补肾活血法治疗强直性脊柱炎临床研究及抗骨化分子机制的探讨
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摘要
强直性脊柱炎(ankylosing spondylitis, AS)的发病机制至今不明,脊柱及外周关节(尤其是髋关节)骨化强直可致患者活动能力丧失,严重影响患者的生活、工作。因此,抑制或延缓骨化发生是AS治疗的关键,然而,没有证据显示目前的治疗方法可以延缓AS骨化的发生发展。中医中药在控制AS病情及延缓AS骨化发生具有巨大的潜能。本论文在导师冯兴华教授补肾活血法治疗AS学术思想的指导下,首先对以补肾活血立法组方的补肾强脊汤治疗AS的疗效与安全性进行系统的综合的评价；然后以体外培养的AS成纤维细胞系统作为研究对象,采用中药血清药理学方法,借助western blotting技术,在蛋白表达层面从BMP/Smad信号传导通路探讨中药复方补肾活血方药抑制AS成纤维细胞向成骨型分化、抗AS骨化作用的可能分子机制。
理论研究
1通过分析古代医籍关于脊强、肾痹、骨痹、大偻等与AS相似疾病的病因病机的论述,对腰痛、足跟痛、肩背痛等AS相关症状病因病机的阐述,总结得出AS的发病当是肾本虚,风、寒、湿、热、瘀血等邪气侵袭人体、痹阻经脉而致。2导师冯兴华教授从事中医风湿病治疗40余年,在强直性脊柱炎的治疗方面享誉盛名,文章从症状学的角度总结导师经验,分析AS发病的原因与机制。导师认为,提炼为“肾虚是AS的根本原因；风、寒、湿、热、瘀血是AS发病的诱因,是标实的表现”,患者先天不足,或后天失养,致使肾虚督空,风、寒、湿、热等外邪乘虚侵袭人体,痹阻筋脉、经络,或内生的湿、热、瘀血阻滞经脉,气血、水湿运行受阻,瘀滞而致生本病。治疗应以“补肾活血”为基本原则。
二临床研究
目的：评价补肾强脊汤治疗强直性脊柱炎的有效性和安全性。分别从AS评价(Assessment in ankylosing spondylitis, ASAS) 20达标率、Bath强直性脊柱炎疾病活动指数(Bath AS disease activity index, BASDAI) 50达标率、Bath强直性脊柱炎功能指数(Bath ankylosing spondylitis functional index, BASFI)、Bath强直性脊柱炎测量学指数(Bath ankylosing spondylitis metrology index, BASMI)等多种疗效评价指标,从脊柱疼痛、晨僵、夜间痛等症状改善情况,从中医证候指标等多个方面与西药柳氮磺胺吡啶比较,评价补肾强脊汤对肾虚瘀阻型AS患者的疗效；并观察其对患者肝肾功能、心血管、血液系统的影响。
方法：采用随机分组、对照,将来自于2008年9月至2009年6月中国中医科学院广安门医院风湿病科门诊的、90例中医辨证为肾虚瘀阻型AS患者分为两组,中药组45例,给予补肾强脊方药煎汤治疗,日两次；西药组45例,给予柳氮磺胺吡啶1g,口服日两次,共治疗24周。以4周、12周、24周为评价点,分别评价并比较两组的ASAS20、BASDAI50达标率。以24周为评价点,分别评价两组治疗前后BASDAI、BASFI、BASMI、脊柱疼痛、晨僵、夜间痛、患者总体评价(PGA)及急性时相反应物血沉(ESR)和C反应蛋白(CRP)的变化情况,以及中医证候指标的改善情况,并比较两组间的差异。
结果：补肾强脊汤在各评价点(4周、12周、24周)的ASAS20达标率分别为27.27%、65.91%、86.36%, BASDAI50达标率分别为6.98%、27.27%、63.64%,均较西药组为高。中医肾虚血瘀证候总有效率为86.36%,高于西药组的58.14%。补肾强脊汤可以有效的改善BASDAI、BASFI、BASMI等的评分,对患者总体评价、脊柱疼痛、夜间痛、晨僵、肌腱端不适等临床症状有明显缓解作用,对ESR、CRP亦有显著改善作用。试验过程中,中药组患者均未出现不良事件。
结论：补肾强脊汤治疗强直性脊柱炎的疗效确切,且起效迅速,疗效持久稳定。在各评价点(4周、12周、24周)的ASAS20达标率分别为27.27%、65.91%、86.36%, BASDAI50达标率分别为6.98%、27.27%、63.64%,均较西药组为高。中医肾虚血瘀证候总有效率为86.36%,高于西药组的58.14%。可以有效的改善AS患者的疾病活动度、躯体功能,改善脊柱疼痛、夜间痛、晨僵、肌腱端不适等相关临床症状。对AS患者颈椎、腰椎、髋关节活动范围都有很好的改善作用。补肾强脊汤治疗AS临床疗效满意,安全性和依从性良好。
三实验研究
