电针足少阳经穴对大鼠骨重塑偶联的“和调”机制

Bone remodelling decoupled gives rise to many bone diseases which lacks bi-regulating therapy. Based on the theory " Foot Shao-yang relates to bone" raised by Neijing , we previously found that electroacupuncture on Foot Shao-yang acupoints seem to harmonize both bone synthesis and degradation of rats, while it's mechanism is unclear. Known that transcriptional gene RANKL and NFATc1 are the most important bone remodel coupling factors, so according to the two factors' active clue in our preliminary trials, we attempt to explore whether electroacupuncture on Foot Shao-yang acupoints can harmonize the rat bone remodel coupling balance via those tow factors. This project will choose the OP rat model , and OP + anti DKK1 antibody (Signal strengthen agent) rat model , and FK506 (Signal blockers) rat model, apply FQ-PCR and ELISA etc. methods, from the coupling factors and their upstream and downstream signal relationship, try to prove if electroacupuncture on rat's Foot Shao-yang acupoints 1. to alter bone tissues 's RANKL and NFATc1 mRNA ; 2. to affect the output of RANKL and the relationship between its quantity and strengthened Wnt signal ; 3. to recover and assist bone-formation course after blocked NFATc1 signal , so that partly elaborate its mechanism on coordinating bone remodeling. It also can supply experimental evidence for verifying the Neijing's principle on bone and Foot Shaoyang meridian; furthermore, it will help us to develop the coordinate therapy strategy on such bone remodelling decoupled diseases, Osteoporosis, Diabetes Chacot joint and so on.

骨重塑失偶联可引发多种骨病，目前尚缺如双向协同性治疗。据《内经》"足少阳与骨相关"，我们前期工作显示电针足少阳经穴可"和调"大鼠骨分解和合成，但其机制有待阐明。已知转录基因RANKL和NFATc1是骨重塑的偶联因子，故在预试发现两因子变化线索基础上，拟进一步探索电针足少阳经穴如何影响此两因子而"和调"大鼠骨重塑偶联过程。本项目以去卵巢大鼠、去卵巢+抗DKK1抗体（信号加强剂）及FK506（信号阻滞剂）处理大鼠为研究对象，采用FQ-PCR等技术，从两因子及上下游信号关系切入，验证大鼠电针足少阳经穴后①骨组织RANKL、NFATc1mRNA改变；②对RANKL产量变化的影响；③受阻滞NFATc1信号被恢复并参与成骨过程，从而较全面地揭示其"和调"的机制。本项目为深入研究"足少阳与骨相关性"原理及新机制提供可靠实验证据；为骨质疏松、糖尿病关节、癌症溶骨损害等骨重塑失偶联骨病开拓协同的防治思路。

骨重塑失偶联可引发多种骨病，亟需开发双向协同性疗法。我们前期发现电针足少阳经穴可平衡OVX大鼠骨分解和合成，但其机制是否与骨重塑的转录基因（RANKL、NFATc1、OPG、CBFa1）有关，却有待于证实和阐明。而本项目进一步使用信号加强剂和阻滞剂，探索电针对相关信号分子、通路及上、下游关系的影响。.实验(1)：电针足少阳经穴+抗DKK1抗体（Wnt信号加强剂）处理.骨组织切片观察和各组骨小梁面积比计量，均显示电针足少阳经穴干预OVX大鼠和OVX +抗DKK1抗体大鼠骨形态明显改善，优于OVX模型组。但OVX+抗DKK1抗体组未见显著骨形成效应。.RT-PCR检测：当仅电针干预，OPG、NFATc1、RANKL、CBFa1表达较正常骨组织分别高0.7、26、1.6、2.4倍；当Wnt信号激动，则分别高2.4、3.8、0.3、2.3倍；Wnt信号激动后再电针干预，则分别高0.14、3.4、1.16、2.2倍。.实验(2)：电针足少阳经穴+FK506（NFATc1信号阻滞剂）处理.骨组织切片观察和各组骨小梁面积比计量，Fk506组明显表现骨质疏松征象。而预针+FK506和FK506+电针组，则骨小梁排列较规则，连续性稍好，粗大，髓腔明显变小。.RT-PCR检测：FK506导致RANKL表达较正常骨组织高10倍，是产生骨质疏松的原因，而这似与CBFa1高表达有关（高31倍强）。预先电针和电针足少阳经穴，都不能翻转因FK506所产生的NFATc1信号受阻滞，但似能阻止CBFa1高表达（分别高1.5倍和5.4倍）而下调RANKL表达（分别0.4和2.8倍）。.结论：本项目从骨形态学研究和基因测定，初步探明：电针足少阳经穴似通过调节NFATc1表达量进而平衡成骨和破骨活动；当抗DDK1抗体激动Wnt通路时，由于上调OPG和下调RANKL导致NFATc1表达随之减少，进而影响到成骨活动；电针又可抑制抗DDK1抗体的效应而加强骨形成。如果NFATc1信号被阻断，则CBFa1升高而致RANKL过表达，则雄性大鼠出现明显骨质疏松；预电针和电针都不能翻转NFATc1信号，但似能阻止CBFa1高表达而下调RANKL。.Sost的变化与电针、信号激动剂和信号阻滞剂使用无关系。.骨重塑过程异常复杂，本次4个受试基因间不仅表现出相互影响而且似存在交互作用，有待今后更深入地研究。