骨保护素与冠状动脉钙化的相关性研究

目的:探讨血清骨保护素(OPG)水平与冠状动脉钙化(Coronary artery calcification,CAC)的相关性。方法:入选50例患者,所有入选患者均冠状动脉16层螺旋CT扫描,根据结果,CAC的患者30例作为实验组,冠脉无钙化者20例作为对照组,在入院24小时内记录各组患者的一般资料(包括性别、年龄、血脂、血糖、血压等),ELISA法检测各组患者血清OPG水平,计算CAC的积分,分析OPG与CAC的相关性。结果:(1)与对照组相比,CAC患者血清OPG均高于对照组,差异有统计学意义(P<0.01);(2)OPG水平与CAC的程度相关,且随着钙化积分的升高而升高,差异有统计学意义(P<0.01);(3)OPG与FPG及LDL水平正相关(P<0.01或P<0.05)。结论:血清OPG浓度与CAC程度密切相关,随着CAC的积分升高而升高,可能参与了CAC的发展,OPG水平与FPG、LDL水平正相关,提示其参与CAC的发展可能与血糖、血脂代谢有关,共同参与了CAC的发展。     

TNFRSF11B gene polymorphisms 1181G > C and 245T > G as well as haplotype CT influence bone mineral density in postmenopausal women

Objectives

Osteoprotegerin (OPG) inhibits osteoclast function by acting as a decoy receptor for receptor activator of nuclear factor-κB ligand (RANKL), thus being an important candidate gene for osteoporosis. Three recent genome-wide association studies also identified the TNFRSF11B gene, coding for OPG, as playing a key role in bone mineral density (BMD) regulation. As variations in the TNFRSF11B gene could alter the susceptibility to osteoporosis, the aim of study was to investigate association of two TNFRSF11B gene polymorphisms with BMD and serum OPG concentration in postmenopausal women.

Study design

478 postmenopausal women were genotyped for the presence of TNFRSF11B gene polymorphisms 245T > G (rs3134069) and 1181G > C (rs2073618). BMDs and serum OPG concentrations were measured.

Results

Two common haplotypes GT and CT occurred in 41.2% and 52.4% of subjects. In osteoporotic postmenopausal women, lumbar spine BMD was associated with polymorphisms 245T > G and 1181G > C, as well as with CT haplotype (p values 0.013, 0.006 and 0.006, respectively). Additionally, femoral neck BMD showed the association with 245T > G (p = 0.047). No other statistically significant associations with BMD were found for the studied SNPs and haplotypes. No association with serum OPG concentration was shown in any of the studied groups.

Conclusions

Our results suggest that, in postmenopausal osteoporosis, polymorphisms 245T > G and 1181G > C, as well as haplotype CT in TNFRSF11B gene influence BMD.     

Effects of coumestrol on expression of bone markers during primary osteoblastic cells differentiation

目的 观察植物雌激素香豆雌酚对成骨细胞增殖分化的作用并探讨其作用机制.方法 从小鼠颅盖骨获得成骨细胞并用0,10-9～10-5 M香豆雌酚孵育48 h,以17β雌二醇为阳性对照,用酶消化法测定碱性磷酸酶及Ⅰ型胶原含量,放免法测定骨钙素含量,RT-PCR法测定OPG及RANKL mRNA表达情况,Western Blot测定OPG蛋白含量.结果 干预48 h,不同浓度香豆雌酚呈剂量依赖性增加碱性磷酸酶及Ⅰ型胶原含量,10-6 M时达到最大效应(P<0.05),但10-5 M效应有所降低,香豆雌酚轻度增加成骨细胞骨钙素含量,各组间无统计学差异.香豆雌酚呈剂量依赖性增加OPG基因及蛋白的表达(P<0.05),轻度降低RANKL基因的表达.结论 香豆雌酚能增加成骨细胞增殖及分化,可能其部分通过OPG/RANKL发挥作用.     

The effect of risedronate treatment on serum cytokines in postmenopausal osteoporosis: a 6-month randomized and controlled study

There is much evidence suggesting that the decline in ovarian function after menopause is associated with spontaneous increases in proinflammatory cytokines. Treatment with risedronate is accompanied by significant changes in bone turnover and bone mineral density. The objective of this study was to determine the effects of risedronate treatment on the level of serum cytokines including receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin among postmenopausal women with osteoporosis. The study group consisted of 61 postmenopausal women with osteoporosis. Patients were randomly divided in two groups: In group 1 (n = 41) postmenopausal women received oral risedronate (35 mg/week), calcium (1,000 mg/day), and vitamin D (400 IU/day) for 12 months. In group 2 (control group; n = 20) patients received only oral calcium (1,000 mg/day) and vitamin D (400 IU/day). Bone mineral density (BMD) of lumbar spine (L1–L4) and proximal femur were determined using dual X-ray absorptiometry at baseline and after one year. Venous blood samples were obtained for determination of serum cytokines including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), RANKL, osteoprotegerin, and markers of bone formation and resorption. Levels of serum cytokines were measured before therapy and after three and 6 months. Markers of bone metabolism were studied before therapy and after 6 months. In group 1 (risedronate plus calcium/vitamin D-treated patients), serum levels of RANKL and IL-1β significantly decreased and the level of osteoprotegerin significantly increased after three and 6 months, but no significant difference was found in TNF-α level. In group 2, however, the level of serum cytokines did not change after three and 6 months. In cases of bone turnover, both markers of bone resorption and formation significantly decreased after 6 months in group 1. In conclusion risedronate could improve osteoporosis by increasing osteoprotegerin and reducing RANKL and IL-1β.     

