冠心舒通胶囊对大鼠心肌缺血再灌注损伤细胞凋亡影响的实验研究
摘要
研究背景：
随着科学技术的进步，临床诊疗水平的提升，先进医学治疗方法冠状动脉再通术（如冠状动脉介入术、药物溶栓及冠状动脉旁路移植术等）在临床中的成熟应用，使缺血受损心肌能够得到及时再通，显著降低了急性心血管不良事件的致残率与病死率。对缺血心肌进行早期再灌注治疗的益处是不可否定的，不但可使缺血的心肌再次获得生机，将梗死面积降至最小，在临床中若冠状动脉闭塞一定时间后血管再得到灌注，心肌细胞则有一定程度再灌注损伤。部分循证医学资料提示，在心肌梗死后4小时对血管进行再通有时则收获不到预期效果，反而促进了心肌细胞的死亡。多年来学者们已经逐步注意到心肌缺血再灌注时所引发的一系列不良的瀑布式连锁性事件。心肌缺血再灌注损伤是冠状动脉再通后的严重并发症，在心肌缺血再灌注过程中容易导致心肌顿抑、冠状动脉无复流、再灌注性心律失常以及心肌细胞死亡等形式的心肌损伤性改变。目前随着心血管分子生物学研究的深入，已经逐步证实心肌缺血再灌注损伤与心肌细胞凋亡之间的密切关系，在缺血再灌注损伤过程中存在细胞凋亡参与，缺血再灌注损伤能够加速不可逆转性细胞凋亡，且心肌细胞凋亡数量与缺血再灌注损伤持续时间的长短有一定关系。因此可以说心肌缺血再灌注损伤是影响急性冠脉病变预后的不良事件。临床对心肌缺血再灌注损伤时细胞凋亡药物干预是目前研究的焦点。
祖国中医药对延续中华民族的千年文明起到了不可取代的作用，不但保证了广大劳动人民的健康与长寿，更为医学界的种种突破发挥着不可磨灭的贡献。中药针对疾病标本同治，且副作用小、纯天然，是我国劳动人民几千年探索的结晶，适合国人的体质，适合国人养生保健与治病强身。诸多临床疾病在西药无计可施之时，学者们也将研究重点转向于中药制剂。分子生物学的进展推动着中药成分及药理机制的研究，单味药物所含成分比较有限，中药复方其所含化学成分比较复杂、药理作用具有多层次、多靶点、多指向、多覆盖的特点，针对临床错综复杂的疾病，中药复方体现出其巨大的临床疗效价值。
步长冠心舒通胶囊是咸阳步长制药有限公司研制、开发、推广的国家三类新蒙药，是传统中医药理论知识、临床实践与蒙医蒙药特有理论相结合开发而成。冠心舒通胶囊主要由广枣、丹参、丁香、冰片、天竺黄等药物组成的中药复方制剂，具有活血化瘀、通经活络、行气止痛功能，主要用于治疗胸痹心痛之心血瘀阻证，症见：胸痛、胸闷、心慌、气短；冠心病、心绞痛见上述症候者。广枣为君药具有行气活血、养心、安神之功，用于气滞血瘀、胸痹作痛、心悸气短、心神不安之症；丹参为臣药具有活血调经、祛瘀止痛、凉血消痈、清心除烦、养血安神之效，临床用于治疗月经不调、经闭痛经、症瘕积聚、疮疡肿痛、热痹疼痛、胸腹刺痛、心烦不眠、心绞痛等病；丁香为佐药具有温中降逆、补肾助阳之功，临床用于心腹冷痛、脾胃虚寒、呃逆呕吐、食少吐泻、肾虚阳痿之症；冰片为佐药具有清香宣散、开窍醒神、清热散毒、明目退翳的功效，临床主治热病高热神昏、中风痰厥惊痫、暑湿蒙蔽清窍、喉痹耳聋、口疮齿肿、疮痈疳痔、目赤肿痛、翳膜遮睛。天竺黄为使药，具有清热豁痰、凉心定惊之功用，主治热病神昏谵妄，中风痰迷不语，小儿惊风抽搐，癫痫，痰热咳嗽。在多年临床应用过程中发现冠心舒通胶囊对抑制心肌缺血再灌注损伤细胞凋亡有一定的作用。冠心舒通胶囊的以往研究，均以临床疗效观察或用药经验总结为主，缺乏系统的疗效机制研究，为了明确冠心舒通胶囊对抑制心肌缺血再灌注损伤细胞凋亡作用机理，为该中药复方防治冠心病、改善再灌注损伤心肌功能，降低临床致残率、死亡率，提升患者生活质量，特进行本项研究以期为临床进一步应用提供可靠的理论依据和实验数据。
目的：
探讨冠心舒通胶囊对大鼠心肌缺血再灌注（I/R）损伤细胞凋亡的影响。材料与方法：
1实验动物的饲养
1.1分组
随机将30只SD级大鼠平均分成3个组别，即假手术组（Sham）、模型组(Model)及冠心舒通胶囊组(Drug)。
1.2饲养及处理
室内温度维持在18-22摄氏度，湿度维持在（55±2）%。假手术组10只大鼠，给予生理盐水灌胃，每日2次，连续灌服5日；模型组10只大鼠，建立心肌缺血再灌注模型前，给予生理盐水灌胃，每日2次，连续灌服5日；冠心舒通胶囊组10只大鼠，建立心肌缺血再灌注模型前，给予冠心舒通胶囊灌胃，每次按1.5g/Kg，溶于2ml生理盐水中，每日2次灌胃，连续灌服5日。
2实验指标测定：
2.2.1心肌缺血再灌注模型的制备
术前对大鼠行心电图检查并记录Ⅱ导联心电图，对心电图异常的大鼠予以剔除。选用浓度为30g/L戊巴比妥钠腹腔注射对大鼠进行麻醉，用量为1.5ml/kg，大鼠麻醉后进行固定，连接ALC-V型号动物呼吸机，同时记录心电图。随后沿大鼠左锁骨中线纵行剪开胸部皮肤约2厘米，选用5号针线结扎大鼠左冠状动脉的前降支，大鼠心肌梗死模型制备成功的心电图为ST段弓背向上抬高。当每组大鼠在心肌梗死30分钟后，松开结扎线，进行再灌注3小时。假手术组中大鼠只做穿线而不结扎左前降支处理。
2.2.2实验取材
2.2.2.1摘取大鼠的心脏，使用冰生理盐水进行清洗，摘除血管、脂肪等多余的组织。应用4%的多聚甲醛对大鼠心肌进行固定4小时，再用30%的蔗糖对心肌组织进行脱水（4摄氏度过夜）。
2.2.2.2剪下结扎线以下的左心室心肌组织，剪碎后置于玻璃研磨器中，加入蛋白裂解液后进行研磨，静置30分钟。将左心室匀浆于4摄氏度的离心机中，进行时速3000转/分钟，连续离心10分钟，抽取上清液后，再次进行离心，选用12000转/分钟，连续离心30分钟，再次取其上清液，每20微升分装，并在-80摄氏度的冰箱中保存备用。
2.2.3观察指标
2.2.3.1心肌细胞凋亡指数的检测
在每一个标本中各取已制备的切片1张，应用末端脱氧核苷酸转移酶所介导dUTP缺口末端标记法进行标记心肌凋亡细胞核中DNA3-OH的末端，并采用原位荧光法对凋亡细胞进行标记与显示。在荧光显微镜高倍（×200）视野下观察每张切片标本，在心肌梗死区域随机地选择4个视野，并计算凋亡的细胞指数（心肌细胞的凋亡指数%=镜下凋亡心肌细胞总数/镜下总细胞数×100%）。
2.2.3.2Western Blot检测Bcl-2、Bax基因蛋白的表达
