Targeting systemically administered proteins to bone by bisphosphonate conjugation

To develop a methodology for bone-specific delivery of proteins, a bone-seeking aminobisphosphonate (aminoBP) was previously conjugated to a model protein, bovine serum albumin (BSA). The conjugates were shown to exhibit a high affinity to bone in vitro and in vivo. This study was conducted to determine whether the systemic delivery of proteins to bone can be increased by aminoBP conjugation. Two model proteins used for this study were BSA and lysozyme (LYZ). For each protein, an unmodified (i.e., control) and aminoBP-conjugated protein were (125)I-labeled and injected into rats, and the organ delivery of the proteins were determined. Intravenous (IV) injection of aminoBP-BSA resulted in a 2.0- to 3.7-fold increased delivery to bones as compared to the control protein in young rats. In osteopenic, ovariectomized rats, aminoBP conjugation enhanced the bone delivery of BSA by 2.2- to 7.5-fold. A 3.7- to 5.6-fold increased delivery was also observed for LYZ after IV injection in normal rats. In addition to IV route of administration, subcutaneous injection was also effective in delivering a higher amount of aminoBP-conjugated proteins to bone. We conclude that conjugating bone-seeking aminoBPs to proteins improved their delivery to mineralized tissues. The proposed targeting approach has the potential to improve the efficacy of recombinant proteins capable of stimulating bone formation by enhancing their localization to bones.     

Bisphosphonate conjugation to proteins as a means to impart bone affinity

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.     

A comparison of the anabolic effects of rat and bovine parathyroid hormone (1-34) in ovariectomized rats

The current study was designed to compare the skeletal effects of comparable doses of rat parathyroid hormone 1-34 (rPTH) and bovine parathyroid hormone 1-34 (bPTH) in ovariectomized (OVX) rats. Female Sprague-Dawley rats were OVX or sham-operated at 6 months of age and maintained untreated for 28 days after surgery. Baseline control and OVX rats were sacrificed at the beginning of treatment. Beginning 28 days post-OVX, the remaining rats were subcutaneously injected daily with rPTH or bPTH at 0, 5, 25, or 50 microg/kg/d for 28 days. Bone area, bone mineral content (BMC), and bone mineral density (BMD) of the distal femoral metaphyses were determined ex vivo using dual energy X-ray absorptiometry. Quantitative bone histomorphometry was performed on undecalcified longitudinal sections of the proximal tibia from each rat. Baseline OVX rats exhibited osteopenia as demonstrated by their significantly reduced femoral BMD and proximal tibia cancellous bone volume compared with those of baseline sham controls. Both rPTH and bPTH restored bone in OVX rats by markedly stimulating bone formation in a dose-dependent manner. However, a difference in potency between the two forms of PTH was evident. The percentage increases of BMC, BMD, cancellous bone volume, trabecular thickness, mineralizing surface, and bone formation rate in the OVX rats treated with bPTH at 5 microg/kg/d were the same as or above those treated with rPTH at the 25 microg/kg/d dose level. A relative potency analysis showed that bPTH was approximately 4- to 6-fold relatively more potent than rPTH in increasing distal femoral BMD as well as cancellous bone volume, mineralizing surface, and bone formation rate of proximal tibial metaphyses at comparable dose levels and a given time. These results may serve as a reference for in vivo study design when rPTH or bPTH are to be the agents for studies on bone anabolism.     

Ovariectomy of 12-month-old rats: effects on osteoprogenitor numbers in bone cell populations isolated from femur and on histomorphometric parameters of bone turnover in corresponding tibia

Ovariectomy (OVX) in rats results in increased bone turnover and decreased bone volume and bone mineral density when measured in the metaphyses of long bones. We have investigated the effects of OVX on changes in the number of progenitors in cell populations derived from the metaphyseal bone of femurs of ovariectomized rats at 12 months of age, by using colony assays, bone nodule assays, and limiting dilution analysis at 1.5 and 9 months post-OVX. We have also measured histomorphometric parameters of bone formation and resorption in the corresponding tibia at the same time-points. A significant increase, as shown by bone nodule assays and limiting dilution analysis, occurs in the number of progesterone- and dexamethasone-responsive osteoprogenitors in cell populations isolated from ovariectomized rats at the 9-month post-OVX time-point. Progesterone-responsive osteoprogenitors are also increased at 1.5 months post-OVX. The number of fibroblast colony-forming units does not change. Histomorphometry has shown that OVX causes an increase in osteoblast surfaces, mineralizing surfaces, and bone formation rate at both 1.5 and 9 months post-OVX. The mineral apposition rate is increased at 1.5 months post-OVX. OVX also increases parameters of bone resorption at both time-points, the net result being a decrease in bone mineral density and cancellous bone volume at 9 months post-OVX. Thus, OVX in rats at 12 months of age is associated with an increase in the number of both progesterone- and dexamethasone-responsive osteoprogenitors 9 months post-OVX; this corresponds with increases in the histomorphometric parameters of bone formation.     

