用于筛查绝经后心血管功能障碍的外源雌激素治疗的活体雌激素缺乏小鼠模型
Summary
临床上,更年期妇女雌激素缺乏可能加重脂质紊乱和动脉粥样硬化的发生率。我们通过在apoE-/-小鼠的双背侧切口,通过双边卵巢切除术建立了体内雌激素缺乏模型。小鼠模型适用于绝经后心血管功能障碍的外源雌激素治疗。
Abstract
绝经后妇女患心血管疾病的风险比绝经前妇女大。与卵巢功能完整的雌性小鼠相比,雌性小鼠在失异时出现卵巢切除术(OVX)增加了主动脉动脉粥样硬化的病变。然而,缺乏涉及雌激素缺乏小鼠的实验室模型,这些小鼠具有动脉粥样硬化的易发性状态。这种缺陷是至关重要的,因为更年期妇女的临床雌激素缺乏可能加剧先前存在的或持续的脂质紊乱和动脉粥样硬化的发生率。在这项研究中,我们通过双背侧切口在脂蛋白E(apoE)-/-小鼠中通过双边卵巢切除术建立了体内雌激素缺乏小鼠模型。然后,我们比较17°-雌二醇和伪原蛋白素(PPD)(植物雌激素)通过薄杉醇传播的内给的影响。我们发现,虽然PPD对降低OVX apoE-/-小鼠的最终体重和血浆TG有一定作用,但它具有抗动脉粥样硬化和心脏保护能力,与17°雌二醇相当。PPD是一种植物雌激素,已报告发挥抗肿瘤的特性。因此,该方法适用于通过口服给雌激素筛选植物雌激素,以取代绝经后妇女的传统激素替代疗法,据报告,这种疗法具有潜在的有害肿瘤遗传能力。通过木耳传播的口服给政是无创的,使其广泛适用于许多患者。本文包含通过 apoE-/-小鼠和 Peroral 17°-雌二醇或植物雌激素激素通过薄荷传播的双背侧切口进行双边卵巢切除术的逐步演示。血浆脂质和心血管功能分析使用超声心动图跟踪。
Introduction
流行病学和临床研究显示,绝经后妇女患心血管疾病的风险比绝经前妇女1、2要大得多。激素替代疗法(HRT)可降低心血管疾病的相对风险至0.37-0.79 3。在其他并发症中,由心血管疾病引起的动脉粥样硬化是全世界4.然而,缺乏涉及雌激素缺乏小鼠出现动脉粥样硬化倾向状态的实验室模型。该协议提供了一个体内雌激素缺乏小鼠模型,用于筛查绝经后心血管功能障碍的外源性雌激素治疗。
先前的研究表明,在高胆固醇饮食中应用OVX在动脉粥样硬化啮齿动物中可以模仿绝经后妇女患有动脉粥样硬化5、6、7、8。一种类似更年期女性动脉粥样硬化状态的可重复和方便的动物模型是外源雌激素研究的基础。在这里,双侧侧切口的双边卵巢切除术被应用于动脉粥样硬化易脂蛋白E敲除(apoE-/-)小鼠9,10。与中腹或背侧切口相比,双背侧切口是一种更容易、更省时的方法,可以避免严重的腹腔粘附和炎症。通过薄荷传播(见材料表)的渗透管理是非侵入性和方便的,使其广泛适用作为一个长期管理模式11。慢释放颗粒植入也很受欢迎6。然而,植入物可能会加重感染的发生率,特别是在接受OVX的小鼠中。其他非侵入性管理模式,如口腔口和供水管理,也有许多缺点。口腔食道通常对小鼠造成压力,并可能导致食管损伤。通过饮用水管理激素是非常有益的;然而,添加DMSO作为乳化剂是不可避免的,因为外源雌激素不溶于水。在这里,我们选择通过长期给政用的薄荷替代17°-雌二醇或植物雌激素激素。
最近,HRT对绝经后妇女心血管系统的有益作用在妇女健康倡议(WHI)试验12中受到质疑。一方面,仅外源雌激素对心血管系统有有益的影响;另一方面,它可以与醋酸甲酰乙酰丙酮结合,增加心血管事件的风险。更严重的是,HRT可能导致乳房和子宫肿瘤进展,这种效果已经明显限制了其使用13,14。更多的兴趣集中在肿瘤细胞15、16、17中缺乏线粒体活性的外源雌激素的心血管保护作用上。对人类和动物的多项研究表明,具有与雌激素结构相似的植物雌激素在心血管保护方面可以发挥有益的作用。
因此,本工作的目标是(i) 通过双背侧切口在 apoE-/-小鼠中通过双边卵巢切除术建立体内雌激素缺乏小鼠模型,以及 (ii) 比较血管保护作用施用17°-雌二醇和伪丙二醇(PPD),通过薄荷传播。17°-雌二醇是一种外源雌激素,属于女性性激素6,11,19。PPD,一种来自迪奥斯科卡植物的类固醇皂苷和植物雌激素,以前曾被报道具有抗肿瘤的特性20。
Protocol
所有动物护理和实验规程均经中国医学科学院动物护理与使用委员会和北京协和医学院批准(许可号:SYXK(北京)2013-0023)。apoE-/-小鼠的起源是C57BL/6J9,10。 1. 通过 apoE 中的双侧切口进行双边卵巢切除术-/-小鼠 在产间(年龄28天),用避免素(三溴二苯醚乙醇;200毫克/千克;腹内)麻醉雌性apoE-/-C57BL/6J小鼠。<strong…
Representative Results
本研究中使用的一种典型的实验治疗方案如图1所示。在产间(年龄28天),雌性apoE-/-C57BL/6J小鼠用非他林(三溴二苯醚乙醇;200毫克/千克;腹内)进行麻醉。小鼠通过1厘米背切口进行双边OVX或假手术。在双边OVX一周后,小鼠被喂食高胆固醇饮食(1.25%胆固醇,0%乔拉)12周。17°-雌二醇(0.1毫克+千克-1)或PPD(0.5毫克+kg-1)在治疗的最后9周内通过薄桃传播?…
Discussion
这里描述的方法类似于在更年期妇女看到的脂质紊乱和动脉粥样硬化的小鼠模型。有充分证据表明,绝经后妇女的雌激素缺乏会加重先前存在或持续的高胆固醇血症的发病率,并逐渐复杂和广泛地出现动脉硬化病变1。为了模拟临床上容易发生动脉粥样硬化的情况,应用了缺乏动脉硬化的小鼠,这是一种可重复和方便的动物来源,用于研究23、24、25。<sup class="xr…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金(81202526至J.X.)、中国国家自然科学基金(81302769至B.S.)、北京市自然科学基金(47144226至B.S.)的支持。科学基金会(201110490325 至 J.X.)和中国教育部博士计划基金会 (201211106120031 至 B.S.)。