从-80摄氏度的冰箱中将心肌匀浆蛋白取出后，对浆液进行复温，蛋白上清液经过SDS-聚丙烯酰胺凝聚电泳作用下转移至硝酸纤维素膜上，用10%的脱脂奶粉TBST液进行封闭1小时，加入一抗（比例：1:1000）（兔抗鼠bcl-2,bax），在室温下进行孵育2小时后，应用TBST液进行3次洗涤，加入二抗在室温条件下孵育2小时，3次洗涤，经过ECL试剂盒后显色，再对其采用凝聚成像系统进行扫描处理，显色后的结果采用Quality one软件对灰度进行分析，观察并计算蛋白相对的表达水平。
结果：
1.心肌细胞凋亡指数
在200倍的荧光显微镜显示下，在大鼠心肌梗死区域呈现绿色荧光的地带为心肌凋亡细胞主要的分布区域，其中药物干预组大鼠的凋亡细胞数目显著少于模型组，二者统计学具有显著的统计学差异（p<0.01）。
2.基因蛋白Bcl-2与Bax的表达水平
与假手术组大鼠比较，模型组大鼠基因蛋白Bax表达增强；基因蛋白Bcl-2表达明显减弱，二者具有显著的统计学差异(P 讨论：
机体组织出现缺血后，对缺血组织进行再灌注治疗后，多种因素导致缺血的组织细胞出现代谢功能障碍及结构破坏，从而使组织损害程度进一步加重。人体的许多组织都存在缺血再灌注损伤的现象，尤其是在大脑及心脏。心肌缺血再灌注损伤是多种因素参加的极其复杂的环节，就目前研究表明较为认同的再灌注损伤机制为：氧自由基产生、钙离子超载、心肌细胞能量代谢障碍、炎症细胞激活、内皮细胞功能障碍以及心肌细胞发生凋亡等。心肌缺血再灌注损伤发生过程中心肌细胞的过度凋亡是再一次导致心肌损伤的重要原因，心肌细胞发生凋亡作为缺血再灌注损伤的早期事件，贯穿于缺血再灌注损伤的病理生理过程。
细胞凋亡是受基因调控的一种程序性的细胞死亡，参与心肌细胞凋亡的主要调控基因有Bc1-2、p21、Fas、p53、热休克蛋白及caspase家族蛋白酶。根据调控基因的不同功能可以分为三个种类： Bc1-2、lAP、EIB等为具有抑制凋亡功能的基因；Fas、p53、Bax等为具有促凋亡功能的基因；C-myc、Bcl家族等为具有双向调控功能的基因。目前研究中，受关注程度最多、功能较明确的具有抑制凋亡功能的基因为Bc1-2，促进细胞凋亡的基因为Bax。基因蛋白Bcl-2及Bax是Bcl-2基因蛋白家族中作用互为对立的重要的调控细胞凋亡基因，Bcl-2对细胞凋亡起抑制作用，Bcl-2高度表达对于线粒体内钙离子稳态起到了维持作用，对线粒体膜电位下降起到阻止作用，并抑制线粒体膜通透性转换孔开放，抑制促进凋亡蛋白质如凋亡诱导因子、细胞色素C的释放，从而防止细胞发生凋亡。Bcl-2还能够与Apaf-1及Caspase9进行结合，并维持Bcl-2的非活化状态，阻断Caspase的级联反应，从而抑制细胞的凋亡。基因蛋白Bax对细胞的凋亡起到促进作用。部分研究结果尚提示，调节心肌细胞凋亡的基因水平不仅仅取决于基因蛋白Bcl-2及Bax自身水平的表达高低，还与基因蛋白Bcl-2及Bax水平比值有关，基因蛋白Bcl-2及Bax的比值反映了细胞出现凋亡的程度。
冠心舒通胶囊以广枣、丹参、丁香、冰片、天竺黄等中药药材以一定的比例所组成，是在现代中医药理论与蒙医蒙药理论相互贯通结合的基础上开发研制所成，冠心舒通胶囊有活血化瘀、通经活络、行气止痛的功效。中药广枣及丹参的药理作用均有多种，例如对实验动物的外周微循环具有改善作用、对炎症反应起到抑制作用以及抗氧化作用等。本实验研究主要通过用药后制备心肌缺血再灌注损伤的模型后，检测大鼠心肌细胞的凋亡指数与基因蛋白Bcl-2及Bax的表达水平，实验结果表明，与假手术组大鼠相比较，模型组大鼠基因蛋白Bax的表达增强，基因蛋白Bcl-2的表达明显降低；与模型组大鼠相比，冠心舒通胶囊组大鼠，心肌细胞凋亡指数、基因蛋白Bax的表达明显下降，基因蛋白Bcl-2的表达明显上调。本实验研究最终证实，冠心舒通胶囊能够明显降低缺血再灌注损伤的心肌细胞发生凋亡，从而表明其具有抗心肌缺血再灌注损伤的作用。
结论：
冠心舒通胶囊可以通过抑制心肌细胞的凋亡，下调基因蛋白Bax表达，上调基因蛋白Bcl-2表达，在心肌缺血再灌注损伤时起到保护心肌细胞的作用。
Background：
With the progress of science and technology,and the ascension of diagnosisand treatment,advanced medical treatment method of coronary arteryrecanalization (such as percutaneous coronary intervention, thrombolysis,coronary artery bypass grafting) is being applicated in clinical perfectly,whichcan supply the blood to the ischemic damaged heart promptly. This technologycan lessen the morbidity and mortality remarkablely of acute cardiovascularadverse events. The benefit of reperfusion therapy for the ischemic myocardialcells is above rubies,which not only can make the ischemic myocardial cellsacquire vitality once again,but also can reduce infarction area of the heart.Whencoronary artery occlusion of blood vessels was reperfused after some time inthe clinical, myocardial celles suffer the reperfusion injury a certain extent.Refer to the evidence-based medicine,repassing the occluded coronary arterycan not harvest the desired effect after4hours of myocardial infarction. Thetreatment promotes the myocardial cell to death.In recent years, many medicinalscholars have