金雀异黄素对类风湿关节炎患者成纤维样滑膜细胞骨保护素／细胞核因子κB受体活化因子／细胞核因子κB受体活化因子配体通路的影响

目的研究金雀异黄素（genistein,Gen）对人类风湿关节炎（rheumatoid arthritis,RA）成纤维样滑膜细胞（fibroblast—like synoviocytes,FLS）骨保护素（osteoprotegerin,OPG）／细胞核因子KB受体活化因子（receptor—activator of nuclear factor kappa bata,RANK）／细胞核因子KB受体活化因子配体（receptor—activator of nuclear factor kappa bata ligand,RANKL）通路的影响及机制。方法培养人RA—FLS,并分为对照组（RA—FLS细胞）、TNF—α组（RA—FLS细胞＋TNF-α 10ng／ml）、TNF—α＋Gen组（RA—FLS细胞＋TNF—α 10ng／ml＋Gen 50μM）、Gen组（RA—FLS细胞＋Gen 50μM）。应用免疫印迹检测Gen作用RA—FLS的RANK、RANKL、OPG、雌激素受体（estrogen receptora,ERa）表达情况,免疫荧光检测ERα细胞内分布情况。结果Gen组、TNF-α组及TNF—α＋Gen组细胞内RANK蛋白表达量较对照组无明显差异（P〉0．05）,但OPG及RANKL蛋白表达明显高于对照组（均P〈0．05）。Gen组OPG／RANKL较对照组增高（P〈0．05）。Gen组、TNF-α组及TNF-α＋Gen组细胞内ERa蛋白表达量与对照组比较,差异无统计学意义（P〉0．05）。免疫荧光结果显示核内ERα表达量增加。结论Gen可能通过与雌激素受体结合,转入核内发挥免疫调节作用,从而调节0PG／RANKI／RANK通路,发挥抑制骨破坏的作用。Gen可以上调人RA—FLS中OPG及RANKL,且对OPG的上调作用更强。     

外周血护骨素和核因子κB受体活化因子配体水平在女性类风湿关节炎中的变化及其与骨质疏松的关系

目的探讨女性类风湿关节炎（RA）患者外周血核因子-κB受体活化因子配体（RANKL）和护骨素（OPG）水平的变化及其与女性RA骨质疏松的相关性。方法采用ELISA法测定55例女性RA患者和53例正常女性外周血中RANKL和OPG水平,采用双能X线骨密度吸收仪测定骨密度。结果（1）和正常女性相比,女性RA患者RANKL水平明显升高,OPG水平明显降低（P〈0．001）。（2）女性RA组中骨质疏松发生率38．2％（21／55）明显高于正常女性组[11．3％（6／53）];这种改变不但体现在已绝经的女性RA患者和已绝经的正常女性,同样体现在未绝经的女性RA患者和未绝经的正常女性（Neck部位骨密度除外）。（3）女性RA患者OPG水平与骨密度呈正直线相关;RANKL与之相反（P〈0．05）。（4）Logistic分析显示：OPG／RANKL比值为女性RA患者中骨质疏松发生的保护因素（OR=0．027,P=0．014,95％CI：0．001～0．478）。结论女性RA患者外周血OPG水平、RANKL水平的变化与骨代谢状态的变化密切相关;外周血OPG／RANKL比为女性RA患者中骨质疏松发生的保护因素。     

RANK RANKL和骨保护素在类风湿关节炎患者外周血和滑液中表达的研究

目的研究骨保护素(OPG)和核因子κB受体活化因子(RANK)及其配体(RANKL)在类风湿关节炎(RA)患者外周血和滑液中的表达,探讨RANKL、RANK和OPG在RA骨破坏中的意义。方法收集活动期RA患者治疗前后外周血、滑液和健康对照,以间接免疫荧光标记,流式细胞术检测RANK、RANKL和OPG在外周血和滑液中CD3+T淋巴细胞、中性粒细胞和CD14+单核细胞的表达率及平均荧光强度。结果①RA组治疗前后外周血RANK、RANKL和OPG在CD3+T淋巴细胞、中性粒细胞和CD14+单核细胞上的表达率差异无统计学意义(P>0.01);RANK的表达强度治疗后高于治疗前(P<0.01)。②RA组外周血CD3+T淋巴细胞上RANKL及RANK的表达率和表达强度明显低于滑液,OPG的表达则高于滑液(P<0.01),RANKL/OPG比率明显增高(P<0.01)。结论RANK表达强度的改变提示其可能与RA炎症的发展和转归有关,而活化的CD3+T淋巴细胞可能通过上调RANK、RANKL的表达,下调OPG表达参与RA破骨细胞的分化和骨损伤的调节机制。     

