补肾舒脊颗粒延缓强直性脊柱炎异位骨化的机制研究

Ectopic ossification is an important pathological manifestation in the later stage of Ankylosing Spondylitis (AS), it is also an important factor inducing poor prognosis or disability. It is thought that the mechanism is associated with endochondral bone formation and membranous bone formation. Both Wnt and BMP pathway play an important role in it. On the basis of inheriting the academic experience of professor Jiao Shu De, we create an effective medicine, Bu Shen Shu Ji granule, to treat AS. In clinical research, we have demonstrated that this granule could postpone the ectopic ossification in AS. In basic research, we have proved that it could modulate DKK1 and BMPs of the serum in AS,which were upstream elements of Wnt and BMP pathway. On basis of these evidences, we put forward the hypothesis that the mechanism of this granule in postponing ectopic ossification is intervening the endochondral bone formation and membranous bone formation in BMSCs through modulating Wnt and BMP pathway. In this research, the ectopic ossification animal model of elder male DBA/1 mice would be introduced in, animal and cell experiments would be conducted to confirm the effect of the granule in modulating the endochondral bone formation and membranous bone formation in BMSCs and the effect in modulating Wnt and BMP pathway. In future study, we would conduct cell blocking experiments to further validate the special targets in these pathway. The research has very important significance for exploring the effect target of this Chinese medicine in treating ectopic ossification of AS, forming treatment methods for early stage to postpone the process of ectopic ossification.

异位骨化是强直性脊柱炎（AS）疾病后期的主要病理特点，是患者预后不良和致残的主要因素。目前认为其机制可能与Wnt和BMP通路介导的间充质干细胞（BMSCs）软骨内成骨及膜内成骨有关。课题组继承焦树德学术思想，创立补肾舒脊颗粒（舒药）治疗AS，临床研究证实能延缓AS异位骨化，前期基础研究显示该药能调节患者血清中Wnt和BMP通路的上游因子DKK1和BMPs的水平，据此提出假说：舒药延缓AS异位骨化的可能机制是调节BMSCs软骨内成骨和膜内成骨的Wnt和BMP通路。本研究引入老龄雄性DBA/1小鼠异位骨化动物模型，采用动物及细胞实验相结合，研究舒药对BMSCs软骨内成骨和膜内成骨的影响，以及对Wnt和BMP通路的调节作用，并运用细胞水平阻断实验进一步验证该通路的特异性。本研究对于探索中药治疗AS异位骨化的作用靶点，形成早期干预治疗方法，延缓异位骨化发生，具有重要意义。

异位骨化是强直性脊柱炎（AS）疾病后期的主要病理特点，其主要机制可能与Wnt和BMP通路介导的间充质干细胞（BMSCs）软骨内成骨及膜内成骨有关。本研究引入异位骨化动物模型，采用动物及细胞实验相结合，研究补肾舒脊颗粒对Wnt/β-catenin和BMP/Smad信号通路的调节作用。并运用体内试验和细胞水平阻断实验进一步验证。研究对于探索中药治疗AS异位骨化的作用靶点，形成早期干预治疗方法，延缓异位骨化发生，具有重要意义。动物实验部分，课题将DBA/1小鼠随机分为模型组、阳性药物组、补肾舒脊颗粒低、中、高剂量组，分别给予生理盐水、洛索洛芬钠水溶液、补肾舒脊颗粒溶液（低、中、高剂量）灌胃。评价补肾舒脊成方对动物模型异位骨化的影响，以及对模型附着点Wnt5a、β-catenin、DKK1、SOST、GSK-3β、BMP4、Smad4、Runx2 mRNA及蛋白表达水平的影响。细胞实验部分，诱导人骨髓间充质干细胞分别向软骨细胞和成骨细胞分化，然后分为空白组、模型组、对照组及中药高、中、低剂量组，分别给予正常大鼠血清、正常大鼠血清、洛索洛芬钠含药血清、补肾舒脊高、中、低剂量含药血清干预，检测各组细胞Wnt5a、β-catenin、DKK1、SOST、GSK-3β、BMP4、Smad4、Runx2 mRNA及蛋白表达水平的变化。在诱导过程中，分别给予Wnt/β-catenin信号通路和BMP4/Smad4信号通路的阻断剂，然后给与上述干预措施，观察补肾舒脊颗粒含药血清对上述指标的影响。结果提示：补肾舒脊颗粒成方可减轻实验动物模型关节肿胀程度、异位骨化；补肾舒脊颗粒通过增加DKK1的表达来抑制Wnt/β-catenin信号通路上Wnt5a、β-catenin、SOST的表达，从而起到延缓强直性脊柱炎异位骨化的作用。补肾舒脊颗粒通过抑制BMP4/Smad4信号通路上BMP4、Smad4、Runx的表达来起到减轻附着点炎症、延缓异位骨化的作用。