中枢NO/NOS在针刺改善心肌缺血中的作用及机制
|
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。
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 model
Success 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 analysis
The 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 group
Method 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 HRV
Microinjection 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.
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。
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 model
Success 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 analysis
The 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 group
Method 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 HRV
Microinjection 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.
引文
[1]R. Takahashi, Q. Deveraux, I. Tammet al. A single BIR domain of XIAP sufficient for inhibiting caspases [J]. J Biol Chem,1998,273 (14):7787-7790.
[2]叶仁高,陆再英,谢毅.内科学[M].北京：人民卫生出版社,2004：273.
[3]R. Datta, E. Oki, K. Endoet al. XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage [J]. J Biol Chem,2000,275 (41) 31733-31738.
[4]金菊林,徐鸿华.心肌梗死与心理应激因素[J].心脑血管病防治,2002,2(2)：7.
[5]J. A. Herd. Cardiovascular response to stress [J]. Physiol Rev,1991; 71:305-331.
[6]A. Schomig, M. Haass, G. Richardt. Catecholamine release and arrhythmias in acute myocardial ischaemia [J]. Eur Heart J,1991,12 Suppl F:38-47.
[7]朱俏萍,李建生,等.急性心肌梗死患者交感神经活性与室性心律失常的关系[J].中国综合临床,2003,19(2)：117-118.
[8]T. Nielsen, T. H. Haghfelt, S. Haunsoet al. [Ischemic heart disease and "integrated" rehabilitation] [J]. Ugeskr Laeger,2000,162 (27):3828.
[9]P. K. Ganguly, K. S. Dhalla, Q. Shao et al. Differential changes in sympathctic activity in left and right ventricle in congestive heart failure myocardial infarction [J]. Am Heart J,1997,133(3):340-349.
[10]沈法荣,金宏义.冠心病心肌缺血发作前后心脏自主神经功能的变化[J].心电学杂志,1997,16(2)：71-72.
[11]R. A. Dampney, J. Horiuchi, T. Tagawa et al. Medullary and supramedullary mechanisms regulating sympathetic vasomotor tone [J]. Acta Physiol Scand, 2003,177 (3):209-218.
[12]A. F. Sved, S. Ito, J. C. Sved. Brainstem mechanisms of hypertension:role of the rostral ventrolateral medulla [J]. Curr Hypertens Rep,2003,5(3):262-268.
[13]R. A. Dampney, M. J. Coleman, M. A. Fonteset al. Central mechanisms underlying short-and long-term regulation of the cardiovascular system [J]. Clin Exp Pharmacol Physiol,2002,29 (4):261-268.
[14]赵明中,陈运贞.实验性心肌缺血大鼠心脏自主神经活性的动态变化及意义[J].中国心脏起搏与心电生理杂志,1998,12(2)：101-103.
[15]强文安,刘枝俏.脑组织一氧化氮合成酶的特性及其与NADPH—diaphorase[J].中国神经免疫学和神经病学杂志,1996,3(4)：191-198.
[16]高博,尹桂山.神经系统中的一氧化氮[J].生物化学与生物物理进展,1999,26(1)：31-34.
[17]J. Oka, M. Imamura, E. Hattaet al. Carrier-mediated norepinephrine release and reperfusion arrhythmias induced by protracted ischemia in isolated perfused guinea pig hearts:effect of presynaptic modulation by alpha(2)-adrenoceptor in mild hypothermic ischemia [J]. J Pharmacol Exp Ther,2002,303 (2):681-687.
[18]J. Y. Chan, S. H. Chan, A. Y. Chang. Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat [J]. Neuropharmacology,2004,46 (8) 1184-1194.
[19]夏春梅,陈军,王锦等.电针诱导心肌缺血大鼠延髓头端腹外侧区nNOS和iNOS差异表达[J].生理学报,2008,60(4)：453-461.
[20]朱桂平,雷达,钟亮尹等.幼龄自发性高血压大鼠血管内皮功能研究[J].岭南心血管病杂志,2004,10(5)：368-370.
[21]晨阳,薛康,李春跃.原发性高血压与应激性高血压大鼠NO/NOS、ET的变化[J].中国医学物理学杂志,2006,23(1)：35-39.
[22]吕娟秀,周培华,王锦等.延髓腹外侧一氧化氮介导电针内关对急性心肌缺血大鼠心功能的作用[J].生理学报,2004,56(4)：503-508.
[23]S. H. Chan, L. L. Wang, J. Y. Chan. Differential engagements of glutamate and GABA receptors in cardiovascular actions of endogenous nNOS or iNOS at rostral ventrolateral medulla of rats [J]. Br J Pharmacol,2003,138(4):584-593.
[24]A. I. Malomouzh, M. R. Mukhtarov, E. E. Nikolskyet al. Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism [J]. J Neurochem, 2007,102 (6):2110-2117.
[25]M. Nakaya, K. Kondo, K. Kaga. Immunohistochemical study of ml-m5 muscarinic receptors and nNOS in human inferior turbinate mucosa [J]. Acta Otolaryngol Suppl,2007 (559):103-107.
[26]H. Fischer, H. Prast, A. Philippu. Adenosine release in the ventral striatum of the rat is modulated by endogenous nitric oxide [J]. Eur J Pharmacol,1995,275 (3): R5-R6.
[27]M. Uematsu, G. R. Gaudette, J. O. Laurikkaet al. Adenosine-enhanced ischemic preconditioning decreases infarct in the regional ischemic sheep heart [J]. Ann Thorac Surg,1998,66 (2):382-387.
[28]J. D. McCully, M. Uematsu, R. A. Parkeret al. Adenosine-enhanced ischemic preconditioning provides enhanced cardioprotection in the aged heart [J]. Ann Thorac Surg,1998,66 (6):2037-2043.
[29]N. Nassar, A. A. Abdel-Rahman. Brainstem phosphorylated extracellular signal-regulated kinase 1/2-nitric-oxide synthase signaling mediates the adenosine A2A-dependent hypotensive action of clonidine in conscious aortic barodenervated rats[J]. J Pharmacol Exp Ther,2008,324 (1):79-85.
[30]林华,易受乡,等.针刺改善冠心病心脏功能的临床观察[J].甘肃中医,2002,15(4)：86-89.
[31]蔡荣林,胡玲,周逸平等.电针对急性心肌缺血家兔心功能及心交感神经电活动的影响[J].针刺研究,2007,32(4)：243-246.
[32]高楠,苏勋庄.针刺改善心肌缺血作用的临床观察[J].上海针灸杂志,1997,16(3)：7-8.
[33]孟竞璧.针刺治疗冠心病的疗效及其作用机理研究[J].中医杂志,2002,43(8)：587-589.
[34]D. Felhendler, B. Lisander. Effects of non-invasive stimulation of acupoints on the cardiovascular system [J]. Complement Ther Med,1999,7(4):231-234.