Effect of zoledronic acid on markers of bone turnover and mineral density in osteoporotic patients with beta-thalassaemia

Osteoporosis has emerged as an important cause of morbidity in patients with thalassemia major. Studies regarding the efficacy of bisphosphonates in thalassemia-induced osteoporosis have yielded conflicting results. We performed this prospective study to evaluate the efficacy of zoledronic acid in osteoporotic patients with thalassemia major. Patients, 29, were given 1 mg zoledronic acid intravenously every 3 months for a total of four doses. Twenty age- and sex-matched healthy blood donors served as controls. Before each infusion and 3 months after the last infusion, we determined serum levels of osteoprotegerin (OPG), N-terminal cross-linking telopeptide of type I collagen (NTX), osteocalcin (OC) and insulin-like growth factor 1 (IGF-1). Bone mineral density (BMD) of the lumbar spine was measured at baseline and after the treatment’s completion. At baseline, OPG did not differ significantly between patients and controls (p=0.2), NTX were higher in patients although not significantly (p=0.139), whereas, OC levels were significantly higher and IGF-1 levels significantly lower in patients than in controls (p<0.001 and p<0.006, respectively). Zoledronic acid administration resulted in a significant decrease in NTX, OC and IGF-1 (p<0.05, p<0.001 and p<0.05, respectively) and in a significant increase in OPG and BMD (p<0.05 for both comparisons). The change in NTX, osteocalcin and IGF-1 became significant as early as 3 months after the first administration of zoledronic acid, while the change in OPG reached significance only after three infusions. Our study supports the effectiveness of bisphosphonates in the treatment of thalassemia-induced osteoporosis.     

IL-17 induces myocardial fibrosis and enhances RANKL/OPG and MMP/TIMP signaling in isoproterenol-induced heart failure

Objective

This study was designed to investigate whether IL-17 can regulate the expression of the MMP/TIMP system, the OPG/RANK/RANKL system, or type-I and type-III collagen fibers in a rat model of isoproterenol-induced heart failure (HF). We also investigated the effect of IL-17 on myocardial fibrosis in this model.

Methods

HF was induced in Wistar–Kyoto rats by hypodermic injection of isoproterenol (ISO) twice every 24 h. After 2 months, the surviving rats were divided into three groups: monoclonal Anti-IL-17 Ab (100 μg/day), IgG (100 μg/day) or PBS were injected five times every 48 h (i.p.). One day after the last injection, all of the rats were sacrificed. H&E and Masson staining were used to evaluate myocardial fibrosis, and immunohistochemistry was used to measure the levels of MMP-1, TIMP-1, TIMP-4, OPG, RANKL, type-I and type-III collagen fibers. We also treated adult rat cardiac fibroblasts with IL-17 (10 ng/ml), IL-17 (10 ng/ml) + OPG (10 ng/ml), IL-17 (10 ng/ml) + anti-RANKL Ab (100 ng/ml), or PBS for 24 h, realtime RT-PCR was used to measure the expressions of MMP-1.

Results

The expressions of MMP-1, RANKL, and type-I and -III collagen fibers decreased, and the expressions of TIMP-1, TIMP-4, and OPG increased in the Anti-IL-17 group compared to controls. H&E and Masson staining revealed that blockade of IL-17 can improve myocardial fibrosis in HF. IL-17 increased the expression of MMP-1 in cardiac fibroblasts, and OPG and anti-IL-17 Ab could inhibit this function partly. Thus, IL-17 was dependent on the RANKL/OPG system to induce MMP-1 partly.

Conclusion

Our study demonstrates the contribution of IL-17 to myocardial fibrosis in isoproterenol-induced HF. IL-17 can regulate the RANKL/OPG and MMP/TIMP systems in this model. The RANKL/OPG system is one of intermediaries between IL-17 and MMP-1 in cardiac fibroblasts. As a harmful cytokine, anti-IL-17 treatment is a potential therapeutic strategy in HF.     

