Gene silencing of the BDNF/TrkB axis in multiple myeloma blocks bone destruction and tumor burden in vitro and in vivo

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF‐κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain‐derived neurotrophic factor (BDNF) was identified as a MM‐derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma–osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM‐derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time‐ and dose‐dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID‐RPMI8226 mice model was constructed using lentiviral short hairpin RNA‐transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma–myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.     

Bone mineral density and prediction of non-osteoporotic disease

It is widely recognized that bone mineral density (BMD) is one of the best predictors of osteoporotic fractures. Sex hormone status clearly affects bone either directly or indirectly and a longer estrogen exposure appears to be a major determinant of postmenopausal BMD. Accordingly, several studies have led to the hypothesis that BMD might represent a marker of the accumulated lifetime exposure of estrogen and therefore be used as a predictor factor of the risk of other postmenopausal conditions such as breast cancer or cardiovascular diseases (CVD).     

心房颤动患者血浆骨保护素、核因子κB受体活化因子配体水平及临床意义

目的探讨心房颤动患者血浆骨保护素(osteoprotegerin,OPG)及核因子-κB受体活化因子配体(receptor activator ofnuclear factor kappa B ligand,RANKL)水平及临床意义。方法采用酶联免疫吸附法检测40例阵发性房颤、39例持续性房颤、43例永久性房颤患者及40例窦性心律对照者血浆中OPG、RANKL水平。结果阵发性房颤组、持续性房颤组、永久性房颤组患者血浆中OPG、RANKL水平明显高于对照组,差异有统计学意义(P<0.05),持续性房颤组、永久性房颤组患者与阵发性房颤组相比血浆中OPG、RANKL水平升高,差异有统计学意义(P<0.05),而持续性房颤组、永久性房颤组患者之间差异无统计学意义(P>0.05)。结论 OPG、RANKL与房颤的发生和持续可能相关。     

非诺贝特对高甘油三酯血症并骨质疏松大鼠骨代谢的影响

目的 探讨非诺贝特对去卵巢骨质疏松并高甘油三酯(TG)血症大鼠股骨中细胞核因子κB受体活化因子配体/护骨素(RANKL/OPG)mRNA表达的影响.方法 将40只3月龄雌性SD大鼠,用果糖饲养复制高TG模型,大鼠共分为去卵巢+果糖组、去卵巢+果糖+非诺贝特(FF)组、去卵巢+普食组、假手术+果糖组.12周后取股骨检测细胞核因子κB受体活化因子配体(RANKL) mRNA、护骨素(OPG)mRNA表达水平.结果 去卵巢+果糖组的TG水平高于去卵巢+普食组(P＜0.01),也高于去卵巢+果糖+FF组(P＜0.01);去卵巢+果糖组RANKL mRNA/OPG mRNA水平,均高于去卵巢+果糖+FF组、去卵巢+普食组和假手术+果糖组(P＜0.01或＜0.05).结论 非诺贝特可通过降低TG而保持RANKL mRNA/OPG mRNA的平衡.     

Implication of Receptor Activator of NF-κB Ligand in Wnt/β-Catenin Pathway Promoting Osteoblast-like Cell Differentiation

Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly in-creased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.     

Effects of Huogu I Formula (活骨I方) on correlated factors of bone regeneration in chickens with steroid-induced necrosis of femoral head

Objective:To study the mechanism of HuoguⅠFormula（活骨Ⅰ方） in treating osteonecrosis of femoral head.Methods:Forty-eight healthy female Leghorn chickens were randomly divided into control group,model group and HuoguⅠgroup,and each group consisted of 16 chickens.At the meantime of model establishment,chickens of the HuoguⅠgroup were administrated with decoction,while the model and control group with distilled water by gavage.At the 8th and 16th week after medication,blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations.Specifically, expressions of bone morphogenetic protein-2（BMP2）,transforming growth factor betal（TGFβ₁）,Smad4 and Smad7 were evaluated by immunohistochemistry,while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand（OPG/RANKL） mRNA was detected by in situ hybridization.Results:Compared with the control group,serum levels of total cholesterol（TC）,triglyceride（TG） and low-density lipoprotein cholesterol （LDL-C） in the model group rose significantly.Positive cell counting of BMP2,TGFβ₁,Smad4 and OPG in femoral head of the model group dropped prominently.Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group,levels of TC,TG and LDL-C in Huogu I group reduced significantly.Positive cell counting of BMP2,TGFβ₁,Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly.Especially at the 8th week,these variations were more significant.Conclusion:HuoguⅠFormula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2,TGFβ₁,Smads and OPG/RANKL of osteoclast in femoral head.     

骨保护素基因启动子单核苷酸多态性与脊髓型颈椎病的相关研究

目的探讨骨保护素基因启动子149、950T/C单核苷酸多态性与脊髓型颈椎病的相关性。方法提取192例脊髓型颈椎病和192例性别年龄匹配的健康人外周血DNA,聚合酶链反应产物根据限制性内切酶作用后不同分解产物和等位基因来判断每例患者骨保护素的基因型。确定对照组、疾病组不同的基因型及等位基因,评价颈椎病严重度以JOA评分,同时采用颈椎MRI评价病变节段,采用χ2检验比较两组的基因型分布差异。采用两元回归分析来确定脊髓型颈椎病的独立风险因子。结果在疾病组和对照组之间,149T/C基因型分布及等位基因频率间差异存在统计学意义。对于950 T/C单核苷酸多态性,两组分布差异并无统计学意义。对于149T/C基因型,其JOA评分、颈椎MRI受累节段两者差异无统计学意义。Logistic回归分析结果 149 T/C SNP、平均桌面工作时间为脊髓型颈椎病发病的独立风险因素。结论骨保护素149 T/C单核苷酸多态性与脊髓型颈椎病的发病有关,而与其严重度无关。950T/C单核苷酸多态性与脊髓型颈椎病的发病无明确相关性。149 T/C SNP、平均桌面工作时间为脊髓型颈椎病发病的独立风险因素。     

