【作者】 姜美燕；

【导师】 朱大年；

【作者基本信息】 复旦大学 ， 生理学， 2011， 博士

【摘要】 研究背景：心血管疾病危害人类健康,心肌缺血是常见的心血管疾病。随着人民生活水平的提高和社会竞争压力的增大,心肌缺血发病率逐年上升并呈低龄化趋势。因此如何找到一种降低心肌缺血死亡率,改善心肌缺血的治疗方法成为国内外研究的热点。近年来,国内外研究表明电针内关穴(EA PC6)对治疗心肌缺血有其独特的疗效,而延髓头端腹外侧区(rostral ventrolateral medulla, RVLM)作为心血管调控的中枢部位也可能参与其中,但其机制非常复杂。1980年,Furchgott等在家兔大动脉条标本中发现一种被他们称为内皮舒张因子(endothelium-derived relaxing factor, EDRF)的物质,1987年又由他自己将这种物质确定为一氧化氮(nitric oxide, NO)。随着研究的深入,人们逐渐认识到,NO作为一种非经典的信使物质,广泛存在于体内各组织器官,参与体内的许多功能活动,具有重要的调节作用。NO在心血管调节中枢RVLM也存在,并且参与RVLM对心血管的调节。另外近年研究证实,RVLM区的腺苷(ADO)及其A2A受体(A2AR)也参与心血管效应。那么,中枢NO/NOS是否参与电针改善心肌缺血的机制呢?其调节作用是否与RVLM有关?A2AR与NO/NOS系统的关系如何?目前这些机制仍不很清楚。目的：本课题在急性心肌缺血(AMI)大鼠模型上,观察电针内关穴对急性心肌缺血大鼠心血管反应是否有积极调整作用,从功能学和形态学的角度,观察AMI组和AMI+EA PC6组大鼠心血管调节中枢RVLM的NOS的表达,来分析电针内关穴对心肌缺血大鼠的心血管作用是否与RVLM区NO/NOS系统有关,并进一步观察RVLM区NO/NOS与ADO/A2AR的关系,分析NO/NOS调节心血管活动的作用途径,更深入的探讨电针内关穴改善心肌缺血的中枢神经递质或调质机制。方法：采用结扎冠状动脉左前降支的方法制备AMI大鼠模型；针刺穴位选择双侧“内关”穴(PC6),针刺对照穴位选择双侧“列缺”穴(LU7)；并监测大鼠平均动脉压(MAP),心率(HR)。采用氯化四唑(2,3,5-triphenyltetrazolium chloride,TTC)染色检测心肌缺血是否成功,苏木精和伊红(HE)染色观察缺血心肌组织病理改变,采用超声心动图观察缺血心脏的室壁结构和心功能变化。采用酶联免疫吸附测定法(ELISA)测定各组大鼠血浆肾上腺素(E)和去甲肾上腺素(NE)的含量,观察心肌缺血后心脏自主神经变化,并通过心率变异性(HRV)和压力感受反射敏感性(BRS)分析系统分析大鼠心脏自主神经功能状态。采用免疫组织化学(IHC)方法检测各组大鼠(Sham组、AMI组、AMI+EA PC6组、AMI+EALU7组)RVLM区nNOS和iNOS的表达；采用蛋白免疫印迹法(western blot)检测各组大鼠(Sham组、AMI组、AMI+EA PC6组)RVLM区nNOS和iNOS蛋白水平的变化。采用硝酸还原酶法检测各组大鼠(Sham组、AMI组、AMI+EA PC6组)RVLM区NO含量及NOS、iNOS活性。采用中枢核团RVLM微量注射的方法观察各组大鼠(正常对照组、Sham组、AMI组、AMI+EA PC6组)RVLM区注射L-Arg (NO的供体),L-NAME(非选择性NOS抑制剂),AG(选择性iNOS抑制剂)和7-NI(选择性nNOS抑制剂),对MAP, HR及HRV各项指标(LF、HF、LF/HF)的影响,及A2AR的拮抗剂(SCH58261)对L-Arg心血管效应的影响。采用酶联免疫吸附测定法(ELISA)观察正常对照组大鼠RVLM区微量注射L-Arg以后RVLM区ADO和ACh变化及微量注射SCH58261+L-Arg后RVLM区ACh的变化;采用western blot检测正常对照组大鼠RVLM微量注射L-Arg后RVLM区A2AR和M1R的蛋白变化及微量注射SCH58261+L-Arg后M1R的蛋白表达变化。结果：(1)AMI模型制备：冠状动脉左前降支结扎后,大鼠立即出现心电图S-T段抬高,结扎线远端心肌发白,收缩力降低,运动减弱,说明AMI模型制备成功。TTC及HE染色显示AMI后心肌组织缺血梗死及EA后的心肌组织缺血情况改善,超声心动图显示AMI后收缩期和舒张期左室内径增大,室间隔厚度变薄,射血分数降低,EA内关穴以后收缩期舒张期左室内径减小,室间隔厚度增厚,射血分数升高,进一步说明AMI模型制备成功和EA的治疗作用。(2)血清E与NE含量测定及HRV和BRS监测：AMI大鼠血清中E与NE比Sham组大鼠血清中E和NE明显增多(P<0.05, n=6), EA PC6后两者含量明显降低。AMI大鼠HRV和BRS分析显示,HRV和BRS的LF、HF降低(P<0.05,n=6),但LF/HF明显升高(P<0.05, n=6), EA PC6后可降低LF/HF比值(P<0.05,n=6),EALU7后LF/HF比值与AMI组没有明显区别(P>0.05,n=4)。(3)免疫组织化学结果显示：AMI大鼠RVLM的nNOS阳性神经元数目与Sham组相比明显升高,iNOS阳性神经元数目与Sham组相比明显降低(P<0.05,n=6), EAPC6后的nNOS阳性神经元数目降低(P<0.05, n=6), iNOS阳性神经元数目升高(P<0.05,n=6),而EALU7后RVLM区nNOS和iNOS免疫阳性神经元数目与AMI组相比无明显变化(P>0.05,n=4)。(4) Western blot结果显示：NOS蛋白在各组大鼠RVLM均有表达,与Sham组相比,AMI组大鼠RVLM的nNOS蛋白表达增多(P<0.05, n=4), iNOS蛋白表达减少(P<0.05, n=4), EAPC6后RVLM的nNOS蛋白表达降低(P<0.05,n=4),iNOS蛋白表达增多(P<0.05,n=4)。(5)用硝酸还原酶法检测各组大鼠NO含量及NOS活性试剂盒检测NOS,iNOS活性,结果显示：AMI组大鼠血浆和RVLM的NO含量与Sham组相比明显增多(P<0.05, n=8), NOS活性也明显增强(P<0.05,n=8),而iNOS活性减弱。EAPC6后AMI组大鼠血浆和RVLM的NO含量增多(P<0.05, n=8), NOS和iNOS活性增强(P<0.05,n=8)。(6)正常大鼠RVLM区微量注射L-Arg能使大鼠MAP、HR以及HRV各项指标(LF, HF, LF/HF)均降低(P<0.05, n=12); RVLM区微量注射L-NAME能使大鼠MAP、HR和HRV各项指标(LF,HF, LF/HF)均升高(P<0.05,n=8)。并且L-Arg能使正常大鼠RVLM区的ADO (P<0.05, n=8), A2AR (P<0.05, n=4)升高,使RVLM区的ACh (P<0.05, n=8), MIR (P<0.05, n=4)降低。预先在正常大鼠RVLM区微量注射A2AR拮抗剂SCH58261, 10min后再微量注射L-Arg, L-Arg的上述功能被阻断(P<0.05,n=10)。