Role of osteoprotegerin/receptor activator of nuclear factor kappa B/receptor activator of nuclear factor kappa B ligand axis in nonalcoholic fatty liver disease

Concomitantly with the increase in the prevalences of overweight/obesity, nonalcoholic fatty liver disease(NAFLD) has worldwide become the main cause of chronic liver disease in both adults and children. Patients with fatty liver display features of metabolic syndrome(Met S), like insulin resistance(IR), glucose intolerance, hypertension and dyslipidemia. Recently, epidemiological studies have linked obesity, Met S, and NAFLD to decreased bone mineral density and osteoporosis, highlighting an intricate interplay among bone, adipose tissue, and liver. Osteoprotegerin(OPG), an important symbol of the receptor activator of nuclear factor-B ligand/receptor activator of nuclear factor kappa B/OPG system activation, typically considered for its role in bone metabolism, may also play critical roles in the initiation and perpetuation of obesityrelated comorbidities. Clinical data have indicated that OPG concentrations are associated with hypertension, left ventricular hypertrophy, vascular calcification, endothelial dysfunction, and severity of liver damage in chronic hepatitis C. Nonetheless, the relationship between circulating OPG and IR as a key feature of Met S as well as between OPG and NAFLD remains uncertain. Thus, the aims of the present review are to provide the existent knowledge on these associations and to discuss briefly the underlying mechanisms linking OPG and NAFLD.

Receptor activator of nuclear factorκB ligand/osteoprotegerin axis and vascular calcifications in patients with chronic kidney disease

Vascular calcifications are commonly observed in patients with chronic kidney disease （CKD） and contri-bute to the excessive cardiovascular morbidity and mortality rates observed in these patients populations. Although the pathogenetic mechanisms are not yet fully elucidated, recent evidence suggests a link between bone metabolism and the development and progression of vascular calcifications. Moreover, accumulating data indicate that receptor activator of nuclear factor κB ligand/osteoprotegerin axis which plays essential roles in the regulation of bone metabolism is also involved in extra-osseous bone formation. Further studies are required to establish the prognostic significance of the above biomarkers as predictors of the presence and severity of vascular calcifications in CKD patients and of cardiovascular morbidity and mortality. Moreover, randomized clinical trials are needed to clarify whether inhibition of osteoclast activity will protect from vascular calcifcations.

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

The effect of triptolide （TP） on the expression of receptor activator of nuclear factor-κB ligand （RANKL） and osteoprotegerin （OPG） was explored in rat adjuvant induced arthritis （AA）. AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate （MTX） at the onset （day 9） of arthritis. On the peak of arthritis （day 24）, the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells （PBMC） was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group （.P〈0.01） ; The expression levels of RANKL in the synovium （P〈0.01） and bone （P〈0.05）, and OPG level in synovium （P〈0.05） were lower in TP group than in AA group （P〈0.05）. In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group （all P〈0.01）. It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

丹参酮ⅡA调节骨关节炎小鼠骨代谢的作用机制

目的探讨丹参酮ⅡA(TanⅡA)通过介导Yes激酶相关蛋白(Yes-associated protein,YAP)、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(eceptor activator of nuclear factor-κB,RANK)/骨保护蛋白(osteoprotegerin,OPG)调节骨关节炎小鼠骨代谢的作用机制。方法建立骨关节炎小鼠模型,将60只小鼠随机分成假手术组、模型组、TanⅡA低剂量组和TanⅡA高剂量组,每组15只,造模成功后灌胃给药,连续4周。HE和番红O固绿染色观察软骨组织病理损伤并进行Mankin评分。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测血清骨碱性磷酸酶(bone alkaline phosphatase,BALP)、骨钙素(osteocalcin,OC)、Ⅰ型胶原交联羧基末端肽(C-telopeptide of typeⅠcollagen,CTX)、白细胞介素(interleukin,IL)-1β、IL-6、IL-8和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)。蛋白质印迹法(Western blotting)检测基质金属蛋白酶(matrix metalloproteinases,MMPs)、YAP、RANK、RANKL和OPG蛋白。结果TanⅡA可改善小鼠软骨组织病理变化并降低Mankin评分。与假手术组比较,模型组BALP、OC水平下降,CTX、TNF-α、IL-6、IL-1β、IL-8、MMP1、MMP3和MMP13水平升高(P<0.05)。与模型组比较,TanⅡA低剂量组、TanⅡA高剂量组BALP、OC水平升高,CTX、TNF-α、IL-6、IL-1β、IL-8、MMP1、MMP3和MMP13水平降低(P<0.05)。与假手术组比较,模型组小鼠软骨组织中YAP、OPG和RANK蛋白水平下降,RANKL蛋白水平升高(P<0.05);与模型组比较,TanⅡA 2组小鼠软骨组织中YAP、OPG和RANK蛋白水平上升,RANKL蛋白水平下降(P<0.05)。结论TanⅡA可能通过介导YAP、RANK/RANKL/OPG信号通路调控骨关节炎。

