基于RANKL/RANK/OPG信号通路探讨土贝母苷甲对糖尿病性骨质疏松大鼠骨质流失的影响

目的 探讨土贝母苷甲对糖尿病性骨质疏松(OP)大鼠骨质流失及核因子κB受体活化因子配体(RANKL)/核因子κB受体活化因子(RANK)/骨保护蛋白(OPG)信号通路的影响。方法 采用高脂高糖饮食+链脲佐菌素腹腔注射法构建大鼠糖尿病性OP模型，将60只Wistar大鼠随机分为对照组、模型组、土贝母苷甲低剂量组(1μmol/L)、土贝母苷甲高剂量组(5μmol/L)、OPG组(3 mg/kg)、土贝母苷甲高剂量+OPG组(5μmol/L土贝母苷甲+3 mg/kg OPG)。酶联免疫吸附(ELISA)法测定大鼠血清核心结合因子α1(CBF-α1)、Ⅰ型前胶原氨基端原肽(PINP)、骨钙素(OC)水平；Micro-CT测定骨密度(BMD)及骨小梁微结构参数；TRAP染色测定股骨表面破骨细胞数量(N.Oc/BS);HE染色观测大鼠骨组织病理变化；qRT-PCR法与Western blot法分别测定骨组织RANKL、RANK、OPG mRNA与蛋白水平。结果 与对照组相比，模型组大鼠血清CBF-α1、PINP、OC水平、股骨最大负荷、断裂挠度、BMD、BV/TV、Tb.N、Tb.Th、OPG m...     

骨碎补总黄酮对骨质疏松模型大鼠OPG/RANKL/RANK轴系统的影响

目的 研究不同剂量骨碎补总黄酮灌胃对骨质疏松模型大鼠雌激素水平及骨保护素(orthopantomography,OPG)、核因子κB受体活化因子(receptor activator of NF-κB,RANK)和核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)表达的影响,探讨其对骨代谢影响的可能机制。方法 通过去卵巢法造成骨质疏松大鼠模型,以戊酸雌二醇片0.1 mg·kg^-1及低、中、高不同剂量骨碎补总黄酮(0.054,0.108,0.216 g·kg^-1·d^-1)喂养3个月后,取动脉血,采用放射免疫法测定雌激素水平,用酶联免疫吸附法(ELISA)检测破骨细胞OPG、RANK和RANKL的表达。结果 与模型组2相比,各给药组雌二醇及OPG的表达量增加(P<0.05),RANK和RANKL的表达量减少(P<0.05),且随着骨碎补总黄酮剂量的增加,雌二醇及OPG表达呈上升趋势,RANK和RANKL表达均呈下降趋势。结论 不同剂量骨碎补总黄酮均影响OPG/RANKL/RANK轴系统,且随骨碎补总黄酮剂量的增加效果越明显,可能是通过调控OPG/RANKL/RANK轴系统使OPG表达增加、RANK和RANKL的表达下降来实现的。     

白芦藜醇对牙周炎大鼠OPG/RANKL/RANK信号通路的影响

目的 考察白芦藜醇(Resveratrol, RSV)对牙周炎大鼠骨保护素(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)(OPG/RANKL/RANK)信号通路相关蛋白的影响。方法 采用不同浓度的RSV对牙周炎大鼠进行干预，采用RT-PCR和Western blot分别检测牙周组织细胞OPG、RANKL、IL-1、IL-6、TNF-α和基质金属蛋白酶8(matrix metalloproteinase 8,MMP-8)的mRNA水平和蛋白表达水平。结果 牙周炎模型组大鼠牙周组织中OPG、RANKL、IL-1、IL-6、TNF-α和MMP-8的mRNA表达强度和蛋白水平高于空白对照组(P<0.05),RSV低剂量组、RSV中剂量组、RSV高剂量组则依次低于牙周炎模型组(P<0.05)。结论 RSV可以降低牙周炎大鼠牙周组织中OPG、RANKL、IL-1、IL-6、TNF-...     