observed the reperfusion injury when myocardial ischemia happenswhich can arouse a series of bad waterfall chain event.Myocardial ischemia-reperfusion injury is one of the complications after coronary arteryrecanalizated,and it is easy to cause myocardial stunning, NO-reflow,reperfusion arrhythmia, cardiac cell death and so on in the process of myocardialischemia reperfusion. With the development of cardiovascular progress in thestudy of molecular biology,we have already step by step confirmed that thereis a close relationship between myocardial Ischemia-reperfusion injury andmyocardial apoptosis at present.Myocardial apoptosis is in the process ofmyocardial Ischemia-reperfusion injury which can accelerate irreversible cell apoptosis,and the number of myocardial apoptosis cells is correlated with thelength of duration of ischemia reperfusion injury.Therefore myocardial Ischemia-reperfusion is the adverse event of affect prognosis of acute coronary arterylesions.Clinical drug intervening myocardial apoptosis is the focus of theresearch。
Traditional Chinese Medicine plays an irreplaceable role in continuationthousands years civilization of the Chinese nation.TCM not only guarantees themasses of working people’s health and longevity,but also plays an indeliblecontribution to the breakthroughs in medical science.Traditional Chinesemedicine which is from all natural can healing disease pathogenesis,and thereis a small side effect.The TCM which is suitable for people's physicalfitness,life cultivation and health preservation, curing diseases and improvinghealth is the crystallization of the working people for thousands of years ofour country.Many clinical diseases donot have therapeutic method of westernmedicine and western medicine.Medical scholars translate their focus on Chinesemateria medica preparation.The process of molecular biology promotes theresearch of Chinese native medicine ingredient and pharmacological mechanism.Single herb is limited on sudocrem.Chinese herbal compound’s chemical componentis very complex and pharmacologic action has the characteristic of multilevel,multiple target point, multiway and comprehensive.Facing the clinicalcomplicated diseases, Chinese herbal compound embodies enormous worth ofcurative effect.
Guan Xin Shu Tong capsule which is prepared, developed, rolled out andpopularized is the three kinds of new Mongolian drug of China in the world.GuanXin Shu Tong capsule is united Traditional Chinese Medicine theory, clinicalpractice and characteristic theory of Traditional Mongolian Medicine.Guan XinShu Tong capsule is a compound Chinese medicine which is made up of fructuschoerospondiatis,the root of red-rooted salvia, clove,borneol