骨质疏松兔松质骨微观结构和微观成分的时间序贯性变化

目的:研究去势手术建立骨质疏松兔模型中松质骨微观结构和微观成分的时间序贯性变化。方法:40只新西兰白兔随机分为假手术组(sham组,n=20)和骨质疏松组(OP组,n=20)。OP组兔子给予去势手术处理,sham组给予假手术处理。分别于术后的0周、4周、6周、8周,利用DXA测量腰椎骨密度(每组每个时间点选择5只动物)。之后处死动物,采集腰椎标本。利用Micro-CT、FTIR、腰椎轴向压缩试验得到松质骨的微观结构、微观成分(骨矿盐晶体和胶原)和宏观力学参数。利用t检验比较同一时间点两组之间的相关参数。结果:OP组BMD逐渐下降,松质骨微观结构逐渐疏松,微观组成属性逐渐改变,宏观力学强度均逐渐下降。FTIR在4周时即检测到OP组腰椎骨矿盐和胶原基质比(P=0.046)、骨矿盐结晶度(P=0.018)、胶原交联比(P=0.006)发生显著性改变,早于BMD和微观结构的变化。OP组腰椎宏观生物力学强度在第8周时达到最低点(P=0.001)。结论:去势手术后,腰椎松质骨骨矿盐晶体和胶原属性最早发生变化,松质骨微观成分和微观结构的改变是导致椎体强度明显改变的原因。FTIR技术可以较早的检测到骨质疏松发生过程中骨组织微观成分的改变。     

Sex steroid hormones do not influence the oxidative burst activity of polymorphonuclear leukocytes from ovariectomized cows in vitro

During the periparturient period, dairy cows are subjected to physiological changes that may induce immunosuppression and an increased susceptibility of the animal to bacterial infections such as mastitis. The incidence of clinical environmental mastitis is high during the last period of gestation, at parturition and during the first month of lactation, suggesting a potential influence of sex steroid hormones. Efficient functioning of polymorphonuclear leukocytes (PMN) is necessary during the early phase of infection to clear the mammary gland from invading pathogens. The purpose of this study was to investigate the effect of sex steroid hormones on the oxidative burst activity of isolated PMN from ovariectomized cows. Ovariectomy was performed to minimize the interference of endogenous estrogen and progesterone levels, which are known to vary extensively during the estrus cycle. Isolated PMN were incubated with different concentrations of 17beta-estradiol, estrone or progesterone. A flow cytometric technique was used to quantify the oxidation of intracellular 2',7'-dichlorofluorescin by the oxidative burst system of PMN following stimulation with phorbol myristate acetate. Staurosporine was used as a positive control for our in vitro model. No statistically significant changes in PMN oxidative burst activity were observed at physiological or pharmacological levels of the three sex steroid hormones. A large variation existed in the oxidative burst activity among cows. In an additional experiment, the expression of estrogen receptor alpha and of progesterone receptor in PMN was evaluated immunohistochemically. No specific staining was detected for both receptors in isolated PMN following incubation with different concentrations of sex steroid hormones.     

Proteome analysis for the identification of in vivo estrogen-regulated proteins in bone

Estrogen deficiency results in a reduced bone mass, which can be prevented by treatment with estrogens. This study used a proteomic approach for the first time to obtain a global perspective of estrogens' effects on whole-bone proteins. Bone proteome profiles were examined in three groups of mice: (1) sham-operated with normal ovarian functions, (2) ovariectomised and (3) ovariectomised with estrogen replacement therapy. Bone proteins extracted from the humerus were separated by 2-DE and visualised by CBB colloidal staining. Spot detection and quantification was done by image analysis. Differentially expressed proteins were identified by MS and database search, using peptide mass fingerprint and peptide sequence analysis. Differential expression analysis in the three experimental groups showed significant changes for 14 proteins. These included proteins related to bone metabolism, cytoskeleton components and energy metabolic pathways. Our data suggest that some proteins related to cytoskeleton and to energy pathways, such as tropomyosins, aconitase 2 and enolase beta, might be new molecular targets responsive to the effects of estrogen. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the pleiotropic effects of estrogens on bone.     

Estrogen alters baseline and inflammatory-induced cytokine levels independent from hypothalamic–pituitary–adrenal axis activity

Although estrogen reduces inflammatory-mediated pain responses, the mechanisms behind its effects are unclear. This study investigated if estrogen modulates inflammatory signaling by reducing baseline or inflammation-induced cytokine levels in the injury-site, serum, dorsal root ganglia (DRG) and/or spinal cord. We further tested whether estrogen effects on cytokine levels are in part mediated through hypothalamic–pituitary–adrenal (HPA) axis activation. Lumbar DRG, spinal cord, serum, and hind paw tissue were analyzed for cytokine levels in 17β-estradiol-(20%) or vehicle-(100% cholesterol) treated female rats following ovariectomy/sham adrenalectomy (OVX), adrenalectomy/sham ovariectomy (ADX) or ADX + OVX operation at baseline and post formalin injection. Formalin significantly increased pro-inflammatory interleukin (IL)-6 levels in the paw, as well as pro- and anti-inflammatory cytokine levels in the DRG, spinal cord and serum in comparison to naïve conditions. Estrogen replacement significantly increased anti-inflammatory IL-10 levels in the DRG. Centrally, estradiol significantly decreased pro-inflammatory tumor necrosis factor (TNF)-α and IL-1β levels, as well as IL-10 levels, in the spinal cord in comparison to cholesterol treatment. At both sites, most estradiol modulatory effects occurred irrespective of pain or surgical condition. Estradiol alone had no influence on cytokine release in the paw or serum, indicating that estrogen effects were site-specific. Although cytokine levels were altered between surgical conditions at baseline and following formalin administration, ADX operation did not significantly reverse estradiol’s modulation of cytokine levels. These results suggest that estrogen directly regulates cytokines independent of HPA axis activity in vivo, in part by reducing cytokine levels in the spinal cord.     

