The Diagnosis And Treatment Of SpA Coxitis And The Establishment Of Animal Model

Objective:To establish the early diagnosis and evaluation system of disease activity for coxitis in patients with Sp A. To observe the effects of TNF-α inhibitors group and the thalidomide group. To establish certain animal models and identification methods for Sp A.Methods:1. Clinical trialInclusion criteria: Enrolled patients fullfill the International diagnostic criteria of the SPA or ASAS. 1) aged 18-45; 2) ASDAS> 3.5; 3) Harris score <70; 4) previously employed a NSAIDs, DMARDs combination therapy for three months ineffectively; 5) MRI demonstration corresponds active coxitis(such as BME, synovitis, capsulitis);6) Treatment with prednisone had to be discontinued at least 4 weeks before initiation of the study; 7) Treatment with TNF-α inhibitors or other biological agents had to be discontinued at least 12 weeks before initiation of the study.Exclusion criteria: tuberculosis or other infections, cancer, pregnancy.Grouping:TNF-α inhibitor group(n=60), thalidomide group(n=70).Clinical evaluation and laboratory parameters assessment were carried out at week 0, 24 and 48.2. Clinical diagnostic assessment1) Clinical efficacy assessmentClinical efficacy assessment: ASAS 20 remission criteria, ASAS 40 remission criteria and ASAS partrial remission criteria.Clinical observation index: ASDAS, BASPGS, BASFI, BASMI, HAQ, Harris score, CRP, ESR.2) MRI assessmentMRI images were reviewed and scored by two experienced radiologists and two rheumatologists. MRI bone marrow edema(BME) score mainly used hip coronal score, each side of the femoral head of the hip were reckon as a circle, which was divided into 8 equal parts. Each part equals 45° and scored 1 point. That is 8 points in total. The acetabulum was divided into inner, middle and outer parts.With each part scored 1 point, the total number was 3 points. Plus another 1 point for the femoral neck, the total number of one side of the hip was 12 points. And the bilateral amount was 24 points.3) Immunology and cytobiology assessmentTh17 and Treg cells were tested mainly by flow cytometry. IL-6, IL-17, IL-23, MMP-3 were tested by using a sandwich ELISA.3. Protein Purification and Identification techonology1) Protein purification: After extraction by guanidinium articular cartilage were centrifuged in cesium chloride density gradient. After removal of the side chain the protein was freeze-dried and sent to determine the chemical composition.2) Protein Identification: The activity of extracted and purified cartilage proteoglycan were tested by Western blot; Superdex 200 molecular sieve was used to purify proteoglycan extracted product.4. Establishment and assessment of Sp A animal modelGrouping: Model group(n=30); Control group(n=30).Articular cartilage originated form patients who had taken the knee-joint replacement surgery. Model mice Highly purified proteoglycans was intraperitoneal injected to the model mice. Valbumin was intraperitoneal injected to the control mice. Before immunization and at the 36 weeks after immunization, the model mice were tested by histopathology and radiology method.Results:1. Open-controlled clinical trialsThe change of clinical remission rate for TNF-α inhibitor group vs thalidomide group at week 24: ASAS20 remission(51.7% vs 38.6%, P=0.1343), ASAS 40 remission(40% vs 22.9%, P=0.0348), ASAS partial remission(33.3% vs 12.8%, P=0.0052). At week 48: ASAS20 remission(91.7% vs 61.4%, P<0.001), ASAS 40 remission(80% vs 42.9%, P<0.001), ASAS partial remission(58.3% vs 20%, P<0.001).Changes in clinical parameters: clinical parameters of TNF-α inhibitor group vs thalidomide group after 24 weeks of treatment: ASDAS(1.599 vs 4.594, P<0.001), BASFI(12.39 vs 31.923, P=0.006), HAQ(5.618 vs 9.121, P=0.018), BASPGS(3.353 vs 6.152, P<0.001), Harris score(83.353 vs 71.05, P<0.001) were significantly different. TNF-α inhibitor group clinical improvement were better than thalidomide group. TNF-α inhibitor group vs thalidomide group after 48 weeks of treatment: HAQ(3.031 vs 6.214, P=0.002), BASPGS(1.531 vs 5.000, P<0.001), Harris score(91.531 vs 86.63, P=0.005) were significantly different. TNF-α inhibitor group clinical improvement were better than thalidomide group. In the TNF-α group: week 24 vs baseline: mean ASDAS(3.696 vs 1.599, P<0.001), BASFI(49.063 vs 12.391, P<0.001), BASPG(8.324 vs 3.353, P<0.001), HAQ(15.59 vs 5.618, P<0.001), Harris score(61.324 vs 83.353, P<0.001) have significant difference; ESR(21.62 