慢性阻塞性肺疾病并骨质疏松患者血清MMP-9、TNF-α与骨代谢异常的相关性研究
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摘要
背景及目的：
慢性阻塞性肺疾病(COPD)是一种常见具有气流受限特征的慢性呼吸系统疾病,致残率、病死率高,且有越来越高的趋势,到2020年,COPD可能成为第三大致死性疾病。骨质疏松症以骨量减少、骨结构退化和骨折风险增加为特征,是COPD重要的系统性合并症之一,其在COPD的发病率明显高于正常人群。在严重骨质疏松病人,肺活量随胸椎压缩性骨折和脊柱后凸增多而逐步下降。以往的研究显示：肺功能下降与骨密度下降在某种程度上存在一定的相关性。但COPD继发骨质疏松的潜在机制尚有待进一步阐明。
COPD以肺外周气腔异常持久扩大,伴肺泡壁结构破坏为特点。蛋白酶-抗蛋白酶系统失衡学说占重要地位,近来研究显示,基质金属蛋白酶(MMPs)及其抑制剂(TIMPs)的平衡在COPD和哮喘患者的肺泡壁细胞外基质的降解和重塑中占重要地位。MMPs是一组由钙离子激活的含锌离子内肽酶,广泛存在于各种结缔组织中,是降解细胞外基质和基底膜过程中必不可少的酶。在COPD发展的不同时期,MMP-9表达水平不同。TIMPs是MMPs的内源性天然抑制因子,MMP-9/TIMP-1的比例,目前被认为是反映气道组织破坏与修复动态平衡的标志。
另一方面,MMPs又是反映骨吸收和骨重建的关键酶。不仅破骨细胞及其前体细胞,单核细胞等也可产生分泌MMP-9,MMP-9促进骨吸收。MMP-9在破骨细胞迁移和骨吸收过程中起重要作用。MMP-9在破骨细胞中特异性表达,可促进骨质疏松形成,可能在破骨性骨吸收中发挥重要作用。TIMP-1可阻止骨吸收。
TNF-α是已经明确的重要的COPD系统性炎症因子,COPD系统性合并症的发生和发展均与其有关。TNF-α亦被证实是骨代谢和重塑的重要调节因子,可能通过协同作用的方式刺激破骨细胞的分化。TNF-α和IL-1β、IL-6等多个血清炎症性细胞因子与高分辨CT下定量评估的肺气肿严重程度之间存在明确的相关性。同时,TNF-α和IL-1β,IL-6等细胞因子已经被证实是骨代谢和重塑的重要调节因子,可以通过协同作用的方式刺激破骨细胞的分化。这些炎症因子一方面对间质细胞作用调节骨诱裂的发生,另一方面还可以直接作用于破骨细胞及其前体细胞,最终结果可以导致破骨细胞活性增强,引起和加速骨质破坏和骨量丢失。除此之外,这些炎症因子还可以与骨质疏松相关蛋白OPG/RANK/RANKL系统发生相互作用,共同调节骨代谢。
骨质疏松相关蛋白OPG/RANK/RANKL系统是骨代谢的重要调节因子,在绝经后骨质疏松的发生发展过程中发挥重要作用。骨质疏松相关蛋白OPG/RANK/RANKL系统是骨质疏松和骨代谢研究领域的重大突破。RANKL和OPG分别是破骨细胞分化的促进因子和抑制因子,二者在体内保持一定的比率,如果二者比率失去平衡,就可引起骨代谢紊乱,产生骨量减少／骨质疏松症。然而MMP-9/TIMP-1、TNF-α、OPG/RANK/RANKL系统在COPD继发骨质疏松发病过程中相互关系及作用,目前尚不明确。
骨质疏松症的发生是破骨细胞和成骨细胞的平衡失调引起。骨转换加速即骨吸收超过骨形成,就导致负钙平衡及脱钙。骨吸收和骨形成的速率可以通过测定血清或尿液中细胞代谢的特有产物而确定。骨代谢标志物分为骨形成标志物和骨吸收标志物,随着对这些标志物认识的不断深入,骨转换标志物可以作为骨密度测定的补充手段,更多的应用于评估骨质量、预测骨折率,以及评价骨质疏松治疗的效果。生化标志物以其无创性、易重复性和及时性的优点而易于采用。
本研究旨在对COPD并骨质疏松患者血清MMP-9及其抑制剂TIMP-1、 TNF-α、OPG/RANK/RANKL系统及骨转换标志物水平定量评估,明确其与COPD患者骨代谢异常的相关性,并进一步探讨其在COPD患者肺功能受损及骨质疏松发生发展过程中的作用及相关性,为COPD并骨质疏松患者的治疗和监测提供依据。
本研究分为二部分：
第1部分：慢性阻塞性肺疾病并骨质疏松患者血清MMP-9、 TNF-α、与OPG/RANK/RANKL系统相关性研究
目的：本研究旨在对COPD并骨质疏松患者血清MMP-9及其抑制剂TIMP-1、TNF-α和OPG/RANK/RANKL系统水平定量评估,明确三者间的相关性,并进一步探讨这三者在COPD患者肺功能下降及骨质疏松发生发展过程中的作用及相关性。
方法：1.病人资料及标本收集：收集从2010年9月到2012年5月我院稳定期COPD男性患者90例,按照GOLD(the Global Initiative for Chronic Obstructive Pulmonary Disease)标准进行COPD诊断和严重度分级。为避免女性绝经后骨质疏松对结果的影响,未纳入女性患者。根据骨密度(BMD)结果,分为COPD正常骨量组、低骨量组、骨质疏松组各30人。均完成细胞因子检测。入组患者要求：年龄超过40岁；确诊COPD稳定期；既往有吸烟史或戒烟者。满足以上入选条件的患者如被证实具有以下任意一条或多条特征时则应退出本研究(排除标准)：1).有严重心脑血管疾病、肝肾功能衰竭、意识障碍；2)明确患有COPD之外的其它呼吸系统疾病,如哮喘、支气管扩张等；3)既往有骨骼系统疾病史,如骨折、骨软化症等；4)风湿性疾病史：如类风湿性关节炎等；5)内分泌代谢疾病：甲状腺功能亢进、甲状旁腺功能亢进等;6)入组3个月内有口服或吸入或静脉使用过糖皮质激素；7)入组1年内有使用影响骨骼代谢的药物,包括已经确诊骨质疏松给予治疗的患者；8)5年内有恶性肿瘤史。所有入组患者均签署知情同意书。
所有入组患者当日完成一般资料和病史采集、肺功能。患者生活质量评估采用慢性阻塞性肺疾病评估测试(COPD Assessment Test, CAT)。患者入组同日完成骨密度测定。对全部受试者正位腰椎L1-4及双侧股骨颈进行骨密度测定,采用双能X线快速全身骨密度测定仪(DXA),BMD测量结果包括绝对值(g/cm2)和T值(表示与参考均值的标准差)两种形式。按照WHO(the World Health Organization)的骨质疏松诊断标准,根据T值做出如下诊断：腰椎L1-4及股骨颈各受检部位与同性别、同种族健康成人的骨峰值相比,①骨量正常：不足1个标准差属正常,即T值≥-1.0；②骨量减少：任一受检部位-2.5 2.细胞因子检测：所有入组90例患者在次日清晨空腹抽取肘静脉血10ml,室温下1000转离心5分钟,留取上清液,-80℃冻存待测。MMP-9,TIMP-1,TNF-α1和OPG/RANK/RANKL的检测采用ELISA法。OPG/RANK/RANKL系统水平检测亦采用ELISA法。
3.统计方法：采用SPSS13.0(SPSS Inc, Chicago,USA)统计软件进行数据分析。计量资料数据以均数±标准差表示。正态检验分析采用Shapiro-Wilk检验,正态分布的计量资料组间比较采用单因素方差分析(one-way ANOVA analyses of variance with α post-hoc Tukey's test),参数之间的相关性分析采用Pearson相关分析。分别以腰椎骨密度、股骨颈骨密度为因变量,以血清MMP-9/TIMP-1、TNF-α、OPG/RANK/RANKL水平等为自变量,采用多元逐步线性回归分析。
p<0.05表明差异有统计学意义。
结果：COPD稳定期并骨质疏松组与正常骨量组、低骨量组相比较,BMI、 CAT评分存在显著性差异(均P<0.01)；而年龄、吸烟指数、FEV1/FVC、FEV1%Pre未见明确统计学差异(P>0.05)。低骨量组及骨质疏松组的腰椎骨密度均较正常骨量组下降,差异具有统计学意义(P<0.05,P<0.01)。低骨量组及骨质疏松组的股骨颈密度较正常骨量组明显下降,有显著性差异(均P<0.01)。