雷公藤甲素对佐剂性关节炎大鼠核因子κB受体激活剂配基表达的影响

目的 探讨雷公藤甲素对佐剂性关节炎（AA）大鼠核因子κB受体激活剂配基（RANKL）和护骨素（OPG）表达的影响及其与抑制关节炎大鼠骨侵蚀的关系。方法 AA大鼠经雷公藤甲素治疗后，测定关节中RANKL、OPG的表达，并测定外周血单个核细胞（PBMC） RANKL mRNA的表达水平和外周血中肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β水平，同时测定胫骨关节端骨密度，并作关节骨侵蚀病理积分。结果 雷公藤甲素组和甲氨蝶呤组骨密度高于模型组（P〈0.01），关节骨侵蚀积分低于模型组（P〈0．01）。雷公藤甲素组大鼠关节滑膜（P〈0.01）及骨组织（P〈0.05）RANKL表达量低于模型组，滑膜OPG表达量低于模型组（P〈0.05）。雷公藤甲素组PBMC中RANKLmRNA表达水平低于模型组（P〈0.01），并且TNF-α和IL-1β水平低于模型组（P〈0．01）。结论 雷公藤甲素可能通过抑制RANKL表达而抑制AA大鼠骨质侵蚀。未见雷公藤甲素对OPG表达有促进作用。     

川芎嗪对类风湿关节炎模型大鼠关节滑液RANK/RANKL/OPG表达的影响

目的 探讨川芎嗪对类风湿关节炎(RA)大鼠关节滑液核因子κB活化受体(RANK)、RANK配体(RANKL)和骨保护素(OPG)表达的影响,以及在RA骨破坏和炎症过程中的作用机制.方法 建立大鼠佐剂性关节炎模型,并分为5组,模型对照组、阳性对照组、川芎嗪高、低剂量组和正常对照组,造模15 d后,连续给药7 d,应用间接免疫荧光标记、流式细胞术检测关节滑液中RANK/RANKL/OPG表达水平和平均荧光强度.结果 与正常对照组相比,模型组CD3+T细胞OPG表达明显降低,RANK,RANKL升高,差异有显著性(P＜0.05),中性粒细胞和单核细胞上变化不明显;经过川芎嗪高剂量治疗后,CD3+T细胞上OPG表达增强,RANK、RANKL减弱,平均荧光强度降低,差异有显著性(P＜0.05),中性粒细胞和单核细胞治疗前后RANK、RANKL、OPG表达变化不明显;低剂量组各指标变化无统计学意义.结论 高剂量川芎嗪主要通过调节关节滑液中CD3+T细胞上RANK/RANKL/OPG表达起到缓解RA骨损伤和关节炎症的作用.     

RANKL／RANK／OPG系统在类风湿性关节炎骨质疏松中的研究进展

由细胞核因子KB受体活化因子配体（RANKL）、细胞核因子KB受体活化因子（RANK）和护骨素（OPG）组成的RANKL／RANK／OPG系统是近些年来发现并且研究愈趋热门的一个在骨调节上发挥重要作用的系统。     

慢阻肺合并骨质疏松患者血清IL-31、IL-33水平与OPG/RANK/RANKL系统相关性研究

目的 探讨血清白细胞介素31(IL-31)、白细胞介素33(IL-33)在慢性阻塞性肺疾病(COPD)合并骨质疏松患者中的表达情况及与骨保护素(OPG)/核因子κB受体活化因子(RANK)/RANK配体(RANKL)系统的关联性。方法 收集2019年6月～2023年1月于惠州市第一人民医院就诊的男性稳定期COPD患者90例为COPD组,并纳入同期健康体检者45例为对照组。COPD组患者根据骨密度检测结果,分为非骨质疏松组(n=49)和骨质疏松组(n=41)。测定病例组、对照组入组当天血清IL-31、IL-33、OPG及RANKL水平。结果 (1)相比正常对照组,COPD组患者血清IL-31、RANKL水平及RANKL/OPG比值明显升高,而血清IL-33水平明显下降(均P<0.05);(2)与COPD非骨质疏松组患者相比,骨质疏松组血清IL-31、RANKL水平及RANKL/OPG比值显著升高,而血清IL-33水平、体重指数(BMI)、骨密度及FEV₁(%)、FEV₁/FVC(%)明显降低(均P<0.05);(3) COPD患者骨...     

