【作者】 闫永吉；

【导师】 张旭；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 背景继发性高血压的最常见原因是原发性醛固酮增多症(Primary aldosteronism, PA),肾上腺醛固酮腺瘤(Aldosterone-producing adenoma, APA)是PA的主要类型。APA的主要治疗方法是腺瘤切除或者患侧肾上腺全切。然而,即使手术,仍有部分患者术后效果不佳,具体机制还未阐明。传统认为,醛固酮在肾上腺皮质球状带合成,通过肾脏引起水钠潴留导致高血压。最近发现,血管也能合成醛固酮,并表达盐皮质激素受体(MR)和11-β-羟基类固醇脱氢酶2(11-β-hydroxysteroid dehydrogenase2,11-β-HSD2)。所以,醛固酮可以直接作用于血管导致血管损伤。研究表明醛固酮可以引起血管氧化应激、炎症和纤维化等。体外实验证实,醛固酮可以刺激血管平滑肌细胞(Vascular smooth muscle cell, VSMC)增殖。Nakamura等报道,在细胞水平,醛固酮可以诱导人主动脉平滑肌细胞MDM2表达。MDM2是一种核蛋白,能够与p53形成复合体,阻止p53介导的细胞凋亡或逆转p53引起的G1期和G2期停滞,促进增殖,抑制其表达后,醛固酮的促VSMC增殖的能力也被抑制。我们前期研究发现,醛固酮灌注的大鼠主动脉MDM2表达增加。然而,在动物水平,醛固酮是否能够诱导主动脉平滑肌细胞增殖及MDM2和p53在其中的作用还需要进一步阐明。除了增殖,最近报道醛固酮还能够诱导多种细胞凋亡。醋酸脱氧皮质酮是另一种盐皮质激素,醋酸脱氧皮质酮-盐大鼠的主动脉内凋亡细胞增加。与正常大鼠相比,转基因Ren2大鼠血浆醛固酮和血管紧张素Ⅱ浓度升高,并且主动脉内凋亡细胞也增多。但是醛固酮单独是否能够诱导主动脉细胞凋亡还未被证实。此外,醛固酮还能够促进细胞外基质沉积。Fibulin家族蛋白是一类细胞外基质蛋白,fibulin-5是fibulin家族的新成员,它在弹力纤维的形成过程中是必不可少的。Fibulin-5表达异常与皮肤松弛症、肺气肿及年龄相关性黄斑退变等疾病密切相关。特发性门静脉高压患者的门静脉大分支fibulin-5表达增加。与之相反,胸主动脉夹层动脉瘤患者主动脉fibulin-5表达减少。相对于野生型细胞,来源于fibulin-5-/-小鼠的VSMC在丝裂原刺激下增殖和迁移能力增加,过表达fibulin-5可以抑制这种现象。研究还发现fibulin-5 mRNA在多种恶性肿瘤中表达缺失,提示fibulin-5在这些肿瘤中可能起抑癌的作用。所以,fibulin-5是一种多功能蛋白。最近发现,fibulin-5是TGF-β1新的靶基因,TGF-β1能够以剂量和时间依赖的方式诱导fibulin-5在成纤维细胞和内皮细胞内表达。综上所述,研究醛固酮对主动脉平滑肌细胞增殖、凋亡和细胞外基质蛋白fibulin-5的作用是十分重要的,能够帮助我们揭示PA的致病机理和APA术后高血压的原因。本课题通过建立APA大鼠模型,检测主动脉细胞增殖和凋亡的状态及相关基因的表达,最后检测主动脉fibulin-5含量和TGF-β1的表达。相较于传统上的非特异性MR拮抗剂螺内酯,依普利酮是一种特异性MR拮抗剂,能增强与MR结合的特异性,减少螺内酯引起的并发症。肼苯哒嗪是外周血管扩张剂,对主动脉影响很小。在本课题中我们还应用依普利酮和肼苯哒嗪干预醛固酮的作用,探讨醛固酮引起血管损伤的发生机制。方法1.32只SD大鼠随机分为4组,每组8只：①空白溶剂设为对照组(60%丙二醇+10%乙醇+30%双蒸水)；②APA组,泵入醛固酮(1μg／h)；③APA+特异性MR阻断剂组,依普利酮(100mg/kg/d);④APA+血管扩张剂组,肼苯哒嗪(25mg/kg/d)。根据分组情况,在ALZET 2004微量渗透泵储药仓内分别注入醛固酮溶液或空白溶剂,然后将泵埋植于大鼠皮下。ALZET 2004微量渗透泵可以持续工作4周,为使醛固酮作用时间达到8周,4周后更换渗透泵。依普利酮和肼苯哒嗪溶于饮用水中,每日灌胃一次。每周测量大鼠尾动脉收缩压一次。8周后,放免法检测血浆醛固酮浓度和肾素活性并测量大鼠主动脉中膜厚度和横截面面积。2.免疫组化和Western印迹法检测主动脉增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)阳性的VSMC数及PCNA的表达水平。随后通过免疫组化、RT-PCR和Western印迹检测MDM2和p53的表达水平。3.通过TUNEL法检测大鼠主动脉细胞凋亡。Western印迹法检测细胞色素c和caspase-3的含量。免疫组织化学和Western印迹法检测Bcl-2和Bax的表达水平。4.免疫组化和Western印迹法检测主动脉fibulin-5及TGF-β1的表达水平。结果1.与对照组相比,三周后APA组大鼠收缩压开始升高,第7周达到顶峰,之后维持在高水平(P<0.05)。服用依普利酮或肼苯哒嗪后,APA大鼠收缩压显著下降,但并不能降至对照组的水平(P<0.05)。泵入醛固酮,无论是否给予依普利酮或肼苯哒嗪,大鼠血浆醛固酮的浓度都明显升高(p<0.01),肾素活性则被显著抑制(p<0.01)。除对照组外其它三组大鼠醛固酮浓度和肾素活性没有显著差异。与对照组相比,APA组主动脉横截面面积和主动脉中膜厚度有增加的趋势,但是差异没有统计学意义(P=0.108；P=-0.068)。2.与对照组大鼠相比,免疫组化结果显示APA大鼠主动脉PCNA阳性VSMC显著增多(P<0.01),Western印迹进一步证实APA大鼠主动脉PCNA表达更多。MDM2在mRNA和蛋白水平表达都上调(P<0.05)；而p53只在蛋白水平表达增加(P<0.05)。3.相较于对照组大鼠,APA大鼠主动脉显示TUNEL阳性细胞显著增多(P<0.05),有细胞色素C和活化的caspase-3增加(P<0.05)；Bax表达上调(P<0.05),Bcl-2表达下降(P<0.05)。4.APA大鼠主动脉与对照组相比,fibulin-5和TGF-β1表达均显著增加(P<0.01)。5.醛固酮的上述作用能够被依普利酮抑制,但不被肼苯哒嗪抑制。结论1.通过在大鼠皮下埋植的微量渗透泵持续灌注醛固酮能够模拟APA的激素释放方式,建立APA模型,为研究APA的致病机理提供了很好的平台。2.在体内,醛固酮通过诱导MDM2表达,逆转p53对细胞周期的阻滞作用,促进VSMC增殖。3.醛固酮通过线粒体途径诱导平滑肌细胞和内皮细胞凋亡。4.醛固酮促使主动脉fibulin-5沉积增加,后者至少部分是通过醛固酮诱导的TGF-β1引起的。5.醛固酮的上述作用是醛固酮与MR结合后对主动脉的直接作用,与醛固酮的血压升高效应无关。更多还原

【Abstract】 BackgroundPrimary aldosteronism (PA), mainly deriving from adrenal aldosterone-producing adenoma (APA), is the most common cause of secondary hypertension. However, even after the resection of adenomas, a part of patients still suffer from hypertension, which is possibly due to irreversible vascular remodeling. Aldosterone is traditionally believed to be produced in the adrenal cortex and elevates BP through renal effects on sodium and water retention via binding to the mineralocorticoid receptor (MR). However, it was recently reported that blood vessels per se produced aldosterone and synthesized MRs and 11-β-hydroxysteroid-dehydrogenase-2, an essential enzyme for aldosterone specifically binding to MRs. Thus, blood vessels possess a complete system for aldosterone activity. a lot of studies have shown that aldosterne induces vascular