[35]S. Ballegaard, T. Muteki, H. Haradaet al. Modulatory effect of acupuncture on the cardiovascular system:a cross-over study [J]. Acupunct Electro ther Res, 1993,18 (2):103-115.
[36]A. Richter, J. Herlitz, A. Hjalmarson. Effect of acupuncture in patients with angina pectoris [J]. Eur Heart J,1991,12 (2):175-178.
[37]X. R. Wang, H. Lin, Z. H. Wang. Protective effects of electroacupuncture and Salviae Miltiorrhizae on myocardial ischemia/reperfusion in rabbits [J]. Acupunct Electrother Res,2003,28 (3-4):175-182.
[38]J. M. Foster, B. P. Sweeney. The mechanisms of acupuncture analgesia [J]. Br J Hosp Med,1987,38 (4):308-312.
[39]J. S. Han. Electroacupuncture:an alternative to antidepressants for treating affective diseases? [J]. Int J Neurosci,1986,29 (1-2):79-92.
[40]M. Arya, I. S. Shergill, M. Williamsonet al. Basic principles of real-time quantitative PCR [J]. Expert Rev Mol Diagn,2005,5 (2):209-219.
[41]J. Kato, K. Meguro, A. Satoet al. The effects of morphine administered into the vertebral artery on the somatosympathetic A-and C-reflexes in anesthetized cats [J]. Neurosci Lett,1992,138 (2):207-210.
[42]A. Sato, Y. Sato, R. F. Schmidt. Modulation of somatocardiac sympathetic reflexes mediated by opioid receptors at the spinal and brainstem level [J]. Exp Brain Res,1995,105 (1):1-6.
[43]W. Zhou, L. W. Fu, S. C. Tjen-A-Looiet al. Afferent mechanisms underlying stimulation modality-related modulation of acupuncture-related cardiovascular responses [J]. J Appl Physiol,2005,98 (3):872-880.
[44]李梦,胡玲,蔡荣林等.电针内关、心俞改善急性心肌缺血大鼠心率变异性的协同作用[J].甘肃中医学院学报,2008,25(1)：12-15.
[45]S. C. Tjen-A-Looi, P. Li, J. C. Longhurst. Medullary substrate and differential cardiovascular responses during stimulation of specific acupoints [J]. Am J Physiol Regul Integr Comp Physiol,2004,287 (4):R852-R862.
[46]B. JOHANSSON. Circulatory responses to stimulation of somatic afferents with special reference to depressor effects from muscle nerves [J]. Acta Physiol Scand Suppl,1962,198:1-91.
[47]W. Zhou, L. W. Fu, S. C. Tjen-A-Looiet al. Afferent mechanisms underlying stimulation modality-related modulation of acupuncture-related cardiovascular responses [J]. J Appl Physiol,2005,98 (3):872-880.
[48]H. F. Guo, J. Tian, X. Wanget al. Brain substrates activated by electroacupuncture (EA) of different frequencies (Ⅱ):Role of Fos/Jun proteins in EA-induced transcription of preproenkephalin and preprodynorphin genes [J]. Brain Res Mol Brain Res,1996,43 (1-2):167-173.
[49]H. F. Guo, J. Tian, X. Wanget al. Brain substrates activated by electroacupuncture of different frequencies (Ⅰ):Comparative study on the expression of oncogene c-fos and genes coding for three opioid peptides [J]. Brain Res Mol Brain Res,1996,43 (1-2):157-166.
[50]Y. L. Huang, M. X. Fan, J. Wanget al. Effects of acupuncture on nNOS and iNOS expression in the rostral ventrolateral medulla of stress-induced hypertensive rats [J]. Acupunct Electrother Res,2005,30 (3-4):263-273.
[51]M. A. Pfeffer, J. M. Pfeffer, M. C. Fishbeinet al. Myocardial infarct size and ventricular function in rats [J]. Circ Res,1979,44 (4):503-512.
[52]Li Peng, Yao Tai. Mechanism of the Modulatory Effect of Acupuncture on Abnormal Cardiovascular Functions [M]. Shanghai:Shanghai Medical University Press,1992.
[53]李忠仁主编.实验针灸学(第1版)[M].北京：中国中医药出版社,2003：329.
[54]M. C. Fishbein, S. Meerbaum, J. Ritet al. Early phase acute myocardial infarct size quantification:validation of the triphenyl tetrazolium chloride tissue enzyme staining technique [J]. Am Heart J,1981,101 (5):593-600.
[55]P. N. Khalil, M. Siebeck, R. Husset al. Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts [J]. J Pharmacol Toxicol Methods,2006,54 (3) 307-312.
[56]G. Paxinos, C. R. Watson. The Rat Brain in Stereotaxic Coordinates,3rd edn [M]. San Diego:Academic Press,1997.
[57]魏泓.医学实验动物学.第一版[M].成都：四川科学技术出版社,1998：323-324.
[58]张润峰,王继生.建立大鼠心肌梗死模型的若干问题探讨[J].山西医科大学学报,2004,35(1)：13-15.
[59]王响英,吴淑燕,李苏安等.大鼠直视气管插管方法[J].上海实验动物科学,2004,24(2)：107-108.
[60]边素艳,刘宏斌,杨庭树.提高急性心肌梗死大鼠模型存活率的实验研究[J].中国康复医学杂志,2005,20(3)：180-182.
[61]许丹,张春来,李霞等.制作大鼠急性心肌梗死模型的实验分析[J].中国煤炭工业医学杂志,2006,9(2)：190-191.
[62]张晔.实用临床针灸学[M].上海：上海医科大学出版社,1998.
[63]陈少宗.针刺间使穴,内关穴对冠心病患者左心功能影响的比较观察[J].针灸临床杂志,1994,10(6)：30-32.
[64]郑关毅,张贵灿.针刺内关穴对冠心病患者左心室功能的影响[J].上海针灸杂志,1997,16(4)：8-9.
[65]薛少敏,李传杰.针刺对冠心病患者左心舒张功能的影响[J].中国针灸,1992,12(3)：43-46.
[66]H. R. Middlekauff. Acupuncture in the treatment of heart failure [J]. Cardiol Rev, 2004,12 (3):171-173.
[67]J. Gao, L. Zhang, Y. Wanget al. Antiarrhythmic effect of acupuncture pretreatment in rats subjected to simulative global ischemia and reperfusion--involvement of adenylate cyclase, protein kinase A, and L-type Ca2+ channel [J]. J Physiol Sci,2008,58 (6):389-396.
[68]J. Yan, X. F. Yang, S. X. Yiet al. [Effect of electroacupuncture of "Neiguan" (PC 6) on Na+-K+-ATPase activity and its gene expression in cardiocyte membrane in rats with myocardial ischemia-reperfusion injury] [J]. Zhen Ci Yan Jiu,2007, 32 (5):296-300.