基于骨免疫学论中医药抑制类风湿关节炎骨破坏的研究进展

类风湿关节炎(RA)是一种以滑膜炎、软骨与骨破坏为主要病理表现的自身免疫性疾病,致残率较高。RA免疫及炎症反应与骨细胞代谢互为影响,其核心环节为破坏机体核因子κB受体活化因子配体/核因子κB受体活化因子/骨保护素(RANKL/RANK/OPG)信号通路的平衡,导致成骨细胞减少,以及破骨细胞凋亡减退及异常活化。西药目前以抑制炎症反应及相关细胞因子分泌,减缓疾病进展,但长期使用其不良反应难以忽视。中医药在防治骨破坏中研究逐步深入,但在基础及临床研究方面仍存在一定局限性。

地舒单抗在骨质疏松初始、序贯、联合治疗等方面的临床研究进展

地舒单抗(denosumab,DMAb)是最早出现的全人源核因子-κB受体活化因子配体[receptor activator of nuclear factor-κB(NF-κB)ligand,RANKL]的单克隆抗体。2020年在我国上市,应用于骨质疏松治疗领域,是抑制骨吸收类抗骨松药物中的新成员。本综述的目的为归纳总结近年围绕DMAb在骨质疏松初始、序贯以及联合治疗方面的临床研究进展,为其应用提供参考。

PTHrP促进RANKL诱导巨噬细胞分化为破骨细胞参与中耳胆脂瘤骨破坏

目的:骨质进行性吸收破坏是中耳胆脂瘤最重要的临床特征之一,可导致一系列颅内外并发症,而目前中耳胆脂瘤骨破坏的机制尚未明确。本研究旨在探究甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHrP)参与中耳胆脂瘤骨破坏的机制。方法:收集后天性中耳胆脂瘤患者的25例胆脂瘤标本和13例外耳道正常皮肤组织标本。采用免疫组织化学染色方法检测PTHrP、核因子κB受体活化因子配体(receptor activator for nuclear factor-kappa B ligand,RANKL)和骨保护素(osteoprotegerin,OPG)在中耳胆脂瘤和外耳道正常皮肤组织中的表达,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法检测中耳胆脂瘤和外耳道正常皮肤组织中是否存在TRAP阳性多核巨噬细胞。选取小鼠单核巨噬细胞RAW264.7细胞进行干预,分为RANKL干预组和PTHrP+RANKL共同干预组,采用TRAP染色法检测2组破骨细胞的生成情况,实时聚合酶链反应(real-time polymerase chain reaction,real-time PCR)检测干预后2组破骨细胞相关基因TRAP、组织蛋白酶K(cathepsin K,CTSK)和活化T细胞核因子1(nuclear factor of activated T cell cytoplasmic 1,NFATc1)的mRNA表达水平,骨吸收陷窝实验检测2组破骨细胞的骨吸收功能。结果:免疫组织化学染色结果显示,PTHrP和RANKL在中耳胆脂瘤组织中的表达均显著增高,OPG表达降低(均P<0.05),且PTHrP的表达与RANKL、RANKL/OPG比值均呈显著正相关,与OPG表达呈显著负相关(分别r=0.385、r=0.417、r=-0.316,均P<0.05)。同时,PTHrP、RANKL的表达水平与中耳胆脂瘤的骨破坏程度均呈显著正相关(分别r=0.413、r=0.505,均P<0.05)。TRAP染色结果显示中耳胆脂瘤上皮周围基质中有大量TRAP阳性细胞,并存在细胞核数量为3个或3个以上的TRAP阳性破骨细胞。RANKL或PTHrP+RANKL联合干预5 d后,与RANKL干预组相比,PTHrP+RANKL联合干预组的破骨细胞数量显著增加(P<0.05),且破骨细胞相关基因TRAP、CTSK和NFATc1的mRNA表达水平均升高(均P<0.05)。骨吸收陷窝扫描电镜结果显示RANKL干预组、PTHrP+RANKL联合干预组的骨片表面均形成骨吸收陷窝;与RANKL干预组相比,PTHrP+RANKL联合干预组的骨片表面骨吸收陷窝数量显著增加(P<0.05),面积也更大。结论:PTHrP可能通过促进RANKL诱导胆脂瘤组织周围基质中的巨噬细胞分化为破骨细胞,参与中耳胆脂瘤骨破坏。

