基于“肝主筋、肾主骨”理论探讨OPG/RANKL/RANK信号轴与绝经后骨质疏松症的筋骨相关性

中医基础理论素有"肝主筋,肾主骨"之说,肝藏血主筋,筋束骨利关节,肾为先天之本,主骨而生髓,肝肾精血同源、筋骨相关,互资互生,二者共同介导机体器官、脏腑的发育、生长与衰退过程,尤其与骨细胞生长代谢机制关系密切。随着中医药分子生物学研究的深入,发现骨保护蛋白(OPG)/核因子-k B受体活化因子(RANK)/核因子-k B受体活化因子配体(RANKL)信号轴在骨代谢中发挥关键作用。本文基于"肝主筋,肾主骨"理论,从绝经后骨质疏松症的病因病机出发,从"肝肾论治,筋骨并重"角度将OPG/RANKL/RANK信号转导系统与骨代谢内环境稳态相结合,为中医药柔肝补肾、调筋养骨机制提供分子生物学、筋骨力学依据支撑,以期进一步指导临床论治。     

OPG/RANKL/RANK系统与软骨及软骨下骨

OPG/RANKL/RANK系统在调节骨代谢和骨重塑方面具有重要作用。成骨细胞表达的核因子-κB受体活化因子配体(RANKL)与破骨细胞表面的核因子-κB受体活化因子(RANK)结合促进破骨细胞分化成熟,骨保护素(OPG)则结合RANKL而抑制这一功能。研究表明,OPG/RANKL/RANK系统在软骨及软骨下骨内也有表达,可能成为类风湿关节炎及骨关节炎的治疗靶点。该文就OPG/RANKL/RANK系统在软骨及软骨下骨内表达及其作用研究进展作一综述。     

基于“脾主肉、肾主骨”理论探讨绝经后骨质疏松症的OPG/RANK/RANKL信号调控机制

中医理论传承"辨证论治"之道,强调人作为机体的整体性与统一性,肾主骨生髓为先天之本,脾主肌肉充四肢为后天之源,先后二天互资互生,共同维持人体正常的生理机能,但缺乏客观的分子生物学依据阐明其作用机理。OPG/RANK/RANKL信号转导系统的发现在骨代谢史上具有里程碑的意义,开创了中医药防治和研究绝经后骨质疏松症的新纪元,骨微观信息的改变就是从脾肾论治对骨代谢调控机制的效应表达。本文基于"脾主肉、肾主骨"理论,就绝经后骨质疏松症的病因病机及治法方药结合OPG/RANK/RANKL信号转导系统在骨代谢中的作用展开综述,旨在为绝经后骨质疏松症的中医药防治从分子生物学水平提供科学依据,以期更好地指导临床。     

RANK/RANKL/OPG系统与人工关节无菌性松动

人工关节置换术是临床上治疗各种终末期关节疾病最常用的有效方法,但人工关节无菌性松动常影响其远期疗效。目前多数学者认为人工关节周围破骨细胞介导的骨吸收,是导致人工关节松动的主要原因。近年许多研究表明核因子-κB受体活化因子（RANK）/RANK配体（RANKL）/骨保护素（OPG）系统与人工关节无菌性松动有密切关系,在体内受多种促骨激素和细胞因子的直接或间接作用,调控RANKL-OPG比值,介导破骨细胞分化、活化和凋亡,从而影响骨代谢。该文就RANK/RANKL/OPG系统功能、磨损颗粒对其作用及其基因治疗研究进展作一综述。     

从脾肾论治骨质疏松症的OPG/RANK/RANKL信号调控机制

中医处方遣药传承"辨证论治"之道,强调机体的整体性和时空性,但往往缺乏客观的分子生物学依据解释其作用机理;OPG/RANK/RANKL系统是骨代谢史上里程碑式的发现,对骨质疏松的中医药防治及研究开创新的纪元,从脾肾论治的效应最终可通过该系统对骨代谢调控的微观信息来表达,可以说OPG/RANKL/RANK系统是广大学者热衷于探究中药机理的切入点和共通点。基于此,本文就骨质疏松症的病因病机及治则方药结合OPG/RANK/RANKL系统对骨代谢的调控机制做一综述,旨在为原发性骨质疏松症的中医药防治提供客观的分子生物学依据。     

