长期雌激素替代对循环和心脏局部肾素—血管紧张素系统的调节和对主动脉和心脏结构的影响
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摘要
心血管疾病是绝经后妇女的头号杀手。女性心脏疾病的发病比男性平均晚10年。绝经后妇女心血管疾病的发病率是同龄的绝经前妇女的4倍以上。雌激素水平的下降，是绝经后妇女最主要的生理变化。大量研究表明，雌激素对预防心血管疾病起着重要作用。然而，2002年美国WHI(Women’Health Initiative)研究表明雌激素和孕激素联合使用对心血管系统没有保护作用，反而增加心肌梗塞、中风和肺栓塞的发病危险性，并建议停止使用。2004年，WHI的单纯雌激素替代实验也因为轻度增加中风的发病风险和无明显心血管保护作用宣布停止。WHI的研究报告在激素替代治疗的支持者和患者中引起轩然大波。许多研究者认为WHI的试验虽有科学严谨的一面，但也有其局限性。比如：实验设计并没有真正保证随机和双盲，选取的病人年龄偏大(平均63岁)。2005年Mendelsohn等在Science杂志上提出要解决这些矛盾，需要对绝经前和绝经后妇女的心血管生理更加深入全面的理解，必须通过持续的基础研究以及新的临床实验，来开展雌激素与心血管疾病关系的研究。
肾素—血管紧张素系统(renin-angiotensin system，RAS)在体液和电解质平衡以及动脉血压调节中具有重要的协调作用。研究发现：(1)血管紧张素原基因的启动子含有雌激素反应元件：(2)女性血浆肾素活性低于同龄男性；(3)雌激素替代能轻度抑制血管紧张素转化酶的活性。提示雌激素可能通过调节肾素—血管紧张素系统，影响机体血压和心血管系统。最近研究认为，机体不仅有一个循环的肾素—血管紧张素系统，在心脏还存在着局部RAS系统，且不依赖于循环中的RAS和血流动力学的改变而独立存在。心脏组织RAS的激活在心脏疾病的发生过程中发挥着重要的调节作用，如高血压性心肌肥厚、心肌梗塞、心率失常及缺血再灌注损伤等。但是，极少文献报道雌激素对心脏局部RAS系统的影响。
心脏与血管功能的发挥与其结构密切相关。17β-雌二醇能降低溶酶体蛋白水解酶活性，增加线粒体F_1F_1-ATP酶活性。这些结果提示我们雌激素可能在维持细胞结构的完整性中起着重要作用，但是这些实验都没有从形态学上阐明雌激素对血管内皮细胞和心肌细胞的影响。
本论文通过构建假手术、去势(模拟绝经后妇女)和雌激素替代组三组SD雌鼠模型，研究去势和长期雌激素替代对循环、心脏RAS系统及心脏、主动脉结构的影响，为理解绝经后妇女心血管疾病的高发、雌激素替代的利弊提供有力的理论依据。第一章：长期雌激素替代治疗对循环RAS系统和胸主动脉超微结构的影响
目的：构建假手术、去势和雌激素替代治疗三组SD雌鼠模型；研究去势和长期雌激素替代对主动脉的内皮依赖性舒张功能和超微结构、血压、循环肾素-血管紧张素系统及调节因素一氧化氮(NO)、心钠素(ANP)的影响
方法：5月龄SD雌鼠50只，随机分成三组：假手术组(16只)、去势组(17只)和雌激素替代组(17只)。手术后，雌激素替代组每日皮下注射17β-雌二醇15μg／kg，假手术组和去势组则每日只注射溶剂。手术前后跟踪检测体重、血清雌二醇、NO和血浆血管紧张素Ⅱ、肾素活性和心钠素等指标。手术13周后，每组取6-8只大鼠行离体血管内皮依赖性舒张功能检测，每组取3只大鼠的胸主动脉行电镜检测。
结果：手术后第3周至第13周，去势组大鼠体重逐渐增加，明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。手术后去势组雌激素水平降至11.20±2.65pg／ml，明显低于假手术组(p＜0.05)和替代(p＜0.05)。去势组的子宫明显萎缩，雌激素替代治疗能抑制子宫萎缩。替代组子宫的重量明显高于去势组(p＜0.05)，与假手术组相比无显著性差异。术后第9和13周，去势组大鼠血压明显高于假手术(p＜0.05)和雌激素替代组(p＜0.05)，假手术组和雌激素替代组无明显差异。术后第9和13周，去势组SD大鼠AngⅡ浓度明显低于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。同时，去势组肾素活性明显高于假手术组(p＜0.05)，雌激素替代能显著抑制去势所致肾素活性的升高(p＜0.05)。术后第5，9和13周，去势组大鼠血清NO浓度明显降低(p＜0.05)，雌激素替代能明显缓解去势所致血清NO浓度的降低(p＜0.05)。术后第5周时，雌激素替代组血清NO浓度高于去势组，但仍明显低于假手术组(p＜0.05)。术后第9周和13周，去势组大鼠血浆ANP浓度明显降低(p＜0.05)，雌激素替代能明显缓解去势所致血浆ANP浓度的降低(p＜0.05)。去势组胸主动脉环对ACh内皮依赖性舒张反应较假手术下降，雌激素替代能显著改善去势诱导的血管内皮依赖性舒张功能障碍(p＜0.05)。去势组大鼠胸主动脉内皮细胞肿胀，甚至坏死，雌激素替代能抑制结构的损伤。
结论：本研究表明雌激素能保护内皮细胞，增高血浆ANP和NO，降低肾素活性，从而预防高血压的发生。但是雌二醇对血压和AngⅡ的作用存在矛盾，机体可能通过负反馈调节最终抑制肾素—血管紧张素系统。
第二章：去势和长期雌激素替代对心脏局部RAS系统的调节
目的：研究去势和长期雌激素替代对心脏AngⅡ、AT1和AT2的影响，并探讨雌激素-NO通路是否参与AT1和AT2的调节
方法：手术13周后，处死上述三组大鼠，放免法检测左心室AngⅡ浓度。利用RT-PCR和Westernblot方法检测AT1和AT2 mRNA和蛋白水平的表达。研究雌激素-NO通路对AT1和AT2的影响：100nM雌激素分别联合NOS阻断剂L-NAME(10μM或100μM)或NO供体SNAP(10μ或100μM)，培养H9c2细胞48 h，检测AT1和AT2的表达。