TSLP、HIF-1α、RANKL在义齿修复后种植体周围炎患者龈沟液中的表达及意义

目的研究胸腺基质淋巴细胞生成素(TSLP)、缺氧诱导因子1α(HIF-1α)、核因子-κB受体活化因子配体(RANKL)在义齿修复后种植体周围炎(PI)患者龈沟液中的表达及意义。方法回顾性选取2019年8月至2023年8月大同市第五人民医院收治的义齿修复患者86例作为研究对象,根据术后3个月是否发生PI将患者分为预后良好组(n=61)和预后不良组(n=25)。比较两组患者的临床资料及术前龈沟液TSLP、HIF-1α及RANKL水平,采用多因素Logistic回归分析对龈沟液TSLP、HIF-1α及RANKL水平与义齿修复患者术后发生PI的关系进行分析,采用受试者操作特征(ROC)曲线分析TSLP、HIF-1α及RANKL水平对义齿修复患者的预后评估价值。结果两组患者临床资料(性别、年龄、病程、义齿种植原因及种植颗数)比较,差异均无统计学意义(P>0.05)。预后良好组患者的龈沟液中TSLP、HIF-1α、RANKL水平分别为(122.57±11.30)ng/L、(417.79±115.43)ng/mL、(116.02±13.45)pg/μL,均明显低于预后不良组[(138.93±12.70)ng/L、(576.55±177.60)ng/mL、(133.24±15.69)pg/μL],差异均有统计学意义(P<0.05)。Logistic回归分析义齿修复患者预后,结果显示龈沟液中TSLP水平升高、HIF-1α水平升高和RANKL水平升高是义齿修复患者术后发生PI的独立危险因素(OR=1.119,95%CI:1.048~1.195;OR=1.007,95%CI:1.002~1.013;OR=1.065,95%CI:1.016~1.117;P<0.05)。ROC曲线分析龈沟液中TSLP、HIF-1α、RANKL水平预测义齿修复患者预后的价值,结果显示曲线下面积(AUC)值分别为0.833、0.786和0.809。其中,RANKL具有最高的特异度(0.852),而HIF-1α具有最高的敏感度(0.800),具有较好的预测价值(P<0.05)。结论龈沟液中TSLP、HIF-1α、RANKL水平升高是义齿修复患者术后并发PI的独立危险因素,且均具有较高的预测义齿修复患者预后的价值。

Imbalance of osteoprotegerin/receptor activator of nuclear factor-κB ligand and oxidative stress in patients with obstructive sleep apnea-hypopnea syndrome

