【作者】 孙要军；

【导师】 康玉明；

【作者基本信息】 山西医科大学 ， 生理学， 2011， 博士

【摘要】 心力衰竭时下丘脑室旁核血管紧张素转换酶和血管紧张素1型受体与NMDA受体相互作用对交感神经活动的影响慢性心力衰竭(简称心衰),是由各种心血管疾病导致心输出量降低并伴有心功能进行性恶化的临床综合征,是缺血性心脏病等多种心血管疾病的主要并发症。探讨其发病机制和寻找有效的防治手段是目前心衰研究的热点课题。而持续的交感神经过度激活是心衰的重要特征,可以促进心衰的恶化。因此降低交感神经的兴奋性成为心衰治疗的关键。近年来研究发现中枢神经体液机制的改变会使心衰时已经存在的水钠潴留和交感神经激活更加显著,使心功能进一步恶化并形成恶性循环。并且交感神经中枢的激活是构成心衰病理机制的重要因素。因此,关于心衰时中枢调控机制的研究日益受到重视。交感神经节前神经元的活动完全受下丘脑室旁核(PVN)和延髓头端腹外侧区(RVLM)等心血管中枢的控制。其中室旁核是调控交感神经传出活动的重要中枢位点。心衰时室旁核神经元被激活,使交感神经活动增强,促进心衰的发展。肾素-血管紧张素系统(RAS)是体内重要的体液调节系统,主要通过血管紧张素Ⅱ(ANGⅡ)与AT1受体(AT1-R)结合起作用。心衰时外周肾素-血管紧张素系统等神经内分泌机制被明显激活并且伴有血流动力学改变和交感神经活动持续增强。而研究证实,心血管中枢PVN、RVLM和孤束核等区域存在血管紧张素转换酶(ACE)和AT1-R的表达,对心血管功能和交感神经的活动具有重要的调节作用。脑内ACE主要介导ANGⅡ的产生,中枢ACE的活性在维持正常大鼠肾交感神经的基础紧张性放电中具有重要作用。给正常大鼠室旁核注入ANGⅡ可明显增强交感神经活动。因此非常有必要对RAS的中枢机制进行更深入的探索。室旁核中含有大量的神经递质对交感神经活动和心血管功能具有重要的调节作用。其中最重要的是谷氨酸能系统。研究证实室旁核中神经元激活之所以与心衰有关,主要是室旁核中谷氨酸能机制激活导致的。室旁核内谷氨酸通过谷氨酸受体参与心血管反射,调控交感神经的传出活动和心血管系统的功能。给正常大鼠PVN内微量注入N-甲基-D-天门冬氨酸(NMDA)时,引起血压升高,心率增快,肾交感神经活动增强。NMDA受体是谷氨酸离子型受体的一个主要亚型,由一个NR1亚基(NMDAR1)和一个或多个NR2亚基组成,其功能主要取决于NR1亚基。心衰时刺激室旁核内NMDA受体可使交感神经放电增加,室旁核微量注入NMDA受体拮抗剂则出现相反的情况。此外,心衰时室旁核中去甲肾上腺素增加,引发的外周交感活动增强与心力衰竭的发生发展密切相关；肾交感神经放电活动的调节也受PVN中GABA的内源性抑制作用所控制,参与交感活动的中枢调节。心衰时室旁核中各种神经递质发生改变,参与心血管反射活动的调节,对交感神经兴奋性产生影响。体外研究发现,对同时表达AT1-R和NMDAR1的神经元使用ANGⅡ处理后,可以观察到神经元NMDAR1蛋白的表达呈剂量依赖性增加,AT1-R阻断剂氯沙坦则可以阻止该现象的发生,使NMDAR1蛋白表达不能上调。神经元生存环境中ANGⅡ增加可以促进神经元内谷氨酸机制激活,NMDAR1蛋白表达增加,中枢神经系统内RAS与谷氨酸能机制之间可能存在相互作用。在本研究中,我们进行了以下工作：采用冠脉结扎术建立大鼠心衰模型,经微型渗透泵给室旁核内持续微量注入不同的药物干预4周后,1、观察心力衰竭时大鼠下丘脑室旁核内ACE和AT1-R的变化及其对交感神经活动的影响；2、观察心衰时下丘脑室旁核神经递质系统中NMD AR1、TH和GAD67的变化及其对外周交感神经活动的影响；3、通过使用血管紧张素1型受体阻滞剂、血管紧张素转换酶抑制剂和NMDA受体拮抗剂研究心衰时室旁核内源性ACE和AT1-R与谷氨酸能机制发生的变化,观察RAS与谷氨酸能机制之间是否存在相互作用,共同影响交感神经的传出活动。第一章心力衰竭时大鼠下丘脑室旁核内血管紧张素转换酶和血管紧张素1型受体的变化对交感神经活动的影响[目的]心力衰竭时肾素-血管紧张素系统激活,交感神经活动持续加强,最终导致心功能不断恶化。下丘脑室旁核(PVN)是调控交感神经传出活动的重要中枢位点和整合部位。本文主要观察心衰时大鼠下丘脑室旁核内ACE和AT1-R的变化及其对心衰发生发展和交感神经活动的影响。[方法]选取雄性SD大鼠48只,首先进行室旁核插管术。1周后通过冠脉结扎术建立大鼠心衰模型或假手术模型,同时室旁核插管连接微型渗透泵分别持续微量注入AT1-R阻滞剂缬沙坦(VAL,0.05μg/h. PVN),血管紧张素转换酶抑制剂(ACEI)赖诺普利(Lisinopril,0.46μg/h. PVN)或空白对照药人工脑脊液(Vehicle,0.11μl/h.PVN)干预。4周后经血流动力学检测观察心功能；电生理记录肾交感神经放电活动；计算肺／体重比(Lung/BW)和右心室／体重比(RVW/BW);免疫组织化学染色观察下丘脑室旁核内ACE表达的变化；ELISA法测定血浆NE和AngⅡ含量；Western blot技术测定下丘脑室旁核内Fra-like和AT1-R蛋白的含量变化。[结果]与假手术组比较,心衰大鼠心功能明显降低：左心室舒张木压(LVEDP)明显升高,±dp/dtmax和左室射血分数(LVEF)明显下降,左室前负荷增加(RVW/BW和Lung/BW明显升高,p<0.05)；肾交感神经放电明显增强；血浆NE和AngⅡ浓度增高(p<0.05)；PVN内Fra-like、ACE和AT1-R表达增加(p<0.05)。与空白对照药治疗的心衰大鼠比较,接受缬沙坦或赖诺普利治疗的心衰大鼠LVEDP、RVW/BW和lung/BW有所降低(P<0.05),±dp/dtmax升高(P<0.05),LVEF无明显改变,PVN区域的Fra-like、ACE和AT1-R表达减少(P<0.05),外周血NE和ANGⅡ下降(P<0.05),肾交感神经放电减少(P<0.05)。[结论]心衰时PVN区域内源性的ACE和AT1-R被明显激活引起交感神经放电增加,心功能明显减退。交感神经放电第二章心力衰竭时大鼠下丘脑室旁核内NMDA受体的改变及其对交感神经活动的影响[目的]室旁核中含有大量的神经递质对交感神经活动和心血管功能具有重要的调节作用。其中最重要的是谷氨酸能系统。此外,心衰时室旁核去甲肾上腺素增加,GABA的内源性抑制作用减弱也参与交感活动的中枢调节。本部分主要探讨心衰时大鼠下丘脑室旁核内NMDAR1、TH和GAD67的变化及其对交感神经活动的影响。[方法]选取雄性SD大鼠36只,首先进行室旁核插管。