| Background:Ankylosing spondylitis (AS), a kind of chronic autoimmune disease, is characterized as the central axis joint pain and stiffness. It often happens more commonly in young men with high rate of disability. Bone destruction is one of the main pathological features, and is the key of pathological change, and hip joint damage is one of the main causes of a sharp decline in quality of life in patients with AS, so the prevention and cure of bone destruction in AS is significant, but there is no effective prevention methods.Research shows that, the osteoclast is the main participants of bone destruction, monocytes macrophages are the precursor cells. RANKL activates the RANKL/RANK signaling pathway in monocytes-macrophages, inducing to osteoclast differentiation, thus inhibiting this signaling pathway is likely to prevent the bone destruction.Preliminary clinical study showed that the Bu-Shen-Qiang-Ji Decoction has obvious anti-inflammatory and analgesic effects in the treatment of AS. In the first National Natural Science Foundation of China (30472277), we explored the molecular mechanism of Bu-Shen-Qiang-Ji Decoction mediated serum on anti-AS ossification, and we accumulated a certain theoretical and experimental basis, which would have provided an important guarantee for the follow-up study. Therefore, under the support of the National Natural Science Foundation of China (81173446), this study continues to explore the Bu-Shen-Qiang-Ji Decoction whether can inhibit bone destruction in AS from clinical and experimental perspective.Objective:Using Bu-Shen-Qiang-Ji Decoction to treat AS patients who were in active period and of kidney deficiency and blood stasis syndrome in Chinese traditional medicine, observe the expression of RANKL and OPG in serum before and after treatment. Using Bu-Shen-Qiang-Ji Decoction mediated serum to interfere monocyte-macrophages (osteoclast precursor cells) which were from hip joint synovial of AS patients, observe the activation feature of RANKL/RANK signal pathway. Explore the preventive and therapeutic effects of Bu-Shen-Qiang-Ji Decoction on AS bone destruction.Methods:1 Clinical researches:Design a prospective, open, self-controlled (before and after treatment) clinical study.60 active AS patients and 30 healthy volunteers (served as control group) were included in the study. All AS patients were of kidney deficiency and blood stasis syndrome in TCM. AS group were treated with Bu-Shen-Qiang-Ji Decoction for 12 weeks. Choose 0 week,4weeks,12 weeks as evaluation points, recorded the symptoms and laboratory indexes, and used ASAS20, BASDAI50, TCM syndrome integral to evaluate the curative effect and safety of Bu-Shen-Qiang-Ji Decoction. Use ELISA to detect the expression of serum RANKL, OPG before and after treatment in patients with AS and normal control group.2 Experimental studies:Monocytes-macrophages of synovial membrane cultured in vitro served as the research object. Synovial tissues were from 5 AS patients and 5 cases of control group. AS group synovium were from patients who underwent hip joint replacement operation. Control group synovium were from 5 patients (excluding autoimmune disease) who had fracture at hip joint and underwent hip joint replacement operation. All tissues were surgery waste and met ethical requirements. Immunohistochemical methods were used to test the protein expression in the tissues. Enzyme digestion and adherent culture were used to separate monocyte -macrophages in synovial tissue and flow cytometry to detect cells. MTT method was used to observe the cell growth. Bu-Shen-Qiang-Ji Decoction mediated serum was used to interfere monocyte-macrophages, and Western blot and RT-PCR were used to measure the expression of signaling molecules in RANKL/RANK signaling pathway and the levels of mRNA of some genes before and after the treatment.Results:1 Clinical research:in the first part,60 AS patients in active period and 30 healthy volunteers as control were included in the study. Comparing the serum level of RANKL and OPG with normal group, RANKL and OPG in AS group increased significantly, P<0.05. The ratio of RANKL/OPG had no significant difference between the two groups, P>0.05. The