vs 5.26, P<0.01) have difference; week 48 vs baseline: HAQ(15.59 vs 3.031, P<0.001) have significant difference; BASPGS(8.324 vs 1.531, P<0.001) have difference. In the thalidomide group: week 24 vs baseline: ASDAS(3.387 vs 2.594, P=0.013), BASFI(47.2 vs 31.9, P<0.001), BASMI(1.576 vs 1.061, P<0.001), BASPGS(8.455 vs 6.152, P<0.001), HAQ(14.64 vs 9.121, P<0.001), Harris score(68.59 vs 71.05,P<0.001) have significant difference, ESR have difference(16.96 vs 7.59, P<0.05). week 48 vs baseline: BASFI(47.2 vs 19.9, P<0.001),BASMI(1.576 vs 0.821, P<0.001), BASPGS(8.455 vs 5.0, P<0.001), HAQ(14.64 vs 6.21, P<0.001), Harris score(68.59 vs 86.63, P<0.001), CRP(1.281 vs 0.461, P<0.01) have significant difference.MRI changes: After 24 weeks of treatment, TNF-α inhibitor group compared with thalidomide group, the MRI hip score was 1.45 vs 4.333(P=0.007). There was a significant difference between two groups. TNF-α inhibitor group scored better than the thalidomide group. However, there was no difference in capsulitis and synovitis treatment. After 48 weeks of treatment, TNF-α inhibitor group compared with thalidomide group, the MRI hip score was 17 vs 26(P=0.011). MRI capsulitis(P=0.011) were significantly different between the two groups. TNF-α inhibitor group performed better than the thalidomide group. Biomarkers change: After 24 weeks of treatment compared with the baseline:TNF-α inhibitors:peripheral serum level of IL-7, IL-23, MMP-3(P<0.001) were significantly different; after 48 weeks of treatment compared to the baseline: IL-6, IL-7, IL-23, MMP-3 levels were significantly decreased(P<0.001); 24 weeks of treatment compared with 48 weeks: IL-6(P<0.001) and MMP-3(P=0.0069) were significantly decreased. Thalidomide group: after 24 weeks of treatment compared with baseline: IL-23(P=0.038) and MMP-3(P=0.0207) were different; After 48 weeks of treatment compared with baseline: MMP-3(P=0.0473) was different; Compare week 24 and 48: IL-23(P=0.0556), IL-6(P=0.2592), IL-17(P=0.9959), MMP-3(P=0.3698) were not statistically different. The change of Th17/Treg rate: 24 weeks and 48 weeks compared to the baseline: the peripheral blood Th17/CD4+ T cells in TNF-α inhibitors group was significantly decreased(P<0.001); Treg/CD4+ T cells was significantly increased(P<0.001); In the thalidomide group: Th17 /CD4+ T cells in peripheral bloodwas decreased(P=0.049), Treg covering the proportion of CD4+ T cells was no significant difference(P=0.057); 24 weeks compared with 48 weeks: TNF-α inhibitor group Th17, Treg accounting for CD4+ T cells was no significant difference(P=0.642, P=0.393); Thalidomide group: Th17 cells accounting for the proportion of CD4+ T cells was significantly decreased(P=0.0012), the percentage of Treg was significantly increased(P=0.001).2) Antigen preparation and establishment of Sp AAnimal model anidine salt extraction and purification of articular cartilage. After removal of the side chain, dialysised and dried,the protein we collected was 10 mg. Model mice were immunized three times with a total amount of 300μg. 36 weeks after vaccination, we made histopathological identification and successfully inducted ankylosing spondylitis inflammatory model of Balb/c mice.3)Histopathology and radiology results in the animal modelAfter 36 weeks, the histopathology of model group showed that spinal ligaments intervertebral joints, narrowing joint space or osteophyte formation. X-ray test results of model mice showed parts of the thoracic and lumbar vertebral articular surface became unsmooth, parts of the vertebral space narrowing, or even disappear and interbody fusing. The control group found no changes.Conclusions:1. We have established the system of early diagnosis and disease active evaluation for coxitis of Sp A. And determined the change of MRI is a sensitive method for early diagnosis and assessment of Sp A coxitis patients. We have confirmed that TNF-α inhibitor or thalidomide plus SSZ have improved clinical parameters in patients of coxitis in Sp A. TNF-α inhibitor group prognosed better than thalidomide group. IL-6, IL-23, IL-17, MMP-3, Th17/Treg participate in the pathogenesis of Sp A, which are the biological markers to determine Sp A prognosis.2. We successfully prepared proteoglycans and provided experimental basis for immunization Sp A mice. We successfully established proteoglycan-induced mouse model of Sp A spondylitis and provided a valuable experimental material for further study of the pathogenesis and clinical treatment of Sp A.