[69]J. Gao, W. Fu, Z. Jinet al. Acupuncture pretreatment protects heart from injury in rats with myocardial ischemia and reperfusion via inhibition of the beta(1)-adrenoceptor signaling pathway [J]. Life Sci,2007,80 (16):1484-1489.
[70]L. Yang, J. Yang, Q. Wanget al. Cardioprotective effects of electroacupuncture pretreatment on patients undergoing heart valve replacement surgery:a randomized controlled trial [J]. Ann Thorac Surg,2010,89 (3):781-786.
[71]X. Song, Z. Tang, Z. Houet al. [The anti-hemorrhagic shock of acupuncture on neiguan and its effect on the cardial pump function and the blood viscosity] [J]. Zhen Ci Yan Jiu,1990,15 (1):30-34.
[72]X. Song, Z. Tang, Z. Houet al. An experimental study on acupuncture anti-hemorrhagic shock [J]. J Tradit Chin Med,1993,13 (3):207-210.
[73]S. B. Wang, S. P. Chen, Y. H. Gaoet al. Effects of electroacupuncture on cardiac and gastric activities in acute myocardial ischemia rats [J]. World J Gastroenterol, 2008,14 (42):6496-6502.
[74]张明金,魏明文,等.针刺内关穴对心率变异性的分析[J].中国针灸,2003,23(3)：169-170.
[75]S. Sakai, E. Hori, K. Umenoet al. Specific acupuncture sensation correlates with EEGs and autonomic changes in human subjects [J]. Auton Neurosci,2007,133 (2):158-169.
[76]C. C. Hsu, C. S. Weng, T. S. Liuet al. Effects of electrical acupuncture on acupoint BL15 evaluated in terms of heart rate variability, pulse rate variability and skin conductance response [J]. Am J Chin Med,2006,34 (1):23-36.
[77]石现,赵永兰,等.针刺调节心脏植物神经的实验研究[J].针刺研究,2002,27(1)：68-70.
[78]Z. Li, C. Wang, A. F. Maket al. Effects of acupuncture on heart rate variability in normal subjects under fatigue and non-fatigue state [J]. Eur J Appl Physiol,2005, 94 (5-6):633-640.
[79]刘金兰,张振莉.电针对急性心肌缺血家兔血浆单胺类神经递质的影响[J].中国针灸,1997,17(10)：604-605.
[80]M. M. Wolf, G. A. Varigos, D. Huntet al. Sinus arrhythmia in acute myocardial infarction [J]. Med J Aust,1978,2(2):52-53.
[81]安威,时安云.心肌毖?再灌注损伤与心肌去甲肾上腺素的释放[J].中国病理生理杂志,1990,6(2)：65-68.
[82]冯义柏.粉防己碱对缺血心肌去甲肾上腺素释放的影响[J].同济医科大学学报,1994,23(2)：147-148.
[83]杨天德,刘桥义.纳络酮对缺血再灌注心肌儿茶酚胺释放的影响[J].中华麻醉学杂志,1994,14(5)：331-333.
[84]张彦周.心肌缺血对心肌去甲肾上腺素的释放[J].国外医学：心血管疾病分册,1998,25(5)：265-267.
[85]N. Nagaya, T. Nishikimi, Y. Gotoet al. Plasma brain natriuretic peptide is a biochemical marker for the prediction of progressive ventricular remodeling after acute myocardial infarction [J]. Am Heart J,1998,135 (1):21-28.
[86]高博,尹桂山.神经系统中的一氧化氮[J].生物化学与生物物理进展,1999,26(1)：31-34.
[87]K. P. Patel, Y. F. Li, Y. Hirooka. Role of nitric oxide in central sympathetic outflow [J]. Exp Biol Med (Maywood),2001,226 (9):814-824.
[88]强文安,刘枝俏.脑组织一氧化氮合成酶的特性及其与NADPH—diaphorase [J].中国神经免疫学和神经病学杂志,1996,3(4)：191-198.
[89]D. S. Bredt. Endogenous nitric oxide synthesis:biological functions and pathophysiology [J]. Free Radic Res,1999,31 (6):577-596.
[90]C. Y. Chen, S. J. Huang, T. M. Yanget al. Effective renal plasma flow response and atrial natriuretic peptide in patients with uncomplicated acute myocardial infarction [J]. Jpn Heart J,1993,34 (1):1-10.
[91]J. L. Zweier, P. Wang, P. Kuppusamy. Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy [J]. J Biol Chem,1995,270 (1):304-307.
[92]K. Akiyama, A. Kimura, H. Suzukiet al. Production of oxidative products of nitric oxide in infarcted human heart [J]. J Am Coll Cardiol,1998,32 (2): 373-379.
[93]M. Araki, M. Tanaka, K. Hasegawaet al. Nitric oxide inhibition improved myocardial metabolism independent of tissue perfusion during ischemia but not during reperfusion [J]. J Mol Cell Cardiol,2000,32 (3):375-384.
[94]牛丽丽,祝善俊.冠心病患者血管内皮细胞损伤血管活性物质失衡对冠脉的调控作用[J].解放军医学杂志,1996,21(4)：282-283.
[95]S. X. Ma. Enhanced nitric oxide concentrations and expression of nitric oxide synthase in acupuncture points/meridians [J]. J Altern Complement Med,2003,9 (2):207-215.
[96]S. X. Ma, J. Ma, G. Moiseet al. Responses of neuronal nitric oxide synthase expression in the brainstem to electroacupuncture Zusanli (ST 36) in rats [J]. Brain Res,2005,1037 (1-2):70-77.
[97]C. Depre, L. Fierain, L. Hue. Activation of nitric oxide synthase by ischaemia in the perfused heart [J]. Cardiovasc Res,1997,33 (1):82-87.
[98]W. Bloch, U. Mehlhorn, A. Krahwinkelet al. Ischemia increases detectable endothelial nitric oxide synthase in rat and human myocardium [J]. Nitric Oxide, 2001,5 (4):317-333.
[99]M. Desrois, T. Caus, P. M. Belleset al. Nitric oxide pathway after long-term cold storage and reperfusion in a heterotopic rat heart transplantation model [J]. Transplant Proc,2005,37 (10):4553-4555.
[100]朱大年.生理学(第七版)[M].北京：人民卫生出版社,2008：115.
[101]Q. Lin, P. Li. Rostral medullary cholinergic mechanisms and chronic stress-induced hypertension [J]. J Auton Nerv Syst,1990,31 (3):211-217.
[102]L. Gui, W. Duan, H. Tianet al. Adenosine A 2A receptor deficiency reduces striatal glutamate outflow and attenuates brain injury induced by transient focal cerebral ischemia in mice [J]. Brain Res,2009,1297:185-193.