瘦素通过细胞因子在RANKL/RANK/OPG系统中的作用对破骨细胞形成的调节

RANKL/RANK/OPG系统是近年来发现的破骨细胞分化过程中的一个重要信号传导通路,是成骨细胞作用于破骨细胞的重要途径。细胞因子与RANKL/RANK/OPG系统关系密切,瘦素通过免疫系统影响细胞因子的分泌进而影响RANKL/RANK/OPG系统的平衡,抑制破骨细胞分化,起到抑制破骨细胞的生成、抑制骨吸收的作用。     

姜黄素对类风湿关节炎破骨细胞分化中核因子κB受体活化因子基因及蛋白表达的影响

目的研究姜黄素对类风湿关节炎( RA)破骨细胞( OC)分化中核因子 κB受体活化因子( RANK)基因及蛋白表达的影响。方法采用核因子 κB受体活化因子配体( RANKL)和巨噬细胞集落刺激因子( M?CSF)诱导 RA病人外周血单个核细胞(PBMC)向 OC分化,给予不同浓度的姜黄素( 2.5、5及 10 μmol/L)进行干预,并设空白对照组。抗酒石酸酸性磷酸酶( TRAP)染色标记成熟 OC并计数;实时荧光定量聚合酶链式反应( Real?time PCR)和蛋白质印迹法( Western Blot)检测各组细胞 RANK基因表达和蛋白水平。结果 RA病人 PBMC经 RANKL诱导后能获得大量 TRAP阳性的 OC,经姜黄素( 2.5、5及 10 μmol/L)干预后, TRAP阳性细胞数明显减少(P＜0.05);与空白对照组相比,姜黄素各浓度组细胞 RANK基因表达显著降低( P＜0.05);姜黄素(2.5、5及 10 μmol/L)各浓度组细胞 RANK蛋白的表达分别为( 0.46±0.09)、(0.36±0.08)和(0.25±0.07)与空白对照组(0.68±0.11)相比均显著降低(F＝21.278,P＝0.000)。结论姜黄素可能通过下调 RANK基因和相关蛋白的表达制RA病人 OC分化。,抑,     

红景天苷抑制破骨细胞分化和极化的初步研究

目的　探究红景天苷抑制破骨细胞分化和极化的作用。方法　体外培养RAW264.7细胞,加入可溶性核因子κB受体活化因子配体（sRANKL）连续培养5 d诱导为破骨细胞。设置对照组（培养基不含红景天苷）和实验组（培养基中红景天苷剂量分别为15、30、60 mg/L）,通过TRAP染色对比诱导分化结果,通过鬼笔环肽染色和茜素红染色观察破骨细胞F-Actin环形成和破骨细胞钙化情况,通过qPCR检测基质金属蛋白酶-9（MMP-9）、非受体酪氨酸激酶（c-Src）、组织蛋白酶K（CK）、整合素β3（Integrin β3） mRNA表达水平,Western blot检测MMP-9和c-Src蛋白表达水平。结果　与对照组相比,各实验组TRAP染色阳性细胞数显著减少,F-Actin环形成减少,而破骨细胞茜素红染色显著加深,钙化加重（均P＜0.05）,且随着红景天苷剂量的升高,变化更加明显。与对照组相比,30和60 mg/L SAL组MMP-9、c-Src、CK mRNA表达水平均降低,同时各实验组MMP-9及c-Src蛋白表达水平显著降低。结论　红景天苷对破骨细胞的分化、极化和骨吸收有一定的抑制作用,其作用机制可能与下调MMP-9和c-Src表达有关。     

转移性骨肿瘤治疗新突破

骨骼是恶性肿瘤最常见的转移部位之一,对转移性骨肿瘤的治疗已成为当今肿瘤学研究的热点之一.骨保护素(OPG)、核因子kB受体活化因子配体(RANKL)及核因子kB受体活化因子(RANK)的骨调节轴即OPG/RANKL/RANK,是影响破骨细胞分化、成熟的重要途径.安进公司丌发的完全人源化RANKL抗体denosumab,可通过阻止RANKL和RANK结合抑制破骨细胞形成,从而实现肿瘤骨转移治疗的新突破.本文详细介绍OPG/RANKL/RANK及denosumab的作用机制以及相关大规模临床研究.     

