补肾活血汤对去卵巢大鼠血管舒缩功能及血清E_2的影响

目的:观察补肾活血汤(bushen huoxue decoction,BHD)对去卵巢大鼠血清雌二醇(estradiol,E2)和血管舒缩功能的影响。方法:56只成年雌性SD大鼠随机分为7组:空白组、假手术组、模型组、去卵巢+BHD高、中、低剂量组及去卵巢+替勃龙组(替勃龙组),每组8只。采用免疫法测定实验大鼠血清E2、促黄体激素(luteinizing hormone,LH)和促卵泡激素(follicle-stimulating hormone,FSH)的含量。观察补肾活血汤对给予去甲肾上腺素(noradrenaline,NE)和乙酰胆碱(acetylcholine,Ach)后的离体大鼠腹主动脉血管环产生舒缩的活性的影响,经HE染色后,用光镜观察腹主动脉结构的变化。结果:与模型组相比,BHD各剂量组可显著升高模型大鼠血清E2的水平(P0.05),显著降低LH和FSH的水平(P0.05),使模型大鼠腹主动脉对缩血管物质的反应降低,加NE后模型组血管张力增加显著(P0.05),血管顺应性较好,舒缩功能得到改善,BHD高剂量组优于BHD中、低剂量组。血管中平滑肌细胞较模型组清晰,组织结构排列整齐、紧密,与替勃龙组无显著差异。结论:BHD可使腹主动脉对缩血管物质NE的反应减轻,血管顺应性增强,血管舒缩功能得到改善,血管结构的破坏程度减轻,其机制可能与增加的E2含量有关。     

Sex differences in circadian timing systems: Implications for disease

Virtually every eukaryotic cell has an endogenous circadian clock and a biological sex. These cell-based clocks have been conceptualized as oscillators whose phase can be reset by internal signals such as hormones, and external cues such as light. The present review highlights the inter-relationship between circadian clocks and sex differences. In mammals, the suprachiasmatic nucleus (SCN) serves as a master clock synchronizing the phase of clocks throughout the body. Gonadal steroid receptors are expressed in almost every site that receives direct SCN input. Here we review sex differences in the circadian timing system in the hypothalamic–pituitary–gonadal axis (HPG), the hypothalamic–adrenal–pituitary (HPA) axis, and sleep–arousal systems. We also point to ways in which disruption of circadian rhythms within these systems differs in the sexes and is associated with dysfunction and disease. Understanding sex differentiated circadian timing systems can lead to improved treatment strategies for these conditions.     

Cryptorchidism and endocrine disrupting chemicals

Prospective clinical studies have suggested that the rate of congenital cryptorchidism has increased since the 1950s. It has been hypothesized that this may be related to environmental factors. Testicular descent occurs in two phases controlled by Leydig cell-derived hormones insulin-like peptide 3 (INSL3) and testosterone. Disorders in fetal androgen production/action or suppression of Insl3 are mechanisms causing cryptorchidism in rodents. In humans, prenatal exposure to potent estrogen diethylstilbestrol (DES) has been associated with increased risk of cryptorchidism. In addition, epidemiological studies have suggested that exposure to pesticides may also be associated with cryptorchidism. Some case-control studies analyzing environmental chemical levels in maternal breast milk samples have reported associations between cryptorchidism and chemical levels. Furthermore, it has been suggested that exposure levels of some chemicals may be associated with infant reproductive hormone levels.     

Effect of Extended 30 μg Ethinyl Estradiol with Continuous Low-Dose Ethinyl Estradiol and Cyclic 20 μg Ethinyl Estradiol Oral Contraception on Adolescent Bone Density: A Randomized Trial