Silicon Supplementation Improves the Bone Mineral Density of Calcium-Deficient Ovariectomized Rats by Reducing Bone Resorption

The purpose of this study was to investigate the effect of silicon (Si) supplementation on bone mineral density (BMD) and bone metabolism parameters relative to calcium (Ca) intake levels in ovariectomized rats. A total of 72 female Wistar rats (6 weeks) were ovariectomized (OVX) and divided into six groups, and Si (500 mg of Si per kilogram of feed) was or was not administered with diets containing various levels of Ca (0.1%, 0.5%, and 1.5%) for 10 weeks. The groups were as follows: (1) Ca-deficient group (0.1% Ca), (2) Ca-deficient with Si supplementation group, (3) adequate Ca group (0.5% Ca), (4) adequate Ca with Si supplementation group, (5) high Ca group (1.5% Ca), and (6) high Ca with Si supplementation group. Si supplementation significantly increased the BMD of the femur and tibia in Ca-deficient OVX rats, while no change was observed with Si supplementation in the BMD of the spine, femur, and tibia in the adequate and high Ca groups. Serum alkaline phosphatase and osteocalcin levels were not affected by Si supplementation or Ca intake levels. C-telopeptide type I collagen levels were significantly decreased as a result of Si supplementation in Ca-deficient OVX rats. In summary, Si supplementation produced positive effects on bone mineral density in Ca-deficient OVX rats by reducing bone resorption. Therefore, Si supplementation may also prove to be helpful in preventing osteoporosis in postmenopausal women whose calcium intake is insufficient.     

Short-term Administration of Water-soluble Silicon Improves Mineral Density of the Femur and Tibia in Ovariectomized Rats

Silicon is important for the proper growth and development of bone and connective tissues. This study was designed to investigate if water-soluble silicon could be used for the treatment of postmenopausal osteoporosis. Silicon (Si 20 mg/kg body weight/day) was administrated orally to 17-week-old ovariectomized (OVX) rats for 4 weeks. Silicon did not alter weight gain in OVX rats. Silicon supplementation significantly increased the bone mineral density of the femur (p < 0.05, vs. OVX control group) and tibia in OVX rats (p < 0.05, vs. OVX control group). Serum alkaline phosphatase and osteocalcin, two bone formation biomarkers tested, were not significantly altered, but urinary calcium and phosphorous excretion tended to decrease with silicon supplementation. OVX rats with silicon supplementation showed a relatively higher serum CTx compared to the nonsupplemented OVX group (p < 0.01, vs. OVX control group). According to these results, short-term soluble silicon supplementation improved bone mineral density in OVX-induced osteoporosis.     

中等强度跑台运动预防去卵巢大鼠骨质疏松

目的观察中等强度跑台运动对去卵巢大鼠骨质疏松的预防作用。方法将30只3月龄未经产雌性SD大鼠随机分为假手术、去卵巢静止和去卵巢运动三个组。去卵巢运动组每周进行4次时间45min、速度18m/min、坡度5°的跑台训练。实验结束时,检测血清雌二醇（E2）、碱性磷酸酶（ALP）、抗酒石酸酸性磷酸酶（TRAP）和骨钙素（BGP）水平以及右侧游离股骨和胫骨的骨密度（BMD）和骨矿物含量（BMC）;同时观察左侧股骨远端和胫骨近端组织形态学变化。结果与假手术组比较,去卵巢静止组大鼠血清ALP活性和BGP含量显著升高,血清TRAP活性和E2含量显著下降,股骨近段和远端以及胫骨近端BMD和BMC显著下降,股骨远端和胫骨近端骨小梁断裂增加、数目减少;与去卵巢静止组比较,去卵巢运动组大鼠血清E2和BGP含量显著上升,股骨三个部位以及胫骨近端BMD和BMC显著增加,股骨远端和胫骨近端骨小梁断裂减少、数目增加。结论中等强度跑台运动能增加去卵巢大鼠血清E2和BGP含量,改善去卵巢大鼠骨组织学结构。     

High-impact exercise strengthens bone in osteopenic ovariectomized rats with the same outcome as Sham rats.