正常大鼠RVLM区微量注射iNOS和nNOS拮抗剂AG和7-NI。AG使MAP、HR、LF、HF、LF/HF比值均升高(P<0.05,n=8),7-NI使MAP、HR、LF、HF、LF/HF比值均降低(P<0.05,n=8)。(7) AMI+EA PC6组RVLM区微量注射L-NAME能阻断EA PC6对AMI大鼠心血管的改善作用(P<0.05,n=7)。(8)各组(Sham组、AMI组、AMI+EA PC6组)大鼠RVLM微量注射L-Arg能使大鼠MAP和HR降低(P<0.05或P<0.01,n=7),并且降低幅度在AMI大鼠更明显。同时L-Arg也能使三组大鼠LF/HF降低,AMI组降低最明显。结论：(1)采用结扎大鼠冠状动脉左前降支的方法,能成功制备急性心肌缺血模型,该模型大鼠外周交感紧张性增高。这可能与AMI大鼠RVLM区nNOS表达上调及iNOS表达及活性下调有关。(2)电针AMI大鼠内关穴,可以降低AMI大鼠交感紧张性,改善大鼠平均动脉压和心率,改善HRV和BRS。电针的调节机制可能是通过调节NO/NOS的表达,调节交感紧张性,改善心肌缺血。(3)正常大鼠RVLM区NO通过增加ADO释放,激活A2AR活性,进而降低ACh释放及M1R活性,从而降低交感兴奋性,影响心功能。(4)电针内关穴改善心肌缺血作用的中枢机制之一可能是通过调节RVLM区NOS的表达及活性,增加ADO释放,激活A2AR活性,进而降低ACh释放及M1R活性,从而调整交感紧张性,改善心肌缺血。更多还原

【Abstract】 Background:Cardiovascular disease represents the world’s largest killers, which affects adult and aged individuals in both Western and Eastern societies. In particular, substantial morbidity and mortality result from ischemic cardiovascular disease. How to reduce mortality and improve myocardial ischemia has become one of the hot spots. In recent years, electroacupuncture (EA) treatment on ischemic cardiomyopathy has shown its own unique effects at home and abroad. The rostral ventrolateral medulla (RVLM) is a key area for regulation of cardiovascular activities. This area contains many neurotransmitters/neuromodulators, such as angiotensin-(1-7), carbon monoxide, adenosine (ADO) and nitric oxide (NO), which are thought to be important in cardiovascular control. NO is acted as a neural transmitter and is involved in central cardiovascular regulation, but the detailed mechanism is not so clear and may be more complex in the central nervous system (CNS) than that in the periphery. Recent studies have confirmed that central ADO and adenosine A2A receptor (A2AR) are involved in the cardiovascular effects by inhibitory effects on sympathetic outflow. Well then, whether medullary NO/NOS system is involved in the therapeutic effect of EA on myocardial ischemia, and whether adenosine mediates NO/NOS to regulating myocardial ischemia. These mechanisms require further study.Objectives and hypothesis:The present study was performed on acute myocardial ischemia (AMI) model of ligating the left anterior descending coronary artery in rats. To investigate the effect of EA on cardiac function in AMI rats, we observed the expressions of NOS in the RVLM. Meanwhile, we investigated the crosstalk of NO/NOS and ADO/A2AR, which subsequently inhibited acetylcholine (ACh)-M1 receptor (M1R) activation, in order to further study the mechanisms underlying the EA. Thus, we tested the hypothesis that the central NO-dependent sympathetic inhibition was mediated through ADO/A2AR and ACh-M1R in the RVLM, and further investigated the central cardiovascular mechanisms of EA on AMI rats. Methods:AMI model was duplicated by ligation of the left anterior descending coronary artery. Bilateral "Neiguan" (PC 6) acupoints were used as the acupoint and bilateral "lieque" (LU 7) acupoint as the control. Mean artery pressure (MAP) and heart rate (HR) were monitored, so as to observe the effects of EA on AMI rats. The ischemic