结果：手术13周后，去势组大鼠左心室AngⅡ明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。去势组大鼠左心室AT1 mRNA和蛋白表达明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。去势组大鼠左心室AT2 mRNA和蛋白表达明显低于假手术组(p＜0.05)和雌激素替代组(p＜0.05)，雌激素替代组的AT2 mRNA和蛋白表达明显高于假手术组(p＜0.05)。细胞实验结果：1 nM和100 nM17β-雌二醇孵育H9c2细胞48 h，AT1 mRNA和蛋白表达明显低于无雌激素(p＜0.05)和0.01 nM雌激素培养组(p＜0.05)；1μM雌二醇(1μM)孵育48 h，AT1 mRNA和蛋白表达明显高于100 nM 17β-雌二醇组(p＜0.05)。1 nM和100 nM 17β-雌二醇孵育H9c2细胞48 h，AT2mRNA和蛋白表达明显高于无雌激素(p＜0.05)和0.01 nM雌激素培养组(p＜0.05)；1μM雌二醇(1μM)孵育48h，AT2 mRNA和蛋白表达有下降的趋势，但与100 nM 17β-雌二醇组相比，差异无统计学意义(p＞0.05)。100 nM 17β-雌二醇加用NOS抑制剂L-NAME(10μM或100μM)较E2组明显增高AT1 mRNA和蛋白表达(p＜0.05)。单纯用100μM L-NAME组的AT1 mRNA和蛋白表达明显高于雌二醇加用NOS抑制剂L-NAME组(p＜0.05)。E2加用NO供体SNAP(100μM)较E2组能明显降低AT1 mRNA表达(p＜0.05)，且其表达明显低于加用10μM SNAP组(p＜0.05)。E2加用L-NAME或SNAP培养H9c2细胞48h的AT2 mRNA表达与单用E2组无显著性差异(p＞0.05)，但明显高于无雌激素培养组(p＜0.05)。
结论：长期雌激素替代能通过影响心脏局部RAS系统，使之适应心肌缺血等不良刺激，保护心脏功能；并且雌激素-NO通路参与了AT1的调节。这对于解释绝经后妇女心血管疾病高发的原因提供了新的思路。
第三章：去势和长期雌激素替代对心肌超微结构的影响
目的：研究去势和长期雌激素替代对左心室心肌细胞超微结构的影响，探讨雌激素是否对心肌细胞的亚细胞结构具有保护作用。
方法：手术13周后，处死上述三组大鼠。快速取左心尖组织，置于4％多聚甲醛或2.5％戊二醛固定液中固定，分别行石蜡和超薄切片，光学显微镜和电子显微镜下观察心肌细胞结构。硫代巴比妥酸比色法测定左心室MDA浓度。
结果：手术13周后，去势组大鼠左心室MDA明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。光学显微镜下：假手术组大鼠左心室心肌细胞呈梭形，肌纤维排列整齐；去势组大鼠与雌激素替代组大鼠心肌细胞形态与假手术组相似，无明显改变。电子显微镜下：去势组大鼠左心室心肌细胞水肿，肌原纤维走向不规则，结构紊乱，部分肌丝甚至局部溶解；线粒体高度肿胀、空泡变。雌激素替代组大鼠左心室心肌细胞肌原纤维较去势组排列整齐，除少部分线粒体肿胀外，大部分正常。
结论：从形态学上阐明长期雌激素缺乏能导致心肌亚细胞结构紊乱，雌激素替代能阻止心肌结构的破坏。雌激素可能通过影响心脏局部RAS系统，在维持亚细胞结构的完整中起着重要作用。
Cardiovascular diseases are the leading cause of death in postmenopausal women. Heart disease develops in women on average 10 years later in life compared with men. The incidence of cardiovascular diseases is 4-fold higher in postmenopausal women than in women of the same age who are premenopausal. The decrease of the estrogen level is the main physiological change of the postmenopausal women. Much research indicates that estrogen plays an important role in preventing the cardiovascular diseases. Unfortunately, the WHI studies showed that HRT caused a higher incidence of myocardial infarction, ischemic stroke and pulmonary emboli than placebo in 2002. The "estrogen only" arm of WHI was also recently stopped because of the slightly high storke risk and the lack of any beneficial CVD effects. The study report of WHI causes a great panic of the upholders and receivers of hormone replacement therapy. Many researchers believe that although the test of WHI is scientific and rigorous, there is some defect in it. For example, random and 'double blind' are not guaranteed in this design of the experiment, and the patients are prone to be older. In 2005, Mendelsohn et al suggested that resolving this controversy would require a more complete understanding of the cardiovascular biololgy that exist between perimenopausal and older women. It is necessary to explore the interactions between estrogen and cardiovascular disease by continuous basic study and new clinical experiment.