骨重建过程中降钙素基因相关肽与RANK/RANKL/OPG作用机制的研究进展

目的总结骨重建过程中降钙素基因相关肽(calcitonin gene-related peptide,CGRP)与核因子κB受体活化因子(receptor activator of nuclear factorκB,RANK)/核因子κB受体活化因子配体(receptor activator of nuclear factorκB ligand,RANKL)/骨保护蛋白(osteoprotegerin,OPG)信号系统作用机制的研究进展,为进一步研究骨相关疾病的预防及治疗提供理论依据。方法广泛查阅国内外近年来骨重建过程中CGRP与RANK/RANKL/OPG信号系统相关文献,并加以分析总结。结果骨重建过程中,CGRP与RANK/RANKL/OPG信号系统发挥着重要调控作用。结论目前对于骨重建过程中CGRP与RANK/RANKL/OPG信号系统的作用机制研究仍不够深入,需进一步研究二者在骨重建过程中的具体作用方式、相互联系,明确作用机制,为临床上骨相关疾病的治疗带来新思路、新方法。     

骨质疏松症大鼠模型生物力学性能和OPG/RANKL/RANK信号通路研究

摘要：目的 研究针刺对骨质疏松症（osteoporosis，OP）模型大鼠生物力学性能和骨保护素（OPG）/核因子-κB受体活化因子（RANKL） /核因子-κB受体活化因子受体（RANK）信号通路的影响，探讨针刺防治OP的作用机制。方法 选取50只雌性SD大鼠，通过摘除卵巢建立OP大鼠模型，分为假手术组、模型组、针刺低剂量组、针刺高剂量组、阿仑膦酸钠组，每组10只。成功建立模型并对大鼠进行适应性喂养2个月后，连续灌胃给药12周，再进行腹主动脉取血并处死大鼠。通过双能X射线仪测定各组大鼠右侧下肢股骨骨矿含量；用酶联免疫吸附法测定法检测血清钙（Ca2+）、磷（P）和OPG、RANKL、RANK的水平；采用AG-IX万能实验机检测股骨的生物力学性能；采用蛋白质印迹法检测骨组织OPG、RANKL、RANK蛋白的表达。结果 与假手术组比较，模型组大鼠骨组织中骨矿含量、血清Ca2+、P、OPG的表达、股骨组织中OPG蛋白含量和股骨最大载荷、刚度及弹性模量均明显降低（P＜0.01）；血清RANKL和RANK的表达及股骨组织中RANKL、RANK蛋白含量明显升高（P＜0.01）。与模型组比较，针刺低剂量组和针刺高剂量组以及阿仑膦酸钠组上述指标均有明显改善（P<0.05或P<0.01），且针刺的作用具有剂量依赖性（P<0.05或P<0.01）。结论 针刺具有明显的抗OP作用，可能与其改善骨组织生物力学特性以及调控OPG/RANKL/RANK通路有关。     

维持性血液透析患者血清骨保护素与肾性骨营养不良的关系

骨保护素（OPG）是近年来在肿瘤坏死因子受体超家族中发现的一种具有调控破骨细胞产生和活化作用的生物活性物质。它与核因子κB活化子受体配体（RANKL,亦称骨保护素配体OPGL）和核因子κB活化子受体（RANK）组成的分子调控系统是体内维持骨代谢平衡的重要分子机制。在正常的骨转化中成骨细胞表面表达RANKL．它与破骨细胞前体或破骨细胞表面的RANK结合后启动了信号转导，使破骨细胞增殖和活化．溶骨活动增强。同时成骨细胞分泌OPG，它与RANK竞争性的抑制RANKL使溶骨作用不致过度强烈。     

RANK/RANKL/OPG系统在骨髓瘤骨病中的作用

多发性骨髓瘤骨病以进行性骨质破坏为主要特征,主要原因是破骨细胞大量生成和激活.核因子κB受体活化因子(RANK)及其配体(RANKL)、骨保护蛋白(OPG)是调节破骨细胞功能和活性的关键性调节因子,在骨髓瘤引起的骨质破坏中发挥重要作用.研究表明骨髓瘤患者局部RANKL/OPG比例增高可能是引起骨吸收的关键因素.临床前研究表明使用重组OPG或RANK可以抑制破骨形成,减少骨吸收,改变骨髓微环境,预防骨髓瘤骨病的形成,间接抑制骨髓瘤的发展.     

骨保护素防治骨质疏松症的研究进展

骨保护素(osteoprotegerin, OPG)、核因子-κB受体活化因子配体(receptor activator of NF-κB ligand, RANKL)、核因子-κB受体活化因子(receptor activator of NF-κB, RANK)是调节骨吸收和骨形成的重要的调控因子.骨质疏松症是多种原因使破骨细胞功能活跃,破坏骨的动态平衡造成的.OPG与RANK竞争性结合RANKL,从而抑制破骨细胞的分化、激活,促进其凋亡.应用重组OPG蛋白和OPG基因治疗的方法 是目前防治骨质疏松症的关注热点.笔者针对这方面的研究现状作简要的综述.     