腰椎骨密度、股骨颈骨密度与BMI显著正相关(分别为r=0.278,P<0.01; r=0.273,P<0.01);腰椎骨密度、股骨颈骨密度与CAT评分显著负相关(分别为r=-0.331,P<0.01; r=-0.486,P<0.01)。
血清MMP-9、MMP-9/TIMP-1比值、TNF-a在COPD正常骨量组、低骨量组、骨质疏松组依次升高,骨质疏松组升高最显著(P<0.05或P<0.01)；血清TIMP-1水平在COPD正常骨量组、低骨量组、骨质疏松组依次升高,但差异无统计学意义(P>0.05)。血清OPG、RANKL、RANKL/OPG比值在COPD正常骨量组、低骨量组、骨质疏松组依次升高,骨质疏松组升高最显著,骨质疏松组较正常骨量组均升高(P<0.01)；但三组间RANK水平无显著性差异(P>0.05)。腰椎骨密度、股骨颈骨密度、FEV1%Pre与血清MMP-9显著负相关(分别为r=-0.432, P<0.01; r=-0.697, P<0.01和r=-0.226,P<0.05)；腰椎骨密度、股骨颈骨密度、FEV1%Pre与RANKL(分别为r=-0.434, P<0.01; r=-0.538, P<0.01和r=-0.325,P<0.01)、RANKL/OPG比率(分别为r=-0.345,P<0.01和r=-0.420,P<0.01和r=-0.459,P<0.05)呈显著负相关；而与其它细胞因子(TIMP-1、TNF-α、OPG、RANK)无显著相关性(P>0.05)。 FEV1%Pre与腰椎骨密度、股骨颈骨密度显著正相关(r=0.338,P<0.01)和(r=0.409, P<0.01); MMP-9与TNF-a呈显著正相关(r=0.370,P<0.001),与OPG呈显著正相关(r=0.306, P<0.01), MMP-9与RANKL/OPG比值显著正相关(r=0.318,P<0.01)。
结论：骨质疏松症是COPD重要的系统性合并症之一,是多种因素综合作用的结果。腰椎骨密度、股骨颈骨密度、FEV1%Pre与血清MMP-9、RANKL、 RANKL/OPG比率显著负相关；而与其它细胞因子(TIMP-1、TNF-α、OPG、RANK)无显著相关性。在男性稳定期COPD患者,升高的基质金属蛋白酶(MMP)-9可能在骨质疏松发挥重要作用,与骨密度下降可能有关。MMP-9、TNF-α、OPG/RANK/RANKL系统在肺实质损伤和骨质疏松的发生发展过程中,有可能发挥协同作用。
第2部分慢性阻塞性肺疾病患者血清MMP-9及其抑制剂-1、TNF-α与骨转换生化指标及骨密度的关系
目的：观察慢性阻塞性肺疾病(COPD)患者血清基质金属蛋白酶(MMP)-9及其抑制剂-1、TNF-α与骨转换生化指标及骨密度(BMD)的关系。
方法：收集稳定期COPD男性患者90例,采用双能X线吸光测定法(DXA)测定骨密度,根据BMD检测结果分为正常骨量组、低骨量组、骨质疏松组(各30例)。各组患者入组当日完成病史采集、肺功能检查、病情严重程度评分(CAT评分),并ELISA法检测血清MMP-9、TIMP-1、TNF-α和血清骨碱性磷酸酶(sBAP)、血清骨钙素(sOC)、血清I型胶原交联C端肽(sCTX)水平。
结果：COPD稳定期并骨质疏松组与正常骨量组、低骨量组相比较,BMI、 CAT评分存在显著性差异(均P<0.01)；而年龄、吸烟指数、FEV1/FVC、 FEV1%Pre未见明确统计学差异(P>0.05)。低骨量组及骨质疏松组的腰椎骨密度、股骨颈密度均较正常骨量组下降,差异具有统计学意义(P<0.05或P<0.01)。血清MMP-9、MMP-9/TIMP-1比值、TNF-α在COPD正常骨量组、低骨量组、骨质疏松组依次升高,骨质疏松组升高最显著(P<0.05或P<0.01)；血清TIMP-1水平在COPD正常骨量组、低骨量组、骨质疏松组依次升高,但差异无统计学意义(P>0.05)。COPD骨质疏松组、低骨量组的血清sBAP、sOC水平低于正常骨量组(P<0.01),而低骨量组与骨质疏松组间未见显著性差异(P>0.05)；COPD骨质疏松组与正常骨量组、低骨量组比较,血清sCTX水平升高,存在显著性差异(均P<0.01),而在低骨量组与正常骨量组间未见显著性差异(P>0.05)。腰椎骨密度、股骨颈骨密度、FEV1%Pre与血清MMP-9显著负相关(分别为r=-0.432, P<0.01; r=-0.697, P<0.01和r=-0.226,P<0.05)；腰椎骨密度、股骨颈骨密度、FEV1%Pre与sBAP显著正相关(r=0.418, P<0.01; r=0.702, P<0.01; r-0.295, P<0.01)、sOC显著正相关((r=0.338, P<0.01; r=0.574, P<0.01; r=0.278, P<0.01)、sCTX呈显著负相关(r=-0.418, P<0.01; r=-0.602, P<0.01; r=-0.243, P<0.05);而与TIMP-1、TNF-α无显著相关性。FEV1%Pre与血清MMP-9显著负相关(r=-0.226, P<0.05); MMP-9/TIMP-1与sBAP、sOC呈显著负相关(r=-0.525, P<0.01; r=-0.460, P<0.01),与sCTX呈显著正相关(r=0.635, P<0.01)。 TIMP-1与sCTX呈显著正相关(r=0.236,P<0.05),与sBAP、sOC无显著相关性(P>0.05)。TNF-α与sBAP呈显著负相关(r=-0.350,P<0.01),与sCTX呈显著正相关(r=0.370,P<0.01)。TNF-α与sOC无显著相关性(P>0.05)。
结论：在男性稳定期COPD患者,升高的MMP-9、TNF-α与骨转换生化指标有一定相关性,在骨质疏松可能发挥重要作用；血清MMP-9/TIMP-1α TNF-α升高可能与COPD患者骨代谢增快有关。
Background and purpose:
Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease characterized by airflow limitation, It has a high and increasing prevalence and mortality. By2020, COPD may become the third most common fatal disease in the world. Osteoporosis, a condition characterized by low bone mass and bone microarchitectural deterioration which predisposes to fragility fracture, is one of the most important systemic complications of COPD. The incidence of osteoporosis among COPD patients is significantly higher than in the general population. Clinical sequelae of serious osteoporosis, including compression fractures and kyphosis, can further reduce vital capacity, thereby further compromising respiratory status in patients with COPD. A correlation between reduced lung function and bone mineral density (BMD) is suggested by some studies, however, the mechanisms between COPD and osteoporosis are not yet fully clear.