The renin-angiotensin system (RAS) is a critical regulator of blood pressure and fluid homeostasis. Studies suggest that: (1) The gene promoter of angiotensinogen contains an estrogen response element; (2) Plasma renin activity is higher in men than in women; (3) The majority of human studies document a mild to moderate suppression of angiotensin-converting enzyme (ACE) activity with estrogen replacement therapy. These studies suggest that estrogen maybe influence blood pressure and cardiovascular systems through affecting renin-angiotensin system. It has been described that all of the components of the renin-angiotensin system are present in cardiac tissue and that angiotensinⅡ(AngⅡ) is produced locally which regulated independent of the circulating RAS. Activation of cardiac RAS is related to the development of heart diseases such as hypertensively myocardial hypertrophy, myocardial infarction, arrhythmia, ischemia-reperfusion injury. But there are few studies on the effect of estrogen on cardiac renin-angiotensin system.
There is a close relationship between the function and structure of blood vessel and heart. Estrogen may decrease lysosomal enzyme activities and increase mitochondrial FOF1-ATPase activity. These results suggest that estrogen may play an important role in maintaining the structural integrity of cells. But these experiments have not explained the influence of estrogen on endothelium and myocardium morphologically.
The paper studies the effect of long-time estrogen replacement on circulating and cardiac RAS system and on the structure of myocardium and aorta, by establishing sham, OVX and OVX＋E2 three SD female rat models. The study will offer theoretical evidence for understanding the high risk of cardiovascular diseases of postmenopausal women and the benefits and risks of estrogen replacement therapy.
Chapter One: Influence of long-term estrogen treatment on circulating renin-angiotensin system and endothelial ultrastructure
Objective: To establish three SD rat models of sham, ovariectomy and estrogen replacement treatment; To study the influence of ovariectomy and long-term 17β-estradiol treatment on circulating renin-angiotensin system (RAS), nitrite oxide (NO), atrial natriuretic peptide (ANP), blood pressure (BP), the ultrastructural characteristics and endothelium-dependent relaxation of thoracic aorta.
Methods: Rats were ovariectomized (OVX, n=17), not ovariectomized (sham, n=16) or ovariectomized and treated with subcutaneous 17β-estradiol (15μg/kg/day, OVX＋E2, n=17). Within 13 weeks after operation, the blood pressure, the serum estrogen, NO, plasma angiotensinⅡ(AngⅡ), ANP and renin activity were monitored. At the 13th week after operation, vasodilator responses to acetylcholine in the aortic rings and ultrastructural characteristics of thoracic aorta were determined.
