
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