area and the micro-structure of ischemic myocardium were detected by tetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. The heart wall structure was shown by the echocardiography. Plasma epinephrine (E) and norepinephrine (NE) were measured by enzyme-linked immunosorbent assay (ELISA). Program of heart rate variability (HRV) and baroreflex sensitivity (BRS) were used for reflecting changes of cardiac autonomic nervous tones. Immunohistochemistry (IHC) and western blot analysis were used to detect the expression of nNOS and iNOS in the RVLM. L-Arg, L-NAME, SCH58261, AG and 7-NI were respectively microinjected into the RVLM. ADO and ACh were measured by ELISA. A2AR and MIR were measured by western blot.Results:1. Preparation of AMI modelSuccess of duplicating AMI model was depended on S-T segment elevating immediately in ECG typical lead II, the myocardium presenting pale and poor weakening movement. And the ischemic myocardium remained pale in color by TTC staining. HE staining showed that AMI cardiac muscle fibers expressed obviously acidophilic alteration. Moreover, the echocardiography showed that left ventricular end-diastolic dimension (LVDd) and left ventricular end-systolic dimension (LVDs) obviously increased, but interventricular septum thickness became thin. All the results demonstrated the success of duplicating AMI model.2. Assay of E and NE and HRV, BRS analysisThe concentrations of E and NE in the serum of AMI rats increased significantly. They were decreased in the EA PC6 group compared with that in the AMI group (P<0.05, n=6). Analysis of HRV and BRS demonstrated that the lower frequency components (LF) and the higher frequency components (HF) of AMI rats decreased, whereas the ratio of LF/HF increased. LF/HF of AMI rats decreased after EA PC6 treatment (P<0.05, n=6). Conversely, EA at the bilateral Lieque acupoints did not alter all parameters of HRVand BRS.3. The expression of nNOS and iNOS positive neurons in the RVLM of each group(1). The numbers of nNOS immunoreactive neurons in the RVLM of AMI group were significantly increased compared with that of the sham operated group (P<0.05, n=6). The number of iNOS immunoreactive neurons in the RVLM of AMI rats was significantly decreased compared with that of the sham operated group (P<0.05, n=6). EA at bilateral "Neiguan" acupoints could decrease the expression of nNOS immunoreactive neurons and increase the expression of iNOS immunoreactive neurons in the RVLM of AMI rats, respectively. Conversely, EA at "Lieque" did not significantly decrease the expression of nNOS in the RVLM and increase the expression of iNOS in the RVLM of AMI rats.