The effect of jump exercise on middle-aged osteopenic rats was investigated. Forty-two 9-mo-old female rats were either sham-operated (Sham) or ovariectomized (OVX). Three months after surgery, the rats were divided into the following groups: Sham sedentary, Sham exercised, OVX sedentary, and OVX exercised. Rats in the exercise groups jumped 10 times/day, 5 days/wk, for 8 wk, with a jumping height of 40 cm. Less than 1 min was required for the jump training. After the experiment, the right tibia and femur were dissected, and blood was obtained from each rat. OVX rats were observed to have increased body weights and decreased bone mass in their tibiae and femurs. Jump-exercised rats, on the other hand, had significantly increased tibial bone mass, strength, and cortical areas. The bone mass and strength of OVX exercised rats increased to approximately the same extent as Sham exercised rats, despite estrogen deficiency or osteopenia. Our data suggest that jump exercise has beneficial effects on lower limb bone mass, strength, bone mineral density, and morphometry in middle-aged osteopenic rats, as well as in Sham rats.     

Effects of Multi-Deficiencies-Diet on Bone Parameters of Peripheral Bone in Ovariectomized Mature Rat

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.     

Analysis of Bacteria,Parasites, and Heavy Metals in Lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis>) and Rocket Salad (<Emphasis Type="Italic">Eruca sativa</Emphasis> L.) Irrigated with Treated Effluent from a Biological Wastewater Treatment Plant

The purpose of this study was to verify the effect of organic gallium on ovariectomized osteopenic rats. Thirty Wistar female rats used were divided into three groups: (1) sham-operation rats (control), (2) ovariectomized (OVX) rats with osteopenia, and (3) OVX rats with osteopenia treated with organic gallium. Treatments were performed over an 8-week period. At sacrifice, the fifth lumbar vertebral body, one tibia, one femur, and the fourth lumbar vertebrae were removed, subjected to micro-CT for determination of trabecular bone structure, and then processed for histomorphometry to assess bone turnover. The femoral neck was used for mechanical compression testing. Treatment with organic gallium increased bone volume in OVX animals. Organic gallium-treated animals had significant increases in trabecular and cortical thickness and bone strength. The plasma total calcium and inorganic phosphate concentrations in OVX rats decreased and bone mineral content in the lumbar vertebrae and femur increased after treatment with organic gallium. These data provide an important proof of concept that organic gallium may represent a powerful approach to treating or reversing severe osteoporosis in humans.     

Comparison of the Therapeutic Effects of Yeast-incorporated Gallium with those of Inorganic Gallium on Ovariectomized Osteopenic Rats

The purpose of this study was to verify the effect of organic gallium on ovariectomized osteopenic rats. Thirty Wistar female rats used were divided into three groups: (1) sham-operation rats (control), (2) ovariectomized (OVX) rats with osteopenia, and (3) OVX rats with osteopenia treated with organic gallium. Treatments were performed over an 8-week period. At sacrifice, the fifth lumbar vertebral body, one tibia, one femur, and the fourth lumbar vertebrae were removed, subjected to micro-CT for determination of trabecular bone structure, and then processed for histomorphometry to assess bone turnover. The femoral neck was used for mechanical compression testing. Treatment with organic gallium increased bone volume in OVX animals. Organic gallium-treated animals had significant increases in trabecular and cortical thickness and bone strength. The plasma total calcium and inorganic phosphate concentrations in OVX rats decreased and bone mineral content in the lumbar vertebrae and femur increased after treatment with organic gallium. These data provide an important proof of concept that organic gallium may represent a powerful approach to treating or reversing severe osteoporosis in humans.     