Background:Obstructive sleep apnea-hypopnea syndrome (OSAHS) is associated with a higher prevalence of osteoporosis.However,the underlying mechanisms linking OSAHS with bone loss are still unclear.The aim of this study was to investigate the changes of receptor activator of nuclear factor-κB ligand (RANKL,an osteoclastogenesis-promoting factor) and osteoprotegerin (OPG,the decoy receptor for RANKL),oxidative stress and bone metabolism markers in OSAHS,in order to understand the potential mechanisms underlying bone loss in OSAHS patients.Methods:Forty-eight male patients with OSAHS,confirmed by polysomnography (PSG) study,were enrolled.Twenty male subjects who were confirmed as not having OSAHS served as the controls.The subjects’bone mineral density (BMD) was assessed in lumbar spine and femoral neck using dual-energy X-ray absorptiometry (DXA).Blood samples were collected from all subjects for measurement of RANKL,OPG,the bone formation marker bone-specific alkaline phosphatase (BAP),the bone resorption marker tartrate-resistant acid phosphatase 5b (TRAP-5b),and total antioxidant capacity (TAOC).Results:The BMD and the T-score of the femoral neck and the lumbar spine were significantly lower in OSAHS patients as compared to the control group (P< 0.05).The serum level of BAP was significantly decreased in the OSAHS group (15.62 ± 5.20 μg/L) as compared to the control group (18.83 ± 5.50 μg/L,t= -2.235,P< 0.05),while the levels of TRAP-5b did not differ between the two groups (t= -1.447,P> 0.05).The serum level of OPG and the OPG/RANKL ratio were lower in the OSAHS group compared to the control group (bothP< 0.05).TAOC level was also decreased significantly in the OSAHS group (P< 0.05).Correlation analysis showed that the TAOC level was positively correlated with BAP in the OSAHS group (r= 0.248,P= 0.04),but there were no correlations between TAOC and the BMD or the T-scores.The correlations between the level of OPG (or the OPG/RANKL ratio) and BMD or TAOC did not reach significance.Conclusion:In OSAHS patients,lower levels of TAOC were associated with decreased bone formation,suggesting a role of oxidative stress in bone loss,while the role of OPG/RANKL imbalance in bone metabolism in OSAHS needs further evaluation .

Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

Background The cannabinoid receptor-2 （CB2） is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist （AM630） on receptor activator of nuclear factor kappa B （RANK） ligand （RANKL）induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase （TRAP） staining using a commercial kit. Total ribonucleic acid （RNA）was isolated and real-time reverse transcriptase-polymerase chain reaction （RT-PCR） was done to examine the expression of RANK, cathepsin K （CPK） and nuclear factor kappa B （NF-κB）. The extracellular signal-regulated kinase （ERK）,phosphorylation of ERK （P-ERK） and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide （MTT） assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis：possible association with inflammatory cells

Background Receptor activator of nuclear factor kappa B （NF-κB） ligand （RANKL） and osteoprotegerin （OPG） have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators （RANKL and OPG） and inflammatory cell infiltration in chronic apical periodontitis.Methods The samples of chronic periapical lesions （n=40） and healthy periapical tissues （n=10） were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68.Results The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues （P＜0.001）. The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration （P＜0.05）. Significantly increased RANKL expression was found with T lymphocytes （CD3＋）, macrophages （CD68＋） and B lymphocytes （CD20＋）infiltration （P＜0.05）. No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration.Conclusions RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

Effect of Wenhua Juanbi Recipe(温化蠲痹方) on Expression of Receptor Activator of Nuclear Factor Kappa B Ligand,Osteoprotegerin,and Tumor Necrosis Factor Receptor Superfamily Member 14 in Rats with Collagen-Induced Arthritis