Results: By 3 weeks and, until the end of the study, OVX had significantly higher weights than other two groups (p＜0.05). E2-treatment maintained uterus weights that were significantly higher than OVX (p＜0.05) and similar with sham rats (p＞0.05). Plasma estradiol levels were significantly lower in the OVX compared with sham (P＜0.05) and OVX＋E2 rats (p＜0.05). At the 9th and 13th week, OVX had significantly higher blood pressure than the other two groups (p＜0.05). Overiectomy led to a significant decrease in plasma AngⅡconcentration and a significant increase in renin activity of OVX rats compared with sham, which could be restored by estrogen treatment (p＜0.05). At the 5th, 9th and 13th week, serum NO of OVX was significantly lower compared with sham, which could be restored by estrogen treatment (p＜0.05). Plasma ANP at the 9th and 13th week was significantly lower in OVX (p＜0.05). Ovariectomy markedly reduced endothelium-dependent relaxation to acetylcholine in the isolated rat thoracic aortic rings (p＜0.05) and chronic estrogen treatment significantly restored the vasodilator responses to acetylcholine (p＜0.05). It was found under electron microscopy that the endothelial cells of OVX rats were swollen, even necrosed, estrogen treatment can inhibit the changes.
Conclusion: These results suggest that estradiol protects from the development of hypertension and posses a protective effect on the endothelium through increasing NO and ANP concentration, and decreasing renin activity. However, there existed the discordance between the effects of estradiol on angiotensinⅡ, and on blood pressure, which may be a negative feedback that ultimately results in overall suppression of the RAS.
Chapter Two: Influence of ovariectomy and long-term estrogen treatment on cardiac renin-angiotensin system
Objective: To study the effect of ovariectomy and long-term estrogen treatment on the expressions of AT1, AT2 and angiotensinⅡin heart; To test whether the expressions of AT1 and AT2 modulated by estrogen-NO pathway.
Methods: 13 weeks after operation, radioimmunoassay was used to analyse AngⅡconcentrations of left ventricles in three group SD rats. RT-PCR and Westernblot were used to analyse the the expressions of AT1 and AT2 in myocardium of the three group SD rats at mRNA and protein levels. To test whether the expressions of AT1 and AT2 modulated by estrogen-NO pathway, H9c2 cells were exposed to 17β-estradiol (100nM) plus L-NAME (10μM or 100μM) and SNAP(10μM or 100μM) respectively for 48 h, then the expressions of AT1 and AT2 was analysed by RT-PCR and Westernblot.
Results: AngⅡconcentration of left ventricles in OVX rats was higher than sham (p＜0.05) and OVX＋E2 rats (p＜0.05). The expression of AT1 was upregulated in the OVX rats at both mRNA and protein levels (p＜0.05), which was prevented by estrogen treatment (p＜0.05). The expression of AT2 of OVX rats was lower than sham (p＜0.05) and OVX＋E2 rats (p＜0.05) at both mRNA and protein levels. AT1 expressions of H9c2 cells cultured with 1 nM or 100 nM E2 for 48h were significantly lower than 0 nM (p＜0.05) or 0.01 nM E2 group (p＜0.05). AT2 expressions of H9c2 cells cultured with 1 nM or 100 nM E2 for 48h were significantly higher than 0 nM (p＜0.05) or 0.01 nM E2 group (p＜0.05). The expressions of AT1 in H9c2 cells pretreated with 100 nM E2 plus L-NAME (10μM or 100μM) for 48hr was significantly higher than 100 nM E2 group (p＜0.05), and significantly lower than the cells pretreated with only 100μM L-NAME at mRNA and protein levels (p＜0.05). The expressions of AT1 in H9c2 cells pretreated with 100 nM E2 plus 100μM SNAP for 48hr was significantly lower than 100 nM E2 group (p＜0.05), and significantly lower than the cells pretreated with 100 nM E2 plus 10μM SNAP at mRNA and protein levels (p＜0.05). The expression of AT2 in H9c2 cells pretreated with 100 nM E2 plus L-NAME (10μM or 100μM)) or SNAP (10μM or 100μM)) had no significant difference compared with 100 nM E2 group (p＞0.05), but higher 0 nM E2 group (p＜0.05).
Conclusion: Long-term estrogen replacement could protect the cardiac function by modulating the cardiac RAS system to adapt to ischemia et al badly stimuli. NOS/NO pathway involved in the decrease of AT1 expression modulated by estrogen. The study offered a new evidence for understanding the high risk of cardiovascular diseases of postmenopausal women.
Chapter Three: Effect of ovariectomy and long-term estrogen treatment on uitrastructure of myocardium
Objective: To study the effect of ovariectomy and long-term estrogen treatment on the on ultrastructure of myocardium in SD female rats.
Methods: At the 13th week, the rats were discected, then left ventricles were removed rapidly and fixed in 4% paraformaldehyde or 2.5% glutaraldehyde respectively. Paraffin block sections (4μm thick) or ultrasections (about 500(?)) were cut, and examined using light microscope and transmission electron microscope. Thiobarbituric acid method was used to analyse MDA concentrations of left ventricles in three group SD rats.
