长期口服雌激素对去卵巢大鼠海马结构内β-淀粉样前体蛋白及其mRNA表达的影响

目的 观察长期口服复方尼尔雌醇和小剂量17β-雌二醇补充治疗对去卵巢大鼠海马结构内β-淀粉样前体蛋白（β-APP）及其mRNA表达的影响。方法 7月龄SD大鼠按体重随机分成5组：正常对照组、假手术组（SHAM）、去卵巢组（OVX）、17β-雌二醇预防干预组（OVX／ERT）和复方尼尔雌醇预防干预组（OVX／NL）连续观察。5组均在去卵巢后35w处死。用免疫组织化学方法（SABC法）显示大鼠海马结构内β-APP，原位杂交方法显示β-APP mRNA。细胞计数及图像分析观察β-APP及其mRNA的变化。结果 OVX组海马结构内各亚区β-APP阳性神经元数量和平均光密度均高于正常组、SHAM组、OVX／ERT组、OVX／NL组（P〈0．05），而β-APP mRNA阳性神经元数量和平均光密度在5组之间均无显著性差异。结论 长期雌激素缺乏导致了大鼠海马结构内β-APP含量的增加，长期口服复方尼尔雌醇和小剂量17β-雌二醇补充治疗可减少β-APP的含量，且二者效果相当。促进β-APP分解、减少β-APP的含量，进而减少Aβ的沉着可能是雌激素神经保护的重要机制之一。     

Age-Related Bone Mineral Density, Accumulated Bone Loss Rate and Prevalence of Osteoporosis at Multiple Skeletal Sites in Chinese Women

We investigated the age-related bone mineral density (BMD), accumulated bone loss rate (ABLR) and the prevalence of osteoporosis at different skeletal sites in Chinese women. BMD was measured at the anteroposterior (AP) spine, supine lateral spine (areal BMD at the midarea [mLat] and the whole region [Lat], volumetric BMD at the middle region [MVD] and total region [TVD]), hip (femoral neck [FN], trochanter [Troc] and Ward’s triangle [Ward’s]) and forearm (radius + ulna ultradistal [RUUD], 1/3 region [RU1/3] and total region [RUT]) using a dual-energy X-ray absorptiometry (DXA) fan-beam bone densitometer (Hologic QDR 4500A) in 2702 females aged from 5 to 96 years old. Data were analyzed by eight different regression models. We found that the cubic regression model was the best for describing age-related changes in BMD. The coefficients of determination (R ²) of the fitting curve were 0.398 to 0.612 (p= 0.000). The data were then analyzed by 5-year age groups. This showed that the earliest peak BMD was at the age of 20–24 years at Troc and Ward’s, and the latest at the age of 40–44 years at RU1/3 and RUT of the distal forearm. Compared with BMD, the ABLRs were highest at Ward’s (−66.2%) and the lowest at RU1/3 of the distal forearm (−31.3%) in subjects over 80 years old. The prevalence of osteoporosis at at least one site in these women was 0.5 ± 0.4% in those 30–39, 4.6 ± 4.4% in those 40–49, 23.9 ± 13.3% in those 50–59, 56.3 ± 20.3% in those 60–69, 71.8 ± 16.7% in those 70–79 and 83.2 ± 12.1% those over 80 years of age, respectively. The prevalence of osteoporosis in these women was 8.6–11.1% at the age of 40–49 and 36.5–40.6% at the age of 50–59 at the lateral spine regions (mLat, Lat, MVD and TVD), and 0.5–3.7% at the age of 40–49 and and 3.9–21.7% at the age of 50–59 years at the other skeletal sites (AP, FN, Troc, Ward’s, RUUD, RU1/3 and RUT). Significant differences were found in the prevalence of osteoporosis between the lateral spine regions and other skeletal sites (p<0.001) at the age of 40–59 years. In summary, we demonstrated significant age-related differences in peak BMD, ABLR and osteoporosis prevalence among various skeletal sites. Our data suggest that the supine lateral spine is the most sensitive site for the diagnosis of osteoporosis, especially in the early menopausal period, although the prevalence of osteoporosis varied with age and with different sites measured. Received: 20 November 2001 / Accepted: 13 February 2002     

Effects of skeletal size of the lumbar spine on areal bone density,volumetric bone density,and the diagnosis of osteoporosis in postmenopausal women in China