Objective： To study the effect of Wenhua Juanbi Recipe（温化蠲痹方, WJR） on expression of receptor activator of nuclear factor kappa B ligand（RANKL）, osteoprotegerin（OPG）, and tumor necrosis factor receptor superfamily member 14（TNFRSF14, also known as LIGHT） in rats with collagen-induced arthritis（CIA）. Methods： CIA rats were generated by subcutaneous injection of bovine collagen type-Ⅱ at the tail base. Sixty CIA rats were randomly assigned（10 animals/group） to： model, methotrexate（MTX）-treated（0.78 mg/kg body weight）, and WJR-treated（22.9 g/kg） groups. Healthy normal rats（n=10） were used as the normal control. Treatments or saline were administered once daily by oral gavage. Rats were sacrificed at day 28 post-treatment and knee synovium and peripheral blood serum were collected. Toe swelling degree and expression of RANKL, OPG, and LIGHT were determined by Western blot and immunohistochemistry. Results： Compared with the normal group, toe swelling degree was significantly increased in the model group（P〈0.01）. After treatment, toe swelling degree decreased significantly in the WJR and MTX groups compared with the model group（P〈0.01）. Compared with the normal group, expression of RANKL and LIGHT were significantly increased and OPG significantly decreased in peripheral blood and synovium of the model group（P〈0.01）. Conversely, RANKL and LIGHT expression were significantly reduced and OPG increased in the WJR and MTX groups compared with the model group（P〈0.01）. No statistically significant difference existed between WJR and MTX groups. Conclusion： WJR likely acts by reducing RANKL expression and increasing OPG expression, thus inhibiting RANKL/RANK interaction and reducing LIGHT expression, thereby inhibiting osteoclast formation/activation to block bone erosion.

骨保护素/核因子κB受体活化因子/核因子κB受体活化因子配体调节骨代谢及其靶向治疗在口腔领域的应用

背景:骨保护素/核因子κB受体活化因子/核因子κB受体活化因子配体是偶联破骨细胞、成骨细胞分化和活化的重要细胞因子,是调节骨代谢的关键因子,影响着免疫系统、骨的再生和重塑,与牙槽骨的生理性及病理性改建密切相关。目的:分析总结骨保护素/核因子κB受体活化因子/核因子κB受体活化因子配体信号通路对牙槽骨改建的影响及其靶向治疗在口腔领域应用研究中的进展。方法:检索中国知网及PubMed数据库收录的相关文献。中文检索词为“骨保护素,抗RANKL抗体,核因子κB受体活化因子配体,牙周炎,正畸牙移动,种植,牙齿萌出,根尖周病变,牙槽骨吸收”,英文检索词为“OPG,anti-RANKL antibody,RANKL,periodontitis,orthodontic tooth movement,implant,tooth eruption,periapical lesion,alveolar bone resorption”,最终纳入63篇文献进行归纳总结。结果与结论:①抗核因子κB受体活化因子配体通过靶向抑制破骨细胞形成和牙槽骨吸收来治疗口腔疾病;②局部和全身抗核因子κB受体活化因子配体治疗可以抑制牙周炎、种植体周围炎、根尖周病变的进展,且其在预防正畸后复发、增强正畸支抗和种植体骨结合方面也发挥重要作用;③核因子κB受体活化因子配体通过靶向促进破骨细胞分化来治疗口腔疾病;④核因子κB受体活化因子配体治疗可以加速正畸牙移动、缩短治疗周期、减少正畸并发症的发生;⑤虽然抗核因子κB受体活化因子配体治疗存在局限性,但是可以通过合理应用如应用前排除危险因素,应用期间定期口腔维护、避免创伤性牙槽手术等措施来规避。