Emphysema is a kind of chronic pulmonary disease characterized by abnormal and permanent airspace enlargement beyond the terminal bronchioles accompanied by destruction of the alveolar walls. The most widely accepted theory of the pathogenesis is a proteinase-antiproteinase imbalance. Recent studies have shown that the balance between matrix metalloproteinases (MMPs) and their inhibitors(TIMPs) plays an important role in the breakdown and remodelling of ECM in the alveolar walls in COPD and asthma patients. MMPs, a group of calcium-activated zinc ion endopeptidases widely found in a variety of connective tissues, play an essential role in the degradation of the extracellular matrix and basement membrane enzyme. They are differentially expressed during different stages of COPD development. The TIMPs are endogenous natural inhibitors of MMPs. The airway MMP-9/TIMP-1ratio is considered a biomarker of airway tissue destruction and of the dynamic equilibrium of repair.
In addition to lung disease, MMPs have been suggested to have a important role in bone remodelling and turnover. MMP-9is produced by osteoclasts and osteoclast precursor cells,the monocytes also. It is essential for recruitment of preosteoclasts into bone and their local migration into the diaphysis. In contrast, TIMP-1prevents bone resorption.
Experimental studies indicate that TNF-α is a multifaceted cytokine and a well-recognized systemic inflammatory factor related to the occurrence and development of a variety of the systemic complications of COPD. TNF-a is also an important regulator of bone metabolism and remodelling. It maybe stimulate osteoclast differentiation in a synergistic manner. TNF-α,IL-1β,\IL-6are all well-established inflammatory cytokines involved in the systemic inflammation of COPD. There is an association between distinct serum inflammatory mediators and quantitative CT measurements of emphysema. Simultaneously, IL-1β, IL-6and TNF-a have been shown to be important modulators of bone metabolism and remodeling by stimulating osteoclast differentiation in a synergistic fashion. These cytokines may not only regulate osteoclastogenesis via their effects on stromal cells, but also act directly on osteoclasts and their precursors. In addition, they also interact with the osteoporosis-related OPG/RANK/RANKL protein system.
The OPG/RANK/RANKL system is an important regulator of bone metabolism, and participates in the development of post-menopausal osteoporosis. This was a major breakthrough in the field of osteoporosis and bone metabolism and opened a new perspective on bone metabolic balance and osteoclast biology. RANKL and OPG are the promoting and inhibitory factors of osteoclast differentiation, respectively. They maintain a certain ratio within the body. Imbalances in this ratio can disrupt bone metabolism, leading to reduced bone mass/osteoporosis among other bone diseases. Whether MMP-9/TIMP-1, TNF-a,the OPG/RANK/RANKL pathway is involved in the pathogenesis of osteoporosis in COPD has not been studied.
Osteoporosis is a metabolic condition characterized by decreased bone mass and strength due to increased bone turnover, which compromises bone architecture and results in increased fracture risk. Bone is a metabolically active tissue that undergoes continuous remodelling by two counter acting processes, namely bone formation and bone resorption. These processes rely on the activity of osteoclasts, osteoblasts and osteocytes. Acceleration of bone turnover, accompanied with a disruption of the coupling between these cellular activities,plays an established role in the pathogenesis of metabolic bone diseases, such as osteoporosis. In the past few decades, Many research has been working on biochemical markers which may reflect the rate of bone turnover. A large number of studies have shown that serum or urine biochemical markers are related to bone loss and fractures, which is important to identify high-risk patients. The biochemical markers of bone turnover and their applications in clinical trials can test the efficacy of drugs and how they supply the measurement of bone mass. Targeted use of biochemical markers can be further optimized to identify high-risk patients, the process of drug development and clinical monitoring of the efficacy of osteoporosis treatment. Biochemical markers are bound to be used widely because of their advantages of non-invasive, easy repeatability and timeliness.
In this study, the correlations among serum MMP-9and its cognate inhibitor TIMP-1, TNF-a, and the OPG/RANK/RANKL system,bone biochemical markers were assessed, and their associations with impairment of lung function and bone mineral density in patients with COPD was evaluated.
This study include two parts:
Part I Analysis of interrelationship of circulating matrix metalloproteinase-9, TNF-a, and OPG/RANK/RANKL system in COPD patients with osteoporosis
Objective:
To study the relationship between matrix metalloproteinase-9(MMP-9), its cognate inhibitor TIMP-1, inflammatory cytokine TNF-a, and OPG/RANK/RANKL system in COPD patients with osteoporosis.
Methods:
1. Patients and sample collection:Ninety male patients diagnosed with clinically stable COPD were enrolled from Sep2010to May2012. Female patients were excluded from the study to avoid the influence of postmenopausal osteoporosis. COPD was diagnosed according to the criteria issued by the Global Initiative for Chronic Obstructive Lung Disease (GOLD). These ninety patients were divided into three groups based on BMD:those with COPD and normal BMD (T-score>-1.0), those with COPD with osteopenia (T score-1.0to-2.4), and those with COPD and osteoporosis (T-score≤-2.5). Each group contained30patients. All patients were more than40years old and all were current or former smokers. The exclusion criteria were as follows:(1)serious cardiovascular and cerebrovasculardiseases, liver or renal function failure,consciousness disorder,(2) history of respiratory diseases other than COPD, such as asthma, bronchiectasis,(3) history of bone disease, such as fractures,osteomalacia,(4) rheumatic diseases,such as rheumatoid arthritis,(5) metabolic, or inflammatory disease,such as hyperthyrosis, hyperparathyroidism,(6) inhaled, oral, or intravenous corticosteroid treatment within the last3months,(7) medication which may influence bone metabolism within the previous one year, including those being treated for osteoporosis,(8) occurrence of malignancy within the previous five years. All subjects provided informed consent before joining the study.
On the recruiting day, all patients completed pulmonary function test and the life quality assessment using the COPD Assessment Test (CAT) and the measurement of BMD. The BMD of lumbar spine (L1-L4) and bilateral femoral neck was measured using dual X-ray absorptiometry(DXA). BMD is here expressed in g/cm2and a T-score, which was used for diagnosis according to the World Health Organization guidelines as follows:normal BMD:T score greater than-1at both sites (lumbar spine and FN); osteopenia:T score less than or equal to-1but greater than-2.5at either site; osteoporosis:T score less than or equal to-2.5at either site. Ninety patients were divided into three groups of30patients each based on BMD: those with COPD and normal BMD, those with COPD with osteopenia, and those with COPD and osteoporosis.