RANK/RANKL/OPG系统及其相关疾病和抗RANKL疗法的研究进展

骨骼是一个动态组织,在生物的整个生命周期中不断地进行构建、吸收、重建,核因子(nuclear factor,NF)κB受体活化因子(receptor activator of NF-κB,RANK)/NF-κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)/护骨素(osteoprotegerin,OPG)系统是调控这一过程的关键系统.RANK/RANKL/OPG属于肿瘤坏死因子及其受体超家族,三者通过调控破骨细胞的分化和活化来影响骨吸收和重建的过程.另外,免疫细胞也参与和调节RANK/RANKL的表达和分泌,而免疫细胞本身亦受到该系统的影响,因此,RANK/RANKL/OPG系统成为骨和免疫之间的重要关联.RANK/RANKL/OPG系统的失衡与多种骨代谢性疾病及免疫系统疾病引发的继发性骨病密切相关,该系统的发现为研究相关疾病的病理机制和治疗方法提供了基础.由此建立的抗RANKL疗法已经开始应用于临床试验和研究.此文对RANK/RANKL/OPG系统、系统相关疾病以及抗RANKL治疗的进展做一综述.     

ERK1/2和p38MAPK介导BAPTA-AM抑制RANKL诱导小鼠骨髓巨噬细胞分化的机制研究

目的探讨BAPTA-AM对RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞能力的影响以及其信号通路机制的研究。方法体外分离培养小鼠骨髓巨噬细胞,建立RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞的实验模型。应用抗酒石酸酸性磷酸酶(TRAP)染色法检测RANKL诱导的小鼠破骨细胞分化和存活的能力及BAPTA-AM对其的抑制作用;采用免疫印迹法(Western blot)测定ERK1/2和p38MAPK蛋白的磷酸化水平。结果 RANKL诱导的小鼠骨髓巨噬细胞胞质内可见多核,并能分化成为破骨细胞。不同浓度的BAPTA-AM(1、2μmol·L-1)对破骨细胞的形成具有明显的抑制作用,且随着浓度增加能明显地降低TRAP阳性细胞数目。RANKL对小鼠骨髓巨噬细胞的ERK1/2和p38MAPK具有明显的激活作用,诱导胞质ERK1/2和p38MAPK磷酸化,而不同浓度的BAPTA-AM可明显地抑制这种磷酸化作用。结论 BAPTA-AM能明显地抑制RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞,这种抑制作用可能是通过ERK1/2和p38MAPK信号蛋白介导。     

柚皮苷对破骨细胞分化和极化影响的初步探究

目的　探究柚皮苷对破骨细胞分化和极化功能的影响。方法　体外培养RAW264.7细胞,设置对照组（培养基不含柚皮苷）和实验组（培养基中柚皮苷剂量分别为2、20、200 μg/L）连续培养7 d,通过抗酒石酸酸性磷酸酶（TRAP）染色对比组间诱导分化结果,通过实时荧光定量逆转录聚合酶链反应（qPCR）对比组间组织蛋白酶K（CK）、基质金属蛋白酶-9（MMP-9）、整合素β3（integrin β3）和非受体酪氨酸激酶（c-src）mRNA表达水平,通过蛋白免疫印迹法对比组间CK、MMP-9、integrin β3、磷酸化产物p-src蛋白表达水平。结果　与对照组相比,各实验组TRAP染色阳性细胞数显著降低（P＜0.05）;与对照组相比,20 μg/L与200 μg/L组CK、MMP-9、integrin β3及c-src mRNA表达水平显著降低（P＜0.05）;与对照组相比,20 μg/L与200 μg/L组CK、MMP-9、integrin β3及p-src蛋白表达水平显著降低（P＜0.05）。结论　柚皮苷对破骨细胞的分化和极化有一定的抑制作用,其作用机制可能与下调integrin β3、c-src及p-src表达有关。     