卵巢激素撤除诱发雌性小鼠抑郁样状态（英文）

目的：建立卵巢激素撤除诱发雌性小鼠抑郁样状态模型,并且探讨其可能的神经化学机制。方法：将小鼠分为假手术组,卵巢摘除组,己烯雌酚治疗组和氟西汀治疗组。动物行卵巢摘除术后开始给药,术后两周进行强迫游泳试验及悬尾试验以考察其抑郁样状态,并利用高效液相结合电化学检测测定下丘脑及海马中去甲肾上腺素（NE）、多巴胺（DA）以及五羟色胺（5-HT）的含量。结果：在强迫游泳试验及悬尾试验中,卵巢摘除小鼠较假手术组小鼠不动时间显著延长。神经递质测定显示,卵巢摘除小鼠下丘脑中NE与DA的含量显著降低,海马中NE与5-HT的含量显著降低。给予己烯雌酚或氟西汀治疗对抗了卵巢摘除所诱导的抑郁样状态,并且缓解了神经递质水平的下降。结论：双侧卵巢摘除诱发的小鼠抑郁样状态可以模拟妇女更年期抑郁的某些症状。本研究对于探讨卵巢激素撤除诱发抑郁状态的神经生化机制可能具有重要的意义。     

The effects of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced bone loss

Flavonoids, a group of polyphenolic compounds abundant in plants, are known to prevent bone loss in ovariectomized (OVX) animal models. Inhibition of osteoclast differentiation and bone resorption is considered as an effective therapeutic approach in the treatment of postmenopausal bone loss. Luteolin, a plant flavonoid, has potent anti-inflammatory properties both in vivo and vitro. In this study, we found that luteolin markedly decreased the differentiation of both bone marrow mononuclear cells and Raw264.7 cells into osteoclasts. Luteolin also inhibited the bone resorptive activity of differentiated osteoclasts. We further investigated the effects of luteolin on ovariectomy-induced bone loss using micro-computed tomography, biomechanical tests and serum markers assay for bone remodeling. Oral administration of luteolin (5 and 20 mg/kg per day) to OVX mice caused significant increase in bone mineral density and bone mineral content of trabecular and cortical bones in the femur as compared to those of OVX controls, and prevented decreases of bone strength indexes induced by OVX surgery. Serum biochemical markers assays revealed that luteolin prevents OVX-induced increases in bone turnover. These data strongly suggest that luteolin has the potential for prevention of bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.     

Involvement of PRIP, phospholipase C-related, but catalytically inactive protein, in bone formation

PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.     

The effects of ovariectomy on binge eating proneness in adult female rats

Ovarian hormones are associated with binge eating in women, however findings are limited by the lack of experimental control inherent in human studies. Animal research that manipulates ovarian hormone status and examines individual differences in extreme binge eating proneness is needed to model clinical phenotypes in humans and to confirm causal effects. The purpose of this study was to examine the effects of adult ovariectomy on overall binge eating risk and extreme binge eating phenotypes using the binge eating resistant (BER)/binge eating prone (BEP) rat model. We predicted that palatable food consumption would significantly increase after ovariectomy in all rats because ovarian hormones generally suppress food intake. If differences in responsiveness to ovarian hormones underlie BER/BEP phenotypes, then differences in binge eating between BER and BEP rats would be eliminated or diminished after ovariectomy. Changes in palatable food (PF) intake were compared in BER and BEP rats before and after ovariectomy in two samples of adult females. Findings were highly similar in the two samples. PF intake increased significantly following ovariectomy in all rats. However, BEP rats consistently consumed larger amounts of PF than BER rats, both before and after ovariectomy. The consistency of findings across two samples of rats provides strong support for activational effects of ovarian hormones on binge eating. However, the immunity of extreme binge eating phenotypes to ovarian hormone ablation suggests that other, earlier mechanisms (e.g., organizational hormone effects or hormone-independent effects) determine the expression of binge eating phenotypes.     

Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells

Parity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24(+)/CD49f(high) mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland.     

Ovariectomy increases neuronal amyloid-beta binding alcohol dehydrogenase level in the mouse hippocampus

Ovarian hormone decline after menopause may influence cognitive performance and increase the risk for Alzheimer's disease (AD) in women. Amyloid-β peptide (Aβ) has been proposed to be the primary cause of AD. In this study, we examined whether ovariectomy (OVX) could affect the levels of cofactors Aβ-binding alcohol dehydrogenase (ABAD) and receptor for advanced glycation endproducts (RAGE), which have been reported to potentiate Aβ-mediated neuronal perturbation, in mouse hippocampus, correlating with estrogen and Aβ levels. Female ICR mice were randomly divided into ovariectomized or sham-operated groups, and biochemical analyses were carried out at 5 weeks after the operation. OVX for 5 weeks significantly decreased hippocampal 17β-estradiol level, while it tended to reduce the hormone level in serum, compared with the sham-operated control. In contrast, OVX did not affect hippocampal Aβ_1-40 level, although it significantly increased serum Aβ_1-40 level. Furthermore, we demonstrated that OVX increased hippocampal ABAD level in neurons, but not astrocytes, while it did not affect RAGE level. These findings suggest that the expression of neuronal ABAD depends on estrogen level in the hippocampus and the increase in serum Aβ and hippocampal ABAD induced by ovarian hormone decline may be associated with pre-stage of memory deficit in postmenopausal women and Aβ-mediated AD pathology.