Results: MDA concentration of left ventricles in OVX rats was higher than sham (p＜0.05) and OVX＋E2 rats (p＜0.05). By the electron microscopy examination of the left ventricles, the sham rats did not reveal abnormal findings of the cardiac myocytes, but in rats of OVX group, there was disarranged and lysis of some myofibrils. Most of mitochondria were markedly abnormal in shape and were swollen. In OVX＋E2 rats, myofibdls arranged regular. Light band and dark band of sarcomere were clearer than OVX rats. Except some mitochondrias were swollen, most were normal.
Conclusion: Ovariectomy may lead to ultralstructural disorder of myocardium, long-term estrogen replacement can protect against the ultralstructural injury. Estrogen plays an important role in maintaining the normal ultralstructure maybe through modulating the cardiac RAS system.
肾素—血管紧张素系统(renin-angiotensin system，RAS)在体液和电解质平衡以及动脉血压调节中具有重要的协调作用。研究发现：(1)血管紧张素原基因的启动子含有雌激素反应元件：(2)女性血浆肾素活性低于同龄男性；(3)雌激素替代能轻度抑制血管紧张素转化酶的活性。提示雌激素可能通过调节肾素—血管紧张素系统，影响机体血压和心血管系统。最近研究认为，机体不仅有一个循环的肾素—血管紧张素系统，在心脏还存在着局部RAS系统，且不依赖于循环中的RAS和血流动力学的改变而独立存在。心脏组织RAS的激活在心脏疾病的发生过程中发挥着重要的调节作用，如高血压性心肌肥厚、心肌梗塞、心率失常及缺血再灌注损伤等。但是，极少文献报道雌激素对心脏局部RAS系统的影响。
心脏与血管功能的发挥与其结构密切相关。17β-雌二醇能降低溶酶体蛋白水解酶活性，增加线粒体F_1F_1-ATP酶活性。这些结果提示我们雌激素可能在维持细胞结构的完整性中起着重要作用，但是这些实验都没有从形态学上阐明雌激素对血管内皮细胞和心肌细胞的影响。
本论文通过构建假手术、去势(模拟绝经后妇女)和雌激素替代组三组SD雌鼠模型，研究去势和长期雌激素替代对循环、心脏RAS系统及心脏、主动脉结构的影响，为理解绝经后妇女心血管疾病的高发、雌激素替代的利弊提供有力的理论依据。第一章：长期雌激素替代治疗对循环RAS系统和胸主动脉超微结构的影响
目的：构建假手术、去势和雌激素替代治疗三组SD雌鼠模型；研究去势和长期雌激素替代对主动脉的内皮依赖性舒张功能和超微结构、血压、循环肾素-血管紧张素系统及调节因素一氧化氮(NO)、心钠素(ANP)的影响
方法：5月龄SD雌鼠50只，随机分成三组：假手术组(16只)、去势组(17只)和雌激素替代组(17只)。手术后，雌激素替代组每日皮下注射17β-雌二醇15μg／kg，假手术组和去势组则每日只注射溶剂。手术前后跟踪检测体重、血清雌二醇、NO和血浆血管紧张素Ⅱ、肾素活性和心钠素等指标。手术13周后，每组取6-8只大鼠行离体血管内皮依赖性舒张功能检测，每组取3只大鼠的胸主动脉行电镜检测。
结果：手术后第3周至第13周，去势组大鼠体重逐渐增加，明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。手术后去势组雌激素水平降至11.20±2.65pg／ml，明显低于假手术组(p＜0.05)和替代(p＜0.05)。去势组的子宫明显萎缩，雌激素替代治疗能抑制子宫萎缩。替代组子宫的重量明显高于去势组(p＜0.05)，与假手术组相比无显著性差异。术后第9和13周，去势组大鼠血压明显高于假手术(p＜0.05)和雌激素替代组(p＜0.05)，假手术组和雌激素替代组无明显差异。术后第9和13周，去势组SD大鼠AngⅡ浓度明显低于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。同时，去势组肾素活性明显高于假手术组(p＜0.05)，雌激素替代能显著抑制去势所致肾素活性的升高(p＜0.05)。术后第5，9和13周，去势组大鼠血清NO浓度明显降低(p＜0.05)，雌激素替代能明显缓解去势所致血清NO浓度的降低(p＜0.05)。术后第5周时，雌激素替代组血清NO浓度高于去势组，但仍明显低于假手术组(p＜0.05)。术后第9周和13周，去势组大鼠血浆ANP浓度明显降低(p＜0.05)，雌激素替代能明显缓解去势所致血浆ANP浓度的降低(p＜0.05)。去势组胸主动脉环对ACh内皮依赖性舒张反应较假手术下降，雌激素替代能显著改善去势诱导的血管内皮依赖性舒张功能障碍(p＜0.05)。去势组大鼠胸主动脉内皮细胞肿胀，甚至坏死，雌激素替代能抑制结构的损伤。
结论：本研究表明雌激素能保护内皮细胞，增高血浆ANP和NO，降低肾素活性，从而预防高血压的发生。但是雌二醇对血压和AngⅡ的作用存在矛盾，机体可能通过负反馈调节最终抑制肾素—血管紧张素系统。
第二章：去势和长期雌激素替代对心脏局部RAS系统的调节
目的：研究去势和长期雌激素替代对心脏AngⅡ、AT1和AT2的影响，并探讨雌激素-NO通路是否参与AT1和AT2的调节
方法：手术13周后，处死上述三组大鼠，放免法检测左心室AngⅡ浓度。利用RT-PCR和Westernblot方法检测AT1和AT2 mRNA和蛋白水平的表达。研究雌激素-NO通路对AT1和AT2的影响：100nM雌激素分别联合NOS阻断剂L-NAME(10μM或100μM)或NO供体SNAP(10μ或100μM)，培养H9c2细胞48 h，检测AT1和AT2的表达。