To understand the effects of skeletal size of the lumbar spine on areal bone mineral density (aBMD), volumetric bone mineral density (vBMD), and the diagnosis of osteoporosis in postmenopausal women, we measured the projected bone area, bone mineral content (BMC), aBMD, and vBMD at the anteroposterior and lateral lumbar spines in a population of 1081 postmenopausal Chinese women, 42 to 86 years of age. The results indicated that, at the anteroposterior and lateral lumbar spine, there were significant positive correlations between bone area and both BMC (r = 0.606; P = 0.000 and r = 0.610; P = 0.000) and aBMD (r = 0.270; P = 0.000 and r = 0.182; P = 0.000), but not vBMD (r = –0.055; P = 0.000 and r = 0.000; P = 0.929). When bone area at the anteroposterior spine changed by ±1 SD, the BMC, aBMD, and vBMD correspondingly changed by 28.2%, 10.1%, and 1.69% on the basis of their respective means. When a variation of ±1 SD was observed in bone area at the lateral spine, BMC and aBMD, correspondingly changed by 25.9% and 6.18% on the basis of their respective means, while vBMD indicated no change. Through comparisons among large-, intermediate-, and small-bone area groups, significant differences were found in the means of subjects heights, weights, BMC, and vBMD at the anteroposterior and lateral lumbar spines, as well as in the detection rates of osteoporosis by aBMD (P = 0.000). Detection rates of osteoporosis by aBMD at the anteroposterior spine and by aBMD at the lateral spine, and by vBMD were 44.1%, 55.5%, and 49.7%, respectively, in the total population; 31.4%, 41.7%, and 53.7%, respectively, in the large-bone area group; 43.3%, 55.9%, and 50.5%, respectively, in the intermediate-bone area group; and 61.7%, 70.0%, and 42.5%, respectively, in the small-bone area group. No significant differences were found in the detection rates of osteoporosis by vBMD among the groups. The results of multiple linear regression revealed that the major factors influencing skeletal size and aBMD of the lumbar spine were height and weight. Therefore, in menopausal women of the same ethnic group and age, the skeletal size of the lumbar spine would have significant influence upon aBMD and the diagnosis of osteoporosis, i.e., the larger the spinal size, the greater the aBMD and the lower the osteoporosis detection rate, while, conversely, the smaller the skeletal size, the smaller the aBMD and the higher the osteoporosis detection rate. When we use aBMD of the lumbar spine to diagnose osteoporosis in a population with different body sizes, we need to take this body size difference into account. When we use vBMD to diagnose osteoporosis, the effect of body size on BMD will diminish.     

Establishment and evaluation of bone mineral density reference databases appropriate for diagnosis and evaluation of osteoporosis in Chinese women

 This study was designed to establish Bone Mineral Density (BMD) Reference Databases for multiple skeletal sites appropriate for the diagnosis and evaluation of osteoporosis (OP) in Chinese women. We recruited 2702 healthy Chinese women, 5–96 years of age, for BMD assessment. BMD values at multiple skeletal sites including anteroposterior (AP) and lateral (Lat) lumbar spine, hip, and forearm were measured by dual-energy X-ray absorptiometry (DXA) using a QDR 4500A device; results were analyzed according to age group using eight regression models. BMD Reference Databases (CWD) were established according to the best regression equation and compared with Hologic reference databases for “Oriental Women” (OWD). Results indicated that the cubic regression model was superior to the quadratic, linear, logarithmic, and exponential regression models, etc. for our purpose, with a determinate coefficient (R ²) of 0.363–0.650 (P = 0.000). We included 1636 female patients, aged 35–86 years, in our tests. In comparison with Hologic Reference Databases, the mean detection rate of OP in the newly established BMD Reference Databases for Chinese Women (CWD) was 16.0% ± 2.68% lower (range, 13.7%–20.5%) at the AP spine, 16.8% ± 11.0% lower (range, 3.5%–32.8%) at the Lat spine (except for L4), 18.7% ± 4.6% lower (range, 12.6%–24.2%) at the hip, and 14.3% ± 6.9% higher (range, 4.7%–24.3%) at the forearm. The difference in detection rates for OP was significant between the two reference databases (P = 0.000), which was consistent with the differences in peak BMD values and the biological variability between them. Based upon our data, we confirmed that the Hologic BMD Reference Databases for Oriental Women (OWD) were not suitable for the diagnosis of OP in Chinese women; the BMD Reference Databases for Chinese Women (CWD) established in this study would provide reliable diagnostic standards for detection of OP in the women of South China. Received: July 9, 2002 / Accepted: December 5, 2002 Offprint requests to: X.-P. Wu     

Femoral neck trabecular microstructure in ovariectomized ewes treated with calcitonin: MRI microscopic evaluation.

