Interleukin-23 may contribute to the pathogenesis of lumbar disc herniation through the IL-23/IL-17 pathway

From August 2014 to January 2015, 29 patients diagnosed with LDH were randomly recruited from the Spine Department of Tianjin Hospital. The enrolled patients should meet all the following criteria: (1) typical osphyalgia or sciatica symptoms, (2) positive Lasegue’s sign or Bragard test, (3) single lumbar disc herniation, and (4) symptoms persist or recur repeatedly, and conservative treatments were failed. In addition, eight persons who had lumbar fusion surgery because of lumbar vertebral fracture were also included in the experiment and used as normal controls. Individuals with one of the following circumstances should be excluded: (1) history of osteoarthritis, spondylosis, spondylolisthesis, acute or chronic inflammatory disease, hypertension, or diabetes mellitus; (2) have taken or are currently taking immunosuppressive drugs; and (3) previous surgery for IVD disease.

Patients with LDH can be divided into two groups by using the method described by Cheng et al [33]. In short, patients in whom the annulus fibrosus was intact and the nucleus pulposus was completely sealed by the annulus fibrosus were categorized as non-ruptured group (NR, 13 patients). Patients in whom the annulus fibrosus was ruptured and the nucleus pulposus was not completely sealed by the annulus fibrosus and thus the nucleus pulposus was exposed to circulation were categorized as ruptured group (R, 16 patients). For a diagnosis of rupture, both MRI evidence of rupture and visual observation of rupture during surgery had to be present.

During surgery, after removal of the intervertebral disc, it was immediately washed by phosphate-buffered solution (PBS) for three times to clean the remained blood. Subsequently, the NP was separated from the AF by using a stereotaxic microscope and then the NP was cut into two parts: one was fixed in 10 % neutral formalin and used for morphological observation, and the other was placed in a cryopreservation tube and preserved at −80 °C for reverse transcription polymerase chain reaction (RT-PCR). The characteristics of the enrolled subjects are shown in Table 1.

IVD tissues obtained from the surgery were fixed in 10 % neutral formalin for 24 h. After that, they were dehydrated in alcohol and transparentized in xylene. Then, they were embedded in paraffin wax and cut into 5-μm sections. Subsequently, the sections were dewaxed to water and stained by hematoxylin and eosin (HE) and sealed with neutral gum. The pathological changes of the intervertebral disc were evaluated by using a light microscope (Nikon, Japan).

IVD specimens obtained from LDH and vertebral fractures were embedded in paraffin, and sections were cut at 4 μm and mounted on slides and dried at 60 °C. Sections were deparaffinized in xylene and rehydrated through graded alcohols to distilled water. Then, the sections were incubated with H₂O₂ for 10 min to eliminate the activity of endogenous peroxidase, followed by incubation for 2.5 h with human IL-23 immunogen affinity purified polyclonal antibody (Abcam, ab115759) diluted 1:200 in blocking buffer. The sections were washed with PBS and incubated for 30 min with HRP-labeled goat anti-rabbit IgG secondary antibodies (Fitzgerald, 43R-1614, USA) in blocking buffer (1:1000). Color was developed with diaminobenzidine, and the sections were counterstained with hematoxylin for 1 min at room temperature to stain the cell nuclei. Sections were imaged by using a microscope (Nikon, Japan) with ×20 and ×40 objective lenses. Human kidney tissue was used as the positive control.

Semiquantitative grading of IL-23 immunoreactivity in immunostained sections was performed by two graders who evaluated eight separate ×20 magnification fields for each tissue sample. The method was used as previously described by Shamji et al [19]. And this strategy can provide the most complete and comprehensive evaluation of the surgical tissue samples.

Scores were given for degree of cytokine immunoreactivity as follows: 0 = no positive cells and 1 = at least one positively labeled cell.

As few annulus fibrosus was found in the dissected intervertebral disc tissues in our experiment, and nucleus pulposus was generally believed to play a central role in the pathogenesis of LDH, thus only the nucleus pulposus was prepared for the RNA extraction. Tissue samples were grinded in liquid nitrogen and homogenized in 1 ml TRIzol ®Reagent (Invitrogen, Carlsbad, CA, USA) per 100 mg of tissue. The purity and concentration of the extracted total RNA were evaluated by an ultraviolet spectrophotometer (Thermo Fisher NanoDrop-1000, USA). According to the manufacturer’s protocol, 1 μg of total RNA was used to synthesize cDNA using ReverTra Ace qRCR RT Kit (Toyobo, Osaka, Japan). Real-time PCR amplifications were performed using gene-specific primers in a final concentration about 0.4 μM and SYBR® Green Realtime PCR Master Mix (TOYOBO, OSAKA, JAPAN) according to the manufacturer’s protocol. The primer sequences used this in this experiment are shown in Table 2. The thermal cycling conditions were as follows: an initial denaturation at 95 °C for 1 min, followed by 40 cycles of 10 s of denaturing at 95 °C, 15 s of annealing at 58 °C, and 20 s of extension at 72 °C. The expression levels of the target genes were normalized to that of β-actin in the same cDNA samples.

The morphology of the intervertebral disc was observed by using a light microscope (Nikon, Japan). Figure 1a showed the representative results of the three groups. According to the histological performance, we can see that IVD tissues from the ruptured group showed more severely degenerative performance than the other two groups, and there were less degenerative changes in the normal group than the non-ruptured group. To be more specific, in the normal control group, the structures of NP and AF were almost normal, and there were less cracks and small cell clusters in this group; however, in the non-ruptured group, the degenerative changes were more severe, cracks can be seen in this group samples, and there were more small cell clusters in the IVD tissues which is one of the features of degeneration. In the ruptured group, the structures of NP and AF were nearly destroyed, more cracks and fissures can be seen in this group, and it was not rare to see the performances of fibrinoid necrosis, small vessels, and lymphocytes infiltration (Fig. 1b).

Representative pictures and statistical results of the immunohistological staining of IL-23 in the IVD specimens were shown in Table 3 and Fig. 2a. We can see that there were nearly no positive expressions of IL-23 in the normal control group, and the immunoreactivity of IL-23 in the ruptured group was much higher than that in the non-ruptured group (p < 0.001). Besides, strong positive expressions can be found around the small vessels and the infiltrated inflammatory cells which were shown in Fig. 2b.

In order to investigate the gene expression of IL-23 and other inflammatory cytokines, we performed RT-PCR. The results showed that the mRNA levels of IL-23, IL-17, IL-1β, and TNF-α were significantly higher in the ruptured group when compared to the non-ruptured group, but no expression differences were observed at IL-6 between the two groups, and all above cytokines are least detected in the normal control group (Fig. 3). The association degree of the gene expression between IL-23 and IL-17 was calculated by using Pearson correlation coefficient, and significant positive correlations were observed between them (r = 0.794, p < 0.01).

The study was approved by the institutional ethics review board of Tianjin Hospital, and written informed consent was obtained from each patient.