以国家自然科学基金为依托,本课题以体外培养AS成纤维细胞系统作为研究对象,采用中药血清药理学方法,应用western blotting技术,研究BMP/Smad信号传导通路在AS成纤维细胞向成骨型分化的作用,并从该通路出发探讨补肾活血方药抗AS骨化作用的分子机制。
1实验一rhBMP-2对体外培养的正常人髋关节囊成纤维细胞增殖及分化的影响
目的：观察不同浓度重组人骨形态发生蛋白(rhBMP-2)作用下,体外培养的正常人髋关节囊成纤维细胞增殖活性及碱性磷酸酶活性,以了解rhBMP-2能否诱导正常人髋关节囊成纤维细胞向成骨型分化,为探索BMP-2在AS骨化发生中的作用提供基础。
方法：在北京大学人民医院骨关节矫形中心协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。应用MTT比色法检测rhBMP-2 (100、200、400ng/ml)刺激成纤维细胞12h、24h、48h、72h,细胞增殖的时效变化；检测不同浓度rhBMP-2(50、100、200、400、800、1600ng/ml)刺激成纤维细胞24h,细胞生长及增殖量效变化；用酶动力学方法检测不同浓度rhBMP-2作用下细胞分泌ALP的活性。
结果：不同浓度的rhBMP-2随刺激时间的增加均对成纤维细胞的增殖有促进作用,并于刺激24小时后增殖进入高峰,与刺激12小时后增殖情况有显著差异(P<0.05),与刺激48小时、72小时后的增殖情况没有显著差异(P>0.05)。但400ng/ml的rhBMP-2刺激72小时细胞增殖明显减少。rhBMP-2在较低浓度时对成纤维细胞的增殖即有显著的促进作用,并随浓度的升高促进作用增强,与空白对照组间差异有显著的统计学意义(P<0.05)。当浓度为200ng/ml时,增殖活性达到最大值,并由此细胞进入增殖的平台期。同时,成纤维细胞的ALP活性与空白对照组相比,活性均有显著增加,差异均具有显著的统计学意义(P<0.05),但各浓度组组间的差异无统计学意义(P>0.05)。
结论：正常人髋关节囊成纤维细胞具有向成骨型分化的潜能,在外源性rhBMP-2的诱导下可以表达成骨表型,并且可以促进细胞增殖。
2实验研究二BMP/Smad信号传导通路在体外培养正常人髋关节囊成纤维细胞的表达情况
目的：验证BMP-2可以开启正常人髋关节囊成纤维细胞向成骨型分化的信号传导通路。观察正常人髋关节囊成纤维细胞在rhBMP-2刺激下,信号蛋白Smad1、Smad4的表达情况,以及Smad1磷酸化的水平,证实正常人髋关节囊成纤维细胞在BMP-2的刺激下BMP/Smad信号传导通路活化。
方法：在北京大学人民医院骨关节矫形中心协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。应用蛋白印迹western blotting法检测在200ng/ml rhBMP-2刺激不同的时间(2h、6h、12h、24h)后,正常成纤维细胞中pSmad1、Smad1及Smad4的表达情况。
结果：正常人髋关节囊成纤维细胞在受到rhBMP-2的刺激后,pSmad1、Smad1、Smad4表达量都较未有受到BMP-2刺激组的表达量要高。pSmad1在受到rhBMP-2刺激6h后表达即显著升高(差异有显著性,P<0.05),随之持续稳定的表达(与12h组、24h组比较,差异没有显著性,P>0.05)。Smad1蛋白受到刺激6h后表达有所增加,与空白对照组及2h组相比差异有显著性(P<0.05),并随时间的增加其表达量逐渐升高。Smad4蛋白在受到rhBMP-2刺激后,表达量明显增加,刺激24小时后表达量增加尤为明显(与空白对照组及2h、6h、12h组对比,P<0.05)。
结论：BMP-2可以刺激正常人髋关节囊成纤维细胞的BMP/Smad信号传导通路活化。正常人髋关节囊成纤维细胞在受到外源性rhBMP-2刺激后信号蛋白Smad1发生磷酸化,Smad1和Smad4表达上调,显示BMP-2启动了正常人髋关节囊成纤维细胞BMP/Smad的级联反应,激活BMP/Smad通路的信号传导。表明正常人髋关节囊成纤维细胞中的BMP/Smad信号传导通路具有进一步活化的潜能,正常人髋关节囊成纤维细胞在rhBMP-2诱导下向成骨型分化可能是由于BMP-2激活了BMP/Smad信号传导通路。
3实验研究三BMP/Smad通路在强直性脊柱炎成纤维细胞中的表达情况
目的：通过与正常人髋关节囊成纤维细胞比较,观察AS髋关节囊成纤维细胞中pSmad1、Smad1及Smad4的表达情况,以了解AS髋关节囊成纤维细胞中BMP/Smad信号传导通路是否处于活化状态。并用rhBMP-2刺激AS成纤维细胞,观察成骨标志基因Cbfa-1的表达变化,证实BMP/Smad信号传导通路活化可以诱导AS成纤维细胞向成骨型转化。