2. The measurement of cytokines(Enzyme-linked immunosorbent assay,ELISA):Fasting blood samples were collected by venipuncture the next morning. These were centrifuged at1000×g for5min at room temperature to produce serum samples, which were stored at-80℃until analysis.High-sensitivity MMP-9(pro-MMP-9), TIMP-1, and TNF-α levels were determined using sandwich ELISA kits. OPG/RANK/RANKL concentrations were measured using ELISA kits too.
3. Statistics:
Data are expressed as the mean±SD and analyzed using the Statistical Package for the Social Sciences, SPSS13.0(SPSS Inc, Chicago, IL, U.S.). Data from the three experimental groups were analyzed with normally distributed test(shapiro-wilk test),one-way ANOVA with a post-hoc Tukey's test, and Pearson's correlation analysis. The relationships among parameters were evaluated using multiple linear stepwise regression analysis. A p-value<0.05was considered statistically significant.
Results:
The age, BMI, smoking index, FEV1/FVC, FEV1%Pre, and CAT scores of the COPD patients were compared between all groups of patients. COPD patients with osteoporosis had significantly lower BMI and higher CAT scores (all P<0.01) than COPD patients with normal BMD and those with low BMD. There was no significant difference in age, smoking index, FEV1/FVC, or FEV1%Pre(P>0.05). As expected, based on the study design, the lumbar spine and femoral neck BMD was significantly lower in the groups of COPD with low BMD (P<0.05, P<0.01) and COPD with osteoporosis (all P<0.01) than in COPD with normal BMD.
Pearson analysis revealed positively correlations between BMI and the lumbar spine BMD (r=0.278, P<0.01) and femoral neck BMD (r=0.273, P<0.01); negative correlations between CAT scores and the lumbar spine BMD (r=-0.331, P<0.01) and femoral neck BMD (r=-0.486, P<0.01).
The serum level of MMP-9, MMP-9/TIMP-1ratio, the serum level of TNF-a increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.05or P<0.01). However, no difference in the serum level of TIMP-1was detected among the three groups(P>0.05). The serum levels of OPG, RANKL, the RANKL/OPG ratio increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.01). However, no difference existed in the level of RANK among these three groups(P>0.05).
Pearson analysis revealed negative correlations between MMP-9serum levels and the lumbar spine BMD (r=-0.432, P<0.01) and femoral neck BMD (r=-0.697, P<0.01) and FEV1%Pre (r=-0.226, P<0.05); between RANKL serum levels and the lumbar spine BMD (r=-0.434, P<0.01) and femoral neck BMD (r=-0.538, P<0.01) and FEV1%Pre (r=-0.325, P<0.01); and between the RANKL/OPG ratio and the lumbar spine BMD (r=-0.345, P<0.01) and femoral neck BMD (r=-0.420, P<0.01) and FEV1%Pre (r=-0.459, P<0.05). Other cytokines, such as TIMP-1, TNF-a, OPG, and RANK, did not correlate with the lumbar spine or femoral neck BMD or FEV1%Pre (P>0.05). FEV1%Pre was positively correlated with the lumbar spine and femoral neck BMD (r=0.338, P<0.01, r=0.409, P<0.01).MMP-9was positively correlated with TNF-a (r-0.370, P<0.001) and OPG(r=0.306, P<0.01) and RANKL/OPG ratio (r=0.318, P<0.01).
Conclusions:
Osteoporosis is one of the most important systemic complications of COPD. Negative correlations between the serum levels of MMP-9,RANKL, the RANKL/OPG ratio and the lumbar spine BMD, femoral neck BMD,FEV1%Pre. Other cytokines, such as TIMP-1, TNF-α, OPG, and RANK, did not correlate with the lumbar spine BMD or femoral neck BMD or FEV1%Pre. MMP-9, TNF-a, and the OPG/RANK/RANKL system may be closely interrelated and may play interactive roles in pathogenesis of osteoporosis in COPD.
Part Ⅱ Analysis of interrelationship of circulating matrix metalloproteinase-9, TNF-a, bone biochemical markers and bone mineral density in COPD patients
Objective:To study the relationship between matrix metalloproteinase-9(MMP-9), its cognate inhibitor TIMP-1, inflammatory cytokine TNF-a, bone biochemical markers and bone mineral density(BMD) in COPD patients.
Methods:Ninety male patients diagnosed with clinically stable COPD were enrolled, who were divided into three groups based on bone mineral density(BMD):those with COPD and normal BMD, those with COPD with low but non-osteoporotic BMD, and those with COPD and osteoporosis. Each group contained30patients.All these patients completed pulmonary function,dual x-ray absorptiometry(DXA) measurements and COPD assessment test (CAT).All these patients were measured of MMP-9, TIMP-1, TNF-a, and serum bone alkaline phosphatase(sBAP)、serum osteocalcin(sOC)、bone cross-linked C-telopeptides of type Icollagen(sCTX) by ELISA.
Results:COPD patients with osteoporosis had significantly lower BMI and higher CAT scores (all P<0.01), than COPD patients with normal or low BMD. There was no significant difference in age, smoking index, FEV1/FVC, or FEV1%Pre (P>0.05). As expected, based on the study design, the lumbar spine and femoral neck BMD was significantly lower in the groups of COPD with low BMD (P<0.05, P<0.01) and COPD with osteoporosis (all P<0.01) than in COPD with normal BMD. The serum level of MMP-9, the MMP-9/TIMP-1ratio, the serum level of TNF-a increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.05or P<0.01).However, no significant difference in the serum level of TIMP-1was detected among the three groups(P>0.05).
The serum levels of BAP、OC were all significantly lower in COPD patients with osteoporosis and those with low BMD than in those with normal BMD (all P <0.01). However, no significant difference in levels of sBAP、sOC was detected between COPD with osteoporosis and in those with low BMD (P>0.05). The level of sCTX was significantly higher in the groups of COPD with osteoporosis than those with low BMD and those with normal BMD(all P<0.01). However, no difference in the level of sCTX was detected between COPD with low BMD and those with normal BMD (P>0.05).
Pearson analysis revealed negative correlations between MMP-9serum levels and the lumbar spine BMD (r=-0.432, P<0.01) and femoral neck BMD (r=-0.697, P<0.01) and FEV1%Pre (r=-0.226, P<0.05);The BMD of lumbar spine and femoral neck、the lung function FEV1%Pre were positively correlated with sBAP level (r=0.418, P<0.01,and r=0.702, P<0.01,and r=0.295, P<0.01, respectively), sOC level (r=0.338, P<0.01,and r=0.574, P<0.01, and r=0.278, P<0.01, respectively), and negative correlated with sCTX level (r=-0.418, P<0.01,and r=-0.602, P<0.01,and r=-0.243, P<0.05, respectively). However, they had no significant correlation with TIMP-1, TNF-a. The ratio of MMP-9/TIMP-1was negative correlated with the levels of sBAP, sOC(r=-0.525, P<0.01; r=-0.460, P<0.01) and positively correlated with sCTX (r=0.635, P<0.01). The level of TIMP-1was positively correlated with sCTX (r=0.236, P<0.05), TNF-a was negative correlated with sBAP(r=-0.350, P<0.01) and positively correlated with sCTX (r=0.370, P<0.01). But no difference existed between the level of TNF-a with sOC (P>0.05).