低强度超声对颅骨溶解模型小鼠磨损颗粒诱导的骨溶解效应的影响

目的:探讨低强度超声在小鼠颅骨溶解模型中对破骨细胞增殖分化的干预,为临床治疗关节松动提供理论依据。方法：选择30只BALB/c小鼠,按随机数字表法分为空白对照组、颗粒组和超声组,每组10只。采用改良磨损颗粒法复制小鼠颅骨溶解模型,在术后15 d获取小鼠颅顶骨骨组织标本,HE染色观察炎细胞渗出量和骨溶解面积变化;扫描电镜检测骨片吸收陷窝面积百分比;抗酒石酸酸性磷酸酶染色（TRAP）观察破骨细胞增殖分化;免疫组织化学染色定位观察损伤骨组织中骨保护素（OPG）的表达变化。结果：HE染色,与空白对照组比较,颗粒组和超声组炎细胞渗出量和骨溶解面积明显增加（P<0.01）;与颗粒组比较,超声组骨溶解面积明显降低（P<0.05）,而2组炎细胞渗出量比较差异无统计学意义（P>0.05）。扫描电镜,超声组骨片吸收陷窝面积百分比显著低于颗粒组（P<0.01）。TRAP染色,超声组破骨细胞数量明显低于颗粒组（P<0.01）。免疫组织化学,超声组OPG阳性细胞数明显高于颗粒组及空白对照组（P<0.05）。结论：低强度超声能抑制小鼠颅骨溶解模型中破骨细胞的增殖分化,有效降低聚乙烯磨损颗粒所致的骨溶解效应,从而增强关节假体周围骨的密度和强度,降低关节松动的发生率。     

高脂饮食诱导肥胖小鼠模型中氧化应激、炎症递质对骨代谢的影响及其可能机制

目的：探讨高脂饮食诱导肥胖小鼠模型中氧化应激和炎症递质对骨代谢的影响及其可能机制。方法：选用5周龄C57BL/6雄性小鼠16只,随机平均分为正常饮食组、高脂饮食组,分别用正常饮食和高脂饮食喂养。于实验16周末,测各组动物体质量,处死后取内脏脂肪并测量内脏脂肪/体质量比值;用ELISA法测量血清抗酒石酸酸性磷酸酶5b（TRACP-5b）、血清骨钙素（OC）、白介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）;分别用硫代巴比妥酸（TBA）法和羟胺法检测胫骨骨组织丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性;用骨密度仪检测右侧股骨骨密度（BMD）;分别用免疫组织化学技术和实时荧光定量PCR（RT-qPCR）技术检测骨组织骨保护素（OPG）和核因子-κB受体活化因子配体（RANKL）蛋白及mRNA的表达;HE染色光镜观察左侧股骨组织形态学改变。结果：与正常饮食组相比,高脂饮食组体质量、内脏脂肪/体质量比值、骨组织RANKL mRNA/OPG mRNA比值、血清TRACP-5b、IL-6和TNF-α水平、骨组织RANKL表达及骨组织MDA均明显升高,差异均具统计学意义（P＜0.05）;而血清OC水平、骨组织OPG表达、骨组织SOD及BMD均明显下降,差异均具统计学意义（P＜0.05）。在组织形态学上,发现高脂饮食组股骨干骺端骨小梁分布稀疏,骨小梁变细及断裂,伴骨髓腔中有大量脂肪浸润。各指标的相关分析结果显示：BMD与骨组织SOD呈正相关（r=0.627, P＜0.01）,与内脏脂肪/体质量比值（r=-0.858,P＜0.01）、骨组织MDA均呈负相关（r=-0.538,P＜0.01）;SOD与OPG mRNA呈显著正相关（r=0.637,P＜0.01）,与TNF-α（r=-0.673,P＜0.01）、IL-6（r=-0.874,P＜0.01）、RANKL mRNA（r=-0.760,P＜0.01）及RANKL/OPG比值（r=-0.768,P＜0.01）呈显著负相关;而MDA与TNF-α、IL-6、OPG mRNA、RANKL mRNA以及RANKL/OPG无显著相关性（P＞0.05）。结     

还原型谷胱甘肽对大鼠动脉钙化模型的影响与作用机制

目的：探讨还原型谷胱甘肽（glutathione GSH）在大鼠动脉钙化模型中,对肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）与骨保护素（osteoprotegerin,OPG）表达的影响及其意义。方法：将雄性SD大鼠30只随机分为正常对照组、钙化组、GSH治疗组,每组10只。钙化组和治疗组分别皮下注射维生素D3注射液50万U/kg·d-1,连续3d,制备大鼠动脉钙化模型,且治疗组在注射维生素D3前3d开始腹腔注射GSH注射液400mg/kg·d-1,直到处死大鼠。结果：治疗组大鼠Von Kossa染色黑染钙盐明显少于钙化组;治疗组TNF-α免疫组化蛋白含量与mRNA表达量明显低于钙化组（P〈0.05）;治疗组OPG免疫组化蛋白含量与mRNA表达量明显高于钙化组（P〈0.05）。结论：GSH在大鼠动脉钙化过程中,可抑制TNF-α的表达,保护大鼠动脉组织,减少OPG的消耗,抑制大鼠动脉钙化的形成。