破骨细胞形成抑制因子研究进展

破骨细胞形成抑制因子（OPG/OCIF）是最近发现的一种参与调节骨密度的糖蛋白,是一个肿瘤坏死因子(TNF)受体超家族的新成员,其氨基酸序列中具有TNF受体结构类似区.成熟的OPG/OCIF具有7个结构域,可分为三个功能区,即：TNF受体结构区、致死结构区和肝素结合区.OPG/OCIF基因定位在8q23～24上,由5个外显子和4个内含子组成,其表达受到与骨的形成和破坏有关因子的调控：如TGF-β1、1,25(OH)₂VD₃、TNF-α等等.OPG/OCIF抑制骨的破坏和吸收机制主要是抑制破骨细胞的存活,引起破骨细胞凋亡和抑制破骨细胞形成.     

miRNAs在破骨细胞中的研究进展

破骨细胞起源于造血干细胞,是体内一种负责骨吸收的骨特异性多核细胞,在骨代谢平衡的调控中起着重要作用。破骨细胞的分化形成及功能活性异常可引起一系列临床疾病,而其分化形成过程受到多种因子的调控,近年来越来越多研究聚焦于miRNAs对破骨细胞分化形成过程的调控作用。因此,本文主要对影响破骨细胞分化形成的相关miRNAs进行综述,为后续相关研究提供参考。     

破骨细胞骨吸收功能的检测方法

破骨细胞是一种多核的,具有骨吸收功能的骨组织细胞,在骨吸收过程中起着至关重要的作用.破骨细胞骨吸收功能的异常会引发一系列的临床病症,如骨质疏松症、关节置换术后假体松动、骨硬化症和牙周病变等.破骨细胞骨吸收功能的进一步研究对于各类骨疾病的防治具有重要的意义.然而破骨细胞骨吸收功能的检测方法一直以来是制约破骨细胞研究的瓶颈之一.为此,围绕破骨细胞骨吸收功能的检测方法做一综述.     

绝经后骨质疏松症TNF-α通过激活NF-κB促进RANKL诱导的破骨细胞形成

本研究检测了绝经后骨质疏松症妇女的肿瘤坏死因子-α(TNF-α)和雌激素水平,并探讨了TNF-α对破骨前体细胞RAW264.7中破骨细胞标志物核因子κB受体激活因子(nuclear factor kappa-B, RANK)、组织蛋白酶K (Cathepsin K, CTSK)和凝血酶受体激活肽(thrombin receptor activating peptide, TRAP)以及核因子-κB (NF-κB)亚基(p65)和NF-κB抑制蛋白(IκBα)的影响。研究结果表明,绝经后骨质疏松症患者的TNF-α水平显著升高,而雌二醇水平显著降低。核因子κB受体激活因子配体(receptor activator for NF-κBligand, RANKL)处理1周后,破骨前体细胞RAW264.7中破骨细胞标志物RANK、CTSK和TRAP的mRNA和蛋白高度表达。与RANKL对照组相比,TNF-α处理可上调RANK、CTSK和TRAP m RNA的表达。但是,仅TNF-α不能诱导培养的RAW264.7细胞分化为破骨细胞成。TNF-α以剂量依赖性方式诱导NF-κB亚基p65和IκBα磷酸化,而NF-κB抑制剂处理则有效降低了RANK和TRAP的表达。本研究结论表明,绝经后骨质疏松症中TNF-α通过激活NF-κB来促进RANKL诱导的破骨细胞形成。     

