从破骨细胞分化调节通路OPG-RANKL-RANK探讨针刺治疗骨质疏松症的作用机理

Research on mechanism of acupuncture action based on signaling transduction becomes one of hot spots of acupuncture experimental study . Although there are some progresses on experimental and clinical studies of effect of acupuncture on OP, the signaling transduction of acupuncture for OP is still unclear.OPG-RANKL-RANK system in osteoclast differentiation and mature mechanism plays a very crucial role in signal transduction pathways. Once the combination of RANKL and RANK will stimulate osteoclast precursor cell differentiation,activation of mature osteoclasts,causing bone resorption, while OPG can be competitive with the membrane receptor RANK binding RANKL, thereby blocking signal transduction of bone resorption.With vivo and vitro experiment,OPG,RANKL will be deceted by Histomorphology,Cytomorphology,Immunohistochemistry,and in Situ Hybridization methods,and effect of acupuncture on osteoclasts OPG-RANKL-RANK system,the role of signal transduction processes and mechanisms will be explored in order to determine whether the acupuncture promoted OPG-RANKL-RANK expression to verify that hypothesis: Acupuncture by regulating the OPG-RANKL-RANK system to inhibit bone resorption, promoting bone formation. This study will reveals ralated meachnisim of the acupuncture treating osteoporosis (OP) at molecular level by regulating OPG-RANKL-RANK signaling pathways,and provide with scientific evidence for OP treating with acupuncture.

基于信号传导途径的针灸作用机制研究是目前针灸实验研究的热点之一。尽管目前针灸治疗骨质疏松症的临床和实验研究取得了一定进展，但对于针灸治疗骨质疏松症的信号传导途径仍未阐明。OPG-RANKL-RANK系统在破骨细胞的分化及成熟机制中起到十分关键的信号传导通路的作用。RANKL一旦与RANK结合就会刺激前体破骨细胞分化，活化成熟的破骨细胞，引起骨吸收，而OPG可与膜受体RANK 竞争性结合RANKL，从而阻断骨吸收信号的传递。本研究拟通过在体针刺干预和与离体骨细胞培养加入针刺血清，以组织形态、细胞形态、免疫组化染色和原位杂交方法检测OPG、RANKL的变化，探讨针刺治疗后OPG-RANKL-RANK破骨细胞分化调节信号传导变化的规律，阐明针刺是否通过调节这一信号通路系统达到治疗的目的，以期从分子水平揭示针刺调控OPG-RANKL-RANK信号通路的治疗机制，为针灸治疗OP临床应用提供科学依据。

目的 针灸治疗骨质疏松症的信号传导途径仍未阐明。OPG-RANKL-RANK系统在破骨细胞的分化及成熟机制中起到十分关键的作用。本研究旨在探究针刺治疗骨质疏松症OPG-RANKL-RANK的变化规律。方法 选用雌性SD大鼠50只，随机分成空白对照组、假手术组、模型组、己烯雌酚组、电针组各10只。行双侧卵巢切除术复制骨质疏松模型。造模饲养13周后，开始治疗。电针穴取“三阴交”、“足三里”、“肾俞”、“脾俞”，每次15min，所有治疗每周连续5天，休息两天，共治疗13周。在体测量全身骨密度、血清E2、IL-1、IL-6表达；检测骨组织形态计量学指标，测量OB及MSC中IL-1β、IL-6、OPG和RANKL蛋白和mRNA表达。离体测量治疗血清对大鼠OB的IL-1β和IL-6、OPG和RANKL蛋白、mRNA表达以及观察对大鼠OC致骨陷窝数目和面积的变化。 结果 在体实验显示，与假手术组比较，模型组大鼠BMD、胫骨TBV%显著降低，胫骨TRS%、TFS%、OSW、MAR和mAR显著升高；血清E2显著降低，IL-1、IL-6均显著升高；胫骨OB及MSC中IL-1β、IL-6、RANKL蛋白、mRNA表达阳性密度均显著升高，OPG蛋白、mRNA表达阳性密度均显著降低；与模型组相比，电针组大鼠胫骨BMD、TBV%明显增高，TRS%、TFS%、OSW、MAR和mAR明显降低；血清E2明显升高，IL-1、IL-6均显著降低；胫骨OB及MSC中IL-1β、IL-6、RANKL蛋白、mRNA表达阳性密度均明显降低，OPG蛋白、mRNA表达阳性密度皆明显增高。离体测量显示，与假手术血清组比较，针刺血清组大鼠OB中 IL-1β、IL-6、RANKL蛋白、mRNA表达阳性密度均显著升高，OPG蛋白、mRNA表达阳性密度均显著降低；与模型血清组相比，针刺血清组大鼠OB中IL-1β、IL-6、RANKL蛋白、mRNA表达阳性密度均明显降低，OPG蛋白、mRNA表达阳性密度皆明显增高；模型血清组骨吸收陷窝数目及面积显著高于空白血清组，电针血清组骨吸收陷窝数目及面积与模型组血清组相比显著降低。结论 针刺治疗可使OPG表达上调，RANKL表达下调，从而明显降低破骨细胞的活性而发挥对骨质疏松症的作用。本研究为针刺治疗OP临床应用提供了科学依据。