| Ankylosing spondylitis (AS) is a chronic inflammatory autoimmune disease.The disease may cause progressive ankylosis of spine and peripheral joints, leading to movement disorder or even permanent loss of activity ability, and seriously affect the patients’quality of life. The pathological osteogenesis is the main reason of cripple in AS patients, and to research drugs which can effectively delay AS ossification is an urgent need. As the start factor of bone growth, bone morphogenetic proteins (BMPs) have the functions of promoting osteoblast differentiation and inducing undifferentiated mesenchymal cells irreversibly differentiate into the bone lineage. Among BMPs, BMP-2is considered to have the strongest osteogenic capacity and can induce osteogenic differentiation alone. Many studies show that BMP signal transduction plays an important role in AS ossification which may be the breakthrough point to explore drugs preventing and treating AS ossification.Ankylosing spondylitis belongs to’Gubi’category of TCM.The traditional Chinese medicine is an important and effective method for the treatment of AS, which maybe has huge potential in controlling and delaying AS ossification. My tutor professor Feng Xinghua has unique features in treating AS, and this thesis based on summarizing his experiences of using prescritions for treating AS. Under the guidance of tutor’s academic thinking of using the therapeutic principle of invigorating kidney and promoting blood circulation in treating AS, we firstly assessed the clinical effect of Bushenhuoxue Decoction in treating AS and observed its intervention to BMP-2in AS patients’serum. And then we established the finite fibroblast line derived from hip joint capsule of AS patients in vitro, and observed the effect of Bushenhuoxue Decoction to BMP signal transduction in fibriblasts using TCM serum pharmacology method and modern molecular biology techniques. The thesis investigated the possible mechanisms of Bushenhuoxue Decoction treanting AS and anti-ossification from clinical and experimental level, in order to reveal the scientific connotation of tutor’s clinical experiments in treating AS.l Summary of tuor’s experiences in treating ASTo summarize professor Feng Xinghua’s thinking of syndrome differentiation of treating AS by collecting the data of AS patients and analyzing his using drugs regularity using the methods of frequency analysis, association rules analysis and cluster analysis. The results of this study objectively reflected professor Feng’s characteristics in treating AS as follows: Firstly, disease differentiation and syndrome differentiation combined as the core, invigorating kidney and promoting blood circulation as the basic treatment principle, and Bu-Shen-Qiang-Ji (BSQJ) Decoction as the core prescription of treating AS; Secondly, symptom differentiation as the supplement, reflected his idea of individual treatment; Thirdly, emphasis on the application of promoting blood circulation herbs.2Clinical ResearchesFirstly, we investigated the clinical significance of BMP-2in the serum of AS patients.60kidney deficiency with blood stasis syndrome AS patients in active period were adopted, and30healthy adults and30AS patients in disease-free period were as control. All people’s BMP-2was detected and the indexes which reflect the disease activity of AS patients were recorded. And then, BMP-2concentration difference of each group, the correlation between BMP-2and other laboratory parameters, the correlation between BMP-2and the indexes which reflect the disease activity were analyzed.The study results showed that:The serum BMP-2of AS patients in active period and disease-free period were both significantly higher than the normal group; BMP-2was positive correlated with CRP, CD62P and the condition evaluation indexes of AS such as BASDAI, BASFI, BASMI; BMP-2was significantly negative correlated with lumbar flexion, lumbar scoliosis and ankle pitch. The results suggested that the serum BMP-2of AS patients could reflect disease activity. In addition, as the important cytokine promoting bone formation, abnormal increase of BMP-2in the serum of AS patients may be associated with pathological osteogenesis which resulting in the rigidity of spinal and peripheral joints and the limit of activity function.Secondly,60kidney deficiency with blood stasis syndrome AS patients in active period were treated. The patients were given Bu-Shen-Qiang-Ji Decoction and acupoint sticking therapy as adjuvant therapy, and12weeks as a course. After treatment, the effect of BSQJ decoction was evaluated by using ASAS20standard, BASDAI50standard and TCM efficacy evaluation standard, and the intervention of BSQJ decoction to BMP-2was also vobserved.The study results showed that:Invigorating kidney and promoting blood flow method treating AS was of definite clinical effect; After12weeks’treatment, ASAS20responsive ratio was85.00%, BASDAI50responsive ratio was68.33%, and the total efficiency of TCM syndrome curative effect was91.67%; Chinese medicine could significantly improve AS patients’BASDAI, BASFI, BASMI, PGA, night pain and so on, and could reduce the disease activity of AS patients, and improve their functional status; Chinese medicine could effectively reduce the pain and improve the functions of axial joints, hip joints and other peripheral joints; patients’CRP, PLT, CD62p was also improved, reflecting a good anti-inflammatory effects of Chinese medicine; Chinese medicine had significant intervention effect to serum BMP-2of AS patients, suggesting that it may delay AS ossification progress through inhibiting BMP-2’s function of osteogenesis; the comprehensive treatment program used in study had good security.The results of clinical studies verified the exact effect of invigorating kidney and promoting blood flow method for treating AS, provied anti-inflammatory effect and anti-ossification potential of BSQJ decoction, and provided a good basis for developing and optimizing AS comprehensive treatment program.3Experimental ResearchesWith the support from National Natural Science Foundation of China, the experimental study using AS fibroblasts cultured in vitro as the object, using TCM serum pharmacology method and techniques of western blotting and real-time PCR, taking BMP/Smads signal transduction pathway as the breakthrough point, aimed to exploring the possible mechanisms of BSQJ treanting AS and anti-ossification and revealing the scientific connotation of invigorating kidney and promoting blood flow method for treating AS, by detecting expression of BMP/Smads pathway signaling molecules in AS fibroblasts before and after rhBMP-2’s inducement and observing the effect of serum containing BSQJ on BMP/Smads signal pathway of AS fibroblasts with and without rhBMP-2’s inducement.The experimental results showed that:Expression of Cbfal in AS fibroblasts was significantly higher than that in normal fibroblasts; compared with the normal fibroblasts, there’s more BMP-specific binding receptors, Smad4and less Smad6in AS fibroblasts, and the phosphorylation level of Smadl,5was higher; exogenous rhBMP-S coule promote the proliferation of fibroblasts, increasing the expression of cbfal and Smad4and enhancing the phosphorylation degree of Smadl,5. The results suggested that the high activation and regulating mechanism disorder of BMP/Smads pathway which can cause abundant expression of Cbfal and result in fibroblasts differentiate into osteoblasts maybe one of the important molecular mechanisms of AS ossification, and BMP-2was an important cytokine causing pathological osteogenesis.The experimental results also showed that:BSQJ-medicated serum could restrain the expression of BMPRII, Smad4, increase the expression of Smad6and decrease the phosphorylation degree of Smadl,5, affecting the transduction of BMP signal and the expression of cbfal; BSQJ-medicated serum could also decrease the expression of cbfal and inhibite rhBMP-2’s inducement to fibroblasts by regulating the expression of BMPRII, Smad4,6and the phosphorylation degree of Smadl,5. The results suggested that BSQJ could suppress fibroblasts differentiate into osteoblasts by regulating BMP/Smads pathway from multiple aspects, and could significantly inhibit BMP-2’s inducement to fibroblasts, which maybe one of the important anti-ossification molecular mechanisms of BSQJ. The experimental results explored the target and mechanism of BSQJ treating AS from the molecular level and also proved that invigorating kidney and promoting blood flow method was an important and effective methods for treating AS from microscopic view. |
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