人骨关节炎软骨细胞上调成骨细胞中骨保护素的作用途径

背景:研究发现上调的刺猬蛋白信号将导致骨关节炎标志物蛇毒蛋白Runx2、聚蛋白多糖酶5、COL10A1以及基质金属蛋白酶13的升高,而抑制刺猬蛋白能减轻骨关节炎的严重程度。猜测骨关节炎软骨细胞能够通过印度刺猬蛋白(Indian hedgehog protein,IHH)信号通路影响成骨细胞来影响骨的形成。目的:探索人骨关节炎软骨细胞对软骨下成骨细胞的影响。方法:收集骨关节炎患者的胫骨平台标本,使用酶解法提取软骨细胞,利用酶预消化+骨块法提取成骨细胞。采用甲苯胺蓝染色和免疫荧光鉴定软骨细胞;采用碱性磷酸酶染色和免疫荧光鉴定成骨细胞,在海藻酸钠珠中以维持软骨细胞表型,将其与成骨细胞共培养,共培养系统中分别加入IHH信号通路的抑制剂(Cyclopamine,10 nmol/L)和激活剂(Purmorphamine,10 nmol/L),48 h后收集各组成骨细胞,利用qRT-PCR检测成骨细胞中Gli1、骨保护素、Runx2、甲状旁腺激素相关肽、碱性磷酸酶、核因子κB受体活化因子配体(receptor activator of NF-kBligand,RANKL)以及骨钙素等基因mRNA的表达;利用Westernblot检测各处理组的成骨细胞中GLi1、骨保护素、RANKL蛋白的表达。结果与结论:①与骨关节炎软骨细胞共同培养时,成骨细胞中GLi1、骨保护素、RUNX2的mRNA表达水平明显增加,而甲状旁腺激素相关肽的mRNA表达水平相对降低(P<0.05);加入IHH抑制剂(Cyclopamine)后,IHH信号通路目的基因Gli1在mRNA和蛋白水平上表达均明显降低(P<0.05),加入IHH信号通路激活剂(Purmorphamine)后,Gli1在mRNA及蛋白水平上表达均明显升高(P<0.05);骨保护素在实验中表现出与Gli1相同的变化趋势。②成骨细胞骨保护素/RANKL比值测定结果显示,与骨保护素的趋势相同。③结果表明,人骨关节炎软骨细胞能够促进成骨细胞中Gli1、骨保护素、Runx2等蛋白的表达;其中骨保护素的上调与IHH信号通路相关;骨关节炎软骨细胞能够通过IHH信号通路上调成骨细胞骨保护素的表达进而上调骨保护素/RANKL的比值,这将有助于软骨下骨中的骨形成。

黄芪补肾活血汤对芳香化酶抑制剂诱导骨质疏松模型小鼠破骨细胞活性的影响

背景:芳香化酶抑制剂尽管显著提高了激素受体阳性乳腺癌患者的临床获益,但其相关的不良事件——骨质疏松严重影响了患者的生活质量,黄芪补肾活血汤能有效预防芳香化酶抑制剂所致骨质疏松的发生,但是其作用机制尚不清楚。目的:探究黄芪补肾活血汤对芳香化酶抑制剂所致骨质疏松模型小鼠破骨细胞活性的影响及机制。方法:选取60只8周龄C57BL/6J雌性小鼠随机分为假手术组、模型组、黄芪补肾活血汤高、中、低剂量组、阳性对照组各10只,除假手术组外,其余组小鼠均切除双侧卵巢联合皮下注射来曲唑构建绝经后芳香化酶抑制剂所致骨质疏松模型,黄芪补肾活血汤高、中、低剂量组分别给予19.24,9.62,4.81 g/(kg·d)黄芪补肾活血汤进行灌胃(1次/d),阳性对照组给予阿仑膦酸钠5 mg/kg灌胃(1次/周)。给药3个月后,Micro-CT检测胫骨骨密度和骨微结构,对股骨进行苏木精-伊红染色、抗酒石酸酸性磷酸酶染色及免疫组化检测核因子κB受体活化因子配体、骨保护素蛋白表达,ELISA检测血清中Ⅰ型胶原交联羧基端肽、抗酒石酸酸性磷酸酶5b水平。结果与结论:①与假手术组相比,模型组小鼠骨密度显著下降、骨小梁形态疏松断裂、血清中Ⅰ型胶原交联羧基端肽、抗酒石酸酸性磷酸酶5b水平显著上升,表明芳香化酶抑制剂所致骨质疏松模型构建成功;②与模型组相比,黄芪补肾活血汤高、中、低剂量组和阳性对照组小鼠骨密度、骨微结构显著改善,骨小梁形态增粗致密,血清中Ⅰ型胶原交联羧基端肽、抗酒石酸酸性磷酸酶5b水平显著下降,破骨细胞数量减少,核因子κB受体活化因子配体蛋白表达下降,骨保护素蛋白表达升高。结果表明,黄芪补肾活血汤可能调控核因子κB受体活化因子配体/核因子κB受体活化因子/骨保护素信号通路抑制破骨细胞活性,改善骨小梁形态和骨微结构,提高骨密度,进而预防芳香化酶抑制剂所致骨质疏松模型的发生发展。