[103]N. Nassar, A. A. Abdel-Rahman. Brainstem adenosine Al receptor signaling masks phosphorylated extracellular signal-regulated kinase 1/2-dependent hypotensive action of clonidine in conscious normotensive rats [J]. J Pharmacol Exp Ther,2009,328 (1):83-89.
[104]N. Nassar, A. A. Abdel-Rahman. Brainstem phosphorylated extracellular signal-regulated kinase 1/2-nitric-oxide synthase signaling mediates the adenosine A2A-dependent hypotensive action of clonidine in conscious aortic barodenervated rats [J]. J Pharmacol Exp Ther,2008,324 (1):79-85.
[105]M. C. Martins-Pinge, M. R. Garcia, D. B. Zoccalet al. Differential influence of iNOS and nNOS inhibitors on rostral ventrolateral medullary mediated cardiovascular control in conscious rats [J]. Auton Neurosci,2007,131 (1-2) 65-69.
[106]R. R. Morrison, M. A. Talukder, C. Ledentet al. Cardiac effects of adenosine in A(2A) receptor knockout hearts:uncovering A(2B) receptors [J]. Am J Physiol Heart Circ Physiol,2002,282 (2):H437-H444.
[107]L. Stella, L. Berrino, A. Filippelliet al. Nitric oxide participates in the hypotensive effect induced by adenosine A2 subtype receptor stimulation [J]. J Cardiovasc Pharmacol,1995,25 (6):1001-1005.
[108]H. Fischer, H. Prast, A. Philippu. Adenosine release in the ventral striatum of the rat is modulated by endogenous nitric oxide [J]. Eur J Pharmacol,1995,275 (3):R5-R6.
[109]L. Stella, L. Berrino, A. Filippelliet al. Nitric oxide participates in the hypotensive effect induced by adenosine A2 subtype receptor stimulation [J]. J Cardiovasc Pharmacol,1995,25 (6):1001-1005.
[110]M. A. Timoteo, L. Oliveira, E. Campesatto-Mellaet al. Tuning adenosine A1 and A2A receptors activation mediates L-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern [J]. Neurochem Int,2008,52 (4-5):834-845.
[111]J. C. Longhurst. Electroacupuncture treatment of arrhythmias in myocardial ischemia [J]. Am J Physiol Heart Circ Physiol,2007,292 (5):H2032-H2034.
[112]M. C. Martins-Pinge, M. R. Garcia, D. B. Zoccalet al. Differential influence of iNOS and nNOS inhibitors on rostral ventrolateral medullary mediated cardiovascular control in conscious rats [J]. Auton Neurosci,2007,131 (1-2) 65-69.
[1]V. L. Dawson, T. M. Dawson, E. D. Londonet al. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures [J]. Proc Natl Acad Sci U S A,1991,88 (14):6368-6371.
[2]C, Martin, R. Schulz, H. Postet al. Microdialysis-based analysis of interstitial NO in situ:NO synthase-independent NO formation during myocardial ischemia [J]. Cardiovasc Res,2007,74 (1):46-55.
[3]G. Heusch, H. Post, M. C. Michelet al. Endogenous nitric oxide and myocardial adaptation to ischemia [J]. Circ Res,2000,87 (2):146-152.
[4]M. R. Duranski, J. J. Greer, A. Dejamet al. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver [J]. J Clin Invest,2005,115(5): 1232-1240.
[5]T. M. Millar, C. R. Stevens, N. Benjaminet al. Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions [J]. FEBS Lett,1998,427 (2):225-228.
[6]I. H. Chae, K. W. Park, H. S. Kimet al. Nitric oxide-induced apoptosis is mediated by Bax/Bcl-2 gene expression, transition of cytochrome c, and activation of caspase-3 in rat vascular smooth muscle cells [J]. Clin Chim Acta,2004,341 (1-2): 83-91.
[7]M. Manderscheid, U. K. Messmer, R. Franzenet al. Regulation of inhibitor of apoptosis expression by nitric oxide and cytokines:relation to apoptosis induction in rat mesangial cells and raw 264.7 macrophages [J]. J Am Soc Nephrol,2001, 12 (6):1151-1163.
[8]A. Berges, L. Van Nassauw, J. P. Timmermanset al. Time-dependent expression pattern of nitric oxide and superoxide after myocardial infarction in rats [J]. Pharmacol Res,2007,55 (1):72-79.
[9]陈应柱,顾永健,包仕尧.刺五加皂甙对谷氨酸毒性神经元凋亡的保护作用[J].中风与神经疾病杂志,2004,21(6)：533-535.
[10]J. K. Bendall, T. Damy, P. Ratajczaket al. Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat [J]. Circulation,2004,110(16): 2368-2375.
[11]S. Gok, S. Vatansever, K. Vuralet al. The role of ATP sensitive K+ channels and of nitric oxide synthase on myocardial ischemia/reperfusion-induced apoptosis [J]. Acta Histochem,2006,108 (2):95-104.
[12]M. Peng, L. Huang, Z. J. Xieet al. Oxidant-induced activations of nuclear factor-kappa B and activator protein-1 in cardiac myocytes [J]. Cell Mol Biol Res, 1995,41 (3):189-197.
[13]Y. Sawa, R. Morishita, K. Suzukiet al. A novel strategy for myocardial protection using in vivo transfection of cis element'decoy'against NFkappaB binding site: evidence for a role of NFkappaB in ischemia-reperfusion injury [J]. Circulation, 1997,96 (9 Suppl):280-284,285.
[14]B. Chandrasekar, J. E. Streitman, J. T. Colstonet al. Inhibition of nuclear factor kappa B attenuates proinflammatory cytokine and inducible nitric-oxide synthase expression in postischemic myocardium [J]. Biochim Biophys Acta,1998,1406 (1):91-106.
[15]兰晓莉,裴著果,张永学.NO-cGMP通路在早期心肌缺血预适应中的作用[J].华中科技大学学报：医学版,2006(6)：731-733.
[16]R. Schulz, M. V. Cohen, M. Behrendset al. Signal transduction of ischemic preconditioning [J]. Cardiovasc Res,2001,52 (2):181-198.
[17]R. Bolli. The late phase of preconditioning [J]. Circ Res,2000,87 (11):972-983.
[18]R. Bolli, B. Dawn, X. L. Tanget al. The nitric oxide hypothesis of late preconditioning [J]. Basic Res Cardiol,1998,93 (5):325-338.
[19]Z. Q. Zhao, J. S. Corvera, M. E. Halkoset al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion:comparison with ischemic preconditioning [J]. Am J Physiol Heart Circ Physiol,2003,285 (2):H579-H588.
[20]A. Tsang, D. J. Hausenloy, M. M. Mocanuet al. Postconditioning:a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway [J]. Circ Res,2004,95 (3):230-232.