andtabasheer.Guan Xin Shu Tong capsule possesses the effects of promoting bloodcirculation to remove blood stasis, clearing and activating the channels and collaterals, promoting qi circulation to relieve pain,which is used to cure thesyndrome of blood stasis in heart.The syndrome contains stethalgia, suppressionin the chest, be nervous, be short of breathof coronary heart disease andangina.Fructus choerospondiatis is the monarch herb which has the effect ofpromoting qi to activate blood, nourishing heart and soothing the nerves,andcan cure the symptom of qi depression to blood stasis,obstruction of pain,heartpalpitations shortness of breath and malaise.The root of red-rooted salviais the ministerial herb which has the effect of promoting blood circulation torestore menstrual flow,removing stasis pain,healing carbuncle, clearing awaythe heart-fire and relieving restlessness and nourishing the blood andtranquilization, and can cure the symptom of abnormal menstruation,amenorrheadysmenorrhea, concretions and gatherings, ulcers sore, hot bi pain,chest pain,upset and nosleeping, angina and so on.Clove is the adjuvant herb which has theeffect of warming middle energizer descend adverse-rising, reinforcing kidneyto strengthen yang,and can cure the symptom of trusted subordinate crymodynia,deficiency-cold in spleen and stomach,hiccups vomiting, eat less and vomitingand diarrhoea,kidney deficiency impotence.Borneol is the adjuvant herb whichhas the effect of faint scent scattering, inducing resuscitation, clearing heatand diffusing poison and removing nebula, and can cure the symptom of high fevercoma, stroke sputum jue chronic ulcerated,dusting confusion by summer-heatand wet, pharyngitis and deaf, aphtha and tooth swollening, carbuncle,sore redswollen eyes, film covering eyes.Tabasheer is the conductant herb which has theeffect of clearing heat and eliminating phlegm,cold heart to calm the frightened,and can cure the symptom of god faint delirium,stroke sputum fan not language,pediatric epilepsy,epilepsia, sputum and heat cough.In the process of many yearsclinical application,we found that Guan Xin Shu Tong capsule can restrainapoptosis in myocardial ischemia-reperfusion injury.Scholars have researchedthe Guan Xin Shu Tong capsule only on clinical curative effcet observationempirical using summary in previous studies which are short of researching ofcurative effect mechanism system.For the sake of expliciting the mechanism of action of Guan Xin Shu Tong capsule restraining apoptosis in myocardial ischemia-reperfusion injury, reducing the clinical morbidity and mortality,enhancingthe level of patients’life,we proceed this research to provide reliable theorybasis and empirical data.