Ovariectomy induces deterioration of the trabecular structure in the femoral neck of ewes, as depicted by MR microscopic imaging. This structural deterioration is prevented by salmon calcitonin treatment. INTRODUCTION: This study evaluated the trabecular (Tb) microarchitecture of an ovariectomy (OVX)-induced osteoporotic model in ewes and determined the effects of salmon calcitonin (sCT), an osteoclast inhibitor, on the Tb structure. This is the first report of OVX-induced changes in the Tb structure in the femoral neck in the ewes and effect of sCT on the microarchitecture. MATERIALS AND METHODS: Ewes (5-8 years old, n = 28) were equally allocated into sham (Sham), OVX injected with vehicle, or OVX injected with sCT at 50 or 100 IU, three injections per week. They were killed 6 months after OVX. The femoral neck was examined with an MR imager at 9.4 T in axial, coronal, and sagittal planes. An internal calibration procedure as a means of standardizing image analysis was used to adjust the segmentation threshold. Data from all three planes were averaged. RESULTS AND CONCLUSIONS: Compared with Sham, OVX induced significant changes (p < 0.0125) in the MRI-derived femoral neck Tb structure: Tb bone volume fraction (BV/TV), -18%; Tb number, -20%; Tb separation, +23%; number of free ends, +28%; number of nodes, -39%; number of Tb branches, -23%; mean length of Tb branches, -19%. Compared with OVX, treatment of sCT at 100 IU significantly improved all the Tb structural parameters to the Sham level (p < 0.0001 approximately p = 0.0281), whereas 50 IU significantly increased the Tb number and the mean length of the Tb branches. BV/TV explained 74% of the variation of compressive stress of the trabecular cylinder cores of the femoral neck. Combining all structural parameters in a multivariate regression analysis significantly improved the explanation to 84%, and adding BMD further improved the predictive ability of the model to 92%. We conclude that OVX induces deterioration of the MRI-derived Tb microstructure in the femoral neck of ewes. sCT treatment prevents OVX-induced changes. The femoral neck microarchitecture significantly correlates with its biomechanical properties. Combining microstructural parameters with BMD further improves the prediction of bone biomechanical properties. The effects of sCT on OVX ewes may help explain reduced fracture risk in postmenopausal osteoporotic women treated with sCT.     

Effects of chitosan-nanoparticle-mediated tight junction opening on the oral absorption of endotoxins

Recently, we reported a pH-responsive nanoparticle (NP) system shelled with chitosan (CS), which could effectively increase the oral absorption of insulin and produce a hypoglycemic effect, presumably due to the CS-mediated tight junction (TJ) opening. It has been often questioned whether CS can also enhance the absorption of endotoxins present in the small intestine. To address this concern, we studied the effect of CS NPs on the absorption of lipopolysaccharide (LPS), the most commonly found toxin in the gastrointestinal tract. To follow their biodistribution by the single-photon emission computed tomography/computed tomography, LPS and insulin were labeled with (99m)Tc-pertechnetate ((99m)Tc-LPS) and (123)iodine ((123)I-insulin), respectively. The (99m)Tc-LPS was ingested 1 h prior to the administration of the (123)I-insulin-loaded NPs to mimic the physiological conditions. The confocal and TEM micrographs show that the orally administered CS NPs were able to adhere and infiltrate through the mucus layer, approach the epithelial cells and mediate to open their TJs. The radioactivity associated with LPS was mainly restricted to the gastrointestinal tract, whereas (123)I-insulin started to appear in the urinary bladder at 3 h post administration. This observation indicates that the insulin-loaded in CS NPs can traverse across the intestinal epithelium and enter the systemic circulation, whereas LPS was unable to do so, probably because of the charge repulsion between the anionic LPS in the form of micelles and the negatively charged mucus layer. Our in vivo toxicity study further confirms that the enhancement of paracellular permeation by CS NPs did not promote the absorption of LPS. These results suggest that CS NPs can be used as a safe carrier for oral delivery of protein drugs.     

Adiponectin stimulates RANKL and inhibits OPG expression in human osteoblasts through the MAPK signaling pathway.