Conclusions:These results suggest that circulating MMP-9,TNF-a and the bone biochemical markers maybe closely interrelated in bone turnover state and may play important roles in pathogenesis of osteoporosis in COPD. Bone biochemical markers suggest increased bone turnover in COPD.
慢性阻塞性肺疾病(COPD)是一种常见具有气流受限特征的慢性呼吸系统疾病,致残率、病死率高,且有越来越高的趋势,到2020年,COPD可能成为第三大致死性疾病。骨质疏松症以骨量减少、骨结构退化和骨折风险增加为特征,是COPD重要的系统性合并症之一,其在COPD的发病率明显高于正常人群。在严重骨质疏松病人,肺活量随胸椎压缩性骨折和脊柱后凸增多而逐步下降。以往的研究显示：肺功能下降与骨密度下降在某种程度上存在一定的相关性。但COPD继发骨质疏松的潜在机制尚有待进一步阐明。
COPD以肺外周气腔异常持久扩大,伴肺泡壁结构破坏为特点。蛋白酶-抗蛋白酶系统失衡学说占重要地位,近来研究显示,基质金属蛋白酶(MMPs)及其抑制剂(TIMPs)的平衡在COPD和哮喘患者的肺泡壁细胞外基质的降解和重塑中占重要地位。MMPs是一组由钙离子激活的含锌离子内肽酶,广泛存在于各种结缔组织中,是降解细胞外基质和基底膜过程中必不可少的酶。在COPD发展的不同时期,MMP-9表达水平不同。TIMPs是MMPs的内源性天然抑制因子,MMP-9/TIMP-1的比例,目前被认为是反映气道组织破坏与修复动态平衡的标志。
另一方面,MMPs又是反映骨吸收和骨重建的关键酶。不仅破骨细胞及其前体细胞,单核细胞等也可产生分泌MMP-9,MMP-9促进骨吸收。MMP-9在破骨细胞迁移和骨吸收过程中起重要作用。MMP-9在破骨细胞中特异性表达,可促进骨质疏松形成,可能在破骨性骨吸收中发挥重要作用。TIMP-1可阻止骨吸收。
TNF-α是已经明确的重要的COPD系统性炎症因子,COPD系统性合并症的发生和发展均与其有关。TNF-α亦被证实是骨代谢和重塑的重要调节因子,可能通过协同作用的方式刺激破骨细胞的分化。TNF-α和IL-1β、IL-6等多个血清炎症性细胞因子与高分辨CT下定量评估的肺气肿严重程度之间存在明确的相关性。同时,TNF-α和IL-1β,IL-6等细胞因子已经被证实是骨代谢和重塑的重要调节因子,可以通过协同作用的方式刺激破骨细胞的分化。这些炎症因子一方面对间质细胞作用调节骨诱裂的发生,另一方面还可以直接作用于破骨细胞及其前体细胞,最终结果可以导致破骨细胞活性增强,引起和加速骨质破坏和骨量丢失。除此之外,这些炎症因子还可以与骨质疏松相关蛋白OPG/RANK/RANKL系统发生相互作用,共同调节骨代谢。
骨质疏松相关蛋白OPG/RANK/RANKL系统是骨代谢的重要调节因子,在绝经后骨质疏松的发生发展过程中发挥重要作用。骨质疏松相关蛋白OPG/RANK/RANKL系统是骨质疏松和骨代谢研究领域的重大突破。RANKL和OPG分别是破骨细胞分化的促进因子和抑制因子,二者在体内保持一定的比率,如果二者比率失去平衡,就可引起骨代谢紊乱,产生骨量减少／骨质疏松症。然而MMP-9/TIMP-1、TNF-α、OPG/RANK/RANKL系统在COPD继发骨质疏松发病过程中相互关系及作用,目前尚不明确。
骨质疏松症的发生是破骨细胞和成骨细胞的平衡失调引起。骨转换加速即骨吸收超过骨形成,就导致负钙平衡及脱钙。骨吸收和骨形成的速率可以通过测定血清或尿液中细胞代谢的特有产物而确定。骨代谢标志物分为骨形成标志物和骨吸收标志物,随着对这些标志物认识的不断深入,骨转换标志物可以作为骨密度测定的补充手段,更多的应用于评估骨质量、预测骨折率,以及评价骨质疏松治疗的效果。生化标志物以其无创性、易重复性和及时性的优点而易于采用。
本研究旨在对COPD并骨质疏松患者血清MMP-9及其抑制剂TIMP-1、 TNF-α、OPG/RANK/RANKL系统及骨转换标志物水平定量评估,明确其与COPD患者骨代谢异常的相关性,并进一步探讨其在COPD患者肺功能受损及骨质疏松发生发展过程中的作用及相关性,为COPD并骨质疏松患者的治疗和监测提供依据。
本研究分为二部分：
第1部分：慢性阻塞性肺疾病并骨质疏松患者血清MMP-9、 TNF-α、与OPG/RANK/RANKL系统相关性研究
目的：本研究旨在对COPD并骨质疏松患者血清MMP-9及其抑制剂TIMP-1、TNF-α和OPG/RANK/RANKL系统水平定量评估,明确三者间的相关性,并进一步探讨这三者在COPD患者肺功能下降及骨质疏松发生发展过程中的作用及相关性。
方法：1.病人资料及标本收集：收集从2010年9月到2012年5月我院稳定期COPD男性患者90例,按照GOLD(the Global Initiative for Chronic Obstructive Pulmonary Disease)标准进行COPD诊断和严重度分级。为避免女性绝经后骨质疏松对结果的影响,未纳入女性患者。根据骨密度(BMD)结果,分为COPD正常骨量组、低骨量组、骨质疏松组各30人。均完成细胞因子检测。入组患者要求：年龄超过40岁；确诊COPD稳定期；既往有吸烟史或戒烟者。满足以上入选条件的患者如被证实具有以下任意一条或多条特征时则应退出本研究(排除标准)：1).有严重心脑血管疾病、肝肾功能衰竭、意识障碍；2)明确患有COPD之外的其它呼吸系统疾病,如哮喘、支气管扩张等；3)既往有骨骼系统疾病史,如骨折、骨软化症等；4)风湿性疾病史：如类风湿性关节炎等；5)内分泌代谢疾病：甲状腺功能亢进、甲状旁腺功能亢进等;6)入组3个月内有口服或吸入或静脉使用过糖皮质激素；7)入组1年内有使用影响骨骼代谢的药物,包括已经确诊骨质疏松给予治疗的患者；8)5年内有恶性肿瘤史。所有入组患者均签署知情同意书。
所有入组患者当日完成一般资料和病史采集、肺功能。患者生活质量评估采用慢性阻塞性肺疾病评估测试(COPD Assessment Test, CAT)。患者入组同日完成骨密度测定。对全部受试者正位腰椎L1-4及双侧股骨颈进行骨密度测定,采用双能X线快速全身骨密度测定仪(DXA),BMD测量结果包括绝对值(g/cm2)和T值(表示与参考均值的标准差)两种形式。按照WHO(the World Health Organization)的骨质疏松诊断标准,根据T值做出如下诊断：腰椎L1-4及股骨颈各受检部位与同性别、同种族健康成人的骨峰值相比,①骨量正常：不足1个标准差属正常,即T值≥-1.0；②骨量减少：任一受检部位-2.5
3.统计方法：采用SPSS13.0(SPSS Inc, Chicago,USA)统计软件进行数据分析。计量资料数据以均数±标准差表示。正态检验分析采用Shapiro-Wilk检验,正态分布的计量资料组间比较采用单因素方差分析(one-way ANOVA analyses of variance with α post-hoc Tukey's test),参数之间的相关性分析采用Pearson相关分析。分别以腰椎骨密度、股骨颈骨密度为因变量,以血清MMP-9/TIMP-1、TNF-α、OPG/RANK/RANKL水平等为自变量,采用多元逐步线性回归分析。
p<0.05表明差异有统计学意义。
结果：COPD稳定期并骨质疏松组与正常骨量组、低骨量组相比较,BMI、 CAT评分存在显著性差异(均P<0.01)；而年龄、吸烟指数、FEV1/FVC、FEV1%Pre未见明确统计学差异(P>0.05)。低骨量组及骨质疏松组的腰椎骨密度均较正常骨量组下降,差异具有统计学意义(P<0.05,P<0.01)。低骨量组及骨质疏松组的股骨颈密度较正常骨量组明显下降,有显著性差异(均P<0.01)。