Materials
| 17β-estradiol, >98% | Sigma-Aldrich | E8875-250MG | Estrogen |
| Disposable syringes (with 25G needles) | Hunan Luzhou Huikang Development Co., Ltd | 0.5*19TWLB | Cardiac bleeding |
| High-cholesterol mouse diet | Huafukang Bio-Technology | N/A | 1.25% cholesterol, 0% cholate |
| High-Resolution In Vivo Micro-Imaging System | VisualSonics | Vevo®770 | Measurements of intima-media thickness and cardiac dysfunction |
| 2-Methyl-2-butanol | Sigma-Aldrich | 152463-250ML | Preparation of avertin |
| Micro Dissecting forceps, Curved 8mm | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Micro Dissecting forceps, Straight 8mm | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Micro Dissecting Scissors, Curved/Sharp 8mm | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Micro Dissecting Scissors, Straight/Sharp 8mm | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Monofilament suture 4-0 1/2 5*12 19mm | Shanghai Pudong Jinhuan Medical Supplies Co., Ltd | R413 | Suture and ligation of the tissues |
| Nut cream (Nutella) | Ferrero | N/A | Medium for peroral 17β-estradiol or PPD |
| OptiVisor optical glass binocular magnifier | Dohegan Optical Company Inc. | N/A | Assistant of identifying the tissues during ovariectomy |
| Phosphate-buffered saline at pH 7.4 | SIGMA | P3813 | Preparing 1 L saline |
| Pro MultiLabel Microplate Reader | Tecan | Infinite M1000 | Plasma TC and TG determination |
| Pseudoprotodioscin | Shanghai Winherb Medical S & T Development | W-0427 | CAS registry no. 102115-79-7 |
| Rimadyl, 50 mg/mL | Pfizer Pharma GmbH | 462986 | Postoperative analgesia after ovariectomy |
| Sesame oil | Sigma-Aldrich | S3547-1L | Dissolving the 17β-estradiol or PPD |
| Solcoseryl Eye-Gel | Menarini, Solco Basle Ltd. | Eye protection during anesthesia | |
| Stereo microscope | MCALON | MCL-6STV | Image of the intimal region of aorta |
| Table model high speed centrifuge | SIGMA | 1-14K | Preparation of plasma |
| Scissors, slight Curve (14cm) | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Scissors, straight Flat (14cm) | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Tissue forceps, serrated, slight Curve (14cm) | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Tissue forceps, serrated, straight Flat (14cm) | Kanghua Medical Equipment Co., Ltd | Surgical tools | |
| Tribromoethanol | Sigma-Aldrich | T48402-5G | Preparation of avertin |
| Triglycerides (TG) assay kit | Institute of Nanjing Jiancheng Biology Engineering | A110-1 | Plasma TG determination |