Our study indicates that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclasts formation in the co-culture of osteoblast and peripheral blood monocytes systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It also suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation. INTRODUCTION: Recently, adiponectin has emerged as an element in the regulation of bone metabolism, but the mechanism remains. This study was undertaken to investigate the action of adiponectin on osteoclastogenesis through revealing RANKL and osteoprotegerin (OPG) expression in osteoblasts and osteoclast formation. MATERIALS AND METHODS: Real-time quantitative PCR and ELISA were used to detect RANKL and OPG mRNA and protein expression in cultured human osteoblasts. The involved signal pathway was studied using mitogen-activated protein kinase (MAPK) inhibitor and adiponectin receptor 1 (AdipoR1) siRNA. The effects of recombinant adiponectin on osteoclasts formation also were examined in the co-culture systems of osteoblast and peripheral blood monocytes (PBMCs) systems or purified CD14 + PBMCs cultures. RESULTS: Our study showed that recombinant adiponectin induced RANKL and inhibited OPG mRNA expression in human osteoblasts in a dose- and time-dependent manner. Adiponectin also increased soluble RANKL and decreased OPG secretion in osteoblasts conditioned media. Suppression of AdipoR1 with siRNA abolished the adiponectin-regulated RANKL and OPG mRNA expression in osteoblasts. Furthermore, pretreatment of osteoblasts with the MAPK inhibitor SB203580 abolished adiponectin-regulated RANKL and OPG mRNA expression. Adiponectin induced osteoclast formation in the co-culture systems of osteoblast and PBMCs systems, and OPG entirely blocked this response. However, adiponectin had no direct effect on the differentiation of osteoclast precursor purified CD14 + PBMCs. CONCLUSIONS: These data indicate that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclast formation in the co-culture of osteoblast and PBMCs systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation.     

Age-related changes in bone biochemical markers and their relationship with bone mineral density in normal Chinese women

Measurements of bone biochemical markers are increasingly being used to evaluate the state of bone turnover in the management of bone metabolic diseases, especially osteoporosis. However, changes in the bone turnover rate vary with age. The aim of this study was to establish the laboratory reference range of serum bone-specific alkaline phosphatase (sBAP), serum type I collagen cross-linked C-terminal telopeptide (sCTx), and urine CTx (uCTx), based on values from 665 healthy Chinese women aged 20–80 years. We measured the levels of sBAP, sCTx, serum alkaline phosphatase (sALP), and uCTx and evaluated the age-related changes and their relationship with bone mineral density (BMD) in the anteroposterior (AP) lumbar spine, hip, and left forearm. We found significant correlations between biochemical markers and age, with coefficients of determination (R ²) of 0.358 for sBAP, 0.126 for sCTx, 0.125 for uCTx, and 0.336 for sALP. The net changes in different biochemical markers were inversely correlated with the rates of BMD loss in the AP lumbar spine. After correction for age, body weight, and height, the levels of the markers had significant negative correlations with the BMD of the AP lumbar spine, femoral neck, and ultradistal forearm. All four biochemical markers had the highest negative correlation with BMD of the AP lumbar spine (partial correlation coefficients of −0.366, −0.296, −0.290, and −0.258 for sBAP, sCTx, uCTx, and sALP, respectively). The mean and SD values of these markers in premenopausal and postmenopausal women with normal BMD values were used as the normal reference ranges. The reference ranges of sBAP, sCTx, and uCTx for pre- vs postmenopausal women were 17.3 ± 6.23 vs 18.9 ± 7.52 U/l, 3.18 ± 1.49 vs 3.23 ± 1.57 nmol/l, and 15.5 ± 11.4 vs 16.2 ± 12.4 nM bone collagen equivalents/mM urinary creatinine, respectively. Levels of the bone formation marker (sBAP) and bone resorption markers (sCTx, uCTx) increased rapidly in women with osteopenia or osteoporosis, indicating that they may be sensitive markers to determine the bone turnover rate in healthy Chinese women.     

Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways.     

The relationship between bone turnover markers and BMD decreasing rates in Chinese middle-aged women

BackgroundThe relationship between bone turnover markers (BTMs) and BMD decreasing rate (BDR) in Chinese women is unclear. Wu investigated the relationship between (BTMs) and BDR at various skeletal sites in Chinese middle-aged women.MethodsA cross-section study of 555 healthy Chinese women over 35–60 years of age. BMD at posteroanterior spine, the left hip, and the left forearm were measured with a DXA. Levels of serum osteocalcin (OC), bone-specific alkaline phosphatase (BAP), cross-linked N-terminal telopeptides of type I collagen (sNTX) and total urinary deoxypyridinoline (uDPD) were determined.ResultsBDR at various skeletal sites had significant negative correlation with serum OC(r = ? 0.395 to ? 0.530), BAP(r = ? 0.297 to ? 0.486), and sNTX(r = ? 0.207 to ? 0.272). After adjustment of age and weight, serum OC, BAP, and sNTX rather than total uDPD still exhibited significant correlations with BDR. Stepwise regression analyses showed that, serum OC and BAP were the significantly negative determinants of BDR. Between 4.7?27.7% and 1.2?16.1% of the changes in BDR were determined by serum OC and BAP, respectively. However, sNTX and total uDPD had no significant effect on BDR at various skeletal sites.ConclusionsThis study indicated the correlation between BTMs and early-stage BDR in Chinese middle-aged women and suggested that serum OC and BAP, rather than sNTX and total uDPD, are the key determining factors of early BMD decreases.