柚皮素对氧化应激所致成骨细胞凋亡的影响及机制研究

目的：观察柚皮素对氧化应激导致的成骨细胞凋亡的影响,并探讨其机制。方法体外分离培养成骨细胞,细胞分空白对照组、单纯 H2 O2作用组、H2 O2＋0 J．1μmol／L 柚皮素组、H2 O2＋1μmol／L柚皮素组、H2 O2＋10μmol／L柚皮素组。 MTT法检测成骨细胞活力;流式细胞术检测成骨细胞凋亡率;荧光显微镜观察活性氧（ ROS）含量;比色法检测丙二醛（ MDA）含量;Real time-PCR和Western-blot检测凋亡相关蛋白Bcl-2、Bax、Caspase-3表达情况。结果与空白对照组比较,单纯H2 O2处理组细胞活性明显降低,凋亡率及ROS、MDA含量明显增加;同时Bcl-2 mR-NA和蛋白表达明显下调,Bax、Caspase-3 mRNA和蛋白表达明显上调（ P ＜0．05）。与单纯H2 O2处理组比较,柚皮素预处理24 h能够明显增强细胞活性,降低细胞凋亡率及ROS、MDA含量,上调Bcl-2 mRNA和蛋白表达,下调Bax、Caspase-3 mRNA和蛋白表达,呈剂量依赖性（ P ＜0．05）。结论柚皮素在氧化应激条件下能够促进成骨细胞增殖,抑制成骨细胞凋亡,其机制与上调Bcl-2表达,下调Bax、Caspase-3表达有关。     

异鼠李素抗骨质疏松作用及机制探讨

目的：研究异鼠李素对去卵巢大鼠致骨质疏松骨的干预作用及机制。方法：取45只11周龄未经产SD雌性大鼠随机分为去卵巢假手术组（Sham）、去卵巢模型组（Model）和异鼠李素治疗组（IH）。用药12周后,micro-CT检测骨微结构形态并进行分析,ELISA法检测血清中抗酒石酸酸性磷酸酶（TRAP）和骨钙素（OC）水平,RT-PCR检测骨组织骨保护素（OPG）和NF-kB受体活化因子配体（RANKligands,RANKL）mRNA的表达,western blotting检测骨组织活化T细胞核因子c1（NFATc1）的表达。结果：与模型组比,异鼠李素可增加松质骨骨矿密度和体积,增加骨小梁厚度和数量,降低骨小梁间距（P<0.01）;增加骨皮质（密质骨）骨矿密度和体积,使骨皮质增厚,骨皮质孔隙率减少。RT-PCR结果显示,异鼠李素显著增加去卵巢大鼠骨组织OPG mRNA的表达,降低RANKL mRNA的表达。ELISA检测显示,异鼠李素显著降低去卵巢大鼠血液TRAP和OC水平。western blotting检测结果显示,异鼠李素可显著降低骨组织NFATc1的表达。结论：异鼠李素具有防治去卵巢大鼠骨质疏松作用,其机制与调控骨组织RANKL/RANK/OPG信号通路有关。     

Swertiamarin,a natural steroid,prevent bone erosion by modulating RANKL/RANK/OPG signaling

