The Effects of Castration and Hormone Replacement on the Cross‐Sectional Area of Pubococcygeus Muscle Fibers in the Female Rat

In this study, we analyzed the effect of ovariectomy and gonadal hormone replacement on the cross‐sectional area of pubococcygeus (Pcm) fibers. It was found that in comparison to intact animals, ovariectomized animals [for 2 or 6 weeks] had an increased cross‐sectional area average in Pcm fibers. Ovariectomy also reduced the percentage of fibers with smaller cross‐sectional area. In ovariectomized animals after 4 weeks of hormone replacement with an empty Silastic capsule or filled with testosterone propionate or dihydrotestosterone, significantly increased the cross‐sectional area average and the percentage of fibers with larger size. However, 17β‐estradiol but not estradiol benzoate treatment reduced the cross‐sectional area average and increased the percentage of Pcm fibers with smaller size. Progesterone did not have an effect on the cross‐sectional area of this muscle. We conclude that Pcm fibers of female rats are sensitive to gonadal hormones, and contrary to male castration, ovariectomy promotes an increase in their cross‐sectional area. Also, we discuss according to other studies that an external mechanism which lies within the neuromuscular periphery could also participate in the modulatory hormonal effect on mass or muscle fiber size. Furthermore, in this process, estradiol is likely to regulate the fiber cross‐sectional area growing produced by androgens. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Chronic intermittent hypoxia aggravates cardiomyocyte apoptosis in rat ovariectomized model

Background  The prevalence of obstructive sleep apnea (OSA) increases after menopause in women, but remains under diagnosed because of social or lifestyle factors. It is important to evaluate the hazards of OSA on cardiovascular disease in menopausal women. We tested the hypothesis that chronic intermittent hypoxia (CIH) may aggravate cardiomyocyte apoptosis in ovariectomized (OVX) Sprague Dawley (SD) rats; the changes of anti-oxidation ability in cardiac muscles may be one of the reasons for cardiomyocyte apoptosis.

Methods  Forty-eight 60-day old female SD rats were randomly divided into a CIH group, OVX group, OVX+CIH (OC) group, and handled control (HC) group, and the rats were exposed either to CIH (nadir O₂ 6%) or handled normoxic controls. The changes of body weight and whole heart weight were measured. Super oxide dismutase (SOD) and malonaldehyde (MDA) were used to evaluate the level of oxidative stress. TdT-mediated dUTP nick end labeling (TUNEL) was used to measure apoptosis in each rat. Western blotting was used to measure apoptosis associated proteins in cardiac muscle samples from each rat.

Results  When compared with the HC and CIH groups, the levels of oxidative stress in the OC and OVX groups were significantly higher. The levels of SOD in the HC, CIH, OC, and OVX groups were (47.99±4.89), (53.60±4.47), (20.99±2.72), and (30.64±3.79) mmol/mg protein; significantly increased in the CIH group (P <0.05) and significantly decreased in the OC (P <0.01) and OVX (P <0.05) groups. The levels of MDA in the HC, CIH, OVX, and OC groups were (1.63±0.20), (1.93±0.77), (3.30±0.39), and (1.95±0.20) mmol/mg protein; it significantly increased in the CIH (P <0.05), OC (P <0.01), and OVX (P <0.05) groups compared with the HC group. Bax protein expression was significantly increased and bcl-2 protein expression was significantly reduced after CIH compared with HC rats (P <0.05). The protein expression of bax and bcl-2 in the OC group was not significantly different from the CIH group, but the ratio of bax/bcl-2 was significantly increased in the OC group (P <0.05); this was associated with severe cardiomycyte apoptosis in the OC group. TUNEL confirmed this observation.

Conclusions  This study found that CIH may induce oxidative stress in OVX rats but not in CIH rats, and cause more severe cardiomyocyte apoptosis in OVX rats compared with CIH rats. This means that OVX rats exposed to CIH suffered more severe cardiac injury compared with CIH rats due to reduced antioxidation. These findings may partly explain the reason why OSA has a worse cardiovascular impact on menopausal women, and emphasize the importance of detection and early treatment of OSA in menopausal patients.