1周后采用冠脉结扎术建立大鼠心衰模型或假手术模型,同时各组经室旁核插管连接微型渗透泵持续给予特异性NMDA受体阻滞剂AP5 (0.02μg/h.PVN)或空白对照药人工脑脊液(vehicle,0.11μl/h. PVN)进行干预。4周后经血流动力学检测观察心功能；电生理记录肾交感神经放电活动；计算肺／体重比(Lung/BW)和右心室／体重比(RVW/BW); ELISA法测定血浆NE和AngⅡ含量；免疫组织化学染色观察下丘脑室旁核内TH表达的变化；使用Western blot技术测定下丘脑室旁核内NMDAR1和GAD67蛋白含量变化。[结果]与假手术大鼠比较,心衰大鼠心功能明显下降：左心室舒张末压(LVEDP)明显升高,±dp/dtmax明显降低,左室射血分数(LVEF)明显下降,(p<0.05),左室前负荷增加(Lung/BW和RVW/BW明显升高,p<0.05)；肾交感神经放电明显增强,血浆NE和AngⅡ的浓度增高(p<0.05)；室旁核内NMDAR1和TH表达增加,GAD67的表达减少(p<0.05)。与空白对照药治疗的心衰大鼠比较,接受AP5治疗后心衰大鼠LVEDP、RVW/BW和lung/BW有所降低(P<0.05),±dp/dtmax升高(P<0.05),LVEF无明显改变,室旁核区域的NMDAR1和TH表达减少,GAD67表达增加(P<0.05),外周血NE和AngⅡ浓度下降(P<0.05),肾交感神经放电活动减少。[结论]心衰时下丘脑室旁核中谷氨酸能机制(NMDAR1)被明显激活,TH活性增强,GAD67活性降低,可以使交感神经的放电增加,促进心力衰竭的发生发展。第三章心力衰竭时下丘脑室旁核血管紧张素转换酶和血管紧张素1型受体与NMDA受体相互作用对交感神经活动的影响[目的]肾素-血管紧张素系统是体内重要的体液调节系统,心血管中枢下丘脑室旁核内存在血管紧张素转换酶(ACE)和血管紧张素1型受体(AT1-R)的表达,对心血管功能和交感神经的活动具有重要的调节作用。同时室旁核内谷氨酸能系统的激活可以增加交感神经放电,影响心血管功能。本部分主要探讨心力衰竭时室旁核内ACE、AT1-R与NMDA受体之间可能存在的相互作用及其对外周交感神经放电活动的影响。[方法]选取雄性SD大鼠60只,首先进行室旁核插管术。1周后采用冠脉结扎术建立大鼠心衰模型或假手术模型,同时各组室旁核插管连接微型渗透泵持续给予AT1-R阻滞剂缬沙坦(VAL,0.05μg/h. PVN),ACEI赖诺普利(Lisinopril,0.46μg/h. PVN),特异性NMDA受体阻滞剂AP5(0.02μg/h.PVN)或空白对照药人工脑脊液(vehicle,0.11μl/h. PVN)干预。4周后经血流动力学检测观察心功能；电生理记录肾交感神经放电活动；计算肺/体重比(Lung/BW)和右心室/体重比(RVW/BW);免疫组化染色观察下丘脑室旁核内ACE和TH表达的变化；ELISA法测定血浆NE和AngⅡ含量；采用Western blot技术测定室旁核内Fra-like、AT1-R、NMDAR1和GAD67蛋白含量的变化。[结果]与假手术组比较,心衰大鼠心功能明显降低,左心室舒张末压(LVEDP)明显升高,左室射血分数(LVEF)明显下降,±dp/dtmax明显降低(p<0.05),左室前负荷增加(Lung/BW和RVW/BW明显升高,p<0.05)；肾交感神经放电明显增强,血浆NE和AngⅡ浓度增高(p<0.05)；室旁核内Fra-like、ACE、AT1-R、NMDAR1和TH的表达增加,GAD67表达减少(p<0.05)。与空白对照药治疗的心衰大鼠比较,接受缬沙坦、赖诺普利或AP5治疗后心衰大鼠LVEDP, RVW/BW和lung/BW有所降低(P<0.05),±dp/dtmax升高(P<0.05),室旁核区域的Fra-like、ACE和AT1-R、NMDAR1和TH表达减少,GAD67表达增多(P<0.05),外周血NE和ANGⅡ下降(P<0.05)；肾交感神经放电活动减少。[结论]心衰时室旁核区域的ACE和AT1-R被明显激活,使交感神经活动增强,并且可以对室旁核内谷氨酸能机制产生影响,ACE和AT1-R的激活可以增强NMDA的作用。在中枢水平针对二者同时采用相应的治疗措施,有望更有效地改善心衰症状,使交感神经放电减少,提高慢性心衰患者的生存率和生活质量。更多还原

【Abstract】 Interaction Between the Angiotensin Converting Enzyme or Angiotensin Type 1 Receptor and NMDA Receptor in the Paraventricular Nucleus Contribute to Sympathoexcitation in Heart FailureCongestive heart failure (CHF) is a malignant clinical symptom accompanied by decreased cardiac output and aggravated heart function in progress. It is the main complication of various kinds of cardiovascular disease especially in coronary artery disease. The elevated sympathetic nerve activity is the most important characteristic symptom of CHF, which is a major factor contributing to the worsen heart function of CHF. Inhibition of sympathoexcitation is the basic therapy for CHF. The mechanism(s) of it remained to be unresolved. It has been considered that left ventricular dynamics and overactive renin-angiotensin system are the cardinal manifestation of CHF. Recent studies pointed to a central mechanism that contributes to the vicious cycle of volume accumulation, elevated sympathetic nerve discharge and declining