Bone erosion is a central feature of rheumatoid arthritis (RA) that is characterized by the infiltration of the synovial lining by osteoclasts and lymphocytes. In the present study, swertiamarin a major secoiridoid glycoside was evaluated for anti-osteoclastogenic property to prevent bone erosion in Freund's complete adjuvant (FCA) induced in-vivo model, in-vitro osteoblast and osteoclasts as well as in co-culture system and in-silico molecular docking analysis. The swertiamarin treatment decreased the expression of TRAP, RANKL, and RANK levels and increased the levels of OPG levels significantly in both in vitro and in vivo models. In in vitro, the compound treatment significantly increased the cell proliferation and ALP levels in osteoblast cells; the high proliferation (153.8600 ± 5.23%) and ALP release (165.6033 ± 4.13%) were observed at 50 μg/ml concentration of swertiamarin treatment. At the same time the treatment decreased the TRAP positive cells in osteoclast cells; the high reductions of TRAP positive cells (39.32 ± 3.19%) were observed at 50 μg/ml of swertiamarin treatment. The treatment modulated the levels of pro-inflammatory cytokines, MMPs and NF-κB levels in osteoblast and osteoclast co-culture system. In in silico analysis swertiamarin had affinity towards the proteins RANK, RANKL and OPG residues with low binding energy − 4.5, − 3.92 and − 5.77 kcal/mol respectively. Thus, the results of this study revealed the anti-osteoclastogenic activity of swertiamarin on the prevention of bone destruction.     

甲氨蝶呤对破骨细胞的作用及机制研究

本文在诱导培养并纯化破骨细胞的基础上，研究甲氨蝶呤对破骨细胞活性及功能的影响，探讨甲氨蝶呤抑制炎症性骨破坏的作用机制。研究采用MTT法测定甲氨蝶呤对破骨细胞增殖的影响；流式细胞术测定甲氨蝶呤对破骨细胞凋亡的影响；TRAP染色和骨吸收陷窝染色计数、骨吸收陷窝面积测定分别观察甲氨蝶呤对破骨细胞活性及功能的影响；ELISA法测定甲氨蝶呤对破骨细胞中MMP-9分泌的影响；RT-PCR法测定甲氨蝶呤对破骨细胞中MMP-9、 RANK基因表达的影响。结果显示，甲氨蝶呤(0.1～10 μmol·L^-1)可抑制破骨细胞增殖，对破骨细胞的活性及骨吸收功能均有显著的抑制作用，并可促进破骨细胞的凋亡。同时，甲氨蝶呤(0.01～10 μmol·L^-1)对破骨细胞中RANK的mRNA表达具有一定的抑制作用；但对MMP-9表达的影响较弱，只在1～10 μmol·L^-1时才对MMP-9的mRNA表达具有抑制作用。以上结果表明，甲氨蝶呤抑制炎症性骨破坏的作用与其对破骨细胞活性及功能的抑制密切相关。     

Olmesartan decreases IL-1β and TNF-α levels; downregulates MMP-2, MMP-9, COX-2, and RANKL; and upregulates OPG in experimental periodontitis

The objective of this study is to investigate the participation of inflammatory and oxidative stress mediators and the effects on the expression of matrix metalloproteinase (MMP)-2, MMP-9, and receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK)/osteoprotegerin (OPG) pathway in the response to treatment with olmesartan, an angiotensin II type 1 receptor blocker. Male Wistar albino rats were randomly divided into five groups of ten rats each: (1) non-ligature with water, (2) ligature with water, (3) ligature with 1 mg/kg olmesartan, (4) ligature with 6 mg/kg olmesartan, and (5) ligature with 10 mg/kg olmesartan. All groups were treated with olmesartan or the vehicle by gavage daily for 10 days. Following the treatment course, the periodontal tissue of the animals was analyzed by histopathology and immunohistochemistry to determine the expression of cyclooxygenase-2 (COX-2), MMP-2, MMP-9, and members of the RANKL/RANK/OPG pathway and by ELISA and spectroscopic assay to determine the levels of interleukin (IL)-1β, IL-10, tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), malonaldehyde (MDA), and glutathione. The concentrations of MPO and MDA were reduced in the group that received 6 mg/kg olmesartan (p?<?0.05). In addition, the group that was treated with 6 mg/kg olmesartan showed a decreased level of IL-1β (p?<?0.05), and all doses of olmesartan resulted in decreased levels of TNF-α. Furthermore, treatment with 6 mg/kg olmesartan led to downregulation of the expression of COX-2, MMP-2, MMP-9, RANKL, and RANK and to upregulation of the expression of OPG. These findings suggest that 6 mg/kg olmesartan reduces the inflammatory process and bone loss by downregulating MMPs and RANKL in osteoblasts and by upregulating OPG.     