腰椎骨密度、股骨颈骨密度与BMI显著正相关(分别为r=0.278,P<0.01; r=0.273,P<0.01);腰椎骨密度、股骨颈骨密度与CAT评分显著负相关(分别为r=-0.331,P<0.01; r=-0.486,P<0.01)。
血清MMP-9、MMP-9/TIMP-1比值、TNF-a在COPD正常骨量组、低骨量组、骨质疏松组依次升高,骨质疏松组升高最显著(P<0.05或P<0.01)；血清TIMP-1水平在COPD正常骨量组、低骨量组、骨质疏松组依次升高,但差异无统计学意义(P>0.05)。血清OPG、RANKL、RANKL/OPG比值在COPD正常骨量组、低骨量组、骨质疏松组依次升高,骨质疏松组升高最显著,骨质疏松组较正常骨量组均升高(P<0.01)；但三组间RANK水平无显著性差异(P>0.05)。腰椎骨密度、股骨颈骨密度、FEV1%Pre与血清MMP-9显著负相关(分别为r=-0.432, P<0.01; r=-0.697, P<0.01和r=-0.226,P<0.05)；腰椎骨密度、股骨颈骨密度、FEV1%Pre与RANKL(分别为r=-0.434, P<0.01; r=-0.538, P<0.01和r=-0.325,P<0.01)、RANKL/OPG比率(分别为r=-0.345,P<0.01和r=-0.420,P<0.01和r=-0.459,P<0.05)呈显著负相关；而与其它细胞因子(TIMP-1、TNF-α、OPG、RANK)无显著相关性(P>0.05)。 FEV1%Pre与腰椎骨密度、股骨颈骨密度显著正相关(r=0.338,P<0.01)和(r=0.409, P<0.01); MMP-9与TNF-a呈显著正相关(r=0.370,P<0.001),与OPG呈显著正相关(r=0.306, P<0.01), MMP-9与RANKL/OPG比值显著正相关(r=0.318,P<0.01)。
结论：骨质疏松症是COPD重要的系统性合并症之一,是多种因素综合作用的结果。腰椎骨密度、股骨颈骨密度、FEV1%Pre与血清MMP-9、RANKL、 RANKL/OPG比率显著负相关；而与其它细胞因子(TIMP-1、TNF-α、OPG、RANK)无显著相关性。在男性稳定期COPD患者,升高的基质金属蛋白酶(MMP)-9可能在骨质疏松发挥重要作用,与骨密度下降可能有关。MMP-9、TNF-α、OPG/RANK/RANKL系统在肺实质损伤和骨质疏松的发生发展过程中,有可能发挥协同作用。
第2部分慢性阻塞性肺疾病患者血清MMP-9及其抑制剂-1、TNF-α与骨转换生化指标及骨密度的关系
目的：观察慢性阻塞性肺疾病(COPD)患者血清基质金属蛋白酶(MMP)-9及其抑制剂-1、TNF-α与骨转换生化指标及骨密度(BMD)的关系。
方法：收集稳定期COPD男性患者90例,采用双能X线吸光测定法(DXA)测定骨密度,根据BMD检测结果分为正常骨量组、低骨量组、骨质疏松组(各30例)。各组患者入组当日完成病史采集、肺功能检查、病情严重程度评分(CAT评分),并ELISA法检测血清MMP-9、TIMP-1、TNF-α和血清骨碱性磷酸酶(sBAP)、血清骨钙素(sOC)、血清I型胶原交联C端肽(sCTX)水平。
结果：COPD稳定期并骨质疏松组与正常骨量组、低骨量组相比较,BMI、 CAT评分存在显著性差异(均P<0.01)；而年龄、吸烟指数、FEV1/FVC、 FEV1%Pre未见明确统计学差异(P>0.05)。低骨量组及骨质疏松组的腰椎骨密度、股骨颈密度均较正常骨量组下降,差异具有统计学意义(P<0.05或P<0.01)。血清MMP-9、MMP-9/TIMP-1比值、TNF-α在COPD正常骨量组、低骨量组、骨质疏松组依次升高,骨质疏松组升高最显著(P<0.05或P<0.01)；血清TIMP-1水平在COPD正常骨量组、低骨量组、骨质疏松组依次升高,但差异无统计学意义(P>0.05)。COPD骨质疏松组、低骨量组的血清sBAP、sOC水平低于正常骨量组(P<0.01),而低骨量组与骨质疏松组间未见显著性差异(P>0.05)；COPD骨质疏松组与正常骨量组、低骨量组比较,血清sCTX水平升高,存在显著性差异(均P<0.01),而在低骨量组与正常骨量组间未见显著性差异(P>0.05)。腰椎骨密度、股骨颈骨密度、FEV1%Pre与血清MMP-9显著负相关(分别为r=-0.432, P<0.01; r=-0.697, P<0.01和r=-0.226,P<0.05)；腰椎骨密度、股骨颈骨密度、FEV1%Pre与sBAP显著正相关(r=0.418, P<0.01; r=0.702, P<0.01; r-0.295, P<0.01)、sOC显著正相关((r=0.338, P<0.01; r=0.574, P<0.01; r=0.278, P<0.01)、sCTX呈显著负相关(r=-0.418, P<0.01; r=-0.602, P<0.01; r=-0.243, P<0.05);而与TIMP-1、TNF-α无显著相关性。FEV1%Pre与血清MMP-9显著负相关(r=-0.226, P<0.05); MMP-9/TIMP-1与sBAP、sOC呈显著负相关(r=-0.525, P<0.01; r=-0.460, P<0.01),与sCTX呈显著正相关(r=0.635, P<0.01)。 TIMP-1与sCTX呈显著正相关(r=0.236,P<0.05),与sBAP、sOC无显著相关性(P>0.05)。TNF-α与sBAP呈显著负相关(r=-0.350,P<0.01),与sCTX呈显著正相关(r=0.370,P<0.01)。TNF-α与sOC无显著相关性(P>0.05)。
结论：在男性稳定期COPD患者,升高的MMP-9、TNF-α与骨转换生化指标有一定相关性,在骨质疏松可能发挥重要作用；血清MMP-9/TIMP-1α TNF-α升高可能与COPD患者骨代谢增快有关。
Background and purpose:
Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease characterized by airflow limitation, It has a high and increasing prevalence and mortality. By2020, COPD may become the third most common fatal disease in the world. Osteoporosis, a condition characterized by low bone mass and bone microarchitectural deterioration which predisposes to fragility fracture, is one of the most important systemic complications of COPD. The incidence of osteoporosis among COPD patients is significantly higher than in the general population. Clinical sequelae of serious osteoporosis, including compression fractures and kyphosis, can further reduce vital capacity, thereby further compromising respiratory status in patients with COPD. A correlation between reduced lung function and bone mineral density (BMD) is suggested by some studies, however, the mechanisms between COPD and osteoporosis are not yet fully clear.