从中医“虚”论治原发性骨质疏松症与OPG/RANK/RANKL信号通路的相关性

原发性骨质疏松症(primary osteoporosis,POP)是现代临床中常见的骨科疾病,其归属于中医学之"骨痿""骨痹""骨枯"等范畴。随着中医药事业的发展,中医学在POP的治疗中应用愈加广泛,且疗效显著,已受到学界的广泛认可。但遗憾的是,其缺乏现代研究理论依据的支撑。随着分子生物学的发展,OPG/RANK/RANKL信号通路的发现对中医学治疗此病具有重要意义。故笔者将从中医"虚"理论出发,并以中医学脏腑辨证理论为基础,借助现代分子生物学的研究成果OPG/RANK/RANKL信号通路,揭示中医治疗POP的OPG/RANK/RANKL信号通路表达机制,以期为中医学治疗POP提供科学理论依据。     

Expression profiles of receptor activator of nuclear factor kappaB ligand, receptor activator of nuclear factor kappaB, and osteoprotegerin messenger RNA in aged and ovariectomized rat bones.

The receptor activator of nuclear factor-kappaB ligand (RANKL; also known as tumor necrosis factor-related activation-induced cytokine [TRANCE], osteoprotegerin ligand [OPGL], and osteoclast differentiation factor [ODF]) is a transmembrane ligand expressed in osteoblasts and bone marrow stromal cells. It binds to RANK, which is expressed in osteoclast progenitor cells, and induces osteoclastogenesis. OPG, a decoy receptor for RANKL, also binds to RANKL, and competitive binding of RANKL with RANK or OPG is thought to regulate bone metabolism. To investigate roles of the RANKL/RANK/OPG system in pathophysiological conditions, the expression of RANKL, RANK, and OPG messenger RNA (mRNA) was analyzed in bones of aged and ovariectomized rats by means of in situ hybridization. In the control 8-week-old male and sham-operated female rat bones, the expression of RANKL mRNA was detected in hypertrophic chondrocytes of the growth plate and some periosteal and endosteal mesenchymal cells. The expression of RANK mRNA was detected in osteoclast-like cells and mononuclear cells in contact with the cortical and trabecular bones. The expression of OPG mRNA was detected in proliferating chondrocytes and osteocytes. In the 2.5-year-old rat bones, the expression of RANKL, RANK, and OPG mRNA tended to decrease except for the endosteal region. In the ovariectomized rat bones, the expression of RANKL, RANK, and OPG mRNA increased, and high expression of OPG mRNA was induced in resting chondrocytes and osteocytes. These results suggest that estrogen deficiency stimulates the RANKL/RANK/OPG system and induces OPG in cells that have been thought to be less important for bone metabolism.     

橙皮苷对去卵巢骨质疏松症大鼠骨质流失和机制的影响

目的研究橙皮苷对去卵巢骨质疏松大鼠骨质流失和骨保护素(osteoprotegerin,OPG)/核因子kappa B配体的受体激活物(receptor activator of nuclear factor kappa B ligand,RANKL)/RANK通路的影响。方法将48只雌性Sprague-Dawley(SD)大鼠随机分为假手术组、去卵巢骨质疏松组、橙皮苷低剂量组、橙皮苷中剂量、橙皮苷高剂量组和雌激素组。检测骨组织骨体积分数(bone volume fraction,BV/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨密度(bone mineral density,BMD)、骨小梁数量(trabecular number,Tb.N)、骨小梁分离度(trabecular separation,Tb.Sp),通过HE染色观察骨组织病理损伤,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测人Ⅰ型胶原C端肽(C-terminal peptide collagen typeⅠ,CTX-Ⅰ)和血清骨钙素(bone glaprotein,BGP)的表达量。运用qRT-PCR和Western bolt分别检测骨组织OPG、RANKL、RANK的mRNA和蛋白表达。结果在橙皮苷治疗后,与去卵巢骨质疏松组相比,骨质疏松标志物BV/TV、BMD、Tb.Th和Tb.N水平升高,Tb.Sp水平降低;大鼠骨小梁数量明显增加,且骨小梁连接更为紧密;骨吸收标志物CTX-Ⅰ水平降低;骨形成标志物BGP水平升高。OPG mRNA相对表达量和蛋白的表达量升高,RANKL和RANK mRNA相对表达量和蛋白的表达量降低。结论橙皮苷能缓解去卵巢骨质疏松大鼠骨质流失并调节OPG/RANKL/RANK信号通路。     

