【作者】 尤行宏；

【导师】 王华；

【作者基本信息】 湖北中医学院 ， 针灸推拿学， 2009， 博士

【摘要】 目的：探讨“双固一通”针法对家兔急性心肌缺血(AMI)的保护作用及其内阿片肽系统的介导机制。方法：健康清洁级日本大耳白兔，随机分为四组：对照组(正常对照，不造模不针刺)、模型组(模型对照，只结扎左冠状动脉造模，不针刺)、电针组(结扎左冠状动脉造模，“双固一通”针法电针关元、足三里、内关穴位治疗)，纳洛酮组(延髓头端腹外侧注射纳洛酮，结扎左冠状动脉造模，“双固一通”针法电针关元、足三里、内关穴位治疗)。实验动物用20％乌拉坦(5ml／kg)行耳缘静脉注射麻醉后，家兔于延髓腹外侧区头端(rVLM)中枢微量注射给药，结扎左冠状动脉前降支制造急性心肌缺血(AMI)模型，以“双固一通”针法电针刺激治疗。在实验中分别于中枢给药前，给药后(即结扎前)、结扎即刻、结扎后5min、15min、30min、45min、60min等时刻同步记录心电图、动脉血压，实验结束后取材。采用光镜和电镜观察心肌组织结构的变化，采用放射免疫测定法检测血浆、延髓、下丘脑等不同组织部位强啡肽、亮-脑啡肽、甲硫-脑啡肽的含量，采用免疫组织化学法检测延髓L-ENK免疫反应阳性细胞的表达，测定其阳性细胞表达率，采用原位杂交方法检测延髓前脑啡肽原mRNA的表达。结果：1．心电图：与正常组相比，模型组和电针组家兔心电图ST段电位显著抬高、T波高耸(P＜0．01)，与结扎前比较差异有显著改变(P＜0．01)，模型组ST段变化在冠脉结扎后15min时达到高峰，随后在整个观察期变化不明显，而“双固一通”针法电针组ST段电位、T波恢复较模型组有显著性差异(P＜0．05)，rVLM内微量注射纳洛酮对电针作用有阻断作用，能在一定程度上减弱电针的心肌缺血保护效应(P＜0．05)。2．平均动脉血压：与正常组相比，模型组和电针组家兔动脉血压在造模后显著降低(P＜0．01)。模型组血压的变化在冠脉结扎后15min时最为显著，随后动脉血压有所恢复，“双固一通”针法电针组血压恢复较快，在结扎后45min后就与最高峰有显著性差异(P＜0．05)，60min后血压与结扎前已无明显差异(P＞0．05)，rVLM内微量注射纳洛酮能减弱电针促进血压的恢复效应，使血压恢复时间延长(P＜0．05)。3．血浆内阿片肽含量的检测：与正常对照组相比较，模型组家兔在结扎冠脉造成心肌缺血损伤后，血浆中强啡肽、亮-脑啡肽、甲硫-脑啡肽的含量均升高(P＜0．01)，“双固一通”针法电针治疗后，电针组家兔血浆内阿片肽含量较模型组明显升高(P＜0．01)。4．脑组织内阿片肽的测定：结扎冠状动脉造模后，家兔延髓和下丘脑组织内三种阿片肽含量较正常对照组升高(P＜0．01)，电针组在“双固一通”针法治疗后，延髓和下丘脑内强啡肽、亮-脑啡肽、甲硫-脑啡肽的含量升高，与模型组比较具有显著性差异(P＜0．01)。5．心肌组织学观察：光镜观察可见，模型组心肌排列稀疏，不规则，细胞水肿，心肌细胞出现明显空泡变性，部分心肌纤维断裂，细胞间隙增宽，可见较多的炎性细胞浸润。“双固一通”针法电针组心肌结构有明显好转，心肌细胞结构大致正常，纳洛酮能减弱电针的效应。6．心肌超微结构观察：电镜观察可见，模型组心肌细胞呈现缺血性损伤改变，心肌细胞肿胀，肌原纤维排列紊乱，肌原纤维断裂并可见闰盘处变性；线粒体明显肿胀，峭断裂；血管内皮细胞肿胀严重，吞饮小泡明显减少，电子密度不均匀，部分内皮细胞核与胞体分离。“双固一通”针法电针组心肌细胞超微结构结构大致正常，纳洛酮能减弱电针对缺血心肌的保护作用。7．延髓L-ENK神经元表达：延髓切片经免疫组织化学染色显示L-ENK免疫反应阳性细胞主要密集分布在背内侧的NTS至延髓VLM的带状区域，包括NTS与VLM之间的网状结构(RF)。心肌缺血模型组在延髓VLM部位L-ENK免疫反应阳性细胞显著增多(P＜0．01)，“双固一通”针法电针治疗后，电针组阳性细胞在延髓VLM的表达更多(P＜0．01)。8．延髓PPEmRNA的表达：延髓PPEmRNA原位杂交显示，各组家兔内阿片肽基因表达部位也位于这些区域。结扎冠脉造模后，模型组在延髓VLM内可见PPEmRNA表达的细胞数增多(P＜0．01)，电针组在“双固一通”针法治疗后，PPEmRNA在延髓VLM内表达的细胞数明显高于模型组(P＜0．01)。结论：1．“双固一通”针法能促进家兔AMI损伤时心电图ST段电位、T波的恢复，改善AMI时动脉血压的变化，对心肌缺血损伤的超微结构变化有较好的保护作用。2．“双固一通”针法改善AMI损伤与外周内阿片肽的参与有关，能促进血浆强啡肽和脑啡肽的释放，调整心肌细胞活动状态，实现心肌缺血损伤的保护作用。3．“双固一通”针法对缺血心肌的保护作用与中枢内阿片肽介导有关，电针刺激能促进延髓和下丘脑中枢阿片肽的释放，降低心肌组织损伤的程度。4．“双固一通”针法改善AMI损伤与rVLM内阿片系统的参与有关，rVLM内注射纳洛酮能减弱电针的作用，具有阻断效应。5．“双固一通”针法能促进延髓L-ENK免疫阳性神经元和PPEmRNA的表达，电针抗心肌缺血损伤作用与其促进肽能神经元的活动，加速脑啡肽基因表达的过程有关。更多还原

【Abstract】 The Research of Endogenous Opioid Peptides Mechanismin the Protective Effect of Electroacupuncture onAcute Myocardial Ischemia RabbitsSpeciality:Acupuncture and MassageAuthor:You XinghongTutor:Wang Hua ProfessorObjectiveTo explore both the effect and the mechanisms of Shuangguyitongtherapy of acupuncture in the rabbits with acute myocardialischemia（AMI）;Yoresearch theroleofendogenousopioidpeptidesof anti-AMI effect of Shuangguyitong therapy of acupuncture on theAMI rabbits.Methods80 rabbits were evenly and randomly divided into 4 groups:blankcontrol group（blank,non-model and non-needling,n=10）;modelgroup（model and non-needling,n=10）;electric needle group（modeland Shuangguyitong therapy of acupuncture electric needlingGuanyuan,Zusanli and Neiguan acupoint,n=10）.naloxone group（microinjection naloxone into rostral ventrolateral medulla,modeland Shuangguyitong therapy of acupuncture electric needlingGuanyuan,Zusanli