Long Term Effect of High Glucose and Phosphate Levels on the OPG/RANK/RANKL/TRAIL System in the Progression of Vascular Calcification in rat Aortic Smooth Muscle Cells

Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK) axis, and TNF-related apoptosis-inducing ligand (TRAIL) participate in vascular calcification process including atherosclerosis, but their contributions under high glucose (HG) and phosphate (HP) condition for a long-term period (more than 2 weeks) have not been fully determined. In this study, we evaluated the effects of HG and HP levels over 2 or 4 weeks on the progression of vascular calcification in rat vascular smooth muscle cells (VSMCs). Calcium deposition in VSMCs was increased in medium containing HG (30 mmol/L D-glucose) with β-glycerophosphate (β-GP, 12 mmol/L) after 2 weeks and increased further after 4 weeks. OPG mRNA and protein expressions were unchanged in HG group with or without β-GP after 2 weeks. However, after 4 weeks, OPG mRNA and protein expressions were significantly lower in HG group with β-GP. No significant expression changes were observed in RANKL, RANK, or TRAIL during the experiment. After 4 weeks of treatment in HG group containing β-GP and rhBMP-7, an inhibitor of vascular calcification, OPG expressions were maintained. Furthermore, mRNA expression of alkaline phosphatase (ALP), a marker of vascular mineralization, was lower in the presence of rhBMP-7. These results suggest that low OPG levels after long term HG and phosphate stimulation might reduce the binding of OPG to RANKL and TRAIL, and these changes could increase osteo-inductive VSMC differentiation, especially vascular mineralization reflected by increased ALP activity during vascular calcification.     

Gu Ling Pian, a traditional Chinese medicine, regulates function and OPG/RANKL synthesis of osteoblasts via the p38 MAPK pathway

Osteoporosis is a common disease that makes bones prone to fracture and can affect both men and women. Many traditional Chinese medicine formulations have the potential effect of preventing osteoporosis. Gu Ling Pian (GLP), a traditional Chinese medicine formulation, comprised of tonifying kidney herbal medicines, has been demonstrated to prevent osteoporosis by increasing bone mineral density, however the exact mechanism has not yet been elucidated. Osteoprotegerin (OPG), a receptor activator of NF-kappaB (RANK), and RANK ligand (RANKL) play critical roles in bone remodelling by regulating the function of osteoclasts. In this study, we investigated the effect of GLP on osteoblasts, namely MG-63 cells. The cell proliferation and differentiation, synthesis of OPG/RANKL and p38 expression were tested on MG-63 cells exposed to serum from rats fed with GLP or not. The results showed that GLP significantly promoted MG-63 cell proliferation and differentiation. Upregulation of OPG and down-regulation of RANKL at the protein and mRNA level were observed in GLP serum treated MG-63 cells using an enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Further, treatment with GLP serum increased the level of p38 phosphorylation but did not affect the total p38 expression. These effects can be blocked by the p38 specific inhibitor SB203580. The results indicate that GLP can effectively promote the proliferation and differentiation of osteoblasts and regulate their OPG/RANKL expression, while the effects may be mediated via the p38 MAPK pathway. The findings suggest that GLP induces bone formation and may be beneficial for patients with osteoporosis.