Purpose：
To function the effect of Guan Xin Shu Tong capsule on apoptosis in myocardialischemia-reperfusion injury in rats.
Material and method：
1.Laboratory animal breeding husbandry
1.1Grouping
Divided average averagely the30SD grade rats into3groups randomly.Theyare the sham group, the model group and the drug group.
1.2Breeding
The rats were breed under the condition of daily.The indoor temperature wasmaintained between18and22centigrade.The humidity was dominated at（55±2）percent.All the rats can drink water with freedom.All the rats were taken inthe solar radiation12hours.The ten rats in the sham group were offered thenormal saline by gavage,2times a day, which was continuous for5days byirrigation.The ten rats in the model group were offered the normal saline bygavage,2times a day, which was continuous for5days by irrigation,before themodel of myocardial ischemia reperfusion was established.The ten rats in thedrug group which was controled by Guan Xin Shu Tong capsule were offered GuanXin Shu Tong capsules by gavage, before the model of myocardial ischemiareperfusion was established.The Guan Xin Shu Tong capsule dose was1.5gram perkilogram which was dissolved in normal saline, every time to be6ml per kilogramby lavage,2times a day, drenched for5days consecutively.
2. Experimental target determination
2.1Preparating the model of myocardial ischemia reperfusion
All the rats were fasted for12hours before the preparation of model,but the rats can be free to drink.Preoperative ECG examination of all the rats andrecording the electrocardiogram of lead II.Eliminate the rats which the Ecg wereabnormal.All the rats were anesthetized by pentobarbital sodium whoseconcentration was30gram per litre.All the rats were used medicinebyintraperitoneal injection.The dose was1.5milliliter per kilogram.Fixed therats which were the state after anesthesia,and were for endotrachealintubation.Animal Ventilator model number is ALC-V which was used to providethe rats to breath.The rats’s breathing rate was set to50to60times per minute,continuing to record electrocardiogram in rats at the same time.The rats’ leftmid-clavicular line were cut open the chest skin about2centimeter.The rats’left anterior descending coronary artery were ligated by surgery line.The modelof myocardial infarction were success when ST segment of electro cardiogramarchupward.When the rats in each group were at the state of myocardial infarctionfor30minutes,the rats were loosen the ligature and cured by reperfusion therapyfor3hours.The rats in the sham group were given the same treatment,but therats were only did the thread without ligation of left anterior descendingbranch.
2.2Draw materials
2.2.1When the rats in each group were at the state of myocardial infarctionfor30minutes,the rats were cured by reperfusion therapy for3hours.the rats’hearts were taken which were washed by ice normal saline and removed blood vessels,fat and other redundant organization.The myocardium in rats was fixed on4hoursby application of4%parafor maldehyde,and the myocardium in rats was sucrosedin the application of30%on myocardial tissue dehydration (4centigrade forthe night).
2.2.2The left ventricular myocardial tissue was cut out following ligation,and put in glass grinding machine after cuting up.The tissue was grinded afterAdding protein pyrolysis liquid,and standed for30minutes calmly.Leftventricular homogenate was put in the centrifuge in4degrees Celsius,and wasto be carried on the speed of3000round per minute for10minutes continuously.The supernatant fluid was extracted and was centrifugal again onthe speed of12000round per minute for30minutes continuously.Then thesupernatant fluid was choosed, and shipmented every20microlitre partially.andsaved in the refrigerator in-80degrees Celsius.