Effect of dietary calcium: Phosphorus ratio on bone mineralization and intestinal calcium absorption in ovariectomized rats

We investigated the effect of dietary calcium:phosphorus (Ca:P) ratio on bone mineralization and intestinal Ca absorption in ovariectomized (OVX) rat models of osteoporosis and sham-operated rats. Thirty 12-wk-old female Wistar rats were divided into three groups of OVX rats and three groups of sham rats. Thirty days after the adaptation period, OVX rats and sham rats were fed a diet formulated Ca:P, 1:0.5, 1:1 or 1:2 (each diet containing 0.5% Ca), respectively for 42 d. In both sham and OVX rats, serum osteocalcin, a marker of bone turnover, was increased by decreasing Ca:P ratio (1:2). In contrast, rats fed the Ca:P = 1:0.5 diet (dietary P restriction) suppressed the increased serum parathyroid hormone, osteocalcin and urinary deoxypyridinoline, and increased Ca absorption in both sham and OVX rats compared to the Ca:P = 1:1 and 1:2 diets. Especially, in OVX rats, the decreased bone mineral density of the fifth lumbar was also suppressed when rats were fed the Ca:P = 1:0.5 diet. These results indicated that the elevation of dietary Ca:P ratio may inhibit bone loss and increase intestinal Ca absorption in OVX rats.     

Manganese Supplementation Improves Mineral Density of the Spine and Femur and Serum Osteocalcin in Rats

The effect of manganese (Mn) supplementation on bone mineral density (BMD) and bone metabolism parameters was determined in ovariectomized Sprague-Dawley rats. Rats were divided into four groups (OVX, OVX+Mn, sham, sham+Mn) and fed with different intake levels of manganese (adequate 0.001% Mn, supplementation 0.01% Mn) for 12 weeks. BMD of the lumbar vertebrae, femur, and tibia were significantly lowered in ovariectomized rats compared to the sham group. In addition, BMD of the lumbar vertebrae was significantly increased by Mn supplementation in the sham groups. Serum C-telopeptide cross-links of type I collagen (CTx), bone resorption biomarker, alkaline phosphatase (ALP), and bone formation biomarkers were not significantly different among the four groups. However, serum osteocalcin, a more sensitive bone formation biomarker, was significantly increased by Mn supplementation. To summarize, Mn supplementation resulted in increased BMD and bone formation. Based on our findings, more research is needed to better understand the effects of manganese supplementation on bone formation and resorption.     

Ovariectomy worsens secondary hyperparathyroidism in mature rats during low-Ca diet

Estrogen deficiency impairs intestinal Ca absorption and induces bone loss, but its effects on the vitamin D-endocrine system are unclear. In the present study, calciotropic hormones levels, renal vitamin D metabolism, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-dependent intestinal calcium absorption, and bone properties in 3-mo-old sham-operated (sham) or ovariectomized (OVX) rats fed either a normal-Ca (NCD; 0.6% Ca, 0.65% P) or a low-Ca (LCD; 0.1% Ca, 0.65% P) diet for 2 wk were determined. LCD increased serum 1,25(OH)2D3 levels in both sham and OVX rats. Serum parathyroid hormone [PTH(1-84)] levels were highest in OVX rats fed LCD. Renal 25-hydroxyvitamin D1alpha-hydroxylase (1-OHase) protein expression was induced in both sham and OVX rats during LCD, while renal 1-OHase mRNA expression was highest in OVX rats fed LCD. Renal vitamin D receptor (VDR) and mRNA expressions in rats were induced by ovariectomy in rats fed NCD but suppressed by ovariectomy in rats fed LCD. The induction of intestinal calcium transporter-1 and calbindin-D9k mRNA expressions by LCD were not altered by ovariectomy. As expected, bone Ca content, cancellous bone mineral density, and bone strength index in proximal metaphysis of rat tibia were reduced by both ovariectomy and LCD (P<0.05) as analyzed by two-way ANOVA. Taken together, the data demonstrate that ovariectomy alters the responses of circulating PTH levels, renal 1-OHase mRNA expression, and renal VDR expression to LCD. These results suggest that estrogen is necessary for the full adaptive response to LCD mediated by both PTH and 1,25(OH)2D3.     