类风湿关节炎破骨细胞的活化机制及其预测指标

类风湿关节炎(rheumatoid arthritis, RA)是一种系统性、慢性、炎性的自身免疫性疾病。骨破坏在RA的发生和发展中占有重要地位,是RA致残致畸的主要原因。破骨细胞(osteoclast, OC)的异常增生与活化对RA骨侵蚀的发病进展具有重要作用。近年来,RA患者骨破坏的研究逐渐增多。本文结合国内外研究对核因子-κB受体活化因子配体(receptor activation for nuclear factor-κB ligand, RANKL)/核激活因子受体(receptor activator for nuclear factor-κB, RANK)/骨保护素(orthopantomography, OPG)信号通路、Wnt信号通路、抗瓜氨酸蛋白抗体(anti-citrulline protein antibody, ACPA)、基质金属蛋白酶(matrixmetalloproteinases,MMPs)、14-3-3η蛋白、基质细胞衍生因子-1(stromalcellderivedfactor-1,SDF-1)、小泛素样修饰物蛋白(smallubiquitin-likemodifierprotein,SUMO)、分泌型卷曲相关蛋白(secreted frizzled-related protein, SFRP)、骨转换标志物等作一阐述,旨在为RA早期骨破坏诊断提供相关依据。     

破骨细胞的起源

破骨细胞长期来被认为来自骨原细胞。近来由于对碳粒标记腹腔巨噬细胞、鹌鹑－鸡胚嵌合体细胞核标记、巨溶酶体细胞质标记等的研究,以及骨硬化病骨髓移植的研究,都有力地证实破骨细胞起源于造血干细胞。目前认为破骨细胞的发育途径是由多能造血干细胞增殖为循环的单核细胞,进入组织后转变为巨噬细胞,最后融合成多核的破骨细胞。     

重组可溶性核因子κB受体活化因子体外抑制甲状旁腺激素诱导的破骨细胞生成

新近发现的核因子κB受体活化因子配基（receptor activator of nuclear factor-κB ligand,RANKL）,核因子κB受体活化因子（receptor activator ofnuclear factor-κB,RANK）／护骨素（osteoprotegerin,OPG）细胞因子系统提高了对破骨细胞生物学和骨稳态分子水平的认识。RANKL与RANK之间的相互作用决定了破骨细胞的分化。本研究通过体外实验评价可溶性RANK （soluble RANK,sRANK）是否可作为RANKL的拈抗剂下调破骨细胞生成和骨吸收陷窝的形成。构建sRANK的原核表达载体,转化入大肠杆菌表达菌株Origami B（DE3）,成功表达了重组蛋白,亲和层析进行纯化。重组sRANK以剂量依赖方式抑制由甲状旁腺激素（parathyroid hormone,PTH）诱导的破骨细胞生成和骨吸收陷窝形成。RT-PCR实验证实,sRANK可显著抑制PTH刺激的小鼠骨髓细胞碳酸苷酶Ⅱ和抗酒石酸酸性磷酸酶mRNA的表达。结果表明,sRANK具有抗骨吸收功能,可能成为一种治疗以骨吸收加强为特征的骨疾病的新方法。     

培养破骨细胞扫描电镜样本制备方法的探讨

目的：探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法：实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果：破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论：牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。     

培养破骨细胞扫描电镜样本制备方法的探讨

目的:探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法:实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果:破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论:牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。     

破骨细胞伪足小体的结构和功能

破骨细胞是一种由造血干细胞分化而来的具有骨吸收功能的多核细胞。破骨细胞的骨吸收功能与其肌动蛋白骨架的完整性有关。研究表明,破骨细胞肌动蛋白骨架的基本结构为伪足小体（podosome）。在破骨细胞分化的不同阶段,伪足小体呈现不同的形态结构。伪足小体的形成过程及结构完整性直接影响着破骨细胞的分化及其骨吸收活性。深入研究伪足小体的结构和功能可为探索破骨细胞的骨吸收机制和寻找骨骼疾病药物作用靶点提供新的思路。该文将围绕破骨细胞伪足小体的结构、功能及其调节机制进行综述。     

Vitamin D and bone

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.     