heart function in CHF. Central neurohumoral excitation induced a sympathetic dysfunction.The activation of sympathetic preganglionic neurons is controlled directly by the paraven-tricular nucleus (PVN) and the rostral ventrolateral medulla (RVLM). PVN of the hypothalamus is an important region that mediate sympathetic nerve outflow. The increased neuronal excitation accompanied by neurohumoral changes in PVN in HF rats. It will contribute to the aggravated heart function and increased sympathetic nerve activity. The renin-angiotensin system is a major component of the neuroendocrin system. ANGⅡ, the central member of RAS, carries out its functions via AT1 receptors. AT1 receptors and angiotensin converting enzyme have been identi-fyied in the PVN, RVLM and nucleus tractus solitarii(NTS). They may play an essential role in regulating sympathetic nerve activity and cardiovascular function. Injection of ANGⅡinto PVN induced an augment in renal sympathetic nerve activity (RSNA). And the brain ACE activity plays an important role in the baseline RSNA in normal rats.It is necessary for us to investigate the central mechanism(s) of CHF.A number of neurotransmitters in PVN play a key role in regulating sympathetic nerve activity and cardiovascular function. The glutamatergic system is the most important one. The increased glutamatergic mechanisms within the PVN induced the overacting of sympathetic drive in CHF. Functional studies have indicated that glutamatergic mechanisms in the PVN are involved in the accommodation of cardiovascular reflexes. It has been reported that functional NMDA receptor is one of the main subtype of ionotropic glutamate receptor, which was composed of at least one NR1 subunit in combination with one or more NR2 subunits. The function of NMDA receptor may be limited to NR1 subunit. Recent studies from Li et al found that in rat with CHF, stimulation of the NMDA receptor in the PVN has been shown to induce increased fire of renal sympathetic nerve. In contrast, microinjection of the NMDA receptor antagonist into the PVN caused significant decreases in BP, HR and RSNA in rats with HF. Furthermore, the norepinephrine level in the PVN was increased during heart failure and influe-nce sympathetic outflow. The basic RSNA is largely due to diminish in basal GABAergic synaptic activity. It is considered that the alterations in neuronal transmissions in the PVN of HF rats play a key role in the regulation of RSNA.In a vitro study, neurons was incubated with ANGⅡ, which have both AT1-R and NMDAR1.can be observed a dose-dependent increase in the expression of NMDAR1 protein, In addition losartan can prevent it, the expression of NMDAR1 can not be upregulated. ANGⅡcan promote the activation of neuronal mechanisms of glutamate, increase NMDAR1 protein expression, There may be interactions