[21]X. M. Yang, S. Philipp, J. M. Downeyet al. Postconditioning's protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation [J]. Basic Res Cardiol, 2005,100 (1):57-63.
[2]叶仁高,陆再英,谢毅.内科学[M].北京：人民卫生出版社,2004：273.
[3]R. Datta, E. Oki, K. Endoet al. XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage [J]. J Biol Chem,2000,275 (41) 31733-31738.
[4]金菊林,徐鸿华.心肌梗死与心理应激因素[J].心脑血管病防治,2002,2(2)：7.
[5]J. A. Herd. Cardiovascular response to stress [J]. Physiol Rev,1991; 71:305-331.
[6]A. Schomig, M. Haass, G. Richardt. Catecholamine release and arrhythmias in acute myocardial ischaemia [J]. Eur Heart J,1991,12 Suppl F:38-47.
[7]朱俏萍,李建生,等.急性心肌梗死患者交感神经活性与室性心律失常的关系[J].中国综合临床,2003,19(2)：117-118.
[8]T. Nielsen, T. H. Haghfelt, S. Haunsoet al. [Ischemic heart disease and "integrated" rehabilitation] [J]. Ugeskr Laeger,2000,162 (27):3828.
[9]P. K. Ganguly, K. S. Dhalla, Q. Shao et al. Differential changes in sympathctic activity in left and right ventricle in congestive heart failure myocardial infarction [J]. Am Heart J,1997,133(3):340-349.
[10]沈法荣,金宏义.冠心病心肌缺血发作前后心脏自主神经功能的变化[J].心电学杂志,1997,16(2)：71-72.
[11]R. A. Dampney, J. Horiuchi, T. Tagawa et al. Medullary and supramedullary mechanisms regulating sympathetic vasomotor tone [J]. Acta Physiol Scand, 2003,177 (3):209-218.
[12]A. F. Sved, S. Ito, J. C. Sved. Brainstem mechanisms of hypertension:role of the rostral ventrolateral medulla [J]. Curr Hypertens Rep,2003,5(3):262-268.
[13]R. A. Dampney, M. J. Coleman, M. A. Fonteset al. Central mechanisms underlying short-and long-term regulation of the cardiovascular system [J]. Clin Exp Pharmacol Physiol,2002,29 (4):261-268.
[14]赵明中,陈运贞.实验性心肌缺血大鼠心脏自主神经活性的动态变化及意义[J].中国心脏起搏与心电生理杂志,1998,12(2)：101-103.
[15]强文安,刘枝俏.脑组织一氧化氮合成酶的特性及其与NADPH—diaphorase[J].中国神经免疫学和神经病学杂志,1996,3(4)：191-198.
[16]高博,尹桂山.神经系统中的一氧化氮[J].生物化学与生物物理进展,1999,26(1)：31-34.
[17]J. Oka, M. Imamura, E. Hattaet al. Carrier-mediated norepinephrine release and reperfusion arrhythmias induced by protracted ischemia in isolated perfused guinea pig hearts:effect of presynaptic modulation by alpha(2)-adrenoceptor in mild hypothermic ischemia [J]. J Pharmacol Exp Ther,2002,303 (2):681-687.
[18]J. Y. Chan, S. H. Chan, A. Y. Chang. Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat [J]. Neuropharmacology,2004,46 (8) 1184-1194.
[19]夏春梅,陈军,王锦等.电针诱导心肌缺血大鼠延髓头端腹外侧区nNOS和iNOS差异表达[J].生理学报,2008,60(4)：453-461.
[20]朱桂平,雷达,钟亮尹等.幼龄自发性高血压大鼠血管内皮功能研究[J].岭南心血管病杂志,2004,10(5)：368-370.
[21]晨阳,薛康,李春跃.原发性高血压与应激性高血压大鼠NO/NOS、ET的变化[J].中国医学物理学杂志,2006,23(1)：35-39.
[22]吕娟秀,周培华,王锦等.延髓腹外侧一氧化氮介导电针内关对急性心肌缺血大鼠心功能的作用[J].生理学报,2004,56(4)：503-508.
[23]S. H. Chan, L. L. Wang, J. Y. Chan. Differential engagements of glutamate and GABA receptors in cardiovascular actions of endogenous nNOS or iNOS at rostral ventrolateral medulla of rats [J]. Br J Pharmacol,2003,138(4):584-593.
[24]A. I. Malomouzh, M. R. Mukhtarov, E. E. Nikolskyet al. Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism [J]. J Neurochem, 2007,102 (6):2110-2117.
[25]M. Nakaya, K. Kondo, K. Kaga. Immunohistochemical study of ml-m5 muscarinic receptors and nNOS in human inferior turbinate mucosa [J]. Acta Otolaryngol Suppl,2007 (559):103-107.
[26]H. Fischer, H. Prast, A. Philippu. Adenosine release in the ventral striatum of the rat is modulated by endogenous nitric oxide [J]. Eur J Pharmacol,1995,275 (3): R5-R6.
[27]M. Uematsu, G. R. Gaudette, J. O. Laurikkaet al. Adenosine-enhanced ischemic preconditioning decreases infarct in the regional ischemic sheep heart [J]. Ann Thorac Surg,1998,66 (2):382-387.
[28]J. D. McCully, M. Uematsu, R. A. Parkeret al. Adenosine-enhanced ischemic preconditioning provides enhanced cardioprotection in the aged heart [J]. Ann Thorac Surg,1998,66 (6):2037-2043.
[29]N. Nassar, A. A. Abdel-Rahman. Brainstem phosphorylated extracellular signal-regulated kinase 1/2-nitric-oxide synthase signaling mediates the adenosine A2A-dependent hypotensive action of clonidine in conscious aortic barodenervated rats[J]. J Pharmacol Exp Ther,2008,324 (1):79-85.
[30]林华,易受乡,等.针刺改善冠心病心脏功能的临床观察[J].甘肃中医,2002,15(4)：86-89.
[31]蔡荣林,胡玲,周逸平等.电针对急性心肌缺血家兔心功能及心交感神经电活动的影响[J].针刺研究,2007,32(4)：243-246.
[32]高楠,苏勋庄.针刺改善心肌缺血作用的临床观察[J].上海针灸杂志,1997,16(3)：7-8.
[33]孟竞璧.针刺治疗冠心病的疗效及其作用机理研究[J].中医杂志,2002,43(8)：587-589.
[34]D. Felhendler, B. Lisander. Effects of non-invasive stimulation of acupoints on the cardiovascular system [J]. Complement Ther Med,1999,7(4):231-234.
[35]S. Ballegaard, T. Muteki, H. Haradaet al. Modulatory effect of acupuncture on the cardiovascular system:a cross-over study [J]. Acupunct Electro ther Res, 1993,18 (2):103-115.