     

Effect of prolonged use of high dose of tibolone on the vagina of ovariectomized rats

The aim of this study was evaluate the effect of prolonged use of high dose of tibolone on the vagina of ovariectomized rats. Bilateral ovariectomy was performed on 14 rats weighing 250 g. Thirty days later, vaginal smears were collected verifying the menopause status by anoestrus cytology. Rats were divided randomly into groups: experimental rats (n = 9) received 1 mg tibolone/day orally and control rats (n = 6) received placebo (carboxymethylcellulose). After 150 days, all rats were sedated and euthanized by cervical displacement. The vagina was removed, fixed in 10% buffered formalin, sampled and processed for paraffin embedding. Histological sections were stained with haematoxylin and eosin, picrosirius red, periodic acid Schiff (PAS) and PAS-diastase, and Weigert's resorcin-fuchsin. Cell proliferation was analysed by immunohistochemistry to detect Ki67. Histomorphometric analyses were performed for epithelial thickness, per cent area of collagen fibres and blood vessels, mast cells and Ki67-positive nuclei per mm of basal membrane. Means and standard error of means were calculated, and data were compared using the Mann-Whitney test, with significance level at P < 0.05. In the vagina, epithelial thickness, number of Ki67-positive nuclei per mm of basal membrane, number of vessels and number of mast cells were significantly higher in the tibolone group when compared with the control group. Furthermore, the content of glycogen and glycoproteins in the vaginal epithelium was modified by tibolone. Tibolone administered in high dose and for a long period has a trophic effect, reversing vaginal atrophy, and has no dysplastic or neoplastic effect in the vagina of ovariectomized rats.     

芍药苷促进小鼠成骨分化抗骨质疏松作用的实验研究

目的 探讨芍药苷对MC3T3-E1成骨细胞分化以及小鼠骨质疏松模型的影响。 方法 体外细胞实验分为对照组、不同剂量芍药苷干预组。通过CCK-8法检测芍药苷对MC3T3-E1成骨细胞活力的影响;采用碱性磷酸酶（ALP）染色以及活性检测芍药苷促进MC3T3-E1成骨分化能力;通过茜素红染色检测芍药苷促矿化能力;运用荧光定量PCR、Western blot检测Runx2、OPG、RANKL、Col1α1的mRNA以及蛋白表达情况。选取8周龄C57BL/6小鼠24只,分为假手术组、骨质疏松模型组、药物干预组。选取各小鼠左侧股骨远端以及胫骨近端的区域进行苏木素-伊红（HE）染色、Runx2免疫组织化学以及micro-CT扫描。 结果 中、高剂量芍药苷干预组可以促进MC3T3-E1细胞ALP的活性（P<0.05）;高剂量芍药苷能够促进MC3T3-E1细胞的矿化能力（P<0.01）;同时中、高剂量能够促进OPG、Runx2蛋白的表达（P<0.05）,抑制RANKL的表达（P<0.05）。与假手术组比较,OVX组骨微结构破坏明显,Runx2蛋白表达显著减少（P<0.01）;芍药苷干预后骨质疏松模型小鼠骨小梁数量以及厚度显著增加（P<0.01）,骨小梁间隔减少明显（P<0.01）,Runx2蛋白提升明显（P<0.01）。 结论 芍药苷能够促进成骨细胞的分化,上调成骨分化基因,改善骨质疏松模型小鼠的骨微结构,具有抗骨质疏松治疗的潜在价值。     

辛伐他汀延缓生酮饮食导致的骨量丢失的实验观察

目的分析辛伐他汀(ST)治疗后小鼠股骨骨微结构、力学性能及骨形态的改变,评价ST对缓解生酮饮食引起的骨量丢失的治疗作用。方法 40只C57小鼠随机分为假手术组、卵巢切除组(OVX)、卵巢切除加辛伐他汀组(OVX+ST)、生酮饮食组(KD)及生酮饮食加辛伐他汀组(KD+ST),每组8只。OVX和OVX+ST组小鼠行双侧卵巢切除,予以常规标准饮食。KD及KD+ST组予以碳水化合物与脂肪比为1∶3的生酮饮食,OVX+ST及KD+ST组予以ST灌胃,剂量为20 mg/(kg·d)。12周处死取材,测量各组小鼠血酮、血糖、血钙、血磷等的水平,分别采用显微CT扫描、三点弯曲试验、有限元分析及HE染色观察股骨骨量微结构、力学性能及骨形态。结果 KD组和KD+ST组小鼠血酮含量为1. 28 mmol/L和1. 48 mmol/L,明显高于假手术组(0. 60 mmol/L)。各组间血糖、血钙或血磷的差异没有统计学意义。辛伐他汀治疗提高了KD+ST组的松质骨骨量、股骨有限元压缩刚度及三点弯曲刚度,其中KD+ST组较KD组股骨远端松质骨骨体积分数从4. 1%上升到6. 6%,有限元压缩刚度从35 N/mm提高到161 N/mm,股骨三点弯曲刚度从44 N/mm提高到60 N/mm,组织学观察也表明KD+ST组的股骨远端松质骨骨小梁较KD组明显增多。结论辛伐他汀治疗提高了KD组小鼠股骨松质骨骨体积分数、三点弯曲刚度、有限元压缩刚度和骨小梁数目,缓解了生酮饮食导致的骨量丢失。     