Study ObjectiveTo compare changes in lumbar spine bone mineral density after 12 months of a 91-day extended regimen or 28-day combined oral contraceptive with those in a healthy reference group not using hormonal contraceptives.DesignPhase 2, multicenter, open-label, randomized, controlled study.SettingForty-five academic centers, clinical research centers, and community practices in the United States.ParticipantsEight hundred twenty-nine postmenarcheal adolescent girls aged 12-18 years.InterventionsAdolescents were randomly assigned to 91-day levonorgestrel (LNG)/ethinyl estradiol (EE) extended regimen (84 days of LNG 150 μg/EE 30 μg with 7 days of EE 10 μg [LNG/EE extended regimen]) or 28 days of LNG/EE (21 days of LNG 100 μg/EE 20 μg with 7 days of placebo [LNG/EE 21/7]) for 12 months. A reference group not seeking hormonal contraception was also evaluated.Main Outcome MeasuresThe primary end point was mean percent change in lumbar spine bone mineral density measured using dual-energy x-ray absorptiometry.ResultsOf 1361 adolescents randomized/enrolled, 829 were included in the primary analysis. Mean changes in lumbar spine bone mineral density were +2.26% with LNG/EE extended regimen, +1.45% with LNG/EE 21/7, and +2.50% in the reference group. Noninferiority of the LNG/EE extended regimen compared with the reference group was shown. A statistically significant treatment difference was found between LNG/EE 21/7 and the reference group (1.05%; 95% confidence interval, 0.61%-1.49%) but not between LNG/EE extended regimen and the reference group (0.23%; 95% confidence interval, −0.20% to 0.67%). No new safety signals were noted.ConclusionCompared with the reference group, bone accrual was statistically significantly lower among LNG/EE 21/7 users but not among LNG/EE 30-μg extended regimen users. Additional research is needed to clarify the clinical relevance of these findings.     

Endocrine disrupting chemicals, 4‐nonylphenol,bisphenol A and butyl benzyl phthalate,impair metabolism of estradiol in male and female rats as assessed by levels of 15α‐hydroxyestrogens and catechol estrogens in urine

Bisphenol A (BPA), 4‐nonylphenol (NP) and butyl benzyl phthalate (BBP), termed endocrine‐disrupting chemicals, are known to mimic estrogen activity. The effects of these chemicals on 17β‐estradiol (E₂) metabolism in vivo in rats were examined. Male and female rats were given NP (250 mg kg^–1 day^–1), BPA (250 μg kg^–1 day^–1) or BBP (500 mg kg^–1 day^–1) by gavage for 14 days, followed by a single intraperitoneal injection of E₂ (5 mg kg^–1) on the final day. The urinary excretion over 72 hours of 2‐hydroxyestrone 1‐N‐acetylcysteine thioether, 2‐hydroxyestrone 4‐N‐acetylcysteine thioether, 4‐hydroxyestrone 2‐N‐acetylcysteine thioether, 2‐hydroxy‐17β‐estradiol (2‐OHE₂), 2‐hydroxyestrone (2‐OHE₁), 4‐hydroxy‐17β‐estradiol, 4‐hydroxyestrone, 15α‐hydroxyestriol (E₄), 15α‐hydroxy‐17β‐estradiol and 15α‐hydroxyestrone was measured. Increases in urinary excretion of 2‐OHE₁ and decreases in E₄ were observed in males treated with NP or BBP. Decreases in urinary excretion of 2‐OHE₂ and E₄ were observed in males treated with BPA. Decreases in urinary excretion of 2‐OHE₁ and 2‐OHE₂ were observed in females treated with BBP. Normalized liver and weights were increased in both sexes treated with NP or BBP. Histologic observations revealed marked changes in the distal tubules and collecting ducts in the kidneys of rats exposed to NP and BBP, and hypertrophy in the hepatocytes of the centrilobular zone of the liver. No BPA‐related effects on organ weight and on liver or kidney histopathology were found. These results suggest that the 14 day oral dosing of NP and BBP disrupted E₂ metabolism, resulting from marked morphological and functional alterations in the liver and kidneys. In addition, BPA could induce metabolic and endocrine disruption.     

Standardization of plasma vitellogenin level for surveying environmental estrogen pollution using the Japanese common goby Acanthogobius flavimanus

Field surveys of the impact of environmental estrogen (EE) pollution in aquatic wildlife have been conducted using vitellogenin (VTG) as a biomarker to evaluate the influence of EE. However, a standard baseline of VTG level that can be used to evaluate EE pollution has not been fully determined. To the best of our knowledge, the present study is the first to determine the standard baseline VTG level for evaluating the biological effects of EE pollution using the Japanese common goby (Acanthogobius flavimanus) as the target model fish. Plasma VTG and estradiol‐17β (E₂) levels associated with the reproductive cycle of wild goby inhabiting an unpolluted environment were measured. Mean plasma VTG and E₂ levels exhibited similar changes, increasing in the yolk vesicle stage and peaking in the tertiary yolk stage in females. However, plasma VTG and E₂ levels showed no significant changes in males, remaining at low levels throughout the reproductive cycle. The highest VTG levels in females and males were 1.6 mg ml^–1 and 124.87 ng ml^–1, respectively. These results indicate that the baseline level (normal level) in males was approximately 130 ng ml^–1 at most. We concluded that the threshold between normal and abnormal levels with a 10% risk rate was 150 ng ml^–1 in the wild male goby. Plasma VTG levels in males captured from Nagasaki Harbor were higher than the threshold in each reproductive developmental stage, indicating the possibility of EE pollution at this site. The biological standard baseline for VTG established in this study is useful for assessing EE pollution in natural waters.     