方法：在北京大学人民医院骨关节矫形中心及北医三院骨科的协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊以及全髋关节置换手术AS患者的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。应用蛋白印迹western blotting法检测AS成纤维细胞和正常成纤维细胞中pSmad1、Smad1和Smad4的表达情况。然后用rhBMP-2刺激AS成纤维细胞48h后,用western blotting方法检测其Cbfa-1表达的变化。
结果：在AS成纤维细胞中pSmad1、Smad1、Smad4的表达都较正常成纤维细胞多(P<0.05)。在rhBMP-2的刺激下,Cbfa-1表达量明显增加(与AS成纤维细胞组相比,差异具有显著性,P<0.05)。
结论：BMP/Smad信号传导通路的活化是AS髋关节囊成纤维细胞向成骨型分化的可能机理之一,可能是AS骨化发生的机制之一。AS髋关节囊成纤维细胞中pSmad1、Smad1、Smad4蛋白表达上调,BMP/Smad信号传导通路在AS成纤维细胞中处于活化状态。而AS成纤维细胞在BMP-2的诱导下Cbfa-1表达升高,表达成骨表型。
4实验研究四补肾活血方药对AS成纤维细胞BMP/Smad信号传导通路的影响
目的：验证补肾活血方药可以通过抑制BMP/Smad信号传导通路活化,抑制相关蛋白pSmad1、Smad1、Smad4的表达而抑制AS成纤维细胞向成骨型分化,从而发挥抗AS骨化作用。
方法：在北京大学人民医院骨关节矫形中心及北医三院骨科的协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊以及全髋关节置换手术AS患者的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。将成纤维细胞分成正常成纤维细胞组、AS成纤维细胞组、AS成纤维细胞+BMP组、AS成纤维细胞+BMP+对照血清组、AS成纤维细胞+BMP+含药血清组,根据分组情况细胞培养48h后,应用western blotting技术检测各组中Cbfa-1、Smad1、Smad4及pSmad1的表达情况。
结果：AS成纤维细胞在受到rhBMP-2的刺激后,成骨标志基因Cbfa-1表达显著提高,而使用了含药血清处理的AS成纤维细胞Cbfa-1的表达则受到抑制,二者的差异具有显著性(P<0.005)。rhBMP-2刺激AS成纤维细胞后,Smadl蛋白明显发生磷酸化,pSmadl的表达量显著增多(P<0.001), Smad1、Smad4表达亦增加；而使用了补肾活血方药含药血清处理的成纤维细胞其pSmad1、Smad1、Smad4的表达较AS+BMP组的表达降低,差异具有显著性(P<0.001),与AS+BMP+对照血清组比较表达量具有显著差异性(P<0.001)。
结论：证实抑制AS髋关节囊成纤维细胞BMP/Smad信号传导通路的活化是补肾活血方药抗AS骨化的分子机制之一。在补肾活血方药含药血清的干预作用下,AS成纤维细胞Smadl与Smad4表达受抑,Smad1磷酸化受限,BMP/Smad通路传导的信号受制,因而Cbfa-1表达减少,成纤维细胞向成骨型分化被遏。
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 Research
Research 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 Research
Objective: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 Research
With 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 One
Objective: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 Two
Objective: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 Three
Objective: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 Four
Objective: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.