| Total cholesterols (TC) assay kit | Institute of Nanjing Jiancheng Biology Engineering | A111-1 | Plasma TC determination |
Riferimenti
- Mozaffarian, D., et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 133 (4), e38-e360 (2016).
- Packard, B. L. Cardiovascular health and disease in women. The New England Journal of Medicine. 329 (4), 247-256 (1993).
- Samaan, S. A., Crawford, M. H. Estrogen and cardiovascular function after menopause. Journal of the American College of Cardiology. 26 (6), 1403-1410 (1995).
- Libby, P. Inflammation in atherosclerosis. Nature. 420 (6917), 868-874 (2002).
- Tremollieres, F. A., et al. Effect of hormone replacement therapy on age-related increase in carotid artery intima-media thickness in postmenopausal women. Atherosclerosis. 153 (1), 81-88 (2000).
- Bourassa, P. A., Milos, P. M., Gaynor, B. J., Breslow, J. L., Aiello, R. J. Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice. Proceedings of the National Academy of Sciences of the United States of America. 93 (3), 10022-10027 (1996).
- Squadrito, F., et al. Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats. Cardiovascular Research. 45 (2), 454-462 (2000).
- Wang, D., et al. Endothelial dysfunction and enhanced contractility in microvessels from ovariectomized rats: roles of oxidative stress and perivascular adipose tissue. Hypertension. 63 (5), 1063-1069 (2014).
- Zhang, S. H., Reddick, R. L., Piedrahita, J. A., Maeda, N. Spontaneous Hypercholesterolemia and Arterial Lesions in Mice Lacking Apolipoprotein E. Science. 258 (5081), 468-471 (1992).
- Nakashima, Y., Plump, A. S., Raines, E. W., Breslow, J. L., Ross, R. ApoE-Deficient Mice Develop Lesions of All Phases of Atherosclerosis Throughout the Arterial Tree. Arteriosclerosis and Thrombosis. 14 (1), 133-140 (1994).
- Ström, J. O., Theodorsson, A., Ingberg, E., Isaksson, I. M., Theodorsson, E. Ovariectomy and 17-estradiol Replacement in Rats and Mice: A Visual Demonstration. Journal of Visualized Experiments. 64 (e4013), 1-4 (2012).
- Rossouw, J. E., et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women’s Health Initiative randomized controlled trial. The Journal of the American Medical Association. 288 (3), 321-333 (2002).
- Kumle, M. Declining breast cancer incidence and decreased HRT use. Lancet. 372 (9639), 608-610 (2008).
- Jager, W., et al. A randomized comparison of triptorelin and tamoxifen as treatment of progressive ovarian cancer. Anticancer Research. 15 (6B), 2639-2642 (1995).