结果：手术13周后，去势组大鼠左心室AngⅡ明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。去势组大鼠左心室AT1 mRNA和蛋白表达明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。去势组大鼠左心室AT2 mRNA和蛋白表达明显低于假手术组(p＜0.05)和雌激素替代组(p＜0.05)，雌激素替代组的AT2 mRNA和蛋白表达明显高于假手术组(p＜0.05)。细胞实验结果：1 nM和100 nM17β-雌二醇孵育H9c2细胞48 h，AT1 mRNA和蛋白表达明显低于无雌激素(p＜0.05)和0.01 nM雌激素培养组(p＜0.05)；1μM雌二醇(1μM)孵育48 h，AT1 mRNA和蛋白表达明显高于100 nM 17β-雌二醇组(p＜0.05)。1 nM和100 nM 17β-雌二醇孵育H9c2细胞48 h，AT2mRNA和蛋白表达明显高于无雌激素(p＜0.05)和0.01 nM雌激素培养组(p＜0.05)；1μM雌二醇(1μM)孵育48h，AT2 mRNA和蛋白表达有下降的趋势，但与100 nM 17β-雌二醇组相比，差异无统计学意义(p＞0.05)。100 nM 17β-雌二醇加用NOS抑制剂L-NAME(10μM或100μM)较E2组明显增高AT1 mRNA和蛋白表达(p＜0.05)。单纯用100μM L-NAME组的AT1 mRNA和蛋白表达明显高于雌二醇加用NOS抑制剂L-NAME组(p＜0.05)。E2加用NO供体SNAP(100μM)较E2组能明显降低AT1 mRNA表达(p＜0.05)，且其表达明显低于加用10μM SNAP组(p＜0.05)。E2加用L-NAME或SNAP培养H9c2细胞48h的AT2 mRNA表达与单用E2组无显著性差异(p＞0.05)，但明显高于无雌激素培养组(p＜0.05)。
结论：长期雌激素替代能通过影响心脏局部RAS系统，使之适应心肌缺血等不良刺激，保护心脏功能；并且雌激素-NO通路参与了AT1的调节。这对于解释绝经后妇女心血管疾病高发的原因提供了新的思路。
第三章：去势和长期雌激素替代对心肌超微结构的影响
目的：研究去势和长期雌激素替代对左心室心肌细胞超微结构的影响，探讨雌激素是否对心肌细胞的亚细胞结构具有保护作用。
方法：手术13周后，处死上述三组大鼠。快速取左心尖组织，置于4％多聚甲醛或2.5％戊二醛固定液中固定，分别行石蜡和超薄切片，光学显微镜和电子显微镜下观察心肌细胞结构。硫代巴比妥酸比色法测定左心室MDA浓度。
结果：手术13周后，去势组大鼠左心室MDA明显高于假手术组(p＜0.05)和雌激素替代组(p＜0.05)。光学显微镜下：假手术组大鼠左心室心肌细胞呈梭形，肌纤维排列整齐；去势组大鼠与雌激素替代组大鼠心肌细胞形态与假手术组相似，无明显改变。电子显微镜下：去势组大鼠左心室心肌细胞水肿，肌原纤维走向不规则，结构紊乱，部分肌丝甚至局部溶解；线粒体高度肿胀、空泡变。雌激素替代组大鼠左心室心肌细胞肌原纤维较去势组排列整齐，除少部分线粒体肿胀外，大部分正常。
结论：从形态学上阐明长期雌激素缺乏能导致心肌亚细胞结构紊乱，雌激素替代能阻止心肌结构的破坏。雌激素可能通过影响心脏局部RAS系统，在维持亚细胞结构的完整中起着重要作用。
Cardiovascular diseases are the leading cause of death in postmenopausal women. Heart disease develops in women on average 10 years later in life compared with men. The incidence of cardiovascular diseases is 4-fold higher in postmenopausal women than in women of the same age who are premenopausal. The decrease of the estrogen level is the main physiological change of the postmenopausal women. Much research indicates that estrogen plays an important role in preventing the cardiovascular diseases. Unfortunately, the WHI studies showed that HRT caused a higher incidence of myocardial infarction, ischemic stroke and pulmonary emboli than placebo in 2002. The "estrogen only" arm of WHI was also recently stopped because of the slightly high storke risk and the lack of any beneficial CVD effects. The study report of WHI causes a great panic of the upholders and receivers of hormone replacement therapy. Many researchers believe that although the test of WHI is scientific and rigorous, there is some defect in it. For example, random and 'double blind' are not guaranteed in this design of the experiment, and the patients are prone to be older. In 2005, Mendelsohn et al suggested that resolving this controversy would require a more complete understanding of the cardiovascular biololgy that exist between perimenopausal and older women. It is necessary to explore the interactions between estrogen and cardiovascular disease by continuous basic study and new clinical experiment.
