从骨形态发生蛋白及其转导通路探讨补肾活血法治疗强直性脊柱炎的抗骨化作用机制研究
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摘要
强直性脊柱炎(ankylosingspondylitis, AS)是一种慢性炎症性自身免疫性疾病,其病变最终会引起脊柱及外周关节(尤其是髋关节)的骨化强直,导致患者活动功能丧失,严重影响生活质量。病理性成骨是AS患者致残的主要原因,抑制或延缓骨化是AS治疗的关键。骨形态发生蛋白(bone morphogenetic proteins, BMPs)作为骨生长的启动因子,具有促进成骨细胞分化,诱导未分化间充质细胞向骨系细胞不可逆分化的能力,其中BMP-2是活性最强且能单独诱导成骨的,众多研究表明BMPs及其信号的转导在AS骨化过程中发挥着重要的作用,是探索延缓AS骨化药物和方法的重要切入点。
强直性脊柱炎属中医学“骨痹”、“腰痛”等范畴,中医药治疗AS具有疗效好、副作用小的优势,在控制AS病情及延缓AS骨化发生方面具有一定的潜能。导师冯兴华教授是我国著名的风湿病专家,在AS治疗方面有其独到之处。本论文以总结导师处方用药经验为基础,并在其补肾活血法治疗AS学术思想的指导下,首先对补肾活血法治疗AS的疗效进行综合评价并观察其对AS患者血清中BMP-2的干预作用；而后以体外培养的AS髋关节囊成纤维细胞作为研究对象,采用中药血清药理学方法及现代分子生物学技术,观察补肾活血方药对BMP信号转导通路的影响,从临床和实验两个不同层面探讨补肾活血法治疗AS、发挥抗AS骨化作用的可能机制,进而揭示导师治疗AS临床经验的科学内涵。
一、导师治疗AS经验总结
通过收集、整理导师门诊治疗AS患者的病例资料,利用频数分析、聚类分析、关联规则分析的方法对导师治疗AS的用药规律进行数据挖掘,对其经验中最核心的方-证、药-证、药-症关系进行梳理归纳,总结其治疗AS的特色包括：辨病与辨证论治相结合为核心,以补肾活血法作为本病的基本治疗大法,补肾强脊汤作为治疗本病的核心处方；辨症论治为辅,体现个体化治疗思路；重视活血化瘀药的应用。
二、临床研究
通过前瞻性的临床研究,首先选取活动期肾虚血瘀证AS患者60例,并以健康成年人、缓解期肾虚血瘀证AS患者各30例作为对照,检测各组血清中BMP-2的浓度,记录AS患者的各项病情评价指标,通过分析各组BMP-2的浓度差异,以及BMP-2与其它实验室指标、AS病情评价指标的相关性,探讨AS患者血清中BMP-2的临床意义。
研究结果显示：AS活动期及缓解期患者血清中BMP-2均显著高于正常组,BMP-2与实验室指标CRP、CD62P成显著正相关,BMP-2与BASDAI、 BASFI、 BASMI等AS病情评价指标呈显著正相关,与腰椎前屈、腰椎侧弯及踝间距等测量指标呈显著负相关,提示AS患者血清中BMP-2与疾病的活动度相关,且BMP-2作为促进骨形成的重要细胞因子,其在AS患者血清中的浓度增加,可能与AS患者病理性成骨,造成脊柱及外周关节骨化强直,活动功能受限有关。
随后我们对纳入的60例活动期肾虚血瘀证AS患者进行治疗,以补肾活血立法,予口服补肾强脊汤加减作为基础治疗,并佐以穴位贴敷疗法,以4周、8周、12周作为评价点,应用国际ASAS20评价标准、BASDAI50评价标准、中医证候疗效评价等多维疗效评价体系,评价补肾活血法治疗AS的临床疗效及安全性,并观察其对血清中BMP-2的干预作用。
研究结果显示：以补肾活血为法治疗AS的疗效确切,治疗12周后,ASAS20的达标率为85.00%,BASDAI50达标率为68.33%,中医证候疗效总有效率为91.67%；补肾活血中药对AS患者的BASDAI、 BASFI、 BASMK、病人总体评价、夜间痛、肌腱端压痛等有明显的改善作用,能够很好的降低患者的疾病活动度,并能明显改善患者的功能状况；对AS患者中轴关节、髋关节以及其它外周关节的疼痛和功能均有一定的改善作用；对AS患者的CRP、PLT、 CD62P有改善作用,体现了很好的抗炎作用；对AS患者血清中BMP-2有明显的干预作用,提示其可能通过抑制BMP-2的成骨作用从而延缓AS骨化进展；研究所采用的补肾活血综合治疗方案安全性好。
临床研究结果验证了补肾活血法治疗AS的确切疗效,证明以补肾活血立法处方的补肾强脊汤有很好的抗炎作用以及抗AS骨化的潜能,具有广泛的推广价值,并为AS综合治疗方案的制定和优化提供良好的依据。
三、实验研究
