| Ankylosing spondylitis is a chronic inflammatory disease of the skeleton and associated soft tissues. Ankylosis of the spine and hip could lead to disability in activity, work and life. As a result, inhibiting or delaying ossification is the key target for treatment. However, there is no definite evidence showing that any treatment can delay ossification occurring in AS patients. Traditional Chinese medicine shows huge potential in delaying ossification. This thesis based on Professor Feng Xinghua's academic thinking of using the therapeutic principle of supplementing kidney and promoting blood circulation in treating AS, firstly assesses the clinical effect and safety of Bushenqiangji Decoction in treating AS. And then study the roles of BMP/Smad signal transductive pathway played in the molecular mechanism of Bushenhuoxue Decoction in inhibiting AS fibroblast differentiating into osteoblast from protein perspective and with western blotting technique and hebal serum pharmacology.1 Theoretical ResearchResearch one:through analyzing the relative discussions about Jiqiang (spinal ankylosis), Shenbi (kidney impediment), Gubi (bone impediment), Dalv, et al of whose clinical manifestation were similar to AS, and the discussions about causes and pathogenesis of AS symptoms, we concluded that the pathogenesis of AS was that because of kidney deficiency, pathogenic wind, cold, dampness, heat and blood stasis invaded human, and caused meridians blocked. Research two:Dig out Professor Feng Xinghua's experience in treating AS with traditional Chinese medicine from the perspective of symptoms and analyze the cause and mechanism of AS. Conclusions were that kidney deficiency was the basic cause for AS; while pathogenic wind, cold, dampness, heat, blood stasis was the incentive. AS patients were weak innately or were lack of good care, causing kidney deficiency and Du meridian vacuity, and exogenous pathogen invaded and blocked the tendons and meridians, or endogenous pathogen blocked qi, blood circulation, and water flowing, then caused AS happen. The key therapeutic principle for AS was supplementing kidney and promoting blood circulation. 2 Clinical ResearchObjective:Assess the effects of Bushenqiangji Decoction (the therapeutic principle was supplementing kidney and promoting blood circulation) in treating AS and its safety. Therapeutic effects of Bushenqiangji Decoction for AS patients with kidney deficiency and blood stasis pattern were assessed from the measurements of ASAS20 response ratio, BASDAI50 response ratio, BASFI, BASMI, et al, from the improvements of clinical symptoms like spinal pain, stiffness, night pain, from syndrome indexes, compared with the effects of sulfasalazine. And the effects of the decoction on patients'liver, kidney, cardiovascular system, and hematological system were evaluated.Methods:90 patients with the pattern of kidney deficiency and blood stasis were outpatients from Rheumatism Department of Guang'anmen hospital from 2008 September to 2009 June. They were randomly divided into two groups.45 patients as the treatment group were treated with Bushenqiangji Decoction, while 45 patients as the control group were treated with sulfasalazine tablets, twice a day, and the course was 24 weeks. The response ratios of ASAS20 and BASDAI50 of the two groups were evaluated after 4 weeks',12 weeks',24 weeks'treatment, and their differences between the two groups were compared. And the changes of Bath ankylosing spondylitis disease activity index (BASDAI), Bath ankylosing spondylitis functional index (BASFI), Bath ankylosing spondylitis metrology index (BASMI), spinal pain, stiffness, night pain, patient global assessment and ESR, CRP were compared after 24-week-treatment.Results:ASAS20 responsive ratio of Bushenqiangji Decoction at 4th week,12th week, and 24th week was 27.27%,65.91%,86.36%, respectively. BASDAI50 responsive ratio was 6.98%?27.27%?63.64% respectively at 4th week,12th week, and 24th week. Syndrome total effective rate of the treatment group was 86.36%, much higher than the control group (58.14%). Bushenqiangji Decoction could improve the scores of BASDAI, BASFI, BASMI and, PGA, and it could alleviate spinal pain, stiffness, night pain and uncomfort in enthesis, and down regulate ESR and CRP. There was no adverse incidence happening in treatment group. Conclusion:Bushenqiangji Decoction was of definite clinical effect, and effected fast, consistently and steadily. It could improve AS patients's disease activity, function, and alleviate clinical symptoms like spinal pain, night pain, stiffness, and enthesis. It could improve the morbility of cervical vertebrae, spinal vertebrae, and hip joint. Bushenqiangji Decoction was effective, safe and reliable.2 Experiment ResearchWith the support from National Natural Science Foundation of China, this research using AS fibroblast system in vitro as the object, and taking the advantage of herbal serum pharmacological method and the technique of western blotting, aimed to study the effect of BMP/Smad signaling pathway on AS fibroblasts differentiating into osteoblasts, and to dig out the molecular mechanisms of Bushenhuoxue Decoction in anti-ossification of AS.2.1 Experiment OneObjective:To investigate the effects of different concentrations of recombinant human bone morphogenetic protein -2 (rhBMP-2) on fibroblast proliferation and alkaline phosphatase (ALP) activity, in order to see if BMP-2 could induce fibroblast from normal patients'(without AS) capsula articularis coxae differentiate into osteoblast.Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from those who got fracture by accidence. Fibroblast proliferation was detected by methyl thiazolyl tetrazolium (MTT) after different concentrations of rhBMP-2 (100,200,400ng/ml) stimulating the cells for 12h,24h, 48h and 72h. And fibroblast alkaline phosphatase was evaluated with the method of enzyme kinetics.Results:Fibroblast proliferation was promoted in the presence of different concentration of rhBMP-2 as time went by. Fibroblast proliferation went to the peak when cells were stimulated by rhBMP-2 after 24h. rhBMP-2 at a low concentration could significantly increased fibroblast proliferation in a dependent way, which was statistically different (P<0.05). With the concentration of rhBMP-2 at 200ng/ml, proliferative activity was increased to the peak, and then increased no more no matter how high the concentration of rhBMP-2 was. In the other hand, compared with the controlled group, ALP activity secreted by the fibroblasts improved obviously with statistically differences (P<0.05).Conclusion:Fibroblast from capsula articularis coxae of normal patients was potential to differentiate into osteoblast. At the stimulation of exogenous rhBMP-2, fibroblast proliferation increased, and fibroblast could differentiate into osteoblast.2.2 Experiment TwoObjective:Verify that BMP-2 could initiate the signal transduction of fibroblast from capsula articularis coxae of normal patients differentiating into osteoblast. Find out the changes of signal proteins pSmadl, Smadl and Smad4 expression after rhBMP-2 stimulating fibroblast for different periods, in order to testify that BMP/Smad signal transduction pathway was activated when fibroblast was stimulated by BMP-2.Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from those who got fracture by accidence. The expression of Smadl, Smad4, pSmadl in normal fibroblast was detected by western blotting after rhBMP-2 affected the cells 2h,6h,12h,24h.Results:Compared with those without rhBMP-2 treatment, pSmadl, Smad1 and Smad4 expressions of fibroblast treated with rhBMP-2 were much higher. The expression of pSmad1 increased immediately after treating rhBMP-2 for 6h (the difference between 6h group and control group was significant,P<0.05), and the expression persisted steadily (the difference between 12h group and 24h group was not obvious, P>0.05). The expression of Smad1 increased after treating rhBMP-2 for 6h, much higher than that of control group and 2h group (P<0.05), and kept on increasing as time goes by. The expression of Smadl increased after treating rhBMP-2 for 24h, much higher than that of control group,2h group,6h group and 12h group (P<0.05).Conclusion:BMP-2 could activate BMP/Smad signal transduction pathway in fibroblast from capsula articularis coxae of normal patients. Exogenous rhBMP-2 could phosphorylate signal protein Smadl, and up-regulate the expression of Smadl and Smad4, indicating that BMP-2 initiated the BMP/Smad signal transduction cascades in fibroblast from capsula articularis coxae of normal patients, activating the BMP/Smad pathway. It was believed that it was potential for BMP/Smad signal transduction pathway in fibroblast from capsula articularis coxae of normal patients to be further activated, and that rhBMP-2 induced fibroblast differentiating into osteoblast was that BMP-2 activated BMP/Smad signal transduction pathway.2.3 Experiment ThreeObjective:Expressions of pSmad1, Smadl and Smad4 in fibroblast from capsula articularis coxae of AS patients were compared with those from normal patients, and found out whether BMP/Smad pathway was activated in AS fibroblast. And then the expressions of Cbfa-1 were detected after AS fibroblast was stimulated by rhBMP-2, in order to testify that activation of BMP/Smad signal transduction pathway could induce AS fibroblast differentiating into osteoblast.Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from those who got fracture by accidence and those AS patients. Expressions of Smad1, Smad4, pSmad1 in normal fibroblast and AS fibroblast were dectected by western blotting, and the expressions of Cbfa-1 after fibroblast was treated with rhBMP-2 for 48h were western blotting.Results:Expressions of pSmad1, Smadl and Smad4 improved, comparing with those from control group (P<0.05). Expression of Cbfa-1 increased significantly with rhBMP-2 stimulation, whose difference between control group and between AS fibroblast without rhBMP-2 stimulation group was obvious (P<0.05).Conclusion:Activation of BMP/Smad signal transduction pathway was the potential mechanisms of AS fibroblast differentiating into osteoblast, and maybe it was one of the ossification mechanisms for AS. pSmad1, Smad1 and Smad4 in AS fibroblast were of high expressions, indicating that the pathway was activated in AS fibroblast. BMP-2 could induce AS fibroblast into osteoblast. 2.4 Experiment FourObjective:Testify Bushenhuoxue Decoction inhibited AS fibroblast differentiating into osteoblast through inhibiting BMP/Smad pathway from activating, and constraining the expression of pSmadl, Smadl and Smad4, so that inhibiting ossification occurring was achieved.Methods:The fibroblasts used in these researches were available with primary tissue culture, by planting capsula articularis coxae biopsies obtained during surgery for hip replacements from those who got fracture by accidence and those AS patients. The fibroblasts were divided into normal fibroblast group, AS fibroblast group, AS fibroblast+BMP group, AS fibroblast+BMP+control serum group, and AS fibroblast+BMP+decoction-containing serum group. All the cells were treated according to the grouping for 48h, and then the expression of pSmad1, Smad1, Smad4 and Cbfa-1 were detected by western blotting.Results:Cbfa-1 expression increased obviously after AS fibroblast was stimulated by rhBMP-2, while its expression of decoction-containing serum treated group was inhibited, the difference between whom was significant (P<0.005). Expressions of pSmad1, Smad1, and Smad4 all increased after AS fibroblast was stimulated by rhBMP-2 (P<0.001), while their expression of decoction-containing serum treated group decreased. The expressions of all these proteins were quite different from each groups (P<0.001).Conclusion:Inhibiting the activation of BMP/Smad signa1 transduction pathway in AS fibroblast was one of the molecular mechanisms of Bushenhuoxue Decoction delaying ossification occurring in AS patients. With the interference of Bushenhuoxue Decoction, the expression of Smadl and Smad4 was inhibited, phosphorylation of Smadl was constrained, and BMP/Smad transductive signal was restrained, so Cbfa-1 expression decreased and differentiation of fibroblast into osteoblast was inhibited. |
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