骨保护素/核因子кB受体活化因子配体、小泛素样修饰蛋白特异性蛋白酶1、脂蛋白相关磷脂酶A2与阻塞性睡眠呼吸暂停低通气综合征病情程度的关系及其预测心血管事件价值分析

目的分析骨保护素(OPG)/核因子кB受体活化因子配体(RANKL)、小泛素样修饰蛋白特异性蛋白酶1(SENP-1)、脂蛋白相关磷脂酶A2(Lp-PLA2)与阻塞性睡眠呼吸暂停低通气综合征(OSAHS)病情程度的关系及各指标预测心血管事件(CVE)价值。方法纳入2021年9月至2022年9月于河北省胸科医院接受治疗的120例OSAHS患者,根据病情分为轻度组、中度组、重度组,比较三组患者的基线资料,采用多元logistics回归分析OPG/RANKL、SENP-1、Lp-PLA2与阻塞性OSAHS病情程度的关系。随访1年,记录120例患者随访期间心血管事件(CVE)的发生情况,将发生CVE的患者纳入CVE组,将未发生CVE的患者纳入非CVE组,比较两组入院时OPG/RANKL、SENP-1、Lp-PLA2水平;采用受试者操作特性(ROC)曲线评估OPG/RANKL、SENP-1、Lp-PLA2预测OSAHS患者CVE发生风险的价值。结果重度组体重指数、颈围、腰围、臀围、RANK、SENP-1、Lp-PLA2高于中度组、轻度组,中度组高于轻度组(P<0.05),OPG、OPG/RANK低于中度组、轻度组,中度组低于轻度组(P<0.05)。多元logistics回归分析结果显示,颈围、颈围、腰围、RANKL、SENP-1、Lp-PLA2高水平是OSAHS患者病情加重的危险因素(OR>1,P<0.05)。OPG、OPG/RANKL高水平是OSAHS患者病情加重的保护因素(OR<1,P<0.05)。CVE组OPG、OPG/RANKL低于非CVE组,RANKL、SENP-1、Lp-PLA2高于非CVE组(P<0.05)。ROC曲线分析结果显示,OPG/RANKL、SENP-1、Lp-PLA2单一及联合检测预测OSAHS患者发生CVE的曲线下面积均>0.70,具有较好预测效能,且联合检测预测效能更高。结论OPG/RANKL、SENP-1、Lp-PLA2水平与OSAHS患者病情严重程度密切相关,并可有效预测OSAHS患者发生CVE的风险。

白藜芦醇激活细胞外信号调节激酶5信号蛋白促进小鼠MC3T3-E1细胞增殖

背景:细胞外信号调节激酶5信号蛋白对生物体的存活不可或缺,白藜芦醇能通过多种途径促进成骨细胞增殖,但其是否能通过细胞外信号调节激酶5信号蛋白调控成骨细胞功能还需进一步验证。目的:探究细胞外信号调节激酶5对MC3T3-E1细胞增殖以及相关分泌蛋白的调控作用,进一步验证白藜芦醇通过激活细胞外信号调节激酶5完成上述过程。方法:小鼠MC3T3-E1前成骨细胞分别用完全培养基、XMD8-92(细胞外信号调节激酶5抑制剂)、表皮生长因子(细胞外信号调节激酶5激活剂)和白藜芦醇单独干预及XMD8-92+表皮生长因子、白藜芦醇+XMD8-92干预后,通过Western blot检测各组细胞内细胞外信号调节激酶5、磷酸化细胞外信号调节激酶5蛋白,增殖相关蛋白Cyclin D1、CDK4、PCNA,以及成骨细胞分泌蛋白骨保护素、核因子κB受体活化因子配体的表达情况,使用细胞免疫荧光染色检测各组细胞外信号调节激酶5、骨保护素和核因子κB受体活化因子配体荧光强度,使用EdU染色检测各组细胞增殖情况。白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间由细胞形态学观察和CCK-8实验确定。结果与结论:①细胞外信号调节激酶5信号蛋白的激活能有效促进MC3T3-E1细胞增殖、上调骨保护素/核因子κB受体活化因子配体比值;②白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间为5μmol/L,24 h;③白藜芦醇可以激活细胞外信号调节激酶5信号蛋白,进而促进成骨细胞增殖,并上调骨保护素/核因子κB受体活化因子配体比值;④研究结果表明,白藜芦醇可以通过激活细胞外信号调节激酶5信号蛋白促进MC3T3-E1细胞增殖,并通过激活细胞外信号调节激酶5信号蛋白上调骨保护素/核因子κB受体活化因子配体比值。