and Neiguan acupoint,n=10）.Afteranesthesia（20% urethane,5ml/kg,i.p.）andmicroinjection naloxoneor sodium chloride into rostral ventrolateral medulla,AMI rabbitmodels were induced by ligating left ventricular branch of coronaryartery,treated by Shuangguyitong therapy of acupuncturestimulated with electric needling Guanyuan,Zusanli and Neiguan acupoint.To record theⅡleadECGand meanarterialpressureat5 min、15 min、30 min、45 min、60 min after acute myocardial ischemiain rabbits.To observe the change of AMI and myocardialultrastructures of rabbit’ sischemicmyocardial cell.Contents ofDynorphin(Dyn A_1-13),methionine enkephalin（M-ENK）and leuthineenkephalin（L-ENK）in brain tissue and plasma were determined byradioimmunoassay.Investigate the distribution of leuthineenkephalin immunoreactive fibers in the medulla byimmunohistochemical method.To explore the preproenkephalin mRNAexpression of medulla by the method of in situ hybridizationhistochemistry after electroacupuncture.Results1.EGG:Compared with blank control group,the changes of T waveand ST segment of ECG differd significantly in model group andelectric needle group after ischemia（P＜0.01）,no significant dataof model group showed that there was the difference in 60 minutes.Shuangguyitong therapy of acupuncture electric needling Guanyuan,Zusanli and Neiguan acupoint could markedly promote recovery of EGGafter ischemia,there was significant difference than model group（P＜0.05）.The protective effect can be weakened by microinjectionwithNaloxoneintorVLM（P＜0.05）,but can’ tbecompletelyblocked.2.Meanarterial pressure:Compared with blank control group,themean arterial pressure depressed significantly in model group andelectric needle group after ischemia（P＜0.01）,Shuangguyitongtherapy of acupuncture electric needling Guanyuan,Zusanli andNeiguan acupoint could markedly promote recovery of mean arterialpressure after ischemia,there was significant difference thanmodel group（P＜0.05）.The effect can be weakened by microinjection with Naloxone into rVLM（P＜0.05）.3.Concentration of endogenousopioidpeptides inplasma:Comparedwith blank control group,Concentration of Dyn A_1-13),M-ENK and L-ENKincreased markedly in plasma in model group（P＜0.01）Theconcentration of Dyn A_1-13),M-ENK and L-ENK in plasma afterShuangguyitong therapy of acupuncture electric needling Guanyuan,Zusanli and Neiguan acupoint increased significantly compared tothe model group（P＜0.01）4.Concentration of endogenous opioidpeptides in brain:Comparedwith blank control group,Concentration of Dyn A_1-13),M-ENK and L-ENKincreased markedly in medulla and hypothalamus in model group（P＜0.01）.The concentration of Dyn A_1-13),M-ENK and L-ENK in plasmaafter Shuangguyitong therapy of acupuncture electric needlingGuanyuan,Zusanli and Neiguan acupoint increased significantlycompared to the model group（P＜0.01）.5.Myocardial tissue injury:Compared with blank control group,tthetissue injury of myocardium examined with optical microscope inmodel group were obvious Microinjection naloxone into rVLM hasno influence to normal rabbit’ s myocardial tissue.Shuangguyitongtherapy of