Emphysema is a kind of chronic pulmonary disease characterized by abnormal and permanent airspace enlargement beyond the terminal bronchioles accompanied by destruction of the alveolar walls. The most widely accepted theory of the pathogenesis is a proteinase-antiproteinase imbalance. Recent studies have shown that the balance between matrix metalloproteinases (MMPs) and their inhibitors(TIMPs) plays an important role in the breakdown and remodelling of ECM in the alveolar walls in COPD and asthma patients. MMPs, a group of calcium-activated zinc ion endopeptidases widely found in a variety of connective tissues, play an essential role in the degradation of the extracellular matrix and basement membrane enzyme. They are differentially expressed during different stages of COPD development. The TIMPs are endogenous natural inhibitors of MMPs. The airway MMP-9/TIMP-1ratio is considered a biomarker of airway tissue destruction and of the dynamic equilibrium of repair.
In addition to lung disease, MMPs have been suggested to have a important role in bone remodelling and turnover. MMP-9is produced by osteoclasts and osteoclast precursor cells,the monocytes also. It is essential for recruitment of preosteoclasts into bone and their local migration into the diaphysis. In contrast, TIMP-1prevents bone resorption.
Experimental studies indicate that TNF-α is a multifaceted cytokine and a well-recognized systemic inflammatory factor related to the occurrence and development of a variety of the systemic complications of COPD. TNF-a is also an important regulator of bone metabolism and remodelling. It maybe stimulate osteoclast differentiation in a synergistic manner. TNF-α,IL-1β,\IL-6are all well-established inflammatory cytokines involved in the systemic inflammation of COPD. There is an association between distinct serum inflammatory mediators and quantitative CT measurements of emphysema. Simultaneously, IL-1β, IL-6and TNF-a have been shown to be important modulators of bone metabolism and remodeling by stimulating osteoclast differentiation in a synergistic fashion. These cytokines may not only regulate osteoclastogenesis via their effects on stromal cells, but also act directly on osteoclasts and their precursors. In addition, they also interact with the osteoporosis-related OPG/RANK/RANKL protein system.
The OPG/RANK/RANKL system is an important regulator of bone metabolism, and participates in the development of post-menopausal osteoporosis. This was a major breakthrough in the field of osteoporosis and bone metabolism and opened a new perspective on bone metabolic balance and osteoclast biology. RANKL and OPG are the promoting and inhibitory factors of osteoclast differentiation, respectively. They maintain a certain ratio within the body. Imbalances in this ratio can disrupt bone metabolism, leading to reduced bone mass/osteoporosis among other bone diseases. Whether MMP-9/TIMP-1, TNF-a,the OPG/RANK/RANKL pathway is involved in the pathogenesis of osteoporosis in COPD has not been studied.
Osteoporosis is a metabolic condition characterized by decreased bone mass and strength due to increased bone turnover, which compromises bone architecture and results in increased fracture risk. Bone is a metabolically active tissue that undergoes continuous remodelling by two counter acting processes, namely bone formation and bone resorption. These processes rely on the activity of osteoclasts, osteoblasts and osteocytes. Acceleration of bone turnover, accompanied with a disruption of the coupling between these cellular activities,plays an established role in the pathogenesis of metabolic bone diseases, such as osteoporosis. In the past few decades, Many research has been working on biochemical markers which may reflect the rate of bone turnover. A large number of studies have shown that serum or urine biochemical markers are related to bone loss and fractures, which is important to identify high-risk patients. The biochemical markers of bone turnover and their applications in clinical trials can test the efficacy of drugs and how they supply the measurement of bone mass. Targeted use of biochemical markers can be further optimized to identify high-risk patients, the process of drug development and clinical monitoring of the efficacy of osteoporosis treatment. Biochemical markers are bound to be used widely because of their advantages of non-invasive, easy repeatability and timeliness.
In this study, the correlations among serum MMP-9and its cognate inhibitor TIMP-1, TNF-a, and the OPG/RANK/RANKL system,bone biochemical markers were assessed, and their associations with impairment of lung function and bone mineral density in patients with COPD was evaluated.
This study include two parts:
Part I Analysis of interrelationship of circulating matrix metalloproteinase-9, TNF-a, and OPG/RANK/RANKL system in COPD patients with osteoporosis
Objective:
To study the relationship between matrix metalloproteinase-9(MMP-9), its cognate inhibitor TIMP-1, inflammatory cytokine TNF-a, and OPG/RANK/RANKL system in COPD patients with osteoporosis.
Methods:
1. Patients and sample collection:Ninety male patients diagnosed with clinically stable COPD were enrolled from Sep2010to May2012. Female patients were excluded from the study to avoid the influence of postmenopausal osteoporosis. COPD was diagnosed according to the criteria issued by the Global Initiative for Chronic Obstructive Lung Disease (GOLD). These ninety patients were divided into three groups based on BMD:those with COPD and normal BMD (T-score>-1.0), those with COPD with osteopenia (T score-1.0to-2.4), and those with COPD and osteoporosis (T-score≤-2.5). Each group contained30patients. All patients were more than40years old and all were current or former smokers. The exclusion criteria were as follows:(1)serious cardiovascular and cerebrovasculardiseases, liver or renal function failure,consciousness disorder,(2) history of respiratory diseases other than COPD, such as asthma, bronchiectasis,(3) history of bone disease, such as fractures,osteomalacia,(4) rheumatic diseases,such as rheumatoid arthritis,(5) metabolic, or inflammatory disease,such as hyperthyrosis, hyperparathyroidism,(6) inhaled, oral, or intravenous corticosteroid treatment within the last3months,(7) medication which may influence bone metabolism within the previous one year, including those being treated for osteoporosis,(8) occurrence of malignancy within the previous five years. All subjects provided informed consent before joining the study.
On the recruiting day, all patients completed pulmonary function test and the life quality assessment using the COPD Assessment Test (CAT) and the measurement of BMD. The BMD of lumbar spine (L1-L4) and bilateral femoral neck was measured using dual X-ray absorptiometry(DXA). BMD is here expressed in g/cm2and a T-score, which was used for diagnosis according to the World Health Organization guidelines as follows:normal BMD:T score greater than-1at both sites (lumbar spine and FN); osteopenia:T score less than or equal to-1but greater than-2.5at either site; osteoporosis:T score less than or equal to-2.5at either site. Ninety patients were divided into three groups of30patients each based on BMD: those with COPD and normal BMD, those with COPD with osteopenia, and those with COPD and osteoporosis.
2. The measurement of cytokines(Enzyme-linked immunosorbent assay,ELISA):Fasting blood samples were collected by venipuncture the next morning. These were centrifuged at1000×g for5min at room temperature to produce serum samples, which were stored at-80℃until analysis.High-sensitivity MMP-9(pro-MMP-9), TIMP-1, and TNF-α levels were determined using sandwich ELISA kits. OPG/RANK/RANKL concentrations were measured using ELISA kits too.