oxidative stress, inflammation and fibrosis, resulting in vascular damage and remodeling.In in vitro studies, aldsoterone was shown to stimulate vascular smooth muscle cell (VSMC) proliferation. MDM2, a mediator that promotes cell proliferation mainly by forming a complex with p53 and inhibiting p53-mediated G1 and G2 cell cycle arrest and apoptosis. Recently, Nakamura et al. reported that aldosterone induced MDM2 expression in aortic smooth muscel cells by mineralocorticoid receptors. Moreover, after suppressing MDM2 expression, the effect of aldosterone on VSMC proliferation was also inhibited. Our previous study showed that aldosterone induced MDM2 expression in the aorta in vivo. However, whether aldosterone induces VSMC proliferation in vivo as well as the roles of MDM2 and p53 in the proliferation effect of aldosterone need to be elucidated. In addition to VSMC proliferation, previous studies have shown that aldsoterone induces vascular oxidative stress, inflammation and fibrosis, resulting in vascular damage and remodeling. Recently, aldosterone was demonstrated to induce the apoptosis of several types of cells. In addition, deoxycorticosterone acetate (DOCA)-salt rats, another animal model of excessive mineralocorticoid, presented with increased apoptotic vascular smooth muscle cells (VSMCs) in the aorta. Moreover, transgenic Ren2 rats with elevated plasma aldosterone concentration and angiotensin II activity exhibited more apoptotic vascular cells in the aorta than normal rats. Nevertheless, whether aldosterone stimulates aortic apoptosis in vivo is unknown.Besides, aldosterone also promotes extracellular matrix (ECM) deposition. Fibulin-5, a secreted (ECM) glycoprotein, is the new member of fibulin family. Recent studies have identified fibulin-5 as an essential protein for elastic fiber assembly. Inactivation of the fibulin-5 gene in mice results in a generalized elastinaopathy affecting those organs with abundant elastic fibers including blood vessels. Fibulin-5 was shown to be involved in phlebosclerosis of major portal vein branches associated with elastic fiber deposition in idiopathic portal hypertension. In contrast, decreased expression of fibulin-5 correlating with reduced elastin in thoracic aortic dissection was found. Compared with wild type cells, VSMCs from fibulin-5-/- mices demonstrated more proliferation and migration under the stimulation of mitogen, which could be inhibited after overexpression of fibulin-5, suggesting that fibulin-5 is a suppressor of proliferation and migration. Studies demonstrated the lack of fibulin-5 mRNA in most malignant tumors, implying that fubulin-5 acts as a suppressor in most cancers. Therefore, fibulin-5 is a multfunctional protein. Fibulin-5 was reported to be a new target gene of TGF-β1 that induces fibulin-5 expression in fibroblasts and endothelial cells.In the current study, we first constructed rat models of APAs by subcutaneously implanting osmotic mini-pumps in rats. After 8 week infusion of aldosterone, aortic morphology was observed. Then we examined the status of aortic proliferation and apoptosis as well as the expressions of proliferation and apoptosis-related genes. Finally, we evaluated the effects of aldosterone on fibulin-5 deposition and TGF-β1 expression in the aorta. Relative to spironolactone, eplerenone is a specific mineralocorticoid receptor (MR) blocker, which can enhance the selectivity of binding MRs and reduce the side effects by spironolactone. Hydralazine is an old drug used for the treatment of hypertension by directly dilating the resistance arteries with minor effects on aortas. In the present study, we take use of the two drugs to interfere the effect of aldosteone on the aorta to explore the