醛固酮对减压神经中ENaC表达的影响
发布时间：2018-03-24 03:18
本文选题：动脉压力反射 切入点：ENaC 出处：《郑州大学》2017年硕士论文
【摘要】：背景介绍心力衰竭(Heart failure,HF)简称心衰,是各种心脏疾病,比如冠心病、高血压以及慢性肾脏病等各种心血管疾病发展的终末阶段。压力反射(baroreflex)又称减压反射,是心血管活动调节的重要机制之一。位于主动脉弓和颈动脉窦的感觉神经末梢感受动脉血压的变化,经减压神经和窦神经传入,到达心血管中枢,通过调节交感神经和迷走神经的功能,平衡心血管活动。1969年Smyth等人首次以Smyth法测定BRS(baaroreflex sensitivity)来表示压力反射的敏感性。心衰动物的压力反射敏感性明显降低,扰乱自主神经平衡,导致交感神经兴奋性增强,副交感神经活动降低,从而使发病率和死亡率增加。据文献报道,减压反射的传入神经敏感性和兴奋性降会低导致减压反射敏感性降低,但至今尚不清楚传入神经对血压变化敏感性降低的机制。研究发现HF引起Na+通道功能降低抑制了减压神经的电活动,而在减压神经末梢上存在着感受血压变化的机械敏感性离子通道,心衰可能是改变了这种机械敏感性离子通道的功能,降低减压神经对血压变化的敏感性。上皮钠通道(Epithelial Sodium Channel,ENa C)属于ENa C/Deg家族,具有阿米洛利敏感性,是该家族中目前研究最为广泛的分子。ENa C是由α、β、γ、δ四种亚基组成的异源多聚体蛋白,主要分布于肾脏等上皮组织中,功能是维持水,钠平衡。通过醛固酮与细胞内的醛固酮受体结合,增加ENa C通道在细胞膜上的表达,调节ENa C介导的Na+细胞转运。在神经节等非上皮组织中,ENa C是机械敏感性离子通道。发生心力衰竭(HF)时醛固酮显著升高,升高正常动物或者人血液中的醛固酮显著降低减压反射的敏感性。已有实验发现减压神经末梢和胞体中有ENa C通道蛋白的分布,而对醛固酮是否影响减压神经中ENa C通道及其可能机制尚未报道。目的本实验模拟心衰引起大鼠机体醛固酮浓度升高,观察减压反射敏感性变化,以及结状神经节的减压神经胞体中ENa C表达变化,以确定血浆醛固酮升高是否影响减压神经中ENa C通道的表达。方法1、采用渗透泵持续灌输醛固酮的方法,制作高醛固酮动物模型。体重200 g的SD雄性大鼠在10%的水合氯醛腹腔麻醉状态下,于腹背后皮下植入Alzet2004微型渗透泵。2、30只健康雄性SD大鼠给予正常饲料,适应喂养1周后,随机分为5组,均喂养4周。所有的大鼠在20%的乌拉坦(0.5 ml/100 g)腹腔麻醉下,(1)正常对照组(CON,n=6):正常饮食和饮水。(2)溶剂组(VEH,n=6):正常饮食和饮水,装有5%甲醇的渗透泵。(3)螺内酯组(SPI,n=6):正常饮食和饮水的基础上,每天以25 mg/kg灌胃。(4)醛固酮组(ALD,n=6):正常饮食和饮水,将装有溶解在5%甲醇的醛固酮的渗透泵,埋于大鼠腹背部皮下。(5)醛固酮+螺内酯组(ALD+SPI,n=6):正常摄食基础上,皮下植入渗透泵,泵中装有溶解在5%甲醇中的醛固酮。醛固酮(0.75μg/h)或仅5%甲醇经微量渗透泵持续皮下恒流输注。螺内酯(25 mg/kg/day)经灌胃。3、测量血浆醛固酮浓度:模型制备后,于实验前,取SD大鼠的动脉血液1.5-2ml。离心机以1000 g离心15分钟,去出上清液,放置-80℃保存。使用大鼠醛固酮试剂盒测量大鼠血浆内醛固酮的浓度。4、血压和心率的测量:用20%的乌拉坦5 ml/kg腹腔麻醉,使用股动脉插管技术,测量血压和心率,每只动物测量3次,取其收缩压的平均值作为基础值。5、动脉压力反射敏感性测定:各组动物于4周后,参照文献按照改良的Smyth方法进行动脉压力反射敏感性检测。股静脉插管,通过股静脉给药,注射硝普钠(SNP)和苯肾上腺素(PE)分别诱发升压反射和降压反射,以反射前后的收缩压变化值与心动周期变化值之比(ΔSBP/ΔCC)作为升压反射敏感性(BRS-SNP)和降压反射敏感性(BRS-PE)指标。6、取五组大鼠的结状神经节(NG),采用免疫组织化学技术检测ENa C-β、ENa C-γ的表达,并使用ENa C-β、ENa C-γ与Neu N蛋白标志的免疫荧光共染色,鉴别ENa C-β、ENa C-γ在结状神经节(NG)中的分布。7、4周后取五个组大鼠的结状神经节(NG)组织,采用Western blot分别检测脊结状神经节(NG)中的ENa C-β和ENa C-γ表达及变化。8、统计学处理:实验数据以均值±标准差(mean±SD)表示,采用Graph Pad Prism5.0统计软件,采用单因素方差分析和t检验,P㩳0.05为差异有统计学意义。结果1.给醛固酮4周后,大鼠体内醛固酮550.9±7.63 pg/ml,明显高于对照组(193.2±4.95 pg/ml,P㩳0.001)。2.升高醛固酮后,大鼠血压(98.14±3.76 mm Hg)、心率(357.8±8.32 beat/min)、体重(412.7±7.65 g)与对照组相比(94.65±3.97 mm Hg,345.7±11.59 beta/min,416.8±10.3 g),无明显改变(P㧐0.05)。3.醛固酮升高后,大鼠减压反射敏感性(BRS)(0.225±0.05 ms/mm Hg)比正常组(1.113±0.04)ms/mm Hg)的明显降低(P㩳0.001)。而醛固酮受体阻断剂螺内酯(0.973±0.05 ms/mm Hg)可以阻断醛固酮对BRS的影响组,使之恢复近正常水平。4.免疫荧光双染确定ENaC-β和ENa C-γ蛋白在NenN标记的神经元胞体(结状神经节)处有表达。5.通过渗透泵维持大鼠机体高醛固酮浓度,采用western blot检测发现:醛固酮组的结状神经节中表达β/γENa C蛋白,正常组为100,ENa C-β和ENa C-γ蛋白的表达量分别为31.51±3.33和27.10±3.52(P㩳0.001)比正常组明显减少,而醛固酮+螺内酯合用组的β/γENa C的表达量分别为97.08±10.08和98.05±11.14(P㧐0.05)与正常组比较,基本恢复,表明醛固酮受体阻断剂螺内酯阻断了醛固酮对ENa C的表达。结论正常大鼠体内,ALD升高,通过与受体途径降低减压反射神经元胞体中ENa C表达,减弱压力反射。
