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Introduction

Intervertebral disc (IVD) degenerative disease is a common disease in clinic which is also the major factor of lumbago (1,2). Currently, the main methods used to treat these diseases in clinic are nucleus pulposus removal, spinal canal decompression and degenerative spinal segmental fusion. However, these methods only relieve patient symptoms for a short period of time and their long-term efficiency is unsatisfactory, and they may cause multiple complications (3–5). Therefore, identifying a new target for the treatment of intervertebral disc degenerative diseases in order to perfect the clinical treatment strategy for the IVD degeneration is required.

Previous findings showed that the Shh-signaling pathway plays an important role in the process of the intervertebral disc development, differentiation and degeneration (6). Furthermore, the P27 gene is a significant gene in the regulation of the Shh-signal pathway (7,8). Shimura-Miura et al (9) cultured a 13-week-old male Sprague Dawley (SD) rat at fasting status for 6 and 48 h after the intervertebral disc nucleus pulposus cell, to induce cell senescence, and found that P27kip1 expression was elevated in nucleus pulposus cell, and cell percentage was significantly increased at G0/G1 stage, while apparently decreased at S stage, indicating that P27kip1 may be involved in the development process of IVD by regulating the cell cycle. To the best of our knowledge, there is currently no study showing that the P27 gene plays a role via the Shh-signaling pathway. On the basis of the successful establishment of mouse P27 gene knockout (P27−/−) of the IVD degeneration model, this experiment primarily investigates the role of P27 gene in the development process of IVD and determines whether P27 gene played a role via the Shh-signaling pathway. Subsequently, the pathogenesis and possible related molecular mechanism of IVD were further revealed genetically to provide an experimental and theoretical basis for the early prevention and treatment of IVD degeneration.

Materials and methods

Experimental animal

P27−/− IVD degeneration model of mice was established by the Animal Experiment Center of Shanghai General Hospital of Nanjing Medical University. Male and female mice were taken out for mating, and genotype identification was performed for their offspring. The 4-week-old wild-type (WT) mice (WT group, n=36) and P27−/− mice (P27−/− group, n=36) in the same brood were obtained. Mice were housed in a temperature controlled room (21±2°C) on a 12:12-h light/dark cycle (lights on at 06:00 a.m.). All mice had free access to water and food. This study was approved by the Animal Ethics Committee of Nanjing Medical University Animal Center.

Material drawing

The mice were sacrificed by the cervical dislocation when they were 4 weeks old. IVD degenerative vertebral bodies of mice were dissected, separated and 4% of paraformaldehyde was utilized to fix them overnight. Conventional dehydration and paraffin embedding were employed. The bodies were sectioned for standby application.

X-ray examination

Prior to sacrifice, at the 4th week, X-ray examination was conducted. The experimental mice inhaled isoflurane and were anesthetized transitorily, and an X-ray examination was carried out using a metal needle to guide by positioning the location of IVD in the mouses tail. The changes of the IVD height were observed, and then the intervertebral disc height index (DHI) was calculated.

Immunohistochemical staining

First conventional deparaffinage was performed for paraffin sections and then immunohistochemical staining was carried out for them, followed by observation under a light microscope (BX-42; Olympus, Tokyo, Japan).

Alkaline phosphatase (ALP) histochemical staining

After paraffin sections were processed by conventional deparaffinage and hydration, 1% MgCl2 Tris-HCl buffer solution was used to culture them at room temperature overnight, and then they were cultured for 2 h under the environment of ALP staining solution at room temperature in the dark. Water was used to wash them for a few minutes, and then methyl green was employed to restain and conventional water was then used to seal the sections.

Western blot analysis

Lysate and 1% (V/V) phenylmethylsulfonyl fluoride, respectively, were added into the IVD vertebral bodies of 4-week-old WT and P27−/− mice [vertebral body weight: lysate and 1% (V/V) phenylmethylsulfonyl fluoride weight =1:20], and the solutions were mixed well, followed by centrifugation at 10,050 × g for 15 min at 4°C. The supernatant was absorbed and full-automatic microplate reader was used to detect the protein concentration. Additionally, 30 mg protein sample was separated by lauryl sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) electrophoresis. The protein was then transferred from the gel onto nitrocellulose membrane and developed via enhanced chemiluminescence, followed by X-ray film exposure and film-developing in a dark room. β-actin was served as internal control.

Statistical analysis

Statistical software, SPSS 20.0 (IBM, SPSS, Armonk, NY, USA), was employed for analysis. The measurement data were presented as mean ± SD. The Chi-square test was used for label data and the paired sampled-test was utilized for the measurement data. For all the tests, a 5% level of significance was used to draw the conclusions.

Results

X-ray examination

At the end of the fourth week of the model establishment, the average DHI% of the WT group was 0.83±0.06 but it was not statistically significant when compared with that at 0 week of the model establishment (P>0.05) (Fig. 1). At the end of the 4th week since the model was established, the average DHI% of the P27−/− group was 0.53±0.03, indicating a significant difference when compared with the average DHI% of P27−/− at 0 week since model establishment (P>0.05) (Fig. 2).

Figure 1. Intervertebral spaces of Co6-7 (red arrow) and Co7-8 (blue arrow) of mice in the WT group were normal at the end of the 4th week. WT, wild-type.

Figure 2. Intervertebral spaces of Co6-7 (red arrow) and Co7-8 (blue arrow) of mice in P27−/− group were obviously decreased at the end of the 4th week. P27−/−, P27 gene knockout.