雌激素受体α和β在不同雌激素干预大鼠骨代谢中的表达

应用雌性大鼠的骨质疏松模型,通过骨密度(BMD)检测、RT-PCR和Westernblot等技术观察去卵巢(Ovariectomy,OVX)、结合性雌激素(ConjugatedEquineEstrogens,CEE)和戊酸雌二醇(EstradiolValerate,EV)对大鼠松质骨骨量以及松质骨中雌激素受体(ER)α和βmRNA和蛋白表达的影响,探讨两受体亚型在介导雌激素参与松质骨代谢的不同作用机制以及不同来源雌激素对ERα和ERβ表达的差异性调节。40只7-8周龄的雌性Sprague-Dawley大鼠,在观察动情周期后随机分成四组:对照组(Control,n=10)、去卵巢组(Ovariectomy,OVX,n=10)、去卵巢后结合性雌激素治疗组(CEE,n=10)和去卵巢后戊酸雌二醇治疗组(EV,n=10)。对照组大鼠行假手术,其余三组行去卵巢手术。术后48天(12个动情周期),对照组和OVX组用生理盐水喂养12天(3个动情周期),CEE组和EV组分别用药物的生理盐水溶液喂养12天。结果显示:在对照组中,大鼠松质骨ERα的蛋白表达水平显著性高于ERβ蛋白表达水平,而ERα的mRNA表达水平显著性低于ERβmRNA水平。与对照组相比,OVX组大鼠松质骨中ERα的蛋白表达水平显著性降低,ERαmRNA表达水平显著性增加,而ERβ蛋白和mRNA的表达水平均显著性增加。与OVX组相比,CEE组大鼠松质骨中ERβ蛋白和mRNA的表达水平均显著性下降,而EV组大鼠松质骨中ERα蛋白表达显著性上升,ERαmRNA表达显著性下降,ERβ蛋白表达显著性下降。此外,OVX大鼠松质骨的骨密度下降均可通过应用CEE和EV得到显著性改善。上述结果提示:⑴ERα可能是大鼠松质骨中优势表达的受体亚型,在介导雌激素参与松质骨代谢中起着主导作用。⑵不同来源雌激素可能侧重不同的ER亚型途径产生骨保护效应。     

The effect of dehydroepiandrosterone administration on intestinal calcium absorption in ovariectomized female rats

[Purpose] Dehydroepiandrosterone (DHEA) administration reportedly recovers osteoporosis, a bone disorder associated with bone deficiency in postmenopausal women. However, the physiological mechanism of DHEA in osteoporosis remains elusive, especially in terms of intestinal calcium absorption. Therefore, we investigated the effect of DHEA administration on calcium absorption in ovariectomized (OVX) female rats using an estrogen receptor antagonist.[Methods] Female Sprague-Dawley rats (n=23, 6 weeks old) were randomized into three groups: OVX control group (OC, n=7), OVX with DHEA treatment group (OD, n=8), and OVX with DHEA inhibitor group (ODI, n=8) for 8 weeks.[Results] Intestinal calcium accumulation, as well as the rate of absorption, demonstrated no significant differences during the experimental period among investigated groups. The bone mineral density (BMD) of the tibia at the proximal metaphysis was higher in the OD group than that in the OC group (p<0.05); however, BMD of the ODI group showed no significant difference from investigated groups. Furthermore, the BMD of the tibia at the diaphysis did not significantly differ among these groups.[Conclusion] We revealed that DHEA administration does not involve intestinal Ca absorption, although this treatment improves BMD levels in OVX rats. These observations indicate that the effect of DHEA on the bone in postmenopausal women is solely due to its influence on bone metabolism and not intestinal calcium absorption.     

Bone microenvironment specific roles of ITAM adapter signaling during bone remodeling induced by acute estrogen-deficiency

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.     

运动与17β-雌二醇对去卵巢大鼠血清17β-雌二醇和后肢骨骨密度的影响

目的：观察跑台运动和17β-雌二醇（E2）对去卵巢大鼠血清E2水平和后肢骨骨密度（BMD）的影响。方法：按体重将120只成年雌性SD大鼠随机分为假手术、假手术运动、去卵巢、去卵巢运动、雌激素、雌激素加运动6个组。假手术运动、去卵巢运动和雌激素加运动组每周进行5次60min、18m／min的平坡跑台运动训练,雌激素和雌激素加运动组每周按体重颈部皮下注射3次E2,每次50μg／kg体重。分别在运动和给药正式处理7和14周时,用放射免疫法检测血清E2水平;用双能X线骨密度仪检测右侧胫骨和股骨BMD的变化。结果：运动和给药正式处理7周时,去卵巢组胫骨近端、股骨近端和远端BMD以及血清E2水平均显著低于假手术组;去卵巢运动加E2组股骨近端和远端BMD显著高于去卵巢组,E2组和去卵巢运动加E2组大鼠血清E2水平显著高于去卵巢组。运动和给药正式处理14周时,去卵巢组大鼠胫骨近端、股骨近端和远端BMD以及血清E2水平均显著低于假手术组,假手术运动组股骨近端BMD显著高于假手术组;去卵巢运动组、E2组和去卵巢运动加E2组大鼠血清E2水平显著高于去卵巢组,去卵巢运动组股骨远端BMD显著高于去卵巢组,E2组和去卵巢运动加E2组胫骨近端、股骨近端和远端BMD均显著高于去卵巢组;去卵巢运动组大鼠胫骨近端BMD和血清E2水平显著低于去卵巢运动加E2组,E2组胫骨近端BMD显著高于去卵巢运动加E2组。结论：E2和较高中等强度跑台运动对去卵巢大鼠股骨和胫骨松质骨骨量减缓的效应是独立的。     