The renin-angiotensin system (RAS) is a critical regulator of blood pressure and fluid homeostasis. Studies suggest that: (1) The gene promoter of angiotensinogen contains an estrogen response element; (2) Plasma renin activity is higher in men than in women; (3) The majority of human studies document a mild to moderate suppression of angiotensin-converting enzyme (ACE) activity with estrogen replacement therapy. These studies suggest that estrogen maybe influence blood pressure and cardiovascular systems through affecting renin-angiotensin system. It has been described that all of the components of the renin-angiotensin system are present in cardiac tissue and that angiotensinⅡ(AngⅡ) is produced locally which regulated independent of the circulating RAS. Activation of cardiac RAS is related to the development of heart diseases such as hypertensively myocardial hypertrophy, myocardial infarction, arrhythmia, ischemia-reperfusion injury. But there are few studies on the effect of estrogen on cardiac renin-angiotensin system.
There is a close relationship between the function and structure of blood vessel and heart. Estrogen may decrease lysosomal enzyme activities and increase mitochondrial FOF1-ATPase activity. These results suggest that estrogen may play an important role in maintaining the structural integrity of cells. But these experiments have not explained the influence of estrogen on endothelium and myocardium morphologically.
The paper studies the effect of long-time estrogen replacement on circulating and cardiac RAS system and on the structure of myocardium and aorta, by establishing sham, OVX and OVX＋E2 three SD female rat models. The study will offer theoretical evidence for understanding the high risk of cardiovascular diseases of postmenopausal women and the benefits and risks of estrogen replacement therapy.
Chapter One: Influence of long-term estrogen treatment on circulating renin-angiotensin system and endothelial ultrastructure
Objective: To establish three SD rat models of sham, ovariectomy and estrogen replacement treatment; To study the influence of ovariectomy and long-term 17β-estradiol treatment on circulating renin-angiotensin system (RAS), nitrite oxide (NO), atrial natriuretic peptide (ANP), blood pressure (BP), the ultrastructural characteristics and endothelium-dependent relaxation of thoracic aorta.
Methods: Rats were ovariectomized (OVX, n=17), not ovariectomized (sham, n=16) or ovariectomized and treated with subcutaneous 17β-estradiol (15μg/kg/day, OVX＋E2, n=17). Within 13 weeks after operation, the blood pressure, the serum estrogen, NO, plasma angiotensinⅡ(AngⅡ), ANP and renin activity were monitored. At the 13th week after operation, vasodilator responses to acetylcholine in the aortic rings and ultrastructural characteristics of thoracic aorta were determined.
Results: By 3 weeks and, until the end of the study, OVX had significantly higher weights than other two groups (p＜0.05). E2-treatment maintained uterus weights that were significantly higher than OVX (p＜0.05) and similar with sham rats (p＞0.05). Plasma estradiol levels were significantly lower in the OVX compared with sham (P＜0.05) and OVX＋E2 rats (p＜0.05). At the 9th and 13th week, OVX had significantly higher blood pressure than the other two groups (p＜0.05). Overiectomy led to a significant decrease in plasma AngⅡconcentration and a significant increase in renin activity of OVX rats compared with sham, which could be restored by estrogen treatment (p＜0.05). At the 5th, 9th and 13th week, serum NO of OVX was significantly lower compared with sham, which could be restored by estrogen treatment (p＜0.05). Plasma ANP at the 9th and 13th week was significantly lower in OVX (p＜0.05). Ovariectomy markedly reduced endothelium-dependent relaxation to acetylcholine in the isolated rat thoracic aortic rings (p＜0.05) and chronic estrogen treatment significantly restored the vasodilator responses to acetylcholine (p＜0.05). It was found under electron microscopy that the endothelial cells of OVX rats were swollen, even necrosed, estrogen treatment can inhibit the changes.
