| Objective: Ankylosing spondylitis (AS) is a chronic inflammatory disea- se, which is characterized by main involvement of axial joints and sacroiliitis, but sometimes peripheral joints are involved. Recent studies showed that osteoporosis or osteopaenia was common in AS, and influenced the prognosis. However, the mechanism of the bone loss in AS was not fully identified yet. Most scholars deem that osteoclast played an important role in it. Receptor activator of nuclear factors-κB ligand(RANKL), receptor activator of nuclear factors-κB(RANK), osteoprotegerin(OPG) were the essential cytokines which regulated the differentiation and maturation of osteoclast. RANKL/RANK/ OPG system was proved to play a central role in the bone destruction of disease. The aim of this study was to investigate the role of RANKL/RANK/ OPG system in bone metabolism of AS through detecting bone mineral density, bone metabolism factors such as OPG, soluble(s) RANKL and the expression of RANKL in peripheral blood T lymphocytes.Methods: Thirty male AS patients and 20 age-and sex-matched normal controls were studied. All of the patients corresponded with the modified New York criteria for AS, without any other bone diseases and never took glucocor- ticoid, bisphosphonates or DMARDs for long term. The clinical datas of AS patients were collected such as age, disease duration, body mass index (BMI), erythrocyte sedimentation rate (ESR), c-reactive protein (CRP), immunoglob- ulin (Ig)A,M,G, complement (C)3,4, serum levels of calcium (Ca), phosphorus (P) and alkaline phosphatase (ALKP), parathyroid hormone (PTH) and human leukocyte antigen (HLA)-B27. BMD were measured by dual-energy X-ray absorptiometry (DEXA) and serum levels of soluble RANKL, OPG, tartrate resistant acid phosphatase 5b (TRACP-5b) and bone alkaline phosphatase (BALP) were measured by avidin biotin peroxidase complex enzyme-linked immunosorbent assay (ABC-ELISA). Flow cytometry was employed for dete- cting the expression of membrane–bound(mb)RANKL on T lymphocytes from freshly peripheral blood. The correlations were analyzed between the above indexes and the clinical related parameters.All statistical analyses were performed by SPSS 13.0 software for Windows. The mean number±standard deviation ( x±s) was used to express the measurement data. The t or t'test was adopted for comparison between groups. Chi-square test was used for the comparison of the enumeration data. Linear correlation analysis was performed for correlationship. P value<0.05 was considered statistically significant.Results: 1 The clinical data of the subjects included in the study: All the subjects were male. In the group of 30 patients with AS, the mean age was (28.30±7.65)years, mean body mass index was (22.98±3.66)Kg/m2, with a mean disease duration of (6.79±6.86)years. The meanage of control group was (30.20±5.20)years. There were no differences between experimental group and control group in age and gender (P>0.05). The mean levels of ESR, CRP and PTH of AS group were (42.53±26.25)mm/h, (35.35±46.53)mg/L and (24.29±10.89)pg/ml.2 Results of bone density measurement:In AS group, the incidence of normal bone mass, osteopaenia and OP were 16.67%, 46.67% and 36.67% respectively. The incidence of osteopaenia in lumbar, femoral neck, greater trochanter and inter trochanter were 53.33%, 16.67%, 43.33% and 26.67% respectively. The incidence of OP in lumbar, femoral neck, greater trochanter and inter trochanter were 13.33%, 0.00%, 26.67% and 6.67% respectively. There were different incidence of osteopaenia and OP in different measure- ment sites (χ2=14.932, P=0.002) and the highest site was greater trochanter (70.00%).3 Serum levels of bone metabolic indexes:The serum levels of sRANKL, OPG, TRACP-5b, BALP and the ratio of sRANKL to OPG in AS patients were (304.19±580.44)pg/ml, (93.76±155.27)pg/ml, (1.87±1.40)U/L, (28.44±14.56)U/L, (2.83±3.60) respectively, and (64.21±23.86)pg/ml, (55.05± 16.67)pg/ml, (0.96±0.17)U/L, (27.77±5.99)U/L, (1.26±0.51)respectively in the control group. The serum levels of sRANKL, TRACP-5b and and the ratios of sRANKL/OPG in AS group were significantly higher than those in normal controls (P<0.05). There were no statistics different in serum levels of OPG and BALP between AS group and control group (P>0.05).4 The correlation between bone metabolic indexes and clinical paramete- rs: According to linear correlation analysis, the levels of sRANKL were positively correlated with OPG (r=0.844, P =0.000);the levels of both sRANKL and OPG were positively correlated with the levels of TRACP-5b (r=0.828, P=0.000; r=0.986, P=0.000, respectively). Except the negative correlation between sRANKL and C4 (r=-0.493, P=0.027), the negative correlation between OPG and C3 (r=-0.339, P=0.144), the positive correlation between BALP and ALKP(r=0.393, P=0.032), the positive correlation between BALP and BMI (r=0.464, P =0.011), there was no linear correlation between the five indexes related to bone metabolism and clinical data, such as ESR, CRP, PLT, Ca, P, ALKP, IgG, IgA, IgM, C3, C4, PTH, BMI, age and disease duration (P>0.05).5 The correlation between BMD and bone metabolic indexes: According to linear correlation analysis, the BMD of femoral neck, greater trochanter and inter trochanter were positively correlated with body mass index (r=0.389, P=0.034;r=0.545, P=0.002;r=0.493, P=0.006, respectively). However there was no correlation between BMD and sRANKL/OPG ratio and serum levels of sRANKL, TRACP-5b, BALP, ALKP, Ca, P, PTH and clinical data, such as ESR, CRP, age and disease duration (P>0.05).6 The percentage of CD4+/RANKL+ and CD8+/RANKL+ cells in periphe- ral blood: The percentage of CD4+/RANKL+ cells in peripheral blood of AS was (2.84±1.96)%, which was significantly higher than those in normal controls (1.24±0.86)%; whereas the percentage of CD8+/RANKL+cells in peripheral blood of AS (0.91±0.94)% tended to be higher than those in normal controls (0.62±0.50)%, but the difference was not statistically significant (P> 0.05). 7 The correlation between the percentage of CD4+/RANKL+ and CD8+/ RANKL+ cell in peripheral blood of AS patients and the laboratory parameters of of disease activity (ESR, CRP): there were no significant linear correlation between the percentage of CD4+/RANKL+, CD8+/RANKL+ cells in peripheral blood of AS patients and the laboratory parameters of disease activity (ESR, CRP) (P>0.05).Conclusions: 1 AS patients had different degree of bone loss and most of them were complicated with osteopaenia or OP. The degree of bone loss was varying in different measurement sites. For a better overall evaluating the BMD, it was necessary to measure BMD in multiple sites and then make a comprehensive analysis. BMI played an important role in maintaining and increasing BMD.2 There was abnormality in bone metabolism of AS, and enhancement of bone resorption was an important cause of bone loss. The imbalance of RANKL/RANK/OPG system might be one of the mechanisms leading to osteoporosis in AS.3 CD4+ T lymphocytes might play an important role in osteoclast differe- ntiation and osteoporosis in AS by up-regulating the expression of RANKL.
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