between RAS and GLU system in the central nervous systemThe purposes of the present study are:(1) investigate the changes of ACE and AT1-R within the PVN in CHF rats and their contribution to RSNA,these rats received different drug treatment through chronic PVN infusion via osmotic minipump after four weeks. (2) observe the changes of NMDAR1, TH and GAD67 in the PVN in CHF rats and their effects on RSNA. (3) through chronic PVN infusion with AT1-R antagonist, ACEI or AP5 via osmotic minipump, observe the changes of AT1-R or ACE in the PVN, the interaction between RAS and glutamate-ergic mechanisms and its influence on sympathoexcitation.ChapterⅠThe Changes of the Angiotensin Converting Enzyme and Angiotensin Type 1 Receptor in the Paraventricular Nucleus Contribute to Sympathoexcitation in Heart FailureAims:The elevated renin-angiotensin system and sympathetic nerve activity, with aggravated heart function are the cardinal manifestation of congestive heart failure (CHF). The paraventricular nucleus (PVN) of the hypothalamus is an important region that mediate sympathetic nerve outflow. The purposes of the present study are to investigate the expression changes of the angiotensin converting enzyme (ACE) and the angiotensin type 1 receptor (AT1-R) within the PVN in CHF rats and its contribution to RSNA. Methods:Adult male Sprague-Dawley rats weighing 230-250g were implanted with PVN cannulae. A week later, HF was induced by ligation of the left anterior descending coronary artery and SHAM rats underwent the same surgery but did not undergo coronary ligation. Subsequently, animals were PVN treated with the AT1-R antagonist Valsartan (0.05μg/h), ACE inhibitor Lisinopril (0.46μg/h) or vehicle (VEH,0.11μl/h) for 4 weeks. After 4 weeks, Left ventricular function was measured by the hemodynamic parameters. Renal sympathetic nerve discharge was recorded by electrophysiological techniques; Lung/body weight ratio (Lung/BW) and right ventricle/ body weight ratio (RVW/BW) was calculated; Immunohistochemical staining was used for expression of ACE in the hypothalamic paraventricular nucleus; Plasma NE and Angll content were measured using ELISA techniques; Fra-like and AT1-R protein levels of the hypothalamic paraventricular nucleus was measured with Western blot technique. Results:HF rats had a decrease in±dp/dtmax and LV ejection fraction (LVEF), a significant increase in left ventricular end diastolic pressure (LVEDP), sympathetic nerve activity,the expression of Fra-LI (markers of activated neurons), AT1-R and ACE in the PVN, and plasma angiotensinⅡ(ANGⅡ) and norepinephrine (NE) levels when compared with SHAM rats. In contrast, PVN treated with Valsartan or Lisinopril attenuated Fra-LI, AT1-R and ACE expression in the PVN compared with VEH-treated HF rats, and recovered cardiac function partly