理论研究
1通过分析古代医籍关于脊强、肾痹、骨痹、大偻等与AS相似疾病的病因病机的论述,对腰痛、足跟痛、肩背痛等AS相关症状病因病机的阐述,总结得出AS的发病当是肾本虚,风、寒、湿、热、瘀血等邪气侵袭人体、痹阻经脉而致。2导师冯兴华教授从事中医风湿病治疗40余年,在强直性脊柱炎的治疗方面享誉盛名,文章从症状学的角度总结导师经验,分析AS发病的原因与机制。导师认为,提炼为“肾虚是AS的根本原因；风、寒、湿、热、瘀血是AS发病的诱因,是标实的表现”,患者先天不足,或后天失养,致使肾虚督空,风、寒、湿、热等外邪乘虚侵袭人体,痹阻筋脉、经络,或内生的湿、热、瘀血阻滞经脉,气血、水湿运行受阻,瘀滞而致生本病。治疗应以“补肾活血”为基本原则。
二临床研究
目的：评价补肾强脊汤治疗强直性脊柱炎的有效性和安全性。分别从AS评价(Assessment in ankylosing spondylitis, ASAS) 20达标率、Bath强直性脊柱炎疾病活动指数(Bath AS disease activity index, BASDAI) 50达标率、Bath强直性脊柱炎功能指数(Bath ankylosing spondylitis functional index, BASFI)、Bath强直性脊柱炎测量学指数(Bath ankylosing spondylitis metrology index, BASMI)等多种疗效评价指标,从脊柱疼痛、晨僵、夜间痛等症状改善情况,从中医证候指标等多个方面与西药柳氮磺胺吡啶比较,评价补肾强脊汤对肾虚瘀阻型AS患者的疗效；并观察其对患者肝肾功能、心血管、血液系统的影响。
方法：采用随机分组、对照,将来自于2008年9月至2009年6月中国中医科学院广安门医院风湿病科门诊的、90例中医辨证为肾虚瘀阻型AS患者分为两组,中药组45例,给予补肾强脊方药煎汤治疗,日两次；西药组45例,给予柳氮磺胺吡啶1g,口服日两次,共治疗24周。以4周、12周、24周为评价点,分别评价并比较两组的ASAS20、BASDAI50达标率。以24周为评价点,分别评价两组治疗前后BASDAI、BASFI、BASMI、脊柱疼痛、晨僵、夜间痛、患者总体评价(PGA)及急性时相反应物血沉(ESR)和C反应蛋白(CRP)的变化情况,以及中医证候指标的改善情况,并比较两组间的差异。
结果：补肾强脊汤在各评价点(4周、12周、24周)的ASAS20达标率分别为27.27%、65.91%、86.36%, BASDAI50达标率分别为6.98%、27.27%、63.64%,均较西药组为高。中医肾虚血瘀证候总有效率为86.36%,高于西药组的58.14%。补肾强脊汤可以有效的改善BASDAI、BASFI、BASMI等的评分,对患者总体评价、脊柱疼痛、夜间痛、晨僵、肌腱端不适等临床症状有明显缓解作用,对ESR、CRP亦有显著改善作用。试验过程中,中药组患者均未出现不良事件。
结论：补肾强脊汤治疗强直性脊柱炎的疗效确切,且起效迅速,疗效持久稳定。在各评价点(4周、12周、24周)的ASAS20达标率分别为27.27%、65.91%、86.36%, BASDAI50达标率分别为6.98%、27.27%、63.64%,均较西药组为高。中医肾虚血瘀证候总有效率为86.36%,高于西药组的58.14%。可以有效的改善AS患者的疾病活动度、躯体功能,改善脊柱疼痛、夜间痛、晨僵、肌腱端不适等相关临床症状。对AS患者颈椎、腰椎、髋关节活动范围都有很好的改善作用。补肾强脊汤治疗AS临床疗效满意,安全性和依从性良好。
三实验研究
以国家自然科学基金为依托,本课题以体外培养AS成纤维细胞系统作为研究对象,采用中药血清药理学方法,应用western blotting技术,研究BMP/Smad信号传导通路在AS成纤维细胞向成骨型分化的作用,并从该通路出发探讨补肾活血方药抗AS骨化作用的分子机制。
1实验一rhBMP-2对体外培养的正常人髋关节囊成纤维细胞增殖及分化的影响
目的：观察不同浓度重组人骨形态发生蛋白(rhBMP-2)作用下,体外培养的正常人髋关节囊成纤维细胞增殖活性及碱性磷酸酶活性,以了解rhBMP-2能否诱导正常人髋关节囊成纤维细胞向成骨型分化,为探索BMP-2在AS骨化发生中的作用提供基础。