- Bhathena, S. J., Velasquez, M. T. Beneficial role of dietary phytoestrogens in obesity and diabetes. The American Journal of Clinical Nutrition. 76 (6), 1191-1201 (2002).
- Wang, L., Qiu, X. M., Hao, Q., Li, D. J. Anti-inflammatory effects of a Chinese herbal medicine in atherosclerosis via estrogen receptor beta mediating nitric oxide production and NF-kappaB suppression in endothelial cells. Cell Death and Disease. 4 (e551), 1-13 (2013).
- Xiao, J., Wang, N. L., Sun, B., Cai, G. P. Estrogen receptor mediates the effects of pseudoprotodiocsin on adipogenesis in 3T3-L1 cells. American Journal of Physiology-Cell Physiology. 299 (1), C128-C138 (2010).
- Guivarc’h, E., et al. Predominant Role of Nuclear Versus Membrane Estrogen Receptor alpha in Arterial Protection: Implications for Estrogen Receptor alpha Modulation in Cardiovascular Prevention/Safety. Journal of the American Heart Association. 7 (13), 1-17 (2018).
- Osako, M. K., et al. Estrogen inhibits vascular calcification via vascular RANKL system: common mechanism of osteoporosis and vascular calcification. Circulation Research. 107 (4), 466-475 (2010).
- Ivanova, A., et al. Screening of some saponins and phenolic components of Tribulus terrestris and Smilax excelsa as MDR modulators. In vivo. 23 (4), 545-550 (2009).
- Xiao, J., Zhu, T., Yin, Y. Z., Sun, B. Notoginsenoside R1, a unique constituent of Panax notoginseng, blinds proinflammatory monocytes to protect against cardiac hypertrophy in ApoE(-/-) mice. European Journal of Pharmacology. 833 (15), 441-450 (2018).
- Sun, B., Xiao, J., Sun, X. B., Wu, Y. Notoginsenoside R1 attenuates cardiac dysfunction in endotoxemic mice: an insight into oestrogen receptor activation and PI3K/Akt signalling. British Journal of Pharmacology. 168 (7), 1758-1770 (2013).
- Adams, M. R., et al. Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. Lack of an effect of added progesterone. Arteriosclerosis. 10 (6), 1051-1057 (1990).
- Keaney, J. F., et al. 17 beta-estradiol preserves endothelial vasodilator function and limits low-density lipoprotein oxidation in hypercholesterolemic swine. Circulation. 89 (5), 2251-2259 (1994).
- Nakashima, Y., Plump, A. S., Raines, E. W., Breslow, J. L., Ross, R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arteriosclerosis, Thrombosis, and Vascular Biology. 14 (1), 133-140 (1994).
- Saleh, T. M., Cribb, A. E., Connell, B. J. Estrogen-induced recovery of autonomic function after middle cerebral artery occlusion in male rats. American Journal of Physiology- Regulatory, Integrative and Comparative Physiology. 281 (5), R1531-R1539 (2001).
- Bingham, D., Macrae, I. M., Carswell, H. V. Detrimental effects of 17beta-oestradiol after permanent middle cerebral artery occlusion. Journal of Cerebral Blood Flow & Metabolism. 25 (3), 414-420 (2005).
- Pignoli, P., Tremoli, E., Poli, A., Oreste, P., Paoletti, R. Intimal plus medial thickness of the arterial wall: a direct measurement with ultrasound imaging. Circulation. 74 (6), 1399-1406 (1986).
- Hodgin, J. B., Maeda, N. Minireview: estrogen and mouse models of atherosclerosis. Endocrinology. 143 (12), 4495-4501 (2002).
- Bush, T. L., et al. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation. 75 (6), 1102-1109 (1987).
- Marsh, M. M., Walker, V. R., Curtiss, L. K., Banka, C. L. Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice. Journal of Lipid Research. 40 (5), 893-900 (1999).
Citazione di questo articolo
Sun, B., Yin, Y., Xiao, J. An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause. J. Vis. Exp. (150), e59536, doi:10.3791/59536 (2019).