淫羊藿醇提物对去卵巢大鼠骨质疏松治疗作用及机制研究

目的评价淫羊藿醇提物对去除卵巢致骨质疏松大鼠的治疗作用,初步探讨其作用机制。方法 SD大鼠切除双侧卵巢,造成骨质疏松大鼠模型。手术4周后,以阳性药依普黄酮片(200 mg/kg)和不同剂量的淫羊藿醇提物(170、85 mg/kg)连续ig给药8周,然后取血清测定其碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(StrACP)活性;采用免疫组化法测定股骨中骨保护素(OPG)、破骨细胞分化因子(RANKL)蛋白表达;采用三点弯曲法测定其胫骨的生物力学性质;最后运用micro-CT对股骨远端的骨小梁参数进行分析,并对其进行三维重建,得到骨小梁的3D图形来直观地说明淫羊藿醇提物对骨质疏松大鼠的作用。结果淫羊藿醇提物高剂量组不仅能够显著降低血清ALP和StrACP活性,还能显著增加OPG蛋白表达,降低RANKL蛋白表达,且能有效地改善胫骨生物力学性质和股骨骨小梁参数。结论淫羊藿醇提物高剂量(170 mg/kg)对骨质疏松大鼠具有明显的治疗作用,其可能是通过促进成骨细胞活性、抑制破骨细胞活性来实现的。     

淫羊藿对去卵巢大鼠骨质疏松的影响

目的: 研究淫羊藿防治去卵巢大鼠骨质疏松症的作用及初步机制。 方法: 雌性7月龄SD大鼠分为正常组,模型组和淫羊藿高、低剂量组共4组(n=8),除正常组行假手术外,其余均手术彻底摘除卵巢,术后进行模型筛选,1周后依照正常组、模型组灌胃生理盐水,淫羊藿高、低剂量组灌胃淫羊藿提取液(剂量以生药量计为50,25 g·kg^-1),连续给药90 d。给药45 d时检测血清钙(Ca²⁺),90 d时检测血清Ca²⁺、血清磷(P)、超氧化物歧化酶(SOD)、丙二醛(MDA)、碱性磷酸酶(ALP)活性、血清雌二醇(E₂)和睾酮(T)含量,并分离胫骨进行病理切片观察。 结果: 与模型组血清Ca²⁺(3.01±0.33) mmol·L^-1相比,给药45 d后低、高剂量淫羊藿组血清Ca²⁺均下降[(2.48±0.39),(2.86±0.34)mmol·L^-1,(P<0.05,P<0.01)],90 d后高剂量淫羊藿组血清Ca²⁺下降更明显(1.76±0.53)mmol·L^-1,(P<0.01)。与模型组相比,淫羊藿能降低血清MDA(8.4±1.3)mmol·L^-1(P<0.01),提高血清P(4.61±0.82)mmol·L^-1(P<0.05),SOD活性(110±13)U·L^-1(P<0.05),碱性磷酸酶活力(4.4±0.89)U·L^-1(P<0.05),雌二醇水平(25.66±9.38) pg·L^-1(P<0.05)和睾酮水平(1.95±1.21) ng·L^-1(P<0.05)。胫骨病理切片显示高剂量淫羊藿能预防骨小梁断裂,Ⅰ级和Ⅱ级病变率占87.5%,无Ⅳ级病变。 结论: 淫羊藿明显提高性激素水平,清除活性氧,可能是其防治骨质疏松症的主要机制。     

去卵巢后雌性大鼠下丘脑视前区NOS阳性神经元形态学和突触素表达的变化

目的：为解释围绝经期的精神、神经症状提供实验依据。方法：采用免疫细胞化学方法显示去卵巢雌性大鼠下丘脑视前内侧区(MPA)和视前外侧区(LPA)内NOS阳性神经元,形态学改变以及突触素表达。结果：(1)去卵巢组和对照组大鼠的视前内、外区内可见大量NOS阳性神经元;(2)去卵巢动物的下丘脑内NOS阳性神经元突起长度比对照组的明显变短,分支明显变少;(3)去卵巢组动物下丘脑内突触素表达比对照组的明显减少。结论：大鼠去卵巢后,由于雌激素分泌减少,导致了NOS阳性神经元的突起减少和突触减少,结果NO介导的神经元信号传导失常,这些变化可能是引发围绝经期的精神、神经症状的原因之一。