基于OPG/RANK/RANKL信号轴探讨中医“瘀证”与骨质疏松症之间的关系

近年来,随着中医学的不断发展,中医药在治疗骨质疏松症(osteoporosis,OP)中的应用愈加广泛,然而由于只有单纯的中医理论指导,而缺乏现代科学依据的支撑,故而广受争议。与此同时,OPG/RANK/RANKL信号轴的发现又是现代分子生物学的一大突破。因此,笔者立足于中西医结合的角度,基于中医脏腑亏虚立论,从OPG/RANK/RANKL信号调控机制探讨中医学从"瘀证"论治骨质疏松症的合理性,从而为中医从"瘀"辨治OP以及临床研究提供科学理论依据。     

基于OPG/RANKL/RANK轴观察加味阳和汤及其拆方对去卵巢骨质疏松大鼠的影响

目的观察加味阳和汤及其拆方对OPG、RANKL、RANK含量的影响,探讨其防治绝经后骨质疏松症可能的作用机制及组方配伍的合理性。方法选取48只雌性SD大鼠,加味阳和汤按君臣佐使关系拆方,将大鼠等量随机分为假手术组(SHAM)、模型组(OVX)、君药+臣药组(A组)、君药+臣药+佐药组(B组)、君药+臣药+佐药+使药组(C组)、戊酸雌二醇组(E2V)。除SHAM组外,均采用去卵巢骨质疏松大鼠模型,干预给药后(灌胃90 d),处死动物后取右侧股骨及胫骨通过双能X射线骨密度仪检测骨密度(bone mineral density,BMD)及骨矿含量(bone mineral content,BMC),取左侧股骨行HE染色观察骨显微结构,检测血清中骨代谢指标ALP、Ca~(2+)、P~(3-)、E2及血清OPG、RANKL、RANK含量。结果与SHAM组相比,OVX组大鼠股骨及胫骨BMD、BMC降低(P0.05),骨小梁变细、间隙增大、结构缺失,血清Ca~(2+)、P~(3-)、E2、OPG水平下降(P0.05),血清ALP、RANKL、RANK水平上升(P0.05);与OVX组比较,除A组大鼠股骨及胫骨BMD、BMC、血清Ca~(2+)、P~(3-)、E2、OPG、RANKL及B组P~(3-)水平无显著差异外(P0.05),各给药组大鼠股骨及胫骨BMD、BMC均显著升高(P0.05),骨小梁增多、间隙减小、结构趋向完整,血清Ca~(2+)、P~(3-)、E2、OPG水平上升(P0.05),血清ALP、RANKL、RANK水平下降(P0.05)。结论加味阳和汤及其拆方通过提高去卵巢骨质疏松大鼠BMD、BMC,降低骨代谢,改善骨显微结构从而发挥治疗作用,调节OPG/RANKL/RANK轴是可能的机制。     

RANKL,a necessary chance for clinical application to osteoporosis and cancer-related bone diseases

Osteoporosis is a common bone disease characterized by reduced bone and increased risk of fracture. In postmenopausal women, osteoporosis results from bone loss attributable to estrogen deficiency. Osteoclast differentiation and activation is mediated by receptor activator of nuclear factor-κB ligand (RANKL), its receptor receptor activator of nuclear factor-κB (RANK), and a decoy receptor for RANKL, osteoprotegerin (OPG). The OPG/RANKL/RANK system plays a pivotal role in osteoclast biology. Currently, a fully human anti-RANKL monoclonal antibody named denosumab is being clinically used for the treatment of osteoporosis and cancer-related bone disorders. This review describes recent advances in RANKL-related research, a story from bench to bedside. First, the discovery of the key factors, OPG/RANKL/RANK, revealed the molecular mechanism of osteoclastogenesis. Second, we established three animal models: (1) a novel and rapid bone loss model by administration of glutathione-S transferase-RANKL fusion protein to mice; (2) a novel mouse model of hypercalcemia with anorexia by overexpression of soluble RANKL using an adenovirus vector; and (3) a novel mouse model of osteopetrosis by administration of a denosumab-like anti-mouse RANKL neutralizing monoclonal antibody. Lastly, anti-human RANKL monoclonal antibody has been successfully applied to the treatment of osteoporosis and cancer-related bone disorders in many countries. This is a real example of applying basic science to clinical practice.     