Fenofibrate induced PPAR alpha expression was attenuated by oestrogen receptor alpha overexpression in Hep3B cells

The physiological regulation of Oestrogen receptor α (ERα) and peroxisome proliferator‐activated receptor alpha (PPARα) in Hepatocellular carcinoma (HCC) remains unknown. The present study we first treat the cells with fenofibrate and further investigated the possible mechanisms of 17β‐estradiol (E₂) and/or ERα on regulating PPARα expression. We also found higher PPARα expression in the tumor area than adjacent areas and subsequently compared PPARα expression in four different hepatic cancer cell lines. Hep3B cells were found to express more PPARα than the other cell lines. Using the PPARα agonist fenofibrate, we found that fenofibrate increased Hep3B cell proliferation efficiency by increasing cell cycle proteins, such as cyclin D1 and PCNA, and inhibiting p27 and caspase 3 expressions. Next, we performed transient transfections and immuno‐precipitation studies using the pTRE2/ERα plasmid to evaluate the interaction between ERα and PPARα. ERα interacted directly with PPARα and negatively regulated its function. Moreover, in Tet‐on ERα over‐expressed Hep3B cells, E₂ treatment inhibited PPARα, its downstream gene acyl‐CoA oxidase (ACO), cyclin D1 and PCNA expression and further increased p27 and caspase 3 expressions. However, over‐expressed ERα plus 17‐β‐estradiol (10⁻⁸ M) reversed the fenofibrate effect and induced apoptosis, which was blocked in ICI/melatonin/fenofibrate‐treated cells. This study illustrates that PPARα expression and function were negatively regulated by ERα expression in Hep3B cells.     

基于开放式三明治酶联免疫分析的雌二醇检测方法的建立

目的:通过建立开放式三明治酶联免疫分析方法（OE-ELISA）,对雌二醇进行检测。方法:通过基因拼接构建质粒后在原核表达系统中制备小分子泛素相关修饰物蛋白-大肠杆菌麦芽糖结合蛋白-抗体轻链可变区（sumo-MBP-VL）和碱性磷酸酶-抗体重链可变区（ALP-VH）融合蛋白,并从产量和可溶性角度对两种蛋白的表达条件进行优化。将得到的两种蛋白纯化后分别作为包被抗体和酶标抗体,建立检测雌二醇的OE-ELISA方法。应用竞争ELISA和OE-ELISA测定不同浓度的雌二醇标准品,绘制标准曲线并计算两种方法的敏感性。结果:在质粒中加入sumo标签后,实现了sumo-MBP-VL融合蛋白的可溶性表达。在Rosetta 宿主菌中37℃诱导后得到大量的ALP-VH和sumo-MBP-VL融合蛋白。OE-ELISA测定雌二醇标准品的标准曲线为y=0.029 6x+1.302 9,r2=0.9961,敏感性为532 pg/mL,优于竞争ELISA 803 pg/mL的敏感性。结论:成功建立了检测雌二醇的OE-ELISA方法。     

基于谱效关系的醋延胡索炮制前后特征成分研究

目的探讨醋延胡索炮制前后HPLC指纹图谱与镇痛药效的相关性,筛选炮制特征成分。方法建立生延胡索与醋延胡索HPLC指纹图谱,对两者化学成分进行差异性分析,以大鼠扭体反应为模型建立评价指标,采用超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛、孕酮、雌二醇、前列腺素E_2(prostaglandin E_2,PGE_2)、前列腺素F_(2a)(prostaglandin F_(2a),PGF_(2a))等指标评价镇痛药效,结合灰色关联度分析法与熵权法研究醋延胡索的谱-效关系,指认醋延胡索炮制特征成分。结果建立了不同批次生延胡索饮片、醋延胡索饮片的HPLC指纹图谱,确定了31个共有峰。生延胡索饮片与醋延胡索饮片均有止痛效果,以醋延胡索止痛效果最为显著。镇痛谱效关联度分析得到峰9、18～21、25、26醋炙后关联度得分有了显著的提升,与醋炙后镇痛疗效呈显著正相关。结论通过谱-效关系明确了醋延胡索炮制特征成分为原阿片碱、盐酸巴马汀、盐酸小檗碱、去氢紫堇碱、延胡索乙素、四氢小檗碱、延胡索甲素,为醋延胡索炮制质量控制提供一定依据。