局部注射不同剂量抗RANKL抗体对大鼠牙槽骨骨质疏松的影响

目的:探究局部注射抗核因子κB受体活化因子配体(receptor activator of nuclear factor kappa-B ligand,RANKL)抗体对大鼠牙槽骨骨质疏松的影响及不同剂量间的比较研究。方法:采用SD大鼠30只(对照组9只,实验组21只),通过皮下注射地塞米松建立大鼠牙槽骨骨质疏松模型,建模5周后进行模型验证,模型验证成功后,将实验组随机分为3组;实验组Ⅱ、Ⅲ于1、3、7 d在大鼠右上第一磨牙腭侧黏骨膜下注射兔抗大鼠RANKL抗体0.1μg/位点和1μg/位点,实验组Ⅰ注射等体积生理盐水。1周后处死取材,采用微计算机断层扫描技术(micro computed tomography,Micro-CT)测量右上第一磨牙区牙槽骨骨密度(bone mineral density,BMD)、骨体积分数(bone volume fraction,BV/TV);牙槽骨组织病理学染色观察组织形态学改变及破骨细胞数目变化;酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测牙龈中RANKL/骨保护素(osteoprotegerin,OPG)比值变化。结果:成功建立大鼠牙槽骨骨质疏松模型。Micro-CT和组织病理学结果显示,注射RANKL抗体后,牙槽骨BMD、BV/TV增加,破骨细胞数目减少,成骨细胞活跃,骨髓腔减小,但高、低剂量抗体相比变化不显著;ELISA结果显示,注射高剂量抗体后牙龈中RANKL/OPG比值显著降低,注射低剂量抗体后RANKL/OPG比值虽降低,但变化不显著。结论:局部注射抗RANKL抗体可以改善大鼠牙槽骨骨质疏松,低剂量即可达到改善效果。

基于OPG/RANK/RANKL信号通路研究补肾强督方对大鼠去卵巢骨质疏松的作用机制

目的:观察补肾强督方干预去卵巢所致骨质疏松(OP)大鼠对OPG/RANK/RANKL信号通路的影响,以探索补肾强督方防治绝经后骨质疏松(PMOP)的机制。方法:选取40只SPF级雌性SD大鼠适应性喂养1周后称重并按照体重大小从低到高依次编号,然后利用随机数字表法分为假手术组(6只)和手术组(34只),手术组中有4只因为手术原因死亡,剩余30只,手术组切除双侧卵巢构建OP大鼠模型后分为模型对照组、阿仑膦酸钠组、补肾强督方高剂量组、补肾强督方中剂量组、补肾强督方低剂量组各6只。假手术组仅切除等重量卵巢旁脂肪组织。药物干预10周后取大鼠左右股骨及腹主动脉采血制备血清,采用酶联免疫吸附法(ELISA)检测血清中核因子κB受体活化因子(RANK)、核因子κB受体活化因子配体(RANKL)、骨保护素(OPG)、肿瘤坏死因子受体相关因子6(TRAF6)、骨钙素(OCN)水平情况,分别采用蛋白质免疫印迹法(Western blot)和实时荧光定量聚合酶链式反应法(RT-PCR)检测骨组织OPG、RANK、RANKL蛋白及mRNA的表达,并进行统计学处理。结果:补肾强督方可以促进血清中OPG含量以及股骨中OPG蛋白及mRNA的表达,抑制血清中RANK、RANKL、TRAF6、OCN含量和股骨中RANK、RANKL蛋白及mRNA的表达。结论:补肾强督方可能通过调控OPG/RANKL/RANK信号通路以及下游TRAF6因子的表达抑制破骨细胞增殖、刺激成骨细胞的表达,动态调节骨代谢的平衡。