(2). Western blot analysis showed that the expressions of nNOS in the RVLM of AMI rats were significantly increased and iNOS was significantly decreased, compared with those of sham operated rats (P<0.05, n=6). After EA PC6 treatment the expressions of nNOS in the RVLM of AMI rats decreased and iNOS in the RVLM of AMI rats increased.4. The NO content and NOS activity in the RVLM of each groupMethod of nitrate reductase was used to detect the NO content and NOS activity. NO content in the RVLM of AMI group was significantly increased compared with that of the sham operated group (P<0.05, n=8). NOS activity was significantly enhanced, while iNOS activity was significantly attenuated. After EA PC6 treatment, NO content was markedly decreased, and the activities of NOS and iNOS were significantly enhanced.5. The effect of L-Arg, L-NAME, SCH58261, AG and 7-NI in the RVLM of normal rats on MAP, HR and power spectral analysis of HRV.L-Arginine (L-Arg, NO donor) was microinjected into the RVLM, and the MAP, HR and LF/HF of the HRV decreased. Whereas pre-treatment of a competitive antagonist of A2AR, SCH58261, the above inhibitory effects of L-Arg were attenuated. Western blot and ELISA showed that following L-Arg microinjection, ADO and A2AR levels increased, whereas ACh and ACh MIR levels decreased significantly in the RVLM along with the cardiovascular responses. And again, the decrease of ACh was abolished by pre-treatment of SCH58261. Moreover, microinjection of NG-nitro-L-Arginine methyl ester (L-NAME, a nonselective nitric oxide synthase (NOS) inhibitor), into the RVLM induced a significant increase in MAP, HR and sympathetic activity shown from the HRV (all of the LF, HF and LF/HF ratio were increased). Aminoguanidine (AG), a competitive antagonist of iNOS, produced increases in MAP, HR and LF/HF.7-nitroindazole sodium salt (7-NI), an antagonist of nNOS, almost produced decreases in MAP, HR and LF/HF.6. The effect of L-Arg, L-NAME, SCH58261, AG and 7-NI in the RVLM of sham, AMI and AMI+EA PC6 rats on MAP, HR and power spectral analysis of HRVMicroinjection of L-NAME into the RVLM in AMI+EA PC6 group induced significant elevation of MAP and HR. The cardiovascular effect of EA was blocked by L-NAME. Microinjection of L-Arg, SCH58261+L-Arg, AG and 7-NI into RVLM in each group (Sham group, AMI group and AMI+EA PC6 group) induced respectively changes of MAP and HR. L-Arg induced the decrease of MAP, HR and HRV, and the decreased extent was more remarkable in AMI rats. This inhibitory effect was attenuated by pre-treatment of SCH58261 in each group. AG produced increases in MAP, HR and LF/HF in all group.7-NI produced decreases in MAP, HR and LF/HF in all group.Conclusions:1. By the method of ligating left anterior descending coronary artery could successfully duplicate the rat model of AMI. Sympathetic outflows of AMI rats increased.2. EA at "neiguan" acupoints on AMI rats could decrease sympathetic outflow. The mechanism was related to the upregulation of nNOS and downregulation of iNOS in the RVLM.3. Our results supported that the central NO/NOS system modulated cardiovascular activities via activation of A2AR, which subsequently inhibited ACh-M1R activation.4. All of these suggested one of the mechanisms of EA PC6 improving AMI. EA PC6 treatment could modulate cardiovascular activity though the central NO/NOS system, which modulated cardiovascular activities via activation of A2AR, which subsequently inhibited ACh-M1R activation.更多还原