by elevating±dp/dtmax and reducing LVEDP. The treatment also decreased RVW/BW and lung/BW and reduced plasma levels of NE and ANGⅡ. Conclusion:These findings confirm that activated AT1-R and ACE in the PVN contribute to the increased sympathetic nerve activity and the pathophysiology of congestive heart failure.ChapterⅡThe Changes of NMDA Receptor in the Paraventricular Nucleus Contribute to Sympathoexcitation in Heart FailureAims:A number of neurotransmitters in the paraventricular nucleus (PVN) play a key role in regulating sympathetic nerve activity and cardiovascular function. The glutamatergic system is the most important one. Furthermore, the norepinephrine level in the PVN is increased and GABAergic synaptic activity was diminishe during heart failure and influence sympathetic outflow. The purposes of the present study are observe the changes of NMDAR1, TH and GAD67 in the PVN in congestive heart failure (CHF) rats and its contribution to sympathoexcitation in heart failure. Methods:Adult male Sprague-Dawley rats were implanted with PVN cannulae and HF was induced by ligation of the left anterior descending coronary artery and SHAM rats did not undergo coronary ligation. Subsequently, animals were PVN treated with the NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5,0.02μg/h) or vehicle (VEH,0.11μl/h) for 4 weeks. After 4 weeks, Left ventricular function was measured by the hemodynamic parameters. Renal sympathetic nerve discharge was recorded by electrophysiological techniques; Lung/body weight ratio (Lung/BW) and right ventricle/ body weight ratio (RVW/BW) was calculated; Immunohistochemical staining was used for expression of TH in the hypothalamic paraventricular nucleus; Plasma NE and Angll content were measured using ELISA techniques; NMDAR1 and GAD67 protein levels of the hypothalamic paraventricular nucleus was measured with Western blot technique. Results:HF rats had a decreased 67-kDa isoform of glutamate decarboxylase (GAD67), a significant increase in the expression of NMD A receptor subunit NR1(NMDAR1) and tyrosine hydroxylase(TH) in the PVN, and in plasma levels of norepinephrine (NE) and angiotensinⅡ(ANGⅡ) when compared with SHAM rats. In contrast, PVN treated with AP5 increased GAD67 in the PVN, and PVN treated with AP5 also attenuated NMDAR1 and TH in this region,reduced plasma levels of norepinephrine, angiotensinⅡ, and decreased LVEDP and increased±dp/dtmax. compared with VEH-treated HF rats. Conclusion:These results suggest that NMDAR1 in the PVN in HF is activated and contribute to the increased sympathetic nerve activity. Treatment with NMD A receptor antagonists will be a potential way to delay the process of HF.ChapterⅢInteraction between the Angiotensin Converting Enzyme or Angiotensin Type 1 Receptor and NMDA Receptor in the Paraventricular Nucleus