2.3Observation target
2.3.1Myocardial apoptosis index
Only1section card was chosen from in each prepared specimen.The terminalof DNA3-OH in the myocardial apoptotic nuclei was signed by the way of terminaldeoxyribonucleotide transferase mediated dUTP Nick end labeling.The apoptoticcells were signed and displayed by fluorescence in situ tag.Apoptotic cell countanalysis: according to Nick end labeling kits related reaction after operationinstructions, the apoptosis of myocardial cells were in the greenfluorescence.All of the nucleated cells present blue fluorescence after colouredby Desktop Application Programming Interface (DAPI).Each biopsy specimens wasobserved under fluorescence microscope (200) at high magnification view.Inmyocardial infarction area four horizons are randomly selected.The number oftotal number of cells and apoptosis cell number were counted in Each themicroscopic view.The index of the apoptotic cells was calculated by formula ofMyocardial cell apoptosis index=Total number of the apoptosis of myocardialcells/Microscopically the total cell number×100%.
2.3.2Detecting the expression of the pretein gene of Bcl-2and Bax by the wayof Western Blot.
Myocardial homogenate protein was taken out from the refrigerator in-80degrees Celsius and was recovered to the room temperature.Protein in thesupernatant was removed to the nitrocellulose blotting membranes by the way ofSDS-condensation of polyacrylamide electrophoresis,and was closed for1hourby10%skimmed milk powder Tris Buffered Saline Tween (TBST) fluid.The firstresistance was Added in the fluid which the proportion was1vs1000. Afterincubation for2hours at room temperature,the protein in the supernatant wadwashed3times by the Tris Buffered Saline Tween fluid.The second resistance was Added in the fluid which was marked by horseradish peroxidase in the roomtemperatureThe fluid was colored after ECL reagent kit,which was in the condensedimaging system adopted to improve the scanning and used Quality one softwareto analyze the gray level,and observatiing and calculating the relativeexpression level of the protein.
Results：
1.Myocardial apoptosis index
Under the fluorescence microscope show that two hundred times，in themyocardial infarction area of rats the primary distribution area of apoptosisof myocardial cells presents green fluorescence strip.The results shows thatthe numbers of the apoptosis cell was significantly reduced of the rats in thegroup intervened by drugs than the rats in model group.There is a obviousdifference between the rats in the group intervened by drugs and the rats inmodel group(p<0.01).
2.Expression level of the gene proteins of Bcl-2and Bax
Compared with the rats in the control group,the enhanced expression of thegene protein of Bax is remarkable, and the expression decreased of the geneprotein of Bcl-2is evident. There is a obvious difference between the rats inthe control group and the rats in model group(p<0.01). Compared with the ratsin the model group,the rats in the group intervened by Guan Xin Shu Tong capsuleshave an enhanced expression of the gene protein of Bax,and there is a obviousdifference between the rats in the model group and the rats in druggroup(p<0.01).Compared with the rats in the model group,the rats in the groupintervened by Guan Xin Shu Tong capsules have a decreased expression of the geneprotein of Bcl-2,which has a difference between the rats in the model group andthe rats in drug group(p<0.05).
Discussion
The body tissues occurred ischemia which were cured by reperfusion turn upmetabolic dysfunction and structural damage of the tissue cells by a number of factors.This pathological process aggravated the extent the tissue damage.Anumber of the organizations in the body exist the phenomenon of the ischemiareperfusion injury. This kind of situation is consist in the brain and heart.Myocardial Ischemia-reperfusion injury is a vital and complicated segment whichis in a variety of factors. The current research suggests that the ischemiareperfusion injury mechanism which most people agree oxygen free radicalsproduced, intracellular calcium overload, myocardial cells energy metabolismdisordered, activation of inflammatory cells, Endothelial cell dysfunction,myocardial cell apoptosis, and so on.In the process of myocardial ischemia-reperfusion injury, the excessive apoptosis of myocardial cells results in thesignificant cause the myocardial cells damage.The cells of cardiac turning upapoptosis is the early event in the course of ischemia reperfusion injuryprogression.This event throughouts the pathology and physiology process ofischemia-reperfusion injury.