3. Statistics:
Data are expressed as the mean±SD and analyzed using the Statistical Package for the Social Sciences, SPSS13.0(SPSS Inc, Chicago, IL, U.S.). Data from the three experimental groups were analyzed with normally distributed test(shapiro-wilk test),one-way ANOVA with a post-hoc Tukey's test, and Pearson's correlation analysis. The relationships among parameters were evaluated using multiple linear stepwise regression analysis. A p-value<0.05was considered statistically significant.
Results:
The age, BMI, smoking index, FEV1/FVC, FEV1%Pre, and CAT scores of the COPD patients were compared between all groups of patients. COPD patients with osteoporosis had significantly lower BMI and higher CAT scores (all P<0.01) than COPD patients with normal BMD and those with low BMD. There was no significant difference in age, smoking index, FEV1/FVC, or FEV1%Pre(P>0.05). As expected, based on the study design, the lumbar spine and femoral neck BMD was significantly lower in the groups of COPD with low BMD (P<0.05, P<0.01) and COPD with osteoporosis (all P<0.01) than in COPD with normal BMD.
Pearson analysis revealed positively correlations between BMI and the lumbar spine BMD (r=0.278, P<0.01) and femoral neck BMD (r=0.273, P<0.01); negative correlations between CAT scores and the lumbar spine BMD (r=-0.331, P<0.01) and femoral neck BMD (r=-0.486, P<0.01).
The serum level of MMP-9, MMP-9/TIMP-1ratio, the serum level of TNF-a increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.05or P<0.01). However, no difference in the serum level of TIMP-1was detected among the three groups(P>0.05). The serum levels of OPG, RANKL, the RANKL/OPG ratio increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.01). However, no difference existed in the level of RANK among these three groups(P>0.05).
Pearson analysis revealed negative correlations between MMP-9serum levels and the lumbar spine BMD (r=-0.432, P<0.01) and femoral neck BMD (r=-0.697, P<0.01) and FEV1%Pre (r=-0.226, P<0.05); between RANKL serum levels and the lumbar spine BMD (r=-0.434, P<0.01) and femoral neck BMD (r=-0.538, P<0.01) and FEV1%Pre (r=-0.325, P<0.01); and between the RANKL/OPG ratio and the lumbar spine BMD (r=-0.345, P<0.01) and femoral neck BMD (r=-0.420, P<0.01) and FEV1%Pre (r=-0.459, P<0.05). Other cytokines, such as TIMP-1, TNF-a, OPG, and RANK, did not correlate with the lumbar spine or femoral neck BMD or FEV1%Pre (P>0.05). FEV1%Pre was positively correlated with the lumbar spine and femoral neck BMD (r=0.338, P<0.01, r=0.409, P<0.01).MMP-9was positively correlated with TNF-a (r-0.370, P<0.001) and OPG(r=0.306, P<0.01) and RANKL/OPG ratio (r=0.318, P<0.01).
Conclusions:
Osteoporosis is one of the most important systemic complications of COPD. Negative correlations between the serum levels of MMP-9,RANKL, the RANKL/OPG ratio and the lumbar spine BMD, femoral neck BMD,FEV1%Pre. Other cytokines, such as TIMP-1, TNF-α, OPG, and RANK, did not correlate with the lumbar spine BMD or femoral neck BMD or FEV1%Pre. MMP-9, TNF-a, and the OPG/RANK/RANKL system may be closely interrelated and may play interactive roles in pathogenesis of osteoporosis in COPD.
Part Ⅱ Analysis of interrelationship of circulating matrix metalloproteinase-9, TNF-a, bone biochemical markers and bone mineral density in COPD patients
Objective:To study the relationship between matrix metalloproteinase-9(MMP-9), its cognate inhibitor TIMP-1, inflammatory cytokine TNF-a, bone biochemical markers and bone mineral density(BMD) in COPD patients.
Methods:Ninety male patients diagnosed with clinically stable COPD were enrolled, who were divided into three groups based on bone mineral density(BMD):those with COPD and normal BMD, those with COPD with low but non-osteoporotic BMD, and those with COPD and osteoporosis. Each group contained30patients.All these patients completed pulmonary function,dual x-ray absorptiometry(DXA) measurements and COPD assessment test (CAT).All these patients were measured of MMP-9, TIMP-1, TNF-a, and serum bone alkaline phosphatase(sBAP)、serum osteocalcin(sOC)、bone cross-linked C-telopeptides of type Icollagen(sCTX) by ELISA.
Results:COPD patients with osteoporosis had significantly lower BMI and higher CAT scores (all P<0.01), than COPD patients with normal or low BMD. There was no significant difference in age, smoking index, FEV1/FVC, or FEV1%Pre (P>0.05). As expected, based on the study design, the lumbar spine and femoral neck BMD was significantly lower in the groups of COPD with low BMD (P<0.05, P<0.01) and COPD with osteoporosis (all P<0.01) than in COPD with normal BMD. The serum level of MMP-9, the MMP-9/TIMP-1ratio, the serum level of TNF-a increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.05or P<0.01).However, no significant difference in the serum level of TIMP-1was detected among the three groups(P>0.05).
The serum levels of BAP、OC were all significantly lower in COPD patients with osteoporosis and those with low BMD than in those with normal BMD (all P <0.01). However, no significant difference in levels of sBAP、sOC was detected between COPD with osteoporosis and in those with low BMD (P>0.05). The level of sCTX was significantly higher in the groups of COPD with osteoporosis than those with low BMD and those with normal BMD(all P<0.01). However, no difference in the level of sCTX was detected between COPD with low BMD and those with normal BMD (P>0.05).
Pearson analysis revealed negative correlations between MMP-9serum levels and the lumbar spine BMD (r=-0.432, P<0.01) and femoral neck BMD (r=-0.697, P<0.01) and FEV1%Pre (r=-0.226, P<0.05);The BMD of lumbar spine and femoral neck、the lung function FEV1%Pre were positively correlated with sBAP level (r=0.418, P<0.01,and r=0.702, P<0.01,and r=0.295, P<0.01, respectively), sOC level (r=0.338, P<0.01,and r=0.574, P<0.01, and r=0.278, P<0.01, respectively), and negative correlated with sCTX level (r=-0.418, P<0.01,and r=-0.602, P<0.01,and r=-0.243, P<0.05, respectively). However, they had no significant correlation with TIMP-1, TNF-a. The ratio of MMP-9/TIMP-1was negative correlated with the levels of sBAP, sOC(r=-0.525, P<0.01; r=-0.460, P<0.01) and positively correlated with sCTX (r=0.635, P<0.01). The level of TIMP-1was positively correlated with sCTX (r=0.236, P<0.05), TNF-a was negative correlated with sBAP(r=-0.350, P<0.01) and positively correlated with sCTX (r=0.370, P<0.01). But no difference existed between the level of TNF-a with sOC (P>0.05).
Conclusions:These results suggest that circulating MMP-9,TNF-a and the bone biochemical markers maybe closely interrelated in bone turnover state and may play important roles in pathogenesis of osteoporosis in COPD. Bone biochemical markers suggest increased bone turnover in COPD.
引文
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