Contribute to Sympathoexcitation in Heart FailureAims:The renin-angiotensin system is a major component of the neuroendocrin system. The angiotensin type 1 receptor (AT1-R) and the angiotensin converting enzyme (ACE) have been identified in the paraventricular nucleus (PVN) and play an essential role in regulating sympathetic nerve activity and cardiovascular function. The glutamatergic system in the PVN also play an important role in regulating sympathetic nerve activity and cardiovascular function. The purposes of the present study are retrieval whether the changes of AT1-R or ACE in the PVN contribute to the activation of glutamatergic mechanisms in congestive heart failure (CHF). And investigate the effect on renal sympathetic nerve activity (RSNA) or the influence to the progre-ssion of CHF. Methods:Adult male Sprague-Dawley rats were implanted with PVN cannulae and CHF was induced by ligation of the left anterior descending coronary artery and SHAM rats did not undergo coronary ligation. Subsequently, animals were PVN treated with AT1-R antagonist Valsartan(0.05μg/h), ACE inhibitor (ACEI) Lisinopril(0.46μg/h), the NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5,0.02μg/h)or vehicle (VEH, 0.11μl/h) for 4 weeks. After 4 weeks, Left ventricular function was measured by the hemodynamic parameters. Renal sympathetic nerve discharge was recorded by electrophysiological techniques; Lung/ body weight ratio (Lung/ BW) and right ventricle/ body weight ratio (RVW/ BW) was calculated; Immunohistochemical staining was used for expression of ACE and TH in the hypothalamic paraventricular nucleus; Plasma NE and AngⅡcontent were measured using ELISA techniques; Fra-like、AT1-R、NMDAR1 and GAD67 protein levels of the hypothalamic paraventricular nucleus was measured with Western blot technique. Results:HF rats had a decrease in±dp/dtmax, LV ejection fraction (LVEF) and 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN, a significant increase in left ventricular end diastolic pressure (LVEDP), sympathetic nerve activity, the expression of Fra-LI (markers of activated neurons), AT1-R, ACE, NMDAR1 and TH in the PVN, and plasma angiotensinⅡ(ANGⅡ) and norepinephrine (NE) levels when compared with SHAM rats. In contrast, PVN treated with Valsartan, Lisinopril or AP5 attenuated Fra-LI, AT1-R, ACE, NMDAR1 and TH in the PVN compared with VEH-treated HF rats, increased GAD67 in the PVN and recovered cardiac function partly by elevating±dp/dtmax and reducing LVEDP. The treatment also decreased RVW/BW and lung/BW and reduced plasma levels of NE and ANGⅡ. Conclusion:These results suggest that NMDAR1 in the PVN in HF is activated by increased AT1-R and ACE. Perhaps combination therapy with AT1-R antagonist or ACEI and NMDA receptor antagonist will be a effective way to delay the process of CHF.更多还原