The cell apoptosis which is regulated by the genes is a kind of programmedcell death.The main control genes which participates in the process of myocardialcell apoptosis include the regulatory gene of Bc1-2, the regulatory gene of p21,the regulatory gene of Fas, the regulatory gene of p53,the heat shock protein,the caspase family of protease,and so on. According to the different functionof regulating the genes can be divided into three categories.The regulatory geneof Bc1-2,the regulatory gene of lAP and the regulatory gene of EIB are providedwith function of inhibition cell apoptosis. The regulatory gene of Fas, theregulatory gene of p53and the regulatory gene of Bax are provided with functionof promoting cell apoptosis.The regulatory gene family of C-myc and theregulatory gene family of Bcl have the function of promoting cell apoptosis andinhibition cell apoptosis.In the present study,the regulatory genes of mostconcern and the functions relatively clear is the gene of Bc1-2,which has thefunction of inhibition cell apoptosis,and the gene of Bax which has the functionof promoting cell apoptosis.The regulatory gene of Bcl-2and the regulatory geneof Bax are the most important regulatory genes in the protein gene family. The regulatory gene of Bcl-2and the regulatory gene of Bax have a relation withmutually opposite effects.The regulatory gene of Bcl-2highly expressed has thefunction of inhibition cell apoptosis,which has the act of maintaining calciumof steady state in the mitochondria,holding back the membrane potential declineof mitochondria, inhibition of mitochondrial membrane permeability transitionpore opening,and inhibition of promoting apoptosis proteins such as the factorapoptosis inducing, the release of cytochrome C, so as to prevent the cellapoptosis.The regulatory gene of Bcl-2also has the fuctions of combining withon the gene of Apaf-1and Caspase9, maintaining the non-antiactive state ofBcl-2and blocking the cascade reaction of the family pretein of Caspase.Generally speaking,the regulatory gene of Bcl-2also has the fuctions of theprocess of inhibit cell apoptosis.The gene protein of Bax has the funtion ofpushing forward myocardial cell apoptosis.Part of the research results indicatethat the gene level of regulation of myocardial cell apoptosis not only dependson the protein gene level expression of Bcl-2and Bax itself,but also the ratioof the levels of gene protein Bcl-2and Bax.The ratio of the levels of geneprotein Bcl-2and Bax reflects the degree of the appearance of cell apoptosis.
Guan Xin Shu Tong capsule is made up of fructus choerospondiatis,the rootof red-rooted salvia, clove,borneol and tabasheer,which is the product ofmutually penetrating combination of the modern theory of Traditional ChineseMedicine and Mongolian medicine and theory.Guan Xin Shu Tong capsule has theeffects of promoting blood circulation to remove blood stasis, clearing andactivating the channels and collaterals, promoting qi circulation to relievepain,which is used to cure the syndrome of blood stasis in heart.The herbalmedicine of fructus choerospondiatis and the root of red-rooted salvia havea variety of pharmacological effects,such as improving the appearance of theperipheral cycle of experimental animals,possessing the funtion of inhibitingthe inflammatory response, having an antioxidant effect.The experiment researchmainly detecte the apoptosis index of rat myocardial cells and the expressionlevel of the protein gene Bcl-2and Bax through preparation of the model of myocardial ischemia-reperfusion injury after the medication
The experimental result shows that compared with the rats in the controlgroup,the enhanced expression of the gene protein of Bax is remarkable, and theexpression decreased of the gene protein of Bcl-2is evident.Compared with therats in the model group,the rats in the group intervened by Guan Xin Shu Tongcapsules have an enhanced expression of the gene protein of Bax and have adecreased expression of the gene protein of Bcl-2.This experimental studyconfirmes that Guan Xin Shu Tong capsule can obviously decrease ischemia-reperfusion injury of myocardial cell apoptosis occurred finally.so thisexperimentalshows that Guan Xin Shu Tong capsule has the role ofwithstandingredients myocardial ischemia-reperfusion injury.
Conclusion：
Guan Xin Shu Tong capsule has the fuction of protecting myocardial cellsby restraining myocardial cell apoptosis, increasing the expression of proteingene Bcl-2and weakening the expression of protein gene Bax.
