Increasing substance P levels in serum and synovial tissues from patients with developmental dysplasia of the hip (DDH)

Clinical data and samples included in this study were obtained from patients underwent surgery treatment in our institution between March 2011 and December 2012. 45 hips in patients (ranges: 35–48 years, mean: 40.4 years) with chronic osteoarthritis combined with DDH (Crowe:I ~ III) were set to DDH&OA. Including criteria were osteoarthritis secondary to DDH, severe degenerative arthritis of grade 4 in the classification of Kellgren and Lawrence[19, 20]. 56 patients with DDH (Crowe:I ~ III, ranges: 21–34 years, mean: 28.5 years) which defined as the hip having a sharp angle <45°and centre edge(CE) angle <20°[21, 22] were included in this cohort. Severe pain sensation and dysfunction in hip joint were common performances for patients in both of these groups. Besides, 31 normal volunteers (ranges: 22–38 years, mean: 33 years) were also recruited in this study to serve as control group.

Synoviums in hip joint were obtained from patients with DDH&OA (n = 35), DDH (n = 20) separately, as shown in Table 1. In our study, patients with DDH&OA were included and served as the advanced stage of DDH, which help to investigate the content of SP and its receptors NK-1R in different stages of DDH. In order to decrease the effect from age and sex, we recruited the participants roughly the age and acquired the synovial tissues for research mainly from female patients.

The diagnosis of DDH was based on impingement test as well as Sharp, CE angles in pelvic anteroposterior radiograph by chief physician (Dr Chen) in clinic. Clinical and laboratory data for the entire groups are presented in Table 1. Variables such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) was measured by clinical standard technology. Synovial tissues of hip joint in DDH&OA were acquired from the operation of Total hip joint arthroplasty (THA). Synovial samples in DDH were obtained during the operation of periacetabular osteotomy (PAO) and osteochondrocyte plasty (OCP) for patients with testify of opening joint capsule, which is characteristic in our department[23, 24]. Samples of synovial in fossa acetabuli collected from operation were cut into pieces of 1 × 1cm² and immerged into RNA Later (Ambion AM7021 USA) for 12 hs and then stored at −80°C for RNA and protein extraction.

Patients recruited in this study had no secondary osteoarthritis (OA) as a consequence of inflammation, rheumatism, osteonecrosis, trauma, gout or tumor. Kidney or thyroid insufficiency was also an exclusion criterion. Drug therapies with OPIATES or NSAIDS were not allowed. Besides, patients with DDH subsequent by slipped epiphysis, traumatic osteoarthritis and those who had experienced orthopedic hip surgery were also excluded.

The study was performed based on the recommendations from the Declaration of Helsinki and was approved by the Ethical Committee of Shanghai Jiaotong University School Medicine Affiliate Xinhua Hospital. Samples prepared from operation were only for testing purposes, and were acknowledged, consented, and signed by patients.

Collected blood samples were allowed to clot for at least 30 min and then centrifuged at 3000 r/min for 15 min to obtain serum. Serum samples were then kept at −80°C before analysis. The concentration of SP in serum was determined by an enzyme linked immunosorbent assay (ELISA) kit (Cayman, catalog NO.583751, USA) according to the manufacturer’s instructions, which provides accurate measurements of SP with a working range of 1.5 to 500 pg/ml. It was verified by plenty of studies that the SP antibody used here recognizes the intact peptide and shows 100% specificity for SP, 93% for SP2-11, and 30% for SP7-11 and was without cross-reactivity with neurokinin A (2.7%) or neurokinin B (0.04%)[25, 26].

The concentration of SP in synovium was evaluated by the same ELISA Kit described above. SP levels in synovium were extracted by two-step 2% acetic-acid[27, 28]. Briefly, 10-50 mg synovial samples were obtained from patients in separate groups as described in Table 1. We cut the tissue into fragments of 1-4 mm³. The first extraction was carried out by 1 ml 2% acetic-acid in 95°C for 15 mins. The supernatant regarded as SP from nerve endings was collected and then quickly frozen in −80°C. The second extraction was implemented by 1 ml 2% acetic-acid in 95°C for 45 mins. Supernatant was extracted and deemed as SP from non-nerve tissue or cells. The levels of SP were normalized to the weight of the tissue samples and expressed as pg/mg.

Assessment of hip joint mobility was performed using Harris hip score (HHS) by Dr Zhu. Along with HHS score, a visual analogue scale (VAS) was also applied for the assessment of pain severity of patients in separate group. In order to determine VAS score, a 100-mm horizontal line without scaling was designed in which 0 was marked as “no pain in hip” and 10 was marked as “unbearable pain”. Patients were then instructed to place a vertical mark reflecting their soreness severity. Scores of both HHS and VAS in different groups were shown in Table 1.