实验研究以国家自然科学基金为依托,将体外培养的AS髋关节囊成纤维细胞系作为研究对象,采用中药血清药理学方法,借助Western blotting、实时定量PCR等现代分子生物学技术,以调控成骨分化的BMP/Smads信号转导通路为切入点,通过检测BMP/Smads信号分子在AS成纤维细胞中的表达情况及rhBMP-2诱导后的通路表达情况,观察补肾活血立法处方的补肾强脊颗粒含药血清对AS成纤维细胞BMP/Smads信号转导通路及rhBMP-2诱导后通路的影响,从分子信号转导层面探讨补肾活血法治疗AS、发挥抗骨化作用的可能机制。
实验研究结果表明：AS成纤维细胞中成骨标志基因Cbfal表达明显高于正常,且存在大量的BMP特异性结合受体,Smad4呈高表达,Smadl及Smad5的磷酸化程度明显高于正常对照组,Smad6的表达量显著低于正常对照组；而外源性rhBMP-2可以促进成纤维细胞增殖,引起成骨标志基因Cbfal、 Smad4表达上调,Smadl及Smad5的磷酸化程度进一步提高。结果提示BMP/Smads信号转导通路高度活化以及通路调控机制发生紊乱,引起成骨标志基因Cbfal大量表达可能是AS成纤维细胞向成骨型分化,导致AS骨化发生的重要分子机制之一,且BMP-2可能是诱导这一通路活化,引起病理性成骨的重要细胞因子。
实验研究结果亦表明：补肾强脊颗粒含药血清可以显著抑制AS成纤维细胞BMPRII和Smad4表达,提高Smad6表达,降低Smadl及Smad5磷酸化程度,从而减少BMP信号的下传,抑制Cbfal的表达；补肾强脊颗粒含药血清亦通过调节BMPRII、 Smad4、 Smad6的表达,以及Smadl.5的磷酸化程度,从而降低Cbfal的表达,抑制rhBMP-2对成纤维细胞向成骨型分化的诱导作用。结果提示以补肾活血立法处方的补肾强脊颗粒能够通过对BMP/Smads信号转导通路多个环节的调节抑制成纤维细胞向成骨型分化,且能明显抑制BMP-2对成纤维细胞向成骨型分化的诱导,这可能是补肾强脊颗粒发挥抗AS骨化作用,延缓AS骨化进展的重要分子机制之一
实验研究结果从分子水平阐释了以补肾活血法立法处方的补肾强脊颗粒治疗AS的作用靶点与作用机制,从微观角度证明了补肾活血法是治疗AS的重要且有效之法。
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 AS
To 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 Researches
Firstly, 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 Researches
With 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.
强直性脊柱炎属中医学“骨痹”、“腰痛”等范畴,中医药治疗AS具有疗效好、副作用小的优势,在控制AS病情及延缓AS骨化发生方面具有一定的潜能。导师冯兴华教授是我国著名的风湿病专家,在AS治疗方面有其独到之处。本论文以总结导师处方用药经验为基础,并在其补肾活血法治疗AS学术思想的指导下,首先对补肾活血法治疗AS的疗效进行综合评价并观察其对AS患者血清中BMP-2的干预作用；而后以体外培养的AS髋关节囊成纤维细胞作为研究对象,采用中药血清药理学方法及现代分子生物学技术,观察补肾活血方药对BMP信号转导通路的影响,从临床和实验两个不同层面探讨补肾活血法治疗AS、发挥抗AS骨化作用的可能机制,进而揭示导师治疗AS临床经验的科学内涵。
一、导师治疗AS经验总结
通过收集、整理导师门诊治疗AS患者的病例资料,利用频数分析、聚类分析、关联规则分析的方法对导师治疗AS的用药规律进行数据挖掘,对其经验中最核心的方-证、药-证、药-症关系进行梳理归纳,总结其治疗AS的特色包括：辨病与辨证论治相结合为核心,以补肾活血法作为本病的基本治疗大法,补肾强脊汤作为治疗本病的核心处方；辨症论治为辅,体现个体化治疗思路；重视活血化瘀药的应用。
二、临床研究
通过前瞻性的临床研究,首先选取活动期肾虚血瘀证AS患者60例,并以健康成年人、缓解期肾虚血瘀证AS患者各30例作为对照,检测各组血清中BMP-2的浓度,记录AS患者的各项病情评价指标,通过分析各组BMP-2的浓度差异,以及BMP-2与其它实验室指标、AS病情评价指标的相关性,探讨AS患者血清中BMP-2的临床意义。
研究结果显示：AS活动期及缓解期患者血清中BMP-2均显著高于正常组,BMP-2与实验室指标CRP、CD62P成显著正相关,BMP-2与BASDAI、 BASFI、 BASMI等AS病情评价指标呈显著正相关,与腰椎前屈、腰椎侧弯及踝间距等测量指标呈显著负相关,提示AS患者血清中BMP-2与疾病的活动度相关,且BMP-2作为促进骨形成的重要细胞因子,其在AS患者血清中的浓度增加,可能与AS患者病理性成骨,造成脊柱及外周关节骨化强直,活动功能受限有关。