Effects of Astragalus membranaceus with Supplemental Calcium on Bone Mineral Density and Bone Metabolism in Calcium-Deficient Ovariectomized Rats

It has been reported that Astragalus membranaceus, an Asian traditional herb, has an estrogenic effect in vitro. To examine the possible role of A. membranaceus extract with supplemental calcium (Ca) on bone status in calcium-deficient (LCa) ovariectomized (OVX) rats, a total of 48 female rats were divided into six groups: (1) normal control, (2) sham operation with LCa (sham-LCa), (3) OVX with LCa (OVX-LCa), (4) A. membranaceus supplementation with OVX-LCa (OVX-MLCa), (5) Ca supplementation with OVX (OVX-Ca), and (6) A. membranaceus and Ca supplementation with OVX (OVX-MCa). A. membranaceus ethanol extract (500 mg/kg BW) and/or Ca (800 mg/kg BW) were administered orally for 8 weeks along with a Ca-deficient diet. Results revealed that Ca supplementation with or without A. membranaceus extract significantly improved bone mineral density, biomechanical strength, and ash weight of the femur and tibia in OVX rats. High Ca with A. membranaceus combination supplementation significantly increased the ash weight of the femur and tibia and decreased urinary Ca excretion compared with supplementation of Ca alone. Uterine weight was not changed by A. membranaceus administration in OVX rats. These results suggest that A. membranaceus extract combined with supplemental Ca may be more protective against the Ca loss of bone than A. membranaceus or supplementation of Ca alone in calcium-insufficient postmenopausal women.     

Dietary ratio of n-6/n-3 PUFAs and docosahexaenoic acid: actions on bone mineral and serum biomarkers in ovariectomized rats

Hypoestrogenic states escalate bone loss in animals and humans. This study evaluated the effects of the amount and ratio of dietary n-6 and n-3 polyunsaturated fatty acids (PUFAs) on bone mineral in 3-month-old sexually mature ovariectomized (OVX) Sprague-Dawley rats. For 12 weeks, the rats were fed either a high-PUFA (HP) or a low-PUFA (LP) diet with a ratio of n-6/n-3 PUFAs of 5:1 (HP5 and LP5) or 10:1 (HP10 and LP10). All diets (modified AIN-93G) provided 110.4 g/kg of fat from safflower oil and/or high-oleate safflower oil blended with n-3 PUFAs (DHASCO oil) as a source of docosahexaenoic acid (DHA). Fatty acid analyses confirmed that the dietary ratio of 5:1 significantly elevated the amount of DHA in the periosteum, marrow and cortical and trabecular bones of the femur. Dual-energy X-ray absorptiometry measurements for femur and tibia bone mineral content (BMC) and bone mineral density showed that the DHA-rich diets (HP5 and LP5) resulted in a significantly lower bone loss among the OVX rats at 12 weeks. Rats fed the LP diets displayed the lowest overall serum concentrations of the bone resorption biomarkers pyridinoline (Pyd) and deoxypyridinoline, whereas the bone formation marker osteocalcin was lowest in the HP groups. Regardless of the dietary PUFA content, DHA in the 5:1 diets (HP5 and LP5) preserved rat femur BMC in the absence of estrogen. This study indicates that the dietary ratio of n-6/n-3 PUFAs (LP5 and HP5) and bone tissue concentration of total long-chain n-3 PUFAs (DHA) minimize femur bone loss as evidenced by a higher BMC in OVX rats. These findings show that dietary DHA lowers the ratio of 18:2n-6 (linoleic acid)/n-3 in bone compartments and that this ratio in tissue correlates with reduced Pyd but higher bone alkaline phosphatase activity and BMC values that favor bone conservation in OVX rats.