心绞痛贴膏穴位贴敷治疗冠心病的基础和临床研究

【作者】 高敏；

【导师】 王凤荣；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床（专业学位）， 2013， 博士

【摘要】 第一部分导师学术观点目的：探讨“心绞痛贴膏”穴位贴敷防治冠心病的中医理论依据。方法：总结整理导师“心绞痛贴膏”穴位贴敷治疗冠心病的中医理论。从“整体观念”的角度出发，分析经“皮部”治疗胸痹心痛的可行性；以“经络学说”中的“腧穴”和“经络”为切入点，分析穴位贴敷治疗胸痹心痛的药物作用路径。结合古代文献以及现代药理研究成果，证明导师“心绞痛贴膏”的组方依据和取穴依据。结论：中医理论支持“心绞痛贴膏”穴位贴敷治疗冠心病具有可行性和有效性。第二部分心绞痛贴膏穴位贴敷预处理对大鼠急性心肌梗死的影响和作用机制目的：探讨“心绞痛贴膏”穴位贴敷预处理对大鼠急性心肌梗死的干预效应及作用机制。方法：将健康Wistar大鼠55只随机分为正常组、模型组、对照组和治疗组，正常组10只，模型组、对照组和治疗组每组15只。在清洁级实验室以普通饲料饲养。对照组予硝酸甘油贴膜0.3mg心前区外贴，每次1贴，每日1次，每次维持24小时，疗程7天；治疗组予心绞痛贴膏（药物组成：川芎、丹参、冰片、乳香、没药、檀香、元胡等）穴位贴敷，每贴含生药量0.1g，每日一次，维持24h，双侧心俞、双侧足三里、神阙取穴，每穴1贴，疗程7天。第5天开始造模，对模型组、对照组和治疗组动物造成急性心肌梗死动物模型。具体方法是第5-7天每日贴敷后立即对模型组、对照组、治疗组动物注射异丙肾上腺素，采用多点皮下注射的方法，异丙肾上腺素用量为5mg/kg，第7天注射肾上腺素后，将各组存活动物用水合氯醛皮下注射麻醉（药量300mg/kg），麻醉成功后查心电图，以ST段明显上抬或下移为造模成功的初步判断标准，然后对各组动物腹主动脉采血、取心脏。光镜下观察心肌病理学形态，电镜下观察心肌超微结构；心电图测量ST段上抬或下移水平；全自动生化仪酶法检测肌酸激酶（Creatine kinase，CK）、肌酸激酶同工酶（Creatine kinase isoenzyme，CK-MB）、乳酸脱氢酶（LactateDehydrogenase，LDH）；免疫组化法测定丙二醛（Malondialdehyde，MDA）、过氧化物歧化酶（Superoxide Dismutase，SOD）、肿瘤坏死因子（Tumor Necrosis Factors TNF-α）、白介素-6（Interleukin，IL-6）、B细胞淋巴瘤/白血病-2B（cell-lymphoma/leukemia-2，Bcl-2）、Bax、半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）、P物质（Substance P，SP）、内皮细胞型一氧化氮合酶（endothelial nitric oxidesynthase，eNOS）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、钙敏感受体(calcium—sensing receptor，CaSR)水平；荧光定量RT-PCR法测定eNOS、iNOS以及CaSR水平。结果：1光镜及电镜下观察：正常组心肌细胞呈正常表现。模型组心肌细胞坏死严重，可见心肌细胞核和心肌纤维排列紊乱、断裂及缺失，交织成网，横纹消失，心肌细胞核固缩，出现空白区，异染色质呈现团块状，相邻细胞闰盘间隙增宽。对照组和治疗组心肌细胞损伤坏死较模型组轻，但也可见少量断裂、缺损及空白区着色不均匀出现。透射电镜下：正常组心肌细胞无变化。模型组心肌线粒体损伤严重,双层膜结构不清，嵴断裂，大多数呈空泡状，部分溶解；细胞核染色质密集，部分胞浆溶解、消失，呈液化性坏死；肌原纤维极度松弛、出现空泡,部分肌丝断裂，闰盘局部性扩张，结构不清、溶解。对照组和治疗组线粒体损伤较轻，双层膜结构尚清晰，部分嵴断裂，细胞核结构基本完整，肌原纤维排列较整齐，可见肌丝断裂。2心电图结果：模型组、对照组和治疗组ST段上抬或下移的绝对值较正常组明显高（P＜0.01）。对照组、治疗组ST段上抬或下移的绝对值均较模型组低，有统计学意义（P＜0.01），治疗组与对照组比较，无统计学意义。3心肌酶学结果：模型组CK、CK-MB较正常组高（P＜0.01），对照组和治疗组CK水平较模型组低，与模型组比较，有统计学意义（P＜0.01）。对照组、治疗组CK-MB较模型组低，与模型组比较，有统计学意义（P＜0.05，P＜0.01）；治疗组CK、CK-MB水平与对照组比较，无统计学意义。各组LDH水平比较，均无统计学意义（P＞0.05）。4免疫组化结果：模型组MDA、TNF-α、IL-6、caspase3、Bax、SP较正常组高（P＜0.05），治疗组上述指标低于模型组（P＜0.05），治疗组TNF-α、Caspase-3、Bax、SP较对照组低（P＜0.05）；模型组SOD、bcl-2较正常组低，治疗组SOD、bcl-2较模型组和对照组高（P＜0.05）。5eNOS、iNOS、CaSR免疫组化及荧光定量PCR结果模型组、对照组、治疗组eNOS较正常组低（P＜0.05），对照组、治疗组eNOS较模型组高（P＜0.05），治疗组eNOS较对照组低（P＜0.05）。模型组、对照组、治疗组iNOS、CaSR较正常组高（P＜0.05），对照组、治疗组iNOS、CaSR较模型组低（P＜0.05），治疗组iNOS较对照组高（P＜0.05），治疗组CaSR较对照组低（P＜0.05）。结论：1心绞痛贴膏穴位贴敷预处理能够减轻AMI心肌损伤坏死程度，从病理学角度观察其作用效果与硝酸甘油贴膜外贴预处理效果相当，具体干预机制与以下4方面相关：通过抑制脂质过氧化、促进SOD生成、减少MDA；抑制TNF-α、IL-6具有抗炎作用；抑制CaSR减轻钙超载；上调eNOS的表达、下调iNOS的表达。2心绞痛贴膏在调节NOS方面效果不及硝酸甘油贴膜，在抑制过氧化损伤、抗炎、抑制钙超载方面的效果优于硝酸甘油贴膜。3心绞痛贴膏穴位贴敷预处理可能具有抑制AMI心肌细胞凋亡的作用，其机制与通过抑制casepase3促进bcl-2表达、抑制Bax的表达以及抑制CaSR有关。4心绞痛贴膏具有镇痛作用，其机制可能与抑制P物质有关。第三部分心绞痛贴膏穴位贴敷治疗气虚血瘀型冠心病不稳定性心绞痛的临床疗效观察目的：观察“心绞痛贴膏”治疗冠心病不稳定性心绞痛气虚血瘀型患者的临床疗效。方法：将60例气虚血瘀型冠心病不稳定性心绞痛患者随机分为治疗组和对照组。其中治疗组30例，对照组30例，对照组给予西医常规治疗，治疗组在西医常规治疗基础上给予“心绞痛贴膏”穴位贴敷，治疗10天。观察治疗前后的心绞痛发作次数、持续时间、程度和胸痛、胸闷、心悸、气短、乏力中医症状。结果：治疗组患者心绞痛症状、心绞痛积分、中医症状的有效率与对照组比较，有统计学意义（P＜0.05）。治疗组心绞痛发作次数、疼痛程度疗效均优于对照组（P=0.005P=0.006）；治疗组胸痛、气短、乏力疗效均优于对照组（P=0.028P=0.000P=0.010）。结论：在常规西药治疗基础上使用心绞痛贴膏，能够缓解气虚血瘀型不稳定性心绞痛患者的心绞痛，改善中医症状，特别是对心绞痛发作次数、疼痛程度、胸痛、气短、乏力有明显的缓解作用。本研究的创新点在于总结了治疗冠心病的特色疗法，从基础研究和临床研究两方面验证了心绞痛贴膏穴位贴敷治疗冠心病的有效性，探索了一种用于减轻AMI程度的新方法，并研究了其作用机制。更多还原