correlation test showed that RANKL, OPG, had no significant correlation with CRP, ESR, the clinical observation indexes (BASDAI, BASDAI, BASMI, PGA, HAQ) and clinical measurements (BASMI) (P>0.05).The second part, the 60 patients enrolled are of kidney deficiency and blood stasis syndrome, and used traditional Chinese medicine Bu-Shen-Qiang-Ji Decoction as the basic treatment for 12 weeks, evaluated the clinical efficacy and safety respectively at 0 week,4 weeks and 12 weeks, and also evaluated its influence on the expression of RANKL, OPG. As a result,4 weeks and 12 weeks later, the standard rate of ASAS20 reached 33.33% and 72.55%, the standard rate of BASDAI50 were 11.67% and 39.22%, and the effective rate of TCM syndrome integral were 45.10% and 79.43%. After treatment, the indicators had increased gradually, and there were no adverse events associated with traditional Chinese medicine.The spinal inflammation score, back pain score, hip pain score, night pain score, tendon index, cervical flexion and extension and rotation, lateral bending and flexion and extension of lumbar spine, intermalleolar distance, expression of CRP and RANKL, all of these improved significantly after the treatment of in 4 weeks,12 weeks, P<0.05. The ratio of RANKL/OPG in the treatment didn’t change significantly.2 Experimental studies:in the first part, monocyte-macrophages were successfully isolated and cultured. Cells were identified by flow cytometry, and the proportion of monocyte-macrophages was 55.3±4.2%. Immunohistochemistry results showed that, the stained area of RANK, TRAP, and CATK in the synovium of AS group increased obviously than the control group, P<0.05. Western blot detection showed that, RANK (RANKL’s receptors on the surface of monocyte-macrophages), TRAF6, NF-κB, JNK, ERK, p38, NFATcl expressed more than the control group, P<0.05. Phosphorylated NF-κB, JNK, ERK were significantly higher than those of the normal group, P<0.05. The expression of TRAP (marker protein of osteoclast), CATK and MMP3 (bone resorption proteins of osteoclast) were higher than those of the normal group, P<0.05. Confirmed by RT-PCR, RANK, TRAP gene expression was significantly increased in AS synovium, compared with the normal group, the difference was significant P<0.05. The second part, after the intervention of Bu-Shen-Qiang-Ji Decoction mediated serum to monocyte-macrophages, the expression of RANK, TRAF6, NF-κB, JNK, ERK, p38, NFATc1, phosphorylated NF-κB, TRAP, CATK and MMP3 were down regulated in the low dose group, middle dose group, high dose group, and the difference was significant compared with the AS group, P<0.05. The efficacy was especially obviously in middle dose group and high dose group.Conclusion:1, The expression of RANKL, OPG in serum increased significantly in active AS patients, and RANKL/OPG had no obvious change. It suggested the possibility of bone destruction and bone formation in active AS patients, but the final outcome depended on the distribution of RANKL and OPG, and also depended on micro environment; 2, Changes of serum RANKL, OPG and RANKL/OPG could not reflect macroscopically the progression of AS. Maybe it could reflect the micro progression, so clinical studies should also include imaging index to explore its meaning; 3, Separation phenomenon between RANKL, OPG expression and inflammatory, indicated that the occurrence of bone destruction was not necessarily caused by inflammation; 4, Bu-Shen-Qiang-Ji Decoction could control AS disease with a great curative effect and had a high safety; 5, Bu-Shen-Qiang-Ji Decoction may prevent and control AS bone destruction through multi targets and angles. The possible mechanisms were that:one side, by tonifying kidney, invigorating the circulation of blood to improve the inflammation, and to eliminate the influence of many factors on RANKL, OPG expression, eventually down regulated the expression of OPG; one side, acted directly on osteoclast precursor cells, inhibited the activation of RANKL/RANK signaling pathway in the cells, reduce the formation of osteoclasts, and ultimately achieved the purpose of prevention and treatment of bone destruction. |
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