mechanisms of aortic damage by aldosterone.Methods1.32 Sprague-Dawley rats, each subcutaneously implanted with an osmotic mini-pump, were randomly divided into 4 groups:vehicle as control; aldosterone (1μg/h); aldosterone plus eplerenone (100 mg/kg/day) and aldosterone plus hydralazine (25 mg/kg/day). Because Alzet 2004 minipump continuously infuses the test substances only for 4 weeks, another minipump was implanted after the fourth week to realize the goal of 8 week effect of aldosterone on rats. Eplerenone and hydralazine were dissolved in drinking water. Systolic blood pressure (SBP) was measured weekly by the tail-cuff method. At the termination of the study, blood was collected for measurements of plasma renin activity (PRA) and aldosterone concontration (PAC) with radioimmunoassay kits. Then, media width (MW) and cross sectional area (MCSA) of the aortas were measured.2. After 8 weeks, the PCNA-positive VSMCs and PCNA expression level in the aorta were examined by immunohistochemistry and Western blotting. In addition, the expressions of MDM2 and p53 genes were examined by immunohistochemistry, RT-PCR and Western blotting.3. Apoptosis was examined by TUNEL assay. The levels of cytochrome c and caspase-3 were determined by Western blotting and the expressions of Bax and Bcl-2 were evaluated by immnuohistochemistry and Western blotting.4. The expressions of fibulin-5 and TGF-β1 were examined by immnuohistochemistry and Western blotting.Results1. Rat models of APAs were successfully established. SBP was similar among the groups at the beginning of the experiment. After 3 weeks, aldosterone-infused rats showed significantly and progressively increased SBP compared with controls (P<0.05). The increase was significantly but incompletely inhibited after treatment with eplerenone or hydralazine (P<0.05). As expected, PAC was significantly increased with significantly depressed PRA in APA rats; and both were unaffected by eplerenone or hydralazine.2. The MW and MCSA of the aorta in APA rats had a trend to increase relative to those of control rats. However, the increase did not achieve statistical significance(P=0.108; P=0.068, respectively).3. Compared with controls, APA rats showed more PCNA-positive cells and higher PCNA expression (p<0.01). Upregulated expressions of both MDM2 mRNA and protein were shown in the aorta, associated with elevated p53 protien (P<0.05).4. Compared with controls, APA rats exhibited elevated aortic vascular cell apoptosis accompanied with higher levels of cytochrome c and activated caspase-3 as well as significantly upregulated Bax and downregulated Bcl-2.4. Relative to control rats, APA rats exhibited increases in fibulin-5 deposition and TGF-β1 expression.5. All the above effects of aldosterone on the aorta were significantly inhibited after co-administration with eplerenone but not with hydralazine.Conclusions1. By subcutaneously implanting osmotic minipumps in rats, the rat models of APAs can be successfully established, providing a good platform for investigating the pathogenesis of APAs.2. Aldosterone promotes aortic VSMC proliferation, which contributes to aldosterone-induced vascular remodeling, at least partially by upregulating the expression of MDM2 that is able to inhibit the effect of cell cylcle arrest of simultaneously elevated p53.3. Aldosterone induces vascular cell apoptosis in vivo, which may contribute to aldosterone-mediated aortic injury.4. Aldosterone directly promotes aortic deposition of fibulin-5 that is at least partially stimulated by aldsoterone-induced TGF-β1.5. All the above effects of aldosterone on the aorta are the direct effects of aldosterone via MRs and independently of the aldosterone-mediated increase of blood pressure.更多还原