【Abstract】 PART I-Academic View of the SupervisorObjective:To discuss the TCM theoretical foundations of Professor Wang Fengrong’s“angina pectoris patch" acupoint stick application in the prevention andcontrol of coronary heart disease (CHD).Methods:The study summarizes the supervisor’s "angina pectoris patch" acupointapplication experience in the treatment of coronary heart disease and analyzesthe feasibility of chest obstruction heartache treatment through “skin” basedon “holistic concept”. It also analyzes the therapy path for acupointapplication on the treatment of chest obstruction heartache taking "acupuncturepoints" and "meridians" from meridian theory as the breakthrough point. The papermines the ancient literature of "Fifty-Two Diseases”,“Medical Origins" and"Theory of Parallel Prose” and traces the historical origins of acupointapplication therapy for coronary heart disease (CHD) and then combines classicalresults of the literature with modern pharmacological research to illustratethat the supervisor’s angina pectoris patch prescription basis and acupointsposition.Conclusion:With the foundational theories of TCM and meridian, the paper proves that the"angina pectoris patch" acupoint application can be used to treat coronary heartdisease (CHD). PART II–Discussion on "angina pectoris patch" acupoint applicationpretreatment on acute myocardial infarction rats and the influence of mechanismObjectives:To discuss the intervention effect of “angina pectoris patch” on acutemyocardial infarction and explore the intervention mechanism of “anginapectoris patch” on acute myocardial infarction from the perspectives of nitricoxide system adjustment and calcium sensitive receptor inhibition, which arebased on the observation of influence of “angina pectoris patch” acupointapplication pretreatment on acute myocardial infarction in the rat myocardialcell morphology, electrocardiogram and myocardial enzymology.Methods:55healthy wistar rats were randomly divided into normal group, model group,control group and treatment group, with10in normal group, and15in each ofthe model group, control group and treatment group. In clean grade laboratory,feeding with ordinary fodder for7days, the control group were given withNitroglycerin Plaster to0.3mg on the area before the heart, each time1stick,1times a day, keep24hours at a time, a total of seven days. Treatment groupwere given angina pectoris patch acupoint application, each patch0.1g, onceper day lasting for24hours, one patch per acupoint,1time per day and7daysin total. Medical modeling started on the4thday causing the model group, controlgroup and treatment group of animals to be acute myocardial infarction animalmodels with the specific method as follows. Between the5thday and the7thday,applying immediately after injection of animals in model group, control groupand treatment group isopropyl adrenaline, adopt the method of multi-pointsubcutaneous injection, isopropyl dosage of epinephrine is5mg/kg, afterepinephrine injection for7days, each live animals with chloral hydrateanesthesia subcutaneously dose (mg/kg), anesthesia after a successful checkelectrocardiogram (ecg), ST segment obvious up or down for building successfulpreliminary judgment standard, abdominal aorta to groups of animals blood, take heart. Lens under the observation of the myocardial pathology morphology andmyocardial ultrastructure were observed under electron microscope. Fullyautomatic biochemical instrument enzymatic detection of Creatine kinaseisoenzyme (CK-MB), Lactate Dehydrogenase (LDH). Immunohistochemical methodtest Malondialdehyde (MDA), Superoxide Dismutase (SOD), Tumor Necrosis factor(TNF-alpha), Interleukin-6(IL-6), B cell lymphoma/leukemia-2B (Bcl-2),Bax, cysteine, aspartic acid proteinase3(Caspase-3), Substance P (SP),endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS)sensitive receptors, calcium (CaSR).Fluorescence quantitative PCR method typeeNOS, iNOS, CaSR.Results:Observed under optical microscope and electron microscope, myocardial cell ofnormal group shows normalshape. Model group myocyte necrosis, interwoven intoa network, horizontal stripes disappear, myocardial cell nucleus and visiblemuscle fibers arranged disorder, fault and loss, myocardial cell nucleuspycnosis, appear blank area, heterochromatin appears crumb, broadeningintercalated disc gap adjacent cells. Necrosis of myocardial cell damage in thecontrol group and treatment group compared with model group, but also a smallfracture and defect and uneven coloring appear blank area. Under the transmissionelectron microscopy (sem): normal group of myocardial cell no change. Myocardialmitochondrial injury model group, double membrane structure is not clear, crestfracture, most are vacuolated, partly dissolved; The nucleus chromatinconcentrated and part of the cytoplasm dissolved and disappeared, and showsliquefactive necrosis; Myofibril extremely flabby, vacuoles, some fracture,myofilament intercalated disc local expansion, structure is not clear, dissolved.Light in the control group and treatment group of mitochondrial damage and doublemembrane structure is clear, part of the crest fracture, the nucleus structurebasic integrity, myofibril arranged neatly, visible myofilament fracture. Model group, western medicine control group and Chinese medicine treatment groupthe absolute value of st-segment up or down significantly higher than normalgroup (P <0.01). Western medicine control group and traditional Chinese medicine(TCM) the absolute value of st-segment up or move down in treatment group werelower than model group, with statistical significance (P <0.01), Chinesemedicine treatment group compared with the western medicine control group,without statistical significance.Control group and treatment group CK level was lower than those of model group,compared with model group, with statistical significance (P <0.01). CK-MBin the control group, treatment group was lower than those of model group,compared with model group, with statistical significance (P <0.05, P <0.01);Level of CK and CK-MB in treatment group compared with control group, withoutstatistical significance. Groups of LDH level comparison, there were nostatistical significance (P>0.05).immunohistochemical results: model group MDA, TNF-α,IL-6, substance P,caspase3, Bax is high in the normal group (P <0.05). the treatment group TNF-α, substance P, caspase3, Bax was lower than those of control group (P <0.05).Model group, control group, treatment group, SOD, the BCL-2was lower thanthose of normal group, the SOD, the Bcl-2higher than the control group, treatmentgroup (P <0.05).ENOS, iNOS，CaSR immunohistochemical results and fluorescent quantitative PCRresults: model group, control group, treatment group of iNOS higher than normalgroup,(P <0.05). The control group, treatment group of iNOS was lower thanthose of model group (P <0.05). INOS treatment group than the control group,2group, with statistical significance (P <0.05). Model group, control