[36]A. Richter, J. Herlitz, A. Hjalmarson. Effect of acupuncture in patients with angina pectoris [J]. Eur Heart J,1991,12 (2):175-178.
[37]X. R. Wang, H. Lin, Z. H. Wang. Protective effects of electroacupuncture and Salviae Miltiorrhizae on myocardial ischemia/reperfusion in rabbits [J]. Acupunct Electrother Res,2003,28 (3-4):175-182.
[38]J. M. Foster, B. P. Sweeney. The mechanisms of acupuncture analgesia [J]. Br J Hosp Med,1987,38 (4):308-312.
[39]J. S. Han. Electroacupuncture:an alternative to antidepressants for treating affective diseases? [J]. Int J Neurosci,1986,29 (1-2):79-92.
[40]M. Arya, I. S. Shergill, M. Williamsonet al. Basic principles of real-time quantitative PCR [J]. Expert Rev Mol Diagn,2005,5 (2):209-219.
[41]J. Kato, K. Meguro, A. Satoet al. The effects of morphine administered into the vertebral artery on the somatosympathetic A-and C-reflexes in anesthetized cats [J]. Neurosci Lett,1992,138 (2):207-210.
[42]A. Sato, Y. Sato, R. F. Schmidt. Modulation of somatocardiac sympathetic reflexes mediated by opioid receptors at the spinal and brainstem level [J]. Exp Brain Res,1995,105 (1):1-6.
[43]W. Zhou, L. W. Fu, S. C. Tjen-A-Looiet al. Afferent mechanisms underlying stimulation modality-related modulation of acupuncture-related cardiovascular responses [J]. J Appl Physiol,2005,98 (3):872-880.
[44]李梦,胡玲,蔡荣林等.电针内关、心俞改善急性心肌缺血大鼠心率变异性的协同作用[J].甘肃中医学院学报,2008,25(1)：12-15.
[45]S. C. Tjen-A-Looi, P. Li, J. C. Longhurst. Medullary substrate and differential cardiovascular responses during stimulation of specific acupoints [J]. Am J Physiol Regul Integr Comp Physiol,2004,287 (4):R852-R862.
[46]B. JOHANSSON. Circulatory responses to stimulation of somatic afferents with special reference to depressor effects from muscle nerves [J]. Acta Physiol Scand Suppl,1962,198:1-91.
[47]W. Zhou, L. W. Fu, S. C. Tjen-A-Looiet al. Afferent mechanisms underlying stimulation modality-related modulation of acupuncture-related cardiovascular responses [J]. J Appl Physiol,2005,98 (3):872-880.
[48]H. F. Guo, J. Tian, X. Wanget al. Brain substrates activated by electroacupuncture (EA) of different frequencies (Ⅱ):Role of Fos/Jun proteins in EA-induced transcription of preproenkephalin and preprodynorphin genes [J]. Brain Res Mol Brain Res,1996,43 (1-2):167-173.
[49]H. F. Guo, J. Tian, X. Wanget al. Brain substrates activated by electroacupuncture of different frequencies (Ⅰ):Comparative study on the expression of oncogene c-fos and genes coding for three opioid peptides [J]. Brain Res Mol Brain Res,1996,43 (1-2):157-166.
[50]Y. L. Huang, M. X. Fan, J. Wanget al. Effects of acupuncture on nNOS and iNOS expression in the rostral ventrolateral medulla of stress-induced hypertensive rats [J]. Acupunct Electrother Res,2005,30 (3-4):263-273.
[51]M. A. Pfeffer, J. M. Pfeffer, M. C. Fishbeinet al. Myocardial infarct size and ventricular function in rats [J]. Circ Res,1979,44 (4):503-512.
[52]Li Peng, Yao Tai. Mechanism of the Modulatory Effect of Acupuncture on Abnormal Cardiovascular Functions [M]. Shanghai:Shanghai Medical University Press,1992.
[53]李忠仁主编.实验针灸学(第1版)[M].北京：中国中医药出版社,2003：329.
[54]M. C. Fishbein, S. Meerbaum, J. Ritet al. Early phase acute myocardial infarct size quantification:validation of the triphenyl tetrazolium chloride tissue enzyme staining technique [J]. Am Heart J,1981,101 (5):593-600.
[55]P. N. Khalil, M. Siebeck, R. Husset al. Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts [J]. J Pharmacol Toxicol Methods,2006,54 (3) 307-312.
[56]G. Paxinos, C. R. Watson. The Rat Brain in Stereotaxic Coordinates,3rd edn [M]. San Diego:Academic Press,1997.
[57]魏泓.医学实验动物学.第一版[M].成都：四川科学技术出版社,1998：323-324.
[58]张润峰,王继生.建立大鼠心肌梗死模型的若干问题探讨[J].山西医科大学学报,2004,35(1)：13-15.
[59]王响英,吴淑燕,李苏安等.大鼠直视气管插管方法[J].上海实验动物科学,2004,24(2)：107-108.
[60]边素艳,刘宏斌,杨庭树.提高急性心肌梗死大鼠模型存活率的实验研究[J].中国康复医学杂志,2005,20(3)：180-182.
[61]许丹,张春来,李霞等.制作大鼠急性心肌梗死模型的实验分析[J].中国煤炭工业医学杂志,2006,9(2)：190-191.
[62]张晔.实用临床针灸学[M].上海：上海医科大学出版社,1998.
[63]陈少宗.针刺间使穴,内关穴对冠心病患者左心功能影响的比较观察[J].针灸临床杂志,1994,10(6)：30-32.
[64]郑关毅,张贵灿.针刺内关穴对冠心病患者左心室功能的影响[J].上海针灸杂志,1997,16(4)：8-9.
[65]薛少敏,李传杰.针刺对冠心病患者左心舒张功能的影响[J].中国针灸,1992,12(3)：43-46.
[66]H. R. Middlekauff. Acupuncture in the treatment of heart failure [J]. Cardiol Rev, 2004,12 (3):171-173.
[67]J. Gao, L. Zhang, Y. Wanget al. Antiarrhythmic effect of acupuncture pretreatment in rats subjected to simulative global ischemia and reperfusion--involvement of adenylate cyclase, protein kinase A, and L-type Ca2+ channel [J]. J Physiol Sci,2008,58 (6):389-396.
[68]J. Yan, X. F. Yang, S. X. Yiet al. [Effect of electroacupuncture of "Neiguan" (PC 6) on Na+-K+-ATPase activity and its gene expression in cardiocyte membrane in rats with myocardial ischemia-reperfusion injury] [J]. Zhen Ci Yan Jiu,2007, 32 (5):296-300.