[Abstract]:Background heart failure (Heart failure HF) is referred to as heart failure, heart diseases, such as coronary artery disease, end stage chronic kidney disease and hypertension and other cardiovascular disease. Baroreflex (baroreflex) also called depressor reflex, is one of the important mechanisms regulating cardiovascular activity. Changes of sensory nerve endings in the aortic arch and carotid artery sinus feeling of arterial blood pressure, after decompression of nerve and sinus nerve afferent, reach the cardiovascular center, through the regulation of sympathetic nerve and vagus nerve function, cardiovascular activity.1969 Smyth balance for the first time in the determination of BRS by Smyth (baaroreflex sensitivity) to represent the baroreflex sensitivity. Baroreflex sensitivity was significantly reduced heart failure animal, disrupting autonomy nerve balance, leading to enhanced excitability of sympathetic nerve and parasympathetic nerve activity decreased, thereby increasing morbidity and mortality. According to the text Reported, afferent nerve sensitivity and excitability of the depressor reflex low lead depressor reflex sensitivity decreased, but it is still not clear on the mechanism of nerve afferent blood pressure reduced susceptibility. The study found that the Na+ channel function is reduced to inhibit the electrical activity of depressor nerve caused by HF, the mechanosensitive ion channels and the change of blood pressure is felt in vacuum on nerve endings, heart failure may be changed this mechanosensitive ion channel function, reduces the sensitivity to changes in blood pressure depressor nerve. The epithelial sodium channel (Epithelial Sodium Channel, ENa C ENa) belongs to the C/Deg family, with amiloride sensitive in the family, is currently the most widely studied molecular.ENa C by alpha beta, gamma, delta four heterologous subunits of multimeric protein mainly distributed in kidney epithelial tissues, function is to maintain the water and sodium balance through aldosterone and fine. Combined with the intracellular mineralocorticoid receptor, increases the expression of ENa C channel in the cell membrane, regulate ENa mediated by C Na+ cells in the ganglion. Other non epithelial tissues, ENa C is a mechanosensitive ion channel. The occurrence of heart failure (HF) when aldosterone increased significantly increased in normal animals or human blood aldosterone significantly reduce the sensitivity. The depressor reflex experiments indicated that the distribution of ENa C protein in decompression nerve terminals and cell bodies, and whether the effects of ENa on aldosterone C nerve decompression channel and its possible mechanism has not yet been reported. The purpose of this experiment simulated the body caused by heart failure aldosterone concentration in rats was observed and ENa depressor reflex sensitivity, C the nodose ganglion neurons in the expression of decompression, to determine whether elevated plasma aldosterone on expression of ENa in C channel depressor nerve. Methods 1, using osmotic pump continuous irrigation 杈撻啗鍥洪叜鐨勬柟娉，
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