前列腺素E2对人成骨样细胞白介素-6,骨保护素配体/骨保护素mRNA表达的影响

目的研究前列腺素E2（PGE2）对人成骨瘤细胞MG-63分泌的细胞因子白介素-6（IL-6）、骨保护素（Osteoprotegerin，OPG）、骨保护索配体（Ligand of receptor activator of NF-κB，RANKL）表达的调节，探讨PGE引发骨吸收的机制。方法体外培养成骨瘤细胞株（MG-63），分别给予不同剂量的PGE2进行干预试验，用钙估法显示成骨细胞碱性磷酸酶的表达；用MTT法测定PGE2对MG-63细胞刺激增殖作用；提取总RNA进行半定量逆转录PCR分析，检测IL-6，OPG，RANKL基因表达水平的改变。结果PGE2减少碱性磷酸酶的表达，抑制MG-63细胞的增殖，并呈剂量依赖性的上调IL-6mRNA，RANKL／OPGmRNA水平。结论PGE2可以通过IL-6，RANKL／OPGmRNA基因水平的上调，促进骨吸收，此可能为PGE2导致溶骨性病变的重要发病机制。     

OPG/RANK/RANKL系统与骨折和类风湿性关节炎

骨保护素(OPG)、细胞核因子-κB受体活化因子(RANK)和RANK配体(RANKL)是偶联成骨细胞、基质细胞和破骨细胞分化、活化及生物活性的3种主要细胞因子,其形成的局部调节体系在骨代谢中起十分重要的作用。本文简要介绍了OPG/RANK/RANKL系统及该系统在骨质疏松性骨折发生中的作用,RANKL/OPG比值与骨折的关系,OPG和RANKL对骨折愈合的作用,血清OPG或RAN-KL水平与骨折的联系,OPG基因多态性与骨折关系的研究结果。另外还介绍其在类风湿性关节炎发病机制中的作用,OPG/RANK/RANKL与滑膜组织的联系,OPG治疗的相关实验进展。     

白细胞介素-23刺激的类风湿关节炎成纤维细胞对人破骨样细胞形成的研究

目的 探讨向细胞介素(IL)-23刺激的类风湿关节炎(RA)滑膜成纤维细胞(FLS)在人破骨样细胞形成中的作用.方法 用不同浓度(1、5和10 ng/ml)的IL-23刺激RA和骨关节炎(OA)患者滑膜FLS 72 h,实时荧光定量聚合酶链反应(real time-PCR)检测RA和OA滑膜FLS核因子κB受体激活剂配体(RANKL)mRNA表达.分离人外周血单核细胞(MN),与IL-23刺激的RA和OA滑膜FLS共培养,通过抗酒石酸酸性磷酸酶(TRAP)染色观察是否有破骨样细胞形成;同时,real time-PCR法检测破骨样细胞形成的分子标志.结果 不同浓度的IL-23均能上调RA滑膜FLS RANKL的表达;但几乎不诱导OA滑膜细胞RANKL的表达.在IL-23刺激的RA FLS和MN共培养体系中能观察到TRAP染色阳性的破骨样细胞形成;并且破骨样细胞形成的分子标志表达增高.而无IL-23刺激的RA FLS或IL-23刺激的OAFLS和MN共培养体系中未见TRAP染色阳性的破骨样细胞的形成.结论 IL-23通过诱导RA滑膜细胞RANKL的表达促进人MN分化衍生为破骨样细胞.     

Cross-talk between the interleukin-6 and prostaglandin E(2) signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of nuclear factor-{kappa}B (RANK) ligand/RANK system

The osteoprotegerin (OPG)/receptor activator of nuclear factor-kappaB ligand (RANKL)/receptor activator of nuclear factor-kappaB (RANK) system is the dominant and final mediator of osteoclastogenesis. Abnormalities of this system have been implicated in the pathogenesis of many skeletal diseases. Cyclooxygenase (COX)-2 and prostaglandin (PG)E(2), a major eicosanoid product of the COX-2-catalyzed pathway, play key roles in normal bone tissue remodeling. PGE(2) exerts its actions by binding and activating the E series of prostaglandin (EP) receptor. Activation of EP(2) and EP(4) receptors is associated with PGE(2)-induced osteoclast differentiation. IL-6, a major proinflammatory cytokine, has also been reported to induce osteoclast differentiation. Although interactions between the COX-2/PGE(2) and IL-6 systems have been described in bone cells, the mechanisms underlying these cooperative signaling pathways and the possible involvement of the OPG/RANKL/RANK system have not been fully elucidated. We demonstrate that COX-2, PGE(2), and IL-6 stimulate osteoblast growth and osteoclast differentiation. Effects on osteoclast differentiation, particularly with IL-6, were most marked when osteoclast precursor cells were grown in coculture with osteoblasts, indicating a possible role of the RANK/RANKL/OPG system. COX-2 and PGE(2) stimulated osteoclastogenesis through inhibition of OPG secretion, stimulation of RANKL production by osteoblasts, and up-regulation of RANK expression in osteoclasts. PGE(2) stimulated IL-6 secretion by bone cells, whereas COX-2 inhibitors decreased this same parameter. IL-6, in turn, increased PGE(2) secretion, COX-2, and EP receptor subtype expression in bone cells. Finally, IL-6 was the mediator of PGE(2)-induced suppression of OPG production by osteoblasts. These findings provide evidence for cross-talk between the PGE(2) and IL-6 signaling enhance osteoclast differentiation via effects on the OPG/RANKL/RANK system in bone cells.     

Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients

OBJECTIVE: To elucidate the direct role of human T cells in the induction of osteoclastogenesis in rheumatoid arthritis (RA), by studying human monocytes and the pathogenetic roles of receptor activator of nuclear factor kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG). METHODS: Synovial tissue obtained at total knee replacement was stained immunohistologically using anti-RANKL, CD3, and CD4 antibodies. Synovial fluid was obtained from patients with RA, osteoarthritis (OA), gout, or trauma. Concentrations of the soluble form of RANKL (sRANKL) and OPG in the synovial fluid were measured by enzyme-linked immunosorbent assay. Activated T cells from peripheral blood mononuclear cells (PBMC) of healthy volunteers were cultured with human monocytes from PBMC. RESULTS: Immunostaining of the synovial tissue of RA patients demonstrated that RANKL-positive cells were detected in a subset of fibroblast-like synoviocytes and infiltrating mononuclear cells. Double immunostaining revealed that RANKL-positive cells were detected in a subset of CD3+ cells and CD4+ cells. An increased concentration of sRANKL and a decreased concentration of OPG were detected in synovial fluid from RA patients. The ratio of the concentration of sRANKL to that of OPG was significantly higher in synovial fluid of RA patients than in synovial fluid of patients with OA or gout. The activated T cells expressing RANKL induced osteoclastogenesis from autologous peripheral monocytes. The role of RANKL in this osteoclastogenetic process was confirmed by dose-dependent inhibition by OPG. CONCLUSION: The present study is the first to demonstrate osteoclastogenesis using human-derived T cells and monocytes. In addition, the present findings suggest that excess production of RANKL by activated T cells increases the level of sRANKL in synovial fluid and may contribute to osteoclastic bone resorption in RA patients.     

重组抗肿瘤坏死因子-α人鼠嵌合单克隆抗体对类风湿关节炎患者RANK/RANKL/骨保护素系统的影响

目的 探讨重组抗肿瘤坏死因子(TNF)-α人鼠嵌合单克隆抗体(Infliximab)对类风湿关节炎(RA)患者外周血核因子κB受体因子(RANK)/核因子κB受体活化因子(RANKL)/骨保护素系统的影响.方法 50例经严格筛选的类风湿关节炎(RA)患者按随机分配原则分为2组.一组患者接受Infliximab(3 mg/kg)+甲氨蝶呤治疗;一组患者接受甲氨蝶呤单独治疗作为对照,分别于0、2、6、14周给药.观察0周与18周Infliximab治疗组与对照组患者相关临床指标的改变,对比外周血中RANK、RANKL mRNA表达情况以及血清中骨保护素蛋白水平的变化.用t检验和x2检验做统计学分析.结果 经Infliximab治疗后,在RA患者关节放射成像中,可以观察到骨破坏程度有减缓趋势.与对照组比较,Infliximab治疗组(病史＞1年)患者骨丢失情况得到控制;Infliximab治疗组(0周:80.25;18周:63.2)与对照组(0周:83.37;18周:30.87)患者外周血中RANK、RANKL mRNA表达水平均下降(P＞0.05);与对照组比较,Infliximab可使RA患者外周血骨保护素/RANKL比值下降趋势减缓.虽然,经甲氨蝶呤或Infliximab+甲氨蝶呤治疗后,对照组[0周:(238±15)pg/ml;18周:(118±l0)pg/ml]和Infliximab治疗组[0周:(223±6)pg/ml;18周:(162±6)pg/ml)]患者血清中骨保护素水平均下降(P＞0.05),但Infliximab治疗组患者血清骨保护素下降趋势得以减缓.结论 RA患者经Infliximab联合甲氨蝶呤治疗后,骨破坏受到抑制,其作用机制可能是部分通过对RANK/RANKL/骨保护素系统的调节来行使的.     