group,treatment group of eNOS was lower than those of normal group (P <0.05). ENOSin control group, treatment group compared with model group (P <0.05); ENOStreatment group was lower than those of control group, the comparison of two groups, there is statistical significance (P <0.05).model group, control groupand treatment group CaSR higher than normal group, with statistical significance(P <0.05). CaSR in control group, treatment group was lower than those of modelgroup (P <0.05); CaSR treatment group was lower than those of control group,the comparison of two groups, there is statistical significance (P <0.05).Conclusion:1angina patch of acupoint sticking pretreatment can reduce AMI necrosis ofmyocardial damage degree, its effect and the effect of nitroglycerin patchattached with pretreatment, the specific intervention mechanism related to thefollowing four aspects: by inhibiting lipid peroxidation, promoting SODgenerated, reduce the MDA; Suppress TNF alpha, IL-6, have anti-inflammatoryeffects; Inhibition of CaSR reduce calcium overload; Increase the expressionof eNOS, cut the expression of iNOS.2angina pectoris patch in regulating NOS aspects effect is not good asNitroglycerin Plaster, in inhibiting peroxidation damage, anti-inflammatory,inhibiting calcium overload effect superior to Nitroglycerin Plaster.3angina pectoris patch acupoint pretreatment may inhibit myocardial cellapoptosis after AMI. The mechanism is inhibited casepase3, promoting bcl-2expression and inhibit the expression of Bax and inhibition of CaSR.4anginapectoris patch has analgesic action, its mechanism may be related to inhibitingsubstance P.PART III-Clinical Curative Effect Observation of "angina pectoris patch"acupoint application therapy in treatment of both Qi deficiency and blood stasissyndrome of coronary heart disease (CHD) unstable angina pectorisObjective:To observe the clinical treatment effect of “angina pectoris patch” on coronaryheart disease (CHD) unstable angina patients with Qi deficiency and blood stasis. Methods:60patients of Qi deficiency and blood stasis syndrome of coronary heart diseaseunstable angina patients, according to random number table method, were randomlydivided into treatment group and control group, with30patients in each group.Control group were given conventional western medicine treatment and thetreatment group, were given "angina pectoris patch" acupoint applicationtreatment for10days, on the basis of the conventional western medicinetreatment. Angina pectoris attack frequency, duration, numbers of pains and TCMsymptoms such as chest pain, chest tightness, palpitations, shortness of breath,fatigue, constipation.Results:Angina pectoris symptoms, Chinese medicine treatment group effective comparedwith control group, was statistical significant (P <0.05). Attack frequencyof angina pectoris, pain degree of curative effect of treatment group weresuperior to control group (P=0.005, P=0.006); Chest pain, shortness of breath,fatigue effect of treatment group were superior to control group (P=0.028,P=0.000, P=0.010).Conclusion:On the basis of conventional western medicine treatment, angina pectoris patch,can ease the qi deficiency blood stasis type of angina, unstable angina patientsimprove TCM symptoms, especially to attack frequency of angina pectoris, andthe degree of pain, chest pain, shortness of breath, fatigue has obvious effect.更多还原