Asiaticoside,a component of Centella asiatica attenuates RANKL-induced osteoclastogenesis via NFATc1 and NF-κB signaling pathways

Identification of natural compounds that inhibit osteoclastogenesis will facilitate the development of antiresorptive treatment of osteolytic bone diseases. Asiaticoside is a triterpenoid derivative isolated from Centella asiatica, which exhibits varying biological effects like angiogenesis, anti-inflammation, wound healing, and osteogenic differentiation. However, its role in osteoclastogenesis remains unknown. Here, we show that Asiaticoside can suppress RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner. Asiaticoside attenuated the expression of osteoclast marker genes including Ctsk, Atp6v0d2, Nfatc1, Acp5, and Dc-stamp. Furthermore, Asiaticoside inhibited RANKL-mediated NF-κB and NFATc1 activities, and RANKL-induced calcium oscillation. Collectively, this study demonstrates that Asiaticoside inhibited osteoclast formation and function through attenuating RANKL-induced key signaling pathways, which may indicate that Asiaticoside is a potential antiresorptive agent against osteoclast-related osteolytic bone diseases.     

Ellagic acid protects ovariectomy-induced bone loss in mice by inhibiting osteoclast differentiation and bone resorption

Osteoporosis is a devastating disease that features reduced bone quantity and microstructure, which causes fragility fracture and increases mortality, especially in the aged population. Due to the long-term side-effects of current drugs for osteoporosis, it is of importance to find other safe and effective medications. Ellagic acid (EA) is a phenolic compound found in nut galls, plant extracts, and fruits, and exhibits antioxidant and antineoplastic effects. Here, we showed that EA attenuated the formation and function of osteoclast dose-dependently. The underlying mechanism was further discovered by western blot, immunofluorescence assay, and luciferase assay, which elucidated that EA suppressed osteoclastogenesis and bone resorption mainly through attenuating receptor activator of nuclear factor-κB (NF-κB) ligand-induced NF-κB activation and extracellular signal-regulated kinase signaling pathways, accompanied by decreased protein expression of nuclear factor of activated T-cells calcineurin-dependent 1 and c-Fos. Moreover, EA inhibits osteoclast marker genes expression including Dc-stamp, Ctsk, Atp6v0d2, and Acp5. Intriguingly, we also found that EA treatment could significantly protect ovariectomy-induced bone loss in vivo. Conclusively, this study suggested that EA might have the therapeutic potentiality for preventing or treating osteoporosis.     

Antiresorptive agents and osteoclast apoptosis

Antiresorptive agents have proven to be effective therapies for the treatment of bone diseases associated with excessive osteoclast activity. Decreased osteoclast formation, inhibition of osteoclast actions, and reduced osteoclast survival represent mechanisms by which antiresorptive agents could act. The goals of this article are to present the evidence that antiresorptive agents can decrease osteoclast survival through apoptosis, to review the mechanisms by which they are thought to activate the apoptotic process, and to consider whether the actions on apoptosis fully account for the antiresorptive effects. As background, the apoptotic process will be briefly summarized together with the evidence that factors that promote osteoclast survival affect steps in the process. Following this, therapeutic agents that are both antiresorptive and can stimulate osteoclast apoptosis will be discussed. Other bone therapeutic agents that are either antiresorptive or apoptotic, but not both, will be described. Finally, newer antiresorptive compounds that elicit apoptosis and could represent potential therapeutic agents will be noted.     

Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway

Nutritional factors influence bone development. Previous studies demonstrated that bone mass significantly increased with suppressed bone resorption in early life of rats fed with AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for 2 weeks. However, the effects of increased phenolic acids in animal serum due to this diet on bone and bone resorption were unclear. This in vitro and in ex vivo study examined the effects of phenolic hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on osteoclastic cell differentiation and bone resorption. We cultured murine osteoclast (macrophage) cell line, RAW 264.7 cells, and hematopoietic osteoclast progenitor cells (isolated from 4-week-old C57BL6/J mice) with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL). Morphologic studies showed decreased osteoclast number with treatment of 2.5% mouse serum from BB diet–fed animals compared with those treated with serum from standard casein diet–fed mice in both RAW 264.7 cell and primary cell cultures. HA and 3-3-PPA, but not 3–4-PPA, had dose-dependent suppressive effects on osteoclastogenesis and osteoclast resorptive activity in Corning osteo-assay plates. Signaling pathway analysis showed that after pretreatment with HA or 3-3-PPA, RANKL-stimulated increase of osteoclastogenic markers, such as nuclear factor of activated T-cells, cytoplasmic 1 and matrix metallopeptidase 9 gene/protein expression were blunted. Inhibitory effects of HA and 3-3-PPA on osteoclastogenesis utilized RANKL/RANK independent mediators. The study revealed that HA and 3-3-PPA significantly inhibited osteoclastogenesis and bone osteoclastic resorptive activity.     