[69]J. Gao, W. Fu, Z. Jinet al. Acupuncture pretreatment protects heart from injury in rats with myocardial ischemia and reperfusion via inhibition of the beta(1)-adrenoceptor signaling pathway [J]. Life Sci,2007,80 (16):1484-1489.
[70]L. Yang, J. Yang, Q. Wanget al. Cardioprotective effects of electroacupuncture pretreatment on patients undergoing heart valve replacement surgery:a randomized controlled trial [J]. Ann Thorac Surg,2010,89 (3):781-786.
[71]X. Song, Z. Tang, Z. Houet al. [The anti-hemorrhagic shock of acupuncture on neiguan and its effect on the cardial pump function and the blood viscosity] [J]. Zhen Ci Yan Jiu,1990,15 (1):30-34.
[72]X. Song, Z. Tang, Z. Houet al. An experimental study on acupuncture anti-hemorrhagic shock [J]. J Tradit Chin Med,1993,13 (3):207-210.
[73]S. B. Wang, S. P. Chen, Y. H. Gaoet al. Effects of electroacupuncture on cardiac and gastric activities in acute myocardial ischemia rats [J]. World J Gastroenterol, 2008,14 (42):6496-6502.
[74]张明金,魏明文,等.针刺内关穴对心率变异性的分析[J].中国针灸,2003,23(3)：169-170.
[75]S. Sakai, E. Hori, K. Umenoet al. Specific acupuncture sensation correlates with EEGs and autonomic changes in human subjects [J]. Auton Neurosci,2007,133 (2):158-169.
[76]C. C. Hsu, C. S. Weng, T. S. Liuet al. Effects of electrical acupuncture on acupoint BL15 evaluated in terms of heart rate variability, pulse rate variability and skin conductance response [J]. Am J Chin Med,2006,34 (1):23-36.
[77]石现,赵永兰,等.针刺调节心脏植物神经的实验研究[J].针刺研究,2002,27(1)：68-70.
[78]Z. Li, C. Wang, A. F. Maket al. Effects of acupuncture on heart rate variability in normal subjects under fatigue and non-fatigue state [J]. Eur J Appl Physiol,2005, 94 (5-6):633-640.
[79]刘金兰,张振莉.电针对急性心肌缺血家兔血浆单胺类神经递质的影响[J].中国针灸,1997,17(10)：604-605.
[80]M. M. Wolf, G. A. Varigos, D. Huntet al. Sinus arrhythmia in acute myocardial infarction [J]. Med J Aust,1978,2(2):52-53.
[81]安威,时安云.心肌毖?再灌注损伤与心肌去甲肾上腺素的释放[J].中国病理生理杂志,1990,6(2)：65-68.
[82]冯义柏.粉防己碱对缺血心肌去甲肾上腺素释放的影响[J].同济医科大学学报,1994,23(2)：147-148.
[83]杨天德,刘桥义.纳络酮对缺血再灌注心肌儿茶酚胺释放的影响[J].中华麻醉学杂志,1994,14(5)：331-333.
[84]张彦周.心肌缺血对心肌去甲肾上腺素的释放[J].国外医学：心血管疾病分册,1998,25(5)：265-267.
[85]N. Nagaya, T. Nishikimi, Y. Gotoet al. Plasma brain natriuretic peptide is a biochemical marker for the prediction of progressive ventricular remodeling after acute myocardial infarction [J]. Am Heart J,1998,135 (1):21-28.
[86]高博,尹桂山.神经系统中的一氧化氮[J].生物化学与生物物理进展,1999,26(1)：31-34.
[87]K. P. Patel, Y. F. Li, Y. Hirooka. Role of nitric oxide in central sympathetic outflow [J]. Exp Biol Med (Maywood),2001,226 (9):814-824.
[88]强文安,刘枝俏.脑组织一氧化氮合成酶的特性及其与NADPH—diaphorase [J].中国神经免疫学和神经病学杂志,1996,3(4)：191-198.
[89]D. S. Bredt. Endogenous nitric oxide synthesis:biological functions and pathophysiology [J]. Free Radic Res,1999,31 (6):577-596.
[90]C. Y. Chen, S. J. Huang, T. M. Yanget al. Effective renal plasma flow response and atrial natriuretic peptide in patients with uncomplicated acute myocardial infarction [J]. Jpn Heart J,1993,34 (1):1-10.
[91]J. L. Zweier, P. Wang, P. Kuppusamy. Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy [J]. J Biol Chem,1995,270 (1):304-307.
[92]K. Akiyama, A. Kimura, H. Suzukiet al. Production of oxidative products of nitric oxide in infarcted human heart [J]. J Am Coll Cardiol,1998,32 (2): 373-379.
[93]M. Araki, M. Tanaka, K. Hasegawaet al. Nitric oxide inhibition improved myocardial metabolism independent of tissue perfusion during ischemia but not during reperfusion [J]. J Mol Cell Cardiol,2000,32 (3):375-384.
[94]牛丽丽,祝善俊.冠心病患者血管内皮细胞损伤血管活性物质失衡对冠脉的调控作用[J].解放军医学杂志,1996,21(4)：282-283.
[95]S. X. Ma. Enhanced nitric oxide concentrations and expression of nitric oxide synthase in acupuncture points/meridians [J]. J Altern Complement Med,2003,9 (2):207-215.
[96]S. X. Ma, J. Ma, G. Moiseet al. Responses of neuronal nitric oxide synthase expression in the brainstem to electroacupuncture Zusanli (ST 36) in rats [J]. Brain Res,2005,1037 (1-2):70-77.
[97]C. Depre, L. Fierain, L. Hue. Activation of nitric oxide synthase by ischaemia in the perfused heart [J]. Cardiovasc Res,1997,33 (1):82-87.
[98]W. Bloch, U. Mehlhorn, A. Krahwinkelet al. Ischemia increases detectable endothelial nitric oxide synthase in rat and human myocardium [J]. Nitric Oxide, 2001,5 (4):317-333.
[99]M. Desrois, T. Caus, P. M. Belleset al. Nitric oxide pathway after long-term cold storage and reperfusion in a heterotopic rat heart transplantation model [J]. Transplant Proc,2005,37 (10):4553-4555.
[100]朱大年.生理学(第七版)[M].北京：人民卫生出版社,2008：115.
[101]Q. Lin, P. Li. Rostral medullary cholinergic mechanisms and chronic stress-induced hypertension [J]. J Auton Nerv Syst,1990,31 (3):211-217.
[102]L. Gui, W. Duan, H. Tianet al. Adenosine A 2A receptor deficiency reduces striatal glutamate outflow and attenuates brain injury induced by transient focal cerebral ischemia in mice [J]. Brain Res,2009,1297:185-193.
[103]N. Nassar, A. A. Abdel-Rahman. Brainstem adenosine Al receptor signaling masks phosphorylated extracellular signal-regulated kinase 1/2-dependent hypotensive action of clonidine in conscious normotensive rats [J]. J Pharmacol Exp Ther,2009,328 (1):83-89.