Synovial tissues from fossa acetabuli were obtained during operation of total hip arthroplasty (THA) or Bernese periacetabular osteotomy (PAO) as previously described from patients of 20 with DDH, 35 with DDH&OA. The tissues were dissected into small pieces of 80 mg and immediately frozen in liquid nitrogen. Frozen tissues (100 mg) were grinded by mortars into granular powder and was dissolved in 2 ml volumes of Trizol reagent kit (Takara, Code: D9108B, Japan). cDNA was synthesized from 2 mg total RNA through reverse transcription using a TaKaRa RNA PCR Kit (TaKaRa, DRR057A, Japan) according to the manufacturer’s protocol. The sequences for primers (TaKaRa, Biotechnology Co. Ltd, Japan) used to amplify mRNA were as follows: TAC1R: F: 5′-GTCGTGTGCATGATCGAATGG-3′, R: 5′-TTGCTCGTGGTAGCGGTCAG-3′; TAC1: F: 5′-GGTACGACAGCGACCAGATCA-3′, R: 5′-CCCGTTTGCCCATTAATCCA-3′. Real-time PCR was carried out using 200 ng of cDNA and PrimeScript® RT-PCR Kit (Takara, DRR064A, Japan) in 96-well plates in a ABI 7500 Sequence Detection System (ABI, Co. Ltd, USA) according to the manufacturer’s instructions. Relative quantification of each gene was normalized to GAPDH. We used the 2^-ΔΔCt (cycle threshold) method to calculate relative gene expression levels as previously described[29]. Results are presented as fold changes in gene expression normalized to GAPDH and relative to control conditions (DDH group). Data of each sample was displayed on triplicate wells during every PCR amplification circulation. Analysis of the results is based on at least three individual experiments.

The synovial samples were dehydrated in a graded series of ethanol and embedded in paraffin for immunohistochemistry. The tissues were cut into sections measuring 5 um and were mounted on anti-off slides. Sections from each group underwent citrate buffer antigen retrieval. After incubated overnight with the primary antibody NK-1R (1:1000) (Abcam, ab75516, UK) and substance P(1:1000) (Abcam, ab14184, UK), slides were rinsed in phosphate buffer saline (3×5 minutes) and incubated with biotinylated secondary antibody (Dako, Ely, UK). Finally, streptavidin-biotin complex (1:2000) (Amersham Life Science Inc, USA) was used for the visualization of the immunoreactions. From every synovium (8 per group), two sections were acquired at different depths and eight microscopic fields from each section were photographed and analyzed. Thus, in every synovial sample of separate group, 16 microscopic fields were collected. Images of the microscopic fields were captured by a camera (DEI 750, Optronics Engineering, USA) attached to the microscope. The immune activity was measured as the area in mm² using the software (Image-Pro plus 6.0, USA) as previously described[30]. The results were expressed as the positive staining area (mm²) in relation to the total area of each microscopic field.

Synovial samples of different groups (150 mg/sample) were grounded into powder with liquid nitrogen. Total protein was extracted using a Western & IP Cell Lysis Kit (Beyotime, Shanghai, China). The concentration of protein in the cell lysate was determined using a microplate reader (Thermo, Finland) according to the manufacturer’s protocol. For each sample, the whole cell extracts equivalent to 40 mg total protein which were loaded into a 10% SDS–polyacrylamide gel, separated by electrophoresis and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. Non-specific binding was blocked by incubating the PVDF membrane with 10 mM TBS, 1.0% Tween, and 10% dehydrated skimmed milk. Following the blocking procedure, the membranes were incubated overnight with antibodies against NK-1R (1:1000) (Abcam, ab75516, UK) in 4°C. The performance of immunoblotting on the same membranes using antibodies against GAPDH (Beyotime, Shanghai, China) was used as a loading control to assess the group differences. Gray-value of respective well in image strip was analyzed by software (Image J, NIH, USA) and normalized to value of GAPDH. Data of each group was acquired by three times of experiments at least.

As expected, patients in DDH&OA group had obvious higher score in VAS (P < 0.05) but lower score in HHS (P < 0.01) compared to that in DDH group. In the correlation detection (Table 2), significant positive correlations were found in group of DDH&OA (Female + Male): SP/HHS: r = −0.496, P < 0.01; SP/VAS: r = 0.512, P < 0.01. DDH&OA (Male): SP/HHS: r = −0.694, P < 0.01. DDH (Male): SP/HHS: r = −0.82, P < 0.01. Furthermore, we also observed a strong correlation of SP in serum with ESR(r = 0.508, p < 0.01) in patient with DDH&OA (Female + Male).

According to (Figure 2D-F), we can observe significantly intensive staining of NK-1R in DDH&OA compared to that in DDH. Besides, staining of NK-1R mainly located in synovial cells as well as capillary vessel wall cells. In addition, western-blot and gray analysis reflected obviously up-regulated NK-1R levels in DDH&OA group compared to that in DDH group (Figure 3A-B). Similarly, through QRT-PCR, we also witnessed analogical tendency about the mRNA expression of NK-1R in these groups (Figure 3D).

IHC staining as well as QRT-PCR were implemented to detect the expression of SP in synovium of hip joint. According to IHC staining and gray value analysis (Figure 2A-C), positive staining of SP was mainly observed in silk-like and nerve bundle-like nerve endings in synovium. And we can easily get stronger staining of SP in DDH&OA compared to DDH group. So it is with the comparison of mRNA expression of SP from synovial tissue between these groups (Figure 3C).