Ligustilide suppresses RANKL-induced osteoclastogenesis and bone resorption via inhibition of RANK expression

Osteoclast (OC) is the only cell involved in bone resorption. Dysfunction of OCs leads to a variety of bone diseases. Ligustilide (LIG) is the main component of the volatile oil isolated and purified from Angelica sinensis. LIG exerts many pharmacological activities, but its effects on osteoclastogenesis and bone resorption are still unclear. Our study showed that LIG inhibited receptor activator of nuclear factor-κB (NF-κB) ligand-induced OC formation and activation in a dose-dependent manner. Additionally, LIG downregulated the messenger RNA (mRNA) expression of OC-specific genes, such as V-ATPase d2, tartrate-resistant acid phosphatase, a dendritic cell-specific transmembrane protein, cathepsin K, and nuclear factor of activated T cells cl. Furthermore, LIG blocked the activation of NF-κB/extracellular signal-regulated kinase/p38/immunoreceptor tyrosine-based activation motif signaling pathways. Crucially, the expression of tumor necrosis factor receptor-associated factor 6 proteins and the expression of receptor activator of NF-κB mRNA were inhibited by LIG. However, LIG did not affect the formation and mineralization of osteoblasts. Collectively, this observation suggests that LIG may serve as a promising agent for the prevention and treatment of diseases caused by abnormal bone resorption.     

Inhibition of Osteocyte Apoptosis Prevents the Increase in Osteocytic Receptor Activator of Nuclear Factor κB Ligand (RANKL) but Does Not Stop Bone Resorption or the Loss of Bone Induced by Unloading

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.     

Inhibition of zoledronic acid derivatives with extended methylene linkers on osteoclastogenesis involve downregulation of JNK and Akt pathways

Bisphosphonates (BPs), especially zoledronic acid (ZOL), are clinically used to treat osteolytic bone lesions. However, serious side-effects may be also induced during the therapeutic process. To improve the BPs drugs, here, we investigated the effects of a series of ZOL derivatives with increasing number of methylene linker between the imidazole ring and the P–C–P backbone named IPrDP, IBDP, IPeDP, and IHDP on cell viability and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation, function and apoptosis induction in mouse bone marrow-derived macrophages (BMMs). Our results suggested that IPeDP and IHDP, which contains 4 and 5 methylene linkers, respectively, exerted lower toxicity on BMMs compared with ZOL, IPrDP, and IBDP, which contains 1, 2, and 3 methylene linkers respectively. At concentrations below cytotoxicity threshold, IPeDP and IHDP possessed strong abilities of antiosteoclast formation, antibone absorption, and inducing osteoclast apoptosis, which were similar to ZOL and more powerful than IPrDP and IBDP. The mechanism behind these effects of IPeDP and IHDP might involve the interference of small GTPases prenylation through suppression of mevalonate pathway. The downregulation of JNK and Akt phosphorylation and subsequent inhibition of the expression of c-Fos and NFATc1 might also be involved. Our results supported the potential usage of IPeDP and IHDP to treat bone-related disorders involving increased osteoclastogenesis. Our attempt to extend the methylene linker between the imidazole ring and the P–C–P backbone of ZOL also reveals some regularities between the structure and properties of the BPs drugs.