[104]N. Nassar, A. A. Abdel-Rahman. Brainstem phosphorylated extracellular signal-regulated kinase 1/2-nitric-oxide synthase signaling mediates the adenosine A2A-dependent hypotensive action of clonidine in conscious aortic barodenervated rats [J]. J Pharmacol Exp Ther,2008,324 (1):79-85.
[105]M. C. Martins-Pinge, M. R. Garcia, D. B. Zoccalet al. Differential influence of iNOS and nNOS inhibitors on rostral ventrolateral medullary mediated cardiovascular control in conscious rats [J]. Auton Neurosci,2007,131 (1-2) 65-69.
[106]R. R. Morrison, M. A. Talukder, C. Ledentet al. Cardiac effects of adenosine in A(2A) receptor knockout hearts:uncovering A(2B) receptors [J]. Am J Physiol Heart Circ Physiol,2002,282 (2):H437-H444.
[107]L. Stella, L. Berrino, A. Filippelliet al. Nitric oxide participates in the hypotensive effect induced by adenosine A2 subtype receptor stimulation [J]. J Cardiovasc Pharmacol,1995,25 (6):1001-1005.
[108]H. Fischer, H. Prast, A. Philippu. Adenosine release in the ventral striatum of the rat is modulated by endogenous nitric oxide [J]. Eur J Pharmacol,1995,275 (3):R5-R6.
[109]L. Stella, L. Berrino, A. Filippelliet al. Nitric oxide participates in the hypotensive effect induced by adenosine A2 subtype receptor stimulation [J]. J Cardiovasc Pharmacol,1995,25 (6):1001-1005.
[110]M. A. Timoteo, L. Oliveira, E. Campesatto-Mellaet al. Tuning adenosine A1 and A2A receptors activation mediates L-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern [J]. Neurochem Int,2008,52 (4-5):834-845.
[111]J. C. Longhurst. Electroacupuncture treatment of arrhythmias in myocardial ischemia [J]. Am J Physiol Heart Circ Physiol,2007,292 (5):H2032-H2034.
[112]M. C. Martins-Pinge, M. R. Garcia, D. B. Zoccalet al. Differential influence of iNOS and nNOS inhibitors on rostral ventrolateral medullary mediated cardiovascular control in conscious rats [J]. Auton Neurosci,2007,131 (1-2) 65-69.
[1]V. L. Dawson, T. M. Dawson, E. D. Londonet al. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures [J]. Proc Natl Acad Sci U S A,1991,88 (14):6368-6371.
[2]C, Martin, R. Schulz, H. Postet al. Microdialysis-based analysis of interstitial NO in situ:NO synthase-independent NO formation during myocardial ischemia [J]. Cardiovasc Res,2007,74 (1):46-55.
[3]G. Heusch, H. Post, M. C. Michelet al. Endogenous nitric oxide and myocardial adaptation to ischemia [J]. Circ Res,2000,87 (2):146-152.
[4]M. R. Duranski, J. J. Greer, A. Dejamet al. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver [J]. J Clin Invest,2005,115(5): 1232-1240.
[5]T. M. Millar, C. R. Stevens, N. Benjaminet al. Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions [J]. FEBS Lett,1998,427 (2):225-228.
[6]I. H. Chae, K. W. Park, H. S. Kimet al. Nitric oxide-induced apoptosis is mediated by Bax/Bcl-2 gene expression, transition of cytochrome c, and activation of caspase-3 in rat vascular smooth muscle cells [J]. Clin Chim Acta,2004,341 (1-2): 83-91.
[7]M. Manderscheid, U. K. Messmer, R. Franzenet al. Regulation of inhibitor of apoptosis expression by nitric oxide and cytokines:relation to apoptosis induction in rat mesangial cells and raw 264.7 macrophages [J]. J Am Soc Nephrol,2001, 12 (6):1151-1163.
[8]A. Berges, L. Van Nassauw, J. P. Timmermanset al. Time-dependent expression pattern of nitric oxide and superoxide after myocardial infarction in rats [J]. Pharmacol Res,2007,55 (1):72-79.
[9]陈应柱,顾永健,包仕尧.刺五加皂甙对谷氨酸毒性神经元凋亡的保护作用[J].中风与神经疾病杂志,2004,21(6)：533-535.
[10]J. K. Bendall, T. Damy, P. Ratajczaket al. Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat [J]. Circulation,2004,110(16): 2368-2375.
[11]S. Gok, S. Vatansever, K. Vuralet al. The role of ATP sensitive K+ channels and of nitric oxide synthase on myocardial ischemia/reperfusion-induced apoptosis [J]. Acta Histochem,2006,108 (2):95-104.
[12]M. Peng, L. Huang, Z. J. Xieet al. Oxidant-induced activations of nuclear factor-kappa B and activator protein-1 in cardiac myocytes [J]. Cell Mol Biol Res, 1995,41 (3):189-197.
[13]Y. Sawa, R. Morishita, K. Suzukiet al. A novel strategy for myocardial protection using in vivo transfection of cis element'decoy'against NFkappaB binding site: evidence for a role of NFkappaB in ischemia-reperfusion injury [J]. Circulation, 1997,96 (9 Suppl):280-284,285.
[14]B. Chandrasekar, J. E. Streitman, J. T. Colstonet al. Inhibition of nuclear factor kappa B attenuates proinflammatory cytokine and inducible nitric-oxide synthase expression in postischemic myocardium [J]. Biochim Biophys Acta,1998,1406 (1):91-106.
[15]兰晓莉,裴著果,张永学.NO-cGMP通路在早期心肌缺血预适应中的作用[J].华中科技大学学报：医学版,2006(6)：731-733.
[16]R. Schulz, M. V. Cohen, M. Behrendset al. Signal transduction of ischemic preconditioning [J]. Cardiovasc Res,2001,52 (2):181-198.
[17]R. Bolli. The late phase of preconditioning [J]. Circ Res,2000,87 (11):972-983.
[18]R. Bolli, B. Dawn, X. L. Tanget al. The nitric oxide hypothesis of late preconditioning [J]. Basic Res Cardiol,1998,93 (5):325-338.
[19]Z. Q. Zhao, J. S. Corvera, M. E. Halkoset al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion:comparison with ischemic preconditioning [J]. Am J Physiol Heart Circ Physiol,2003,285 (2):H579-H588.
[20]A. Tsang, D. J. Hausenloy, M. M. Mocanuet al. Postconditioning:a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway [J]. Circ Res,2004,95 (3):230-232.
[21]X. M. Yang, S. Philipp, J. M. Downeyet al. Postconditioning's protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation [J]. Basic Res Cardiol, 2005,100 (1):57-63.