方法：在北京大学人民医院骨关节矫形中心协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。应用MTT比色法检测rhBMP-2 (100、200、400ng/ml)刺激成纤维细胞12h、24h、48h、72h,细胞增殖的时效变化；检测不同浓度rhBMP-2(50、100、200、400、800、1600ng/ml)刺激成纤维细胞24h,细胞生长及增殖量效变化；用酶动力学方法检测不同浓度rhBMP-2作用下细胞分泌ALP的活性。
结果：不同浓度的rhBMP-2随刺激时间的增加均对成纤维细胞的增殖有促进作用,并于刺激24小时后增殖进入高峰,与刺激12小时后增殖情况有显著差异(P<0.05),与刺激48小时、72小时后的增殖情况没有显著差异(P>0.05)。但400ng/ml的rhBMP-2刺激72小时细胞增殖明显减少。rhBMP-2在较低浓度时对成纤维细胞的增殖即有显著的促进作用,并随浓度的升高促进作用增强,与空白对照组间差异有显著的统计学意义(P<0.05)。当浓度为200ng/ml时,增殖活性达到最大值,并由此细胞进入增殖的平台期。同时,成纤维细胞的ALP活性与空白对照组相比,活性均有显著增加,差异均具有显著的统计学意义(P<0.05),但各浓度组组间的差异无统计学意义(P>0.05)。
结论：正常人髋关节囊成纤维细胞具有向成骨型分化的潜能,在外源性rhBMP-2的诱导下可以表达成骨表型,并且可以促进细胞增殖。
2实验研究二BMP/Smad信号传导通路在体外培养正常人髋关节囊成纤维细胞的表达情况
目的：验证BMP-2可以开启正常人髋关节囊成纤维细胞向成骨型分化的信号传导通路。观察正常人髋关节囊成纤维细胞在rhBMP-2刺激下,信号蛋白Smad1、Smad4的表达情况,以及Smad1磷酸化的水平,证实正常人髋关节囊成纤维细胞在BMP-2的刺激下BMP/Smad信号传导通路活化。
方法：在北京大学人民医院骨关节矫形中心协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。应用蛋白印迹western blotting法检测在200ng/ml rhBMP-2刺激不同的时间(2h、6h、12h、24h)后,正常成纤维细胞中pSmad1、Smad1及Smad4的表达情况。
结果：正常人髋关节囊成纤维细胞在受到rhBMP-2的刺激后,pSmad1、Smad1、Smad4表达量都较未有受到BMP-2刺激组的表达量要高。pSmad1在受到rhBMP-2刺激6h后表达即显著升高(差异有显著性,P<0.05),随之持续稳定的表达(与12h组、24h组比较,差异没有显著性,P>0.05)。Smad1蛋白受到刺激6h后表达有所增加,与空白对照组及2h组相比差异有显著性(P<0.05),并随时间的增加其表达量逐渐升高。Smad4蛋白在受到rhBMP-2刺激后,表达量明显增加,刺激24小时后表达量增加尤为明显(与空白对照组及2h、6h、12h组对比,P<0.05)。
结论：BMP-2可以刺激正常人髋关节囊成纤维细胞的BMP/Smad信号传导通路活化。正常人髋关节囊成纤维细胞在受到外源性rhBMP-2刺激后信号蛋白Smad1发生磷酸化,Smad1和Smad4表达上调,显示BMP-2启动了正常人髋关节囊成纤维细胞BMP/Smad的级联反应,激活BMP/Smad通路的信号传导。表明正常人髋关节囊成纤维细胞中的BMP/Smad信号传导通路具有进一步活化的潜能,正常人髋关节囊成纤维细胞在rhBMP-2诱导下向成骨型分化可能是由于BMP-2激活了BMP/Smad信号传导通路。
3实验研究三BMP/Smad通路在强直性脊柱炎成纤维细胞中的表达情况
目的：通过与正常人髋关节囊成纤维细胞比较,观察AS髋关节囊成纤维细胞中pSmad1、Smad1及Smad4的表达情况,以了解AS髋关节囊成纤维细胞中BMP/Smad信号传导通路是否处于活化状态。并用rhBMP-2刺激AS成纤维细胞,观察成骨标志基因Cbfa-1的表达变化,证实BMP/Smad信号传导通路活化可以诱导AS成纤维细胞向成骨型转化。