Result of histology

In the WT group, normal IVD staining indicated that round or elliptic nucleus pulposus tissue was evident. The collagen lamellae arrangement was normal and the boundary between nucleus pulposus and annulus fibrosus was clear. In addition, nucleus pulposus cell showed a star-like shape, and annulus fibrosus cells were fibroblast-like, which was located in the collagen fiberboard room (Fig. 3).

Figure 3. (A) Normal intervertebral disc staining in the WT group. (B) IVD staining in P27−/− group. WT, wild-type; IVD, intervertebral disc; P27−/−, P27 gene knockout.

In the P27−/− group, mouse IVD staining indicated that puncturing side annulus fibrosus was fractured with needle-tip puncture trace. Furthermore, the interlamellar architecture of annulus fibrosus distributed in disorder, showing wavy-like shape and radial direction and concentric circle-like fracture image was evident. IVD nucleus pulposus was irregularly reduced to the small volume, extracellular matrix staining in nucleus pulposus became thin, and its boundary with annulus fibrosus was unclear (Fig. 4).

Figure 4. ALP histochemical staining result. (A) IVD ALP staining micrograph. (B) Columnar statistical figure of IVD ALP positive area rate. ALP, alkaline phosphatase; IVD, intervertebral disc. **P<0.01.

Influence of P27 deficiency on mouse IVD bone mass

ALP histochemical staining revealed that the ALP-positive area of mice in the P27−/− group was significantly increased compared with that of mice in the WT group (Fig. 4A and B). In addition, where Col-I immumohistochemical staining revealed that Col-I-positive area of mice in the P27−/− group was apparently increased compared with that of mice in the WT group (Fig. 5A and B).

Figure 5. Col-I immumohistochemical staining result. (A) IVD Col-I staining micrograph (B) Columnar statistical figure of intervertebral disc Col-I positive area rate. IVD, intervertebral disc.**P<0.01.

Influence of P27 deficiency on the Shh-signal pathway

The statistical significant increase in Smo-positive cell rate of mice in the P27−/− group was found compared to mice in the WT group (Fig. 6A and B). In order to further observe the influence of P27 deficiency on the changes of Shh-signal pathway, western blot analysis was used to detect the changes in the protein expression levels of Shh, Patched, Smoothened and Gli2 in IVD. It was found that the protein expression levels of Shh, Patched, Smoothened and Gli2 of mice in the P27−/− group were markedly increased compared to mice in the WT group (Fig. 6C).

Figure 6. Shh-signal pathway expression of mice in the two groups. (A) IVD Smoothened staining micrograph. (B) Columnar statistical figure of Smo-positive cell rate. (C) Western blot analysis result of protein expression levels of Shh, Patched, Smoothened and Gli2 in IVD. IVD, intervertebral disc.**P<0.01.

Discussion

The Shh-signal pathway is one of the Hedgehog pathways that regulate the development and differentiation of vertebrate entoderm, which is a highly conservative morphogenesis pathway of the medial axis organ development existing in both Drosophila melanogaster and vertebrate (10–13). Membrane proteins Ptch and Smo control Shh-signal transmission towards the cell, and receptor Ptch negatively regulates the Shh-signaling pathway (14–16). Receptor Smo is coded by proto-oncogene Smoothened which is homologous with G-protein-coupled receptor and consists of a single peptide chain of seven transmembrane regions, whereas the Ptch affects its function. Transcription factors of Shh-signal pathway belong to the Gli gene family, of which Glil, Gli2 and G1i3, with zinc finger, have been identified (17–20).

Previous findings have shown that Shh can induce notochord and apical plate cells into the IVD and bony vertebral body in the process of mouse embryonic development. However, when Shh is deficient, notochord and apical plate cells cannot normally develop into the IVD and bony vertebral body (21). This shows that Shh-signal pathway plays a key role in the development process of the IVD. It has been shown that the IVD nucleus pulposus cell can also excrete Shh in a 4-day-old mouse, and the activated Shh-signal pathway can thus interact with signal pathways, including TGF-β, BMP and Wnt, as well as regulate the expression of transcription factors, such as P27, SOX9, type-I collagen protein, type-II collagen protein and chondroitin sulfate, and as a result, regulate the growth and development of IVD (22). This finding indicates that the Shh-signal pathway is involved in the whole process of IVD occurrence and may play a crucial role in it. We also found that the protein expression levels of Shh, Patched, Smoothened and Gli2 of mice in the P27−/− group were increased compared to those of mice in the WT group, suggesting that P27 deficiency can activate the Shh-signaling pathway.

ALP belongs to a critical enzyme in the process of osteoblast differentiation and can be used to measure osteoblast activity, whereas type-I collagen belongs to osteoblast product, and can be used to evaluate the osteoblast-differentiated degree. In the present study, ALP histochemical staining demonstrated that the ALP-positive area of mice in the P27−/− group was significantly increased compared to mice in the WT group. Furthermore, the Col-I immunohistochemical staining showed that the Col-I-positive area of mice in the P27−/− group was significantly increased compared to mice in the WT group. These results indicate that P27 deficiency can induce an increase in osteoblast bone formation in the intervertebral disc.

In conclusion, P27 deficiency activates the expression of the Shh-signal pathway and promotes the proliferation of osteoblast. This plays a role in promoting IVD degeneration, which provides a scientific and reliable experimental basis for the treatment of IVD degeneration-related diseases in clinical practice.

References