Elevated levels of osteoprotegerin (OPG) and hepatocyte growth factor (HGF) in rheumatoid arthritis

Rheumatic diseases are often associated with changes in bone metabolism. Excessive production and release of cytokines and other growth factors due to inflammation, e.g. tumor necrosis factor-alpha (TNF-alpha), receptor activator of NF-kappaB ligand (RANKL), interleukins such as IL-1 and IL-6, may cause alterations in bone homeostasis leading to bone degradation. Other components such as osteoprotegerin (OPG) and possibly the ligand-receptor pair hepatocyte growth factor (HGF) and c-met may counteract this destruction, we have measured the levels of OPG, and HGF c-met, in serum, synovial fluid (SF), and cartilage from patients with rheumatoid arthritis (RA) and other arthritides. We found a) elevated levels of both OPG and HGF in SF from RA patients relative to arthritides of other causes, b) increased levels of both OPG and HGF in SF from seropositive RA patients (RA+) compared to seronegative RA patients (RA-), c) elevated levels or both OPG and HGF in serum from RA patients compared to healthy controls, d) no correlation between severity of inflammation and levels of OPG or HGF, and e) presence of HGF c-met in both cartilage and synovial tissue. The most significant elevations of OPG and HGF were found in patients with RA, the rheumatic disease most frequently associated with the development of secondary osteoporosis.     

Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathies and osteoarthritis and normal controls

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.     

Osteoprotegerin and the receptor activator of NF-kappa B ligand in the serum and synovial fluid. A comparison of patients with longstanding rheumatoid arthritis and osteoarthritis

We examined OPG and soluble RANKL in the serum (sOPG, sRANKL) and synovial fluid (synOPG, synRANKL) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). OPG and RANKL were measured in 85 patients (44 with RA, 41 patients with OA) in serum and synovial fluid as well. For measuring of OPG and RANKL ELISA tests were used. The results of OPG and RANKL were compared with clinical and radiological scores. We found a negative correlation for OPG and RANKL in synovial fluids: not only for the whole group of patients (P< 0.003, r=–0.32), but also for the subgroups (RA: P<0.04, r=–0.28, OA: P<0.002, r=–0.54). SRANKL and synRANKL were positively correlated in the whole group (P<0.01, r=0.25) and in the OA group (P<0.02, r=0.35); the RA group was showing a trend (P<0.063, r=0.24), however. Serum OPG was lower in RA, synOPG higher in OA. The difference between the two patient groups was only significant for synOPG (P<0.03, r=0.056), but not for sOPG (P<0.09, r=0.19), sRANKL (P<0.43, r=0.85) or synRANKL (P<0.11, r=0.22). The synOPG:synRANKL ratio was significantly correlated with the Larsen score (P<0.004, r=0.38). Synovial OPG is significantly decreased in rheumatoid joints, whereby synovial RANKL is increased. Lower synOPG could reflect a lower protective effect on bone, thus leading to an earlier and more pronounced bone destruction in RA. However, the effect of different mediators for joint destruction in RA and OA seems not to be important to the pathophysiological changes in the joints. The upregulation of serum OPG might be the result of the inflammation; in contrast, an upregulation of RANKL could not be found in the serum of patients with RA and OA.     

Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis

OBJECTIVE: Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF)-blocking agents, including etanercept and infliximab, has resulted in reductions in the radiographic progression of RA. However, the exact mechanism by which this protection occurs has not been determined. In order to add to such knowledge, we investigated the effect of anti-TNF therapy on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) in synovial tissue. METHODS: The expression of OPG and RANKL in synovial biopsy specimens was evaluated by immunohistochemistry. Serial synovial biopsy specimens were obtained from 18 patients with RA, before and after treatment with etanercept (9 patients) or infliximab (9 patients). Biopsy specimens were evaluated by double-blind semiquantitative analysis and image analysis. The in vitro effect of TNF antagonists on the RANKL/OPG expression in osteoblasts and endothelial cells was evaluated by Western blotting. Statistical analysis was performed using Wilcoxon's signed rank test, followed by the Bonferroni correction for multiple comparisons of paired samples. The results of in vitro experiments were evaluated by one-way analysis of variance, with Tukey's post hoc test. RESULTS: Treatment with both infliximab and etanercept increased the expression of OPG in synovial tissue. After 8 weeks of treatment, neither infliximab nor etanercept influenced RANKL expression. In both groups of patients, the RANKL:OPG ratio decreased following therapy. In vitro, both of the TNF antagonists mimicked the in vivo effect, inducing a decrease in the RANKL:OPG ratio in TNF-primed osteoblasts and endothelial cells. CONCLUSION: Therapy with TNF antagonists in RA modulates the OPG/RANKL system, a potential mechanism that could explain the retardation of radiographic damage observed following anti-TNF therapy.