随后我们对纳入的60例活动期肾虚血瘀证AS患者进行治疗,以补肾活血立法,予口服补肾强脊汤加减作为基础治疗,并佐以穴位贴敷疗法,以4周、8周、12周作为评价点,应用国际ASAS20评价标准、BASDAI50评价标准、中医证候疗效评价等多维疗效评价体系,评价补肾活血法治疗AS的临床疗效及安全性,并观察其对血清中BMP-2的干预作用。
研究结果显示：以补肾活血为法治疗AS的疗效确切,治疗12周后,ASAS20的达标率为85.00%,BASDAI50达标率为68.33%,中医证候疗效总有效率为91.67%；补肾活血中药对AS患者的BASDAI、 BASFI、 BASMK、病人总体评价、夜间痛、肌腱端压痛等有明显的改善作用,能够很好的降低患者的疾病活动度,并能明显改善患者的功能状况；对AS患者中轴关节、髋关节以及其它外周关节的疼痛和功能均有一定的改善作用；对AS患者的CRP、PLT、 CD62P有改善作用,体现了很好的抗炎作用；对AS患者血清中BMP-2有明显的干预作用,提示其可能通过抑制BMP-2的成骨作用从而延缓AS骨化进展；研究所采用的补肾活血综合治疗方案安全性好。
临床研究结果验证了补肾活血法治疗AS的确切疗效,证明以补肾活血立法处方的补肾强脊汤有很好的抗炎作用以及抗AS骨化的潜能,具有广泛的推广价值,并为AS综合治疗方案的制定和优化提供良好的依据。
三、实验研究
实验研究以国家自然科学基金为依托,将体外培养的AS髋关节囊成纤维细胞系作为研究对象,采用中药血清药理学方法,借助Western blotting、实时定量PCR等现代分子生物学技术,以调控成骨分化的BMP/Smads信号转导通路为切入点,通过检测BMP/Smads信号分子在AS成纤维细胞中的表达情况及rhBMP-2诱导后的通路表达情况,观察补肾活血立法处方的补肾强脊颗粒含药血清对AS成纤维细胞BMP/Smads信号转导通路及rhBMP-2诱导后通路的影响,从分子信号转导层面探讨补肾活血法治疗AS、发挥抗骨化作用的可能机制。
实验研究结果表明：AS成纤维细胞中成骨标志基因Cbfal表达明显高于正常,且存在大量的BMP特异性结合受体,Smad4呈高表达,Smadl及Smad5的磷酸化程度明显高于正常对照组,Smad6的表达量显著低于正常对照组；而外源性rhBMP-2可以促进成纤维细胞增殖,引起成骨标志基因Cbfal、 Smad4表达上调,Smadl及Smad5的磷酸化程度进一步提高。结果提示BMP/Smads信号转导通路高度活化以及通路调控机制发生紊乱,引起成骨标志基因Cbfal大量表达可能是AS成纤维细胞向成骨型分化,导致AS骨化发生的重要分子机制之一,且BMP-2可能是诱导这一通路活化,引起病理性成骨的重要细胞因子。
实验研究结果亦表明：补肾强脊颗粒含药血清可以显著抑制AS成纤维细胞BMPRII和Smad4表达,提高Smad6表达,降低Smadl及Smad5磷酸化程度,从而减少BMP信号的下传,抑制Cbfal的表达；补肾强脊颗粒含药血清亦通过调节BMPRII、 Smad4、 Smad6的表达,以及Smadl.5的磷酸化程度,从而降低Cbfal的表达,抑制rhBMP-2对成纤维细胞向成骨型分化的诱导作用。结果提示以补肾活血立法处方的补肾强脊颗粒能够通过对BMP/Smads信号转导通路多个环节的调节抑制成纤维细胞向成骨型分化,且能明显抑制BMP-2对成纤维细胞向成骨型分化的诱导,这可能是补肾强脊颗粒发挥抗AS骨化作用,延缓AS骨化进展的重要分子机制之一
实验研究结果从分子水平阐释了以补肾活血法立法处方的补肾强脊颗粒治疗AS的作用靶点与作用机制,从微观角度证明了补肾活血法是治疗AS的重要且有效之法。
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 AS
To 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 Researches
Firstly, 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 Researches
With 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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