electroacupuncture could obviously reduce the degree ofmyocardium ischemic injury of myocardial ultrastructures inrabbits.The effect can be weakened by microinjection with Naloxonein rVLM.6.Myocardial ultrastructures:Microinjectionnaloxone into rVLMhas no influence to normal rabbit’ s myocardial ultrastructures.Electroacupuncture has significant protective effect to acutemyocardium ischemic in rabbits,results indicated thatShuangguyitong therapy of electroacupuncture could obviously reduce the degree of myocardium ischemic injury of myocardialultrastructures in rabbits.In the electroacupuncture group,lessinjury of myocardial tissue,normal run of myocardial fiber,a smallamount mitochondrial vacuolation and no obvious dissolution andnecrosis of myocardium were found.The protective effect can beweakened by microinjection with Naloxone in rVLM.7.L-ENK immunoreactive neurons in the medulla tissue:L-ENKimmunoreactive neurons were mainly distributed in the nucleitractus solitarii（NTS）,ventrolateral medulla（VLM）and thereticular formation betweenNTS andVLM,forming an arc band zonefrom dorsomedial part to ventrolateral part of medulla.Comparedwith blank control group,these structures ofL-ENK immunoreactivebodies and dibers were increased markedly in medulla in model group（P＜0.01）.After Shuangguyitong therapy of acupuncture electricneedlingGuanyuan,ZusanliandNeiguanacupoint,the expression ofL-ENK immunoreactive bodies were increased significantly comparedto the model group（P＜0.01）.8.PPEmRNA containing neurons in the medulla tissue:PPEmRNAcontaining neurons were also densely seen in the medullastructures.Compared with blank control group,these structures ofPPEmRNA containing neurons were increased markedly in medulla inmodel group（P＜0.01）.After Shuangguyitong therapy of acupunctureelectric needling Guanyuan,Zusanli and Neiguan acupoint,theexpression of PPEmRNA containing neurons were increasedsignificantly compared to the model group（P＜0.01）Comclusions1.Shuangguyitong therapy of acupuncture electric needlingGuanyuan,Zusanli and Neiguan acupoint could markedly promote recovery of the T wave and ST segment of ECG and mean arterialpressure after myocardial ischemia and could obviously reduce thedegree of myocardium ischemic injury of myocardial tissue inrabbits.2.The protective effect of Shuangguyitong therapy ofelectroacupuncture in ameliorating acute myocardium ischemic inrabbits was mediated by endogenous opioid peptides in plasma.3.Shuangguyitong therapy of acupuncture could protect ischemicmyocardium cell after myocardial ischemia.The effect ofameliorating acute myocardium ischemic in rabbits was mediated byendogenous opioid peptides in brain.4.The effect of Shuangguyitong therapy of electroacupuncture inameliorating acute myocardium ischemic in rabbits was mediated byopioid receptors in rVLM.5.Shuangguyitong therapy of acupuncture electric needlingGuanyuan,Zusanli and Neiguan acupoint can significantly improvethe expression of L-ENK immunoreactive neurons and PPEmRNAcontaining neurons in the VLM.The protective effect ofelectroacupuncture was mediated by opioid receptors in VLM.更多还原