OPG/membranous--RANKL complex is internalized via the clathrin pathway before a lysosomal and a proteasomal degradation

The members of the OPG/RANK/RANKL (osteoprotegerin/receptor activator of nuclear factor kappaB/RANK ligand) triad are involved in various osteolytic pathologies such as bone tumors. Although many studies described the use of OPG during the treatment of bone diseases, its bioavailability and the mechanism by which the cells control the extracellular OPG remains blurred. The present work uses a strongly RANKL expressing cellular model to assess the becoming and the bioavailability of exogenous OPG in the context of its interactions with RANKL. The human kidney cell line 293, which initially expresses neither OPG nor RANKL, was stably transfected by the full length of mouse transmembranous form of RANKL (293RL). When OPG is incubated with 293RL cells, the extracellular concentration of OPG was strongly decreased in a time-dependent manner. The OPG disappearance was not inhibited by the addition of several proteases inhibitors, thus excluding any extracellular protease degradation. Contrary to previous results obtained on myeloma cells, which strongly express syndecan-1, the OPG disappearance was unaffected by the use of an antibody against syndecan-1. However, this event was abolished by an antibody against RANKL. These results, not necessarily conflicting, could be in relation with the expression level of the receptors in the two cellular models. In this context, an internalization process was put forward. Confocal microscopy demonstrated via the clathrin pathway an internalization of OPG mediated by RANKL. After being internalized, OPG was then degraded by the proteasome and the lysosome. A similar internalization phenomenon was also observed in osteoblast cells expressing physiologically RANKL, thus validating our data observed on 293RL cells. Western blotting analysis revealed that the half-life of RANKL was greatly reduced in the presence of OPG, pointing out that OPG binding to RANKL induces an enhancement of the ligand internalization. By the light of these results, the inhibitory effect of OPG on bone resorption can be explained not only by a decoy receptor function, competitor inhibitor of the RANK/RANKL binding, but also by the modulation of the RANKL half-life induced by OPG. Reciprocally, this modulation contributes to reduce the bioavailability of OPG.     

The RANKL/RANK/OPG pathway

Understanding of osteoclast formation and activation has advanced considerably since the discovery of the RANKL/RANK/OPG system in the mid 1990s. Osteoblasts and stromal stem cells express receptor activator of NF-jB ligand (RANKL), which binds to its receptor, RANK, on the surface of osteoclasts and their precursors. This regulates the differentiation of precursors into multinucleated osteoclasts and osteoclast activation and survival both normally and in most pathologic conditions associated with increased bone resorption. Osteoprotegerin (OPG) is secreted by osteoblasts and osteogenic stromal stem cells and protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from interacting with RANK. The RANKL/OPG ratio in bone marrow is thus an important determinant of bone mass in normal and disease states. RANKL/RANK signaling also regulates lymph node formation and mammary gland lactational hyperplasia in mice, and OPG protects large arteries of mice from medial calcification. This article reviews the roles of the RANKL/RANK/OPG system in bone and other tissues.     

The Role of RANK/RANKL/OPG Signalling Pathways in Osteoclastogenesis in Odontogenic Keratocysts, Radicular Cysts, and Ameloblastomas

The aim of this study was to evaluate the immunohistochemical expression of molecules involved in osteoclastogenesis, including the receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) in odontogenic keratocysts (OKCs), which has been named as a keratocystic odontogenic tumour by the WHO, and compare their expression with radicular cysts and ameloblastomas. RANK is a member of tumour necrosis factor receptor family and it is activated by RANK ligand. OPG binds to RANKL and inactivates it. The imbalance of these factors could cause the differential bone resorption activity in some diseases and tumours. The expression of these molecules was evaluated in ameloblastomas (n = 20), OKCs (n = 20), and radicular cysts (n = 20) by immunohistochemistry. Immunohistochemical reactivity for RANK, RANKL, and OPG was detected in neoplastic and nonneoplastic epithelium and connective tissue cells. RANK showed the greatest expression in OKCs followed by ameloblastomas, with the lowest expression seen in radicular cysts. Expression of RANKL was detected in all lesions and no significant differences were observed between groups. OPG was expressed very low in all groups. In the stroma, the number of RANK positive cells was higher in OKCs when compared with ameloblastomas and radicular cysts but radicular cyst had higher numbers of RANKL positive cells in the stroma than ameloblastomas. The molecular system of RANK/RANKL/OPG is variably expressed in OKCs, radicular cysts, and ameloblastomas and this system may be involved in the osteoclastogenic mechanisms in OKCs and ameloblastomas. Advanced studies could further clarify the role of RANK, RANKL, and OPG in mediating tumour associated bone osteolysis.