Conclusion: These results suggest that estradiol protects from the development of hypertension and posses a protective effect on the endothelium through increasing NO and ANP concentration, and decreasing renin activity. However, there existed the discordance between the effects of estradiol on angiotensinⅡ, and on blood pressure, which may be a negative feedback that ultimately results in overall suppression of the RAS.
Chapter Two: Influence of ovariectomy and long-term estrogen treatment on cardiac renin-angiotensin system
Objective: To study the effect of ovariectomy and long-term estrogen treatment on the expressions of AT1, AT2 and angiotensinⅡin heart; To test whether the expressions of AT1 and AT2 modulated by estrogen-NO pathway.
Methods: 13 weeks after operation, radioimmunoassay was used to analyse AngⅡconcentrations of left ventricles in three group SD rats. RT-PCR and Westernblot were used to analyse the the expressions of AT1 and AT2 in myocardium of the three group SD rats at mRNA and protein levels. To test whether the expressions of AT1 and AT2 modulated by estrogen-NO pathway, H9c2 cells were exposed to 17β-estradiol (100nM) plus L-NAME (10μM or 100μM) and SNAP(10μM or 100μM) respectively for 48 h, then the expressions of AT1 and AT2 was analysed by RT-PCR and Westernblot.
Results: AngⅡconcentration of left ventricles in OVX rats was higher than sham (p＜0.05) and OVX＋E2 rats (p＜0.05). The expression of AT1 was upregulated in the OVX rats at both mRNA and protein levels (p＜0.05), which was prevented by estrogen treatment (p＜0.05). The expression of AT2 of OVX rats was lower than sham (p＜0.05) and OVX＋E2 rats (p＜0.05) at both mRNA and protein levels. AT1 expressions of H9c2 cells cultured with 1 nM or 100 nM E2 for 48h were significantly lower than 0 nM (p＜0.05) or 0.01 nM E2 group (p＜0.05). AT2 expressions of H9c2 cells cultured with 1 nM or 100 nM E2 for 48h were significantly higher than 0 nM (p＜0.05) or 0.01 nM E2 group (p＜0.05). The expressions of AT1 in H9c2 cells pretreated with 100 nM E2 plus L-NAME (10μM or 100μM) for 48hr was significantly higher than 100 nM E2 group (p＜0.05), and significantly lower than the cells pretreated with only 100μM L-NAME at mRNA and protein levels (p＜0.05). The expressions of AT1 in H9c2 cells pretreated with 100 nM E2 plus 100μM SNAP for 48hr was significantly lower than 100 nM E2 group (p＜0.05), and significantly lower than the cells pretreated with 100 nM E2 plus 10μM SNAP at mRNA and protein levels (p＜0.05). The expression of AT2 in H9c2 cells pretreated with 100 nM E2 plus L-NAME (10μM or 100μM)) or SNAP (10μM or 100μM)) had no significant difference compared with 100 nM E2 group (p＞0.05), but higher 0 nM E2 group (p＜0.05).
Conclusion: Long-term estrogen replacement could protect the cardiac function by modulating the cardiac RAS system to adapt to ischemia et al badly stimuli. NOS/NO pathway involved in the decrease of AT1 expression modulated by estrogen. The study offered a new evidence for understanding the high risk of cardiovascular diseases of postmenopausal women.
Chapter Three: Effect of ovariectomy and long-term estrogen treatment on uitrastructure of myocardium
Objective: To study the effect of ovariectomy and long-term estrogen treatment on the on ultrastructure of myocardium in SD female rats.
Methods: At the 13th week, the rats were discected, then left ventricles were removed rapidly and fixed in 4% paraformaldehyde or 2.5% glutaraldehyde respectively. Paraffin block sections (4μm thick) or ultrasections (about 500(?)) were cut, and examined using light microscope and transmission electron microscope. Thiobarbituric acid method was used to analyse MDA concentrations of left ventricles in three group SD rats.
Results: MDA concentration of left ventricles in OVX rats was higher than sham (p＜0.05) and OVX＋E2 rats (p＜0.05). By the electron microscopy examination of the left ventricles, the sham rats did not reveal abnormal findings of the cardiac myocytes, but in rats of OVX group, there was disarranged and lysis of some myofibrils. Most of mitochondria were markedly abnormal in shape and were swollen. In OVX＋E2 rats, myofibdls arranged regular. Light band and dark band of sarcomere were clearer than OVX rats. Except some mitochondrias were swollen, most were normal.
Conclusion: Ovariectomy may lead to ultralstructural disorder of myocardium, long-term estrogen replacement can protect against the ultralstructural injury. Estrogen plays an important role in maintaining the normal ultralstructure maybe through modulating the cardiac RAS system.
引文
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