方法：在北京大学人民医院骨关节矫形中心及北医三院骨科的协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊以及全髋关节置换手术AS患者的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。应用蛋白印迹western blotting法检测AS成纤维细胞和正常成纤维细胞中pSmad1、Smad1和Smad4的表达情况。然后用rhBMP-2刺激AS成纤维细胞48h后,用western blotting方法检测其Cbfa-1表达的变化。
结果：在AS成纤维细胞中pSmad1、Smad1、Smad4的表达都较正常成纤维细胞多(P<0.05)。在rhBMP-2的刺激下,Cbfa-1表达量明显增加(与AS成纤维细胞组相比,差异具有显著性,P<0.05)。
结论：BMP/Smad信号传导通路的活化是AS髋关节囊成纤维细胞向成骨型分化的可能机理之一,可能是AS骨化发生的机制之一。AS髋关节囊成纤维细胞中pSmad1、Smad1、Smad4蛋白表达上调,BMP/Smad信号传导通路在AS成纤维细胞中处于活化状态。而AS成纤维细胞在BMP-2的诱导下Cbfa-1表达升高,表达成骨表型。
4实验研究四补肾活血方药对AS成纤维细胞BMP/Smad信号传导通路的影响
目的：验证补肾活血方药可以通过抑制BMP/Smad信号传导通路活化,抑制相关蛋白pSmad1、Smad1、Smad4的表达而抑制AS成纤维细胞向成骨型分化,从而发挥抗AS骨化作用。
方法：在北京大学人民医院骨关节矫形中心及北医三院骨科的协助下,取因外伤骨折需行全髋关节置换手术患者(男性,18-55岁)的髋关节囊以及全髋关节置换手术AS患者的髋关节囊,采用组织块培养法培养人髋关节囊成纤维细胞。将成纤维细胞分成正常成纤维细胞组、AS成纤维细胞组、AS成纤维细胞+BMP组、AS成纤维细胞+BMP+对照血清组、AS成纤维细胞+BMP+含药血清组,根据分组情况细胞培养48h后,应用western blotting技术检测各组中Cbfa-1、Smad1、Smad4及pSmad1的表达情况。
结果：AS成纤维细胞在受到rhBMP-2的刺激后,成骨标志基因Cbfa-1表达显著提高,而使用了含药血清处理的AS成纤维细胞Cbfa-1的表达则受到抑制,二者的差异具有显著性(P<0.005)。rhBMP-2刺激AS成纤维细胞后,Smadl蛋白明显发生磷酸化,pSmadl的表达量显著增多(P<0.001), Smad1、Smad4表达亦增加；而使用了补肾活血方药含药血清处理的成纤维细胞其pSmad1、Smad1、Smad4的表达较AS+BMP组的表达降低,差异具有显著性(P<0.001),与AS+BMP+对照血清组比较表达量具有显著差异性(P<0.001)。
结论：证实抑制AS髋关节囊成纤维细胞BMP/Smad信号传导通路的活化是补肾活血方药抗AS骨化的分子机制之一。在补肾活血方药含药血清的干预作用下,AS成纤维细胞Smadl与Smad4表达受抑,Smad1磷酸化受限,BMP/Smad通路传导的信号受制,因而Cbfa-1表达减少,成纤维细胞向成骨型分化被遏。
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 Research
Research 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 Research
Objective: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 Research
With 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 One
Objective: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 Two
Objective: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 Three
Objective: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 Four
Objective: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.
引文
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