唑来膦酸对兔成骨细胞分化及骨保护素分泌的影响

目的 探讨不同浓度的唑来膦酸对成骨细胞分化的影响及其对骨保护素(osteoprotegerin,OPG)分泌的促进作用,以期为唑来膦酸的进一步局部应用提供理论依据.方法 利用组织块培养法进行成骨细胞原代及传代培养.原代培养3d后对细胞爬片行碱性磷酸酶(alkaline phosphatase,ALP)染色,原代培养18 d后将细胞爬片用钙结节染色并鉴定;将唑来膦酸加入含传代第3代成骨细胞悬液的培养液中使其终浓度为0(对照组)、10-5、10-6、10-7、10-8及10-9 mol/L(5个实验组),收集细胞上清液进行ALP检测,用放射免疫测定法测定骨钙素的含量;培养3 d后收集细胞,结合蛋白质印迹法测定细胞分泌的OPG.结果 成骨细胞ALP染色阳性,钙结节茜素红染色呈橘红色钙化结节,唑来膦酸浓度为10-6、10-7、10-8及10-9 mol/L的实验组可促进骨钙素含量增加[分别为(2.80±0.51)、(3.20±0.33)、(4.70±0.35)、(3.40±0.36)μg/L]、ALP活性增强[分别为(5.91±0.35)、(7.62±0.33)、(10.00±0.38)、(8.91±0.29)U/L]、OPG表达升高[分别为(526 955.7±64 068.9)、(661 447.9±75 223.4)、(753 083.3±70 300.0)、(655 184.1±63 401.1)],与对照组[骨钙素含量为(2.40±0.35)μg/L、ALP活性为(4.28±0.36)U/L、OPG表达为(64 713.7±28 715.6)]相比差异均有统计学意义(P＜0.01),并在10-8mol/L组达峰值.结论 唑来膦酸可以提高成骨细胞活性,促进OPG分泌.     

Relationship of Calcification of Atherosclerotic Plaque and Arterial Stiffness to Bone Mineral Density and Osteoprotegerin in Postmenopausal Women Referred for Osteoporosis Screening

Arterial calcification leading to increased arterial stiffness, a powerful risk factor for cardiovascular disease, may underlie the association of osteoporosis with cardiovascular disease in postmenopausal women. Osteoprotegerin (OPG), an indirect inhibitor of osteoclastogenesis, may be involved in arterial calcification. We examined relationships between calcification of subclinical atherosclerotic plaque and arterial stiffness with bone mineral density (BMD) and OPG in a group of 54 postmenopausal women referred for routine osteoporosis screening by dual-energy X-ray absorptiometric scanning of the lumbar spine and hip. Presence of calcified and noncalcified plaque in carotid and femoral arteries was examined using ultrasonography. Pulse wave velocity (PWV), a measure of arterial stiffness, was determined by sequential tonometry over the carotid and femoral region. Fifty-nine percent of osteoporotic women had calcified (echogenic) plaque at one or more sites compared with 42% and 20% for women with osteopenia and normal BMD, respectively (P = 0.04). There was a significant negative correlation between PWV and hip BMD (r = -0.35, P = 0.01), which remained significant when age, mean arterial pressure, and serum lipids were taken into account (P = 0.05). No significant relationships were observed between serum concentrations of OPG and lumbar spine or total hip BMD or with the number of arterial sites with calcified or noncalcified plaque. However, there was a strong correlation between OPG and PWV (r = 0.44, P = 0.001), which remained significant when adjusted for age (P = 0.01). These findings suggest that decreased BMD is associated with arterial calcification and stiffening and raise the possibility that OPG is a marker of arterial stiffening, independent of any association with BMD.     

Bisphosphonate Treatment Increases the Size of the Mandibular Condyle and Normalizes Growth of the Mandibular Ramus in Osteoprotegerin-Deficient Mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. OPG-deficient (OPG–/–) mice develop severe osteoporosis caused by significant enhancement of bone resorption by osteoclasts. We investigated the effect of administering bisphosphonate on mandibular growth and development in OPG–/– mice. Eight-week-old male OPG–/– mice and wild-type (WT) mice were administered bisphosphonate (1.25 mg/kg body weight) intraperitoneally once every 3 days for 30 days. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG–/– mice with bisphosphonate than in those without bisphosphonate. The volume of the whole condyle and the mandibular length in OPG–/– mice without bisphosphonate were significantly smaller than in WT mice without bisphosphonate. Bisphosphonate treatment of the OPG–/– mice resulted in an increase in the volume of the mandibular condyle and mandibular ramus length. In fact, the mandibular ramus length in OPG–/– mice with bisphosphonate was similar to the length in WT mice without bisphosphonate. Histologically, the surface irregularity of the mandibular condyle that was observed in the OPG–/– mice without bisphosphonate tended to be less marked in the OPG–/– mice with bisphosphonate, and the proportion of the area of the cartilage layer relative to the whole condyle was significantly larger in OPG–/– mice with bisphosphonate than in those without bisphosphonate. In conclusion, bisphosphonate treatment results in an increase in mandibular condylar dimensions and normalization of mandibular ramus growth.     

破骨细胞骨吸收的分子机制

破骨细胞性骨吸收是在骨的微环境内进行的复杂分子生物学反应过程,涉及到众多蛋白质和调控因子的参与。有关破骨细胞的活化,骨基质的吸收,骨吸收的调控等方面,现有的数据还不够充分。本文综述了金属基质蛋白酶(M atrix m eta lloprote inases,MM P s)在破骨细胞移行和骨基质吸收方面的重要作用,以及破骨细胞分化因子(R eceptor activator of NF-κB-ligand,RANKL)和护骨素(O steoprotegerin,OPG)在骨吸收调控网络中的地位。