随着科学技术的进步，临床诊疗水平的提升，先进医学治疗方法冠状动脉再通术（如冠状动脉介入术、药物溶栓及冠状动脉旁路移植术等）在临床中的成熟应用，使缺血受损心肌能够得到及时再通，显著降低了急性心血管不良事件的致残率与病死率。对缺血心肌进行早期再灌注治疗的益处是不可否定的，不但可使缺血的心肌再次获得生机，将梗死面积降至最小，在临床中若冠状动脉闭塞一定时间后血管再得到灌注，心肌细胞则有一定程度再灌注损伤。部分循证医学资料提示，在心肌梗死后4小时对血管进行再通有时则收获不到预期效果，反而促进了心肌细胞的死亡。多年来学者们已经逐步注意到心肌缺血再灌注时所引发的一系列不良的瀑布式连锁性事件。心肌缺血再灌注损伤是冠状动脉再通后的严重并发症，在心肌缺血再灌注过程中容易导致心肌顿抑、冠状动脉无复流、再灌注性心律失常以及心肌细胞死亡等形式的心肌损伤性改变。目前随着心血管分子生物学研究的深入，已经逐步证实心肌缺血再灌注损伤与心肌细胞凋亡之间的密切关系，在缺血再灌注损伤过程中存在细胞凋亡参与，缺血再灌注损伤能够加速不可逆转性细胞凋亡，且心肌细胞凋亡数量与缺血再灌注损伤持续时间的长短有一定关系。因此可以说心肌缺血再灌注损伤是影响急性冠脉病变预后的不良事件。临床对心肌缺血再灌注损伤时细胞凋亡药物干预是目前研究的焦点。
祖国中医药对延续中华民族的千年文明起到了不可取代的作用，不但保证了广大劳动人民的健康与长寿，更为医学界的种种突破发挥着不可磨灭的贡献。中药针对疾病标本同治，且副作用小、纯天然，是我国劳动人民几千年探索的结晶，适合国人的体质，适合国人养生保健与治病强身。诸多临床疾病在西药无计可施之时，学者们也将研究重点转向于中药制剂。分子生物学的进展推动着中药成分及药理机制的研究，单味药物所含成分比较有限，中药复方其所含化学成分比较复杂、药理作用具有多层次、多靶点、多指向、多覆盖的特点，针对临床错综复杂的疾病，中药复方体现出其巨大的临床疗效价值。
步长冠心舒通胶囊是咸阳步长制药有限公司研制、开发、推广的国家三类新蒙药，是传统中医药理论知识、临床实践与蒙医蒙药特有理论相结合开发而成。冠心舒通胶囊主要由广枣、丹参、丁香、冰片、天竺黄等药物组成的中药复方制剂，具有活血化瘀、通经活络、行气止痛功能，主要用于治疗胸痹心痛之心血瘀阻证，症见：胸痛、胸闷、心慌、气短；冠心病、心绞痛见上述症候者。广枣为君药具有行气活血、养心、安神之功，用于气滞血瘀、胸痹作痛、心悸气短、心神不安之症；丹参为臣药具有活血调经、祛瘀止痛、凉血消痈、清心除烦、养血安神之效，临床用于治疗月经不调、经闭痛经、症瘕积聚、疮疡肿痛、热痹疼痛、胸腹刺痛、心烦不眠、心绞痛等病；丁香为佐药具有温中降逆、补肾助阳之功，临床用于心腹冷痛、脾胃虚寒、呃逆呕吐、食少吐泻、肾虚阳痿之症；冰片为佐药具有清香宣散、开窍醒神、清热散毒、明目退翳的功效，临床主治热病高热神昏、中风痰厥惊痫、暑湿蒙蔽清窍、喉痹耳聋、口疮齿肿、疮痈疳痔、目赤肿痛、翳膜遮睛。天竺黄为使药，具有清热豁痰、凉心定惊之功用，主治热病神昏谵妄，中风痰迷不语，小儿惊风抽搐，癫痫，痰热咳嗽。在多年临床应用过程中发现冠心舒通胶囊对抑制心肌缺血再灌注损伤细胞凋亡有一定的作用。冠心舒通胶囊的以往研究，均以临床疗效观察或用药经验总结为主，缺乏系统的疗效机制研究，为了明确冠心舒通胶囊对抑制心肌缺血再灌注损伤细胞凋亡作用机理，为该中药复方防治冠心病、改善再灌注损伤心肌功能，降低临床致残率、死亡率，提升患者生活质量，特进行本项研究以期为临床进一步应用提供可靠的理论依据和实验数据。
目的：
探讨冠心舒通胶囊对大鼠心肌缺血再灌注（I/R）损伤细胞凋亡的影响。材料与方法：
1实验动物的饲养
1.1分组
随机将30只SD级大鼠平均分成3个组别，即假手术组（Sham）、模型组(Model)及冠心舒通胶囊组(Drug)。
1.2饲养及处理
室内温度维持在18-22摄氏度，湿度维持在（55±2）%。假手术组10只大鼠，给予生理盐水灌胃，每日2次，连续灌服5日；模型组10只大鼠，建立心肌缺血再灌注模型前，给予生理盐水灌胃，每日2次，连续灌服5日；冠心舒通胶囊组10只大鼠，建立心肌缺血再灌注模型前，给予冠心舒通胶囊灌胃，每次按1.5g/Kg，溶于2ml生理盐水中，每日2次灌胃，连续灌服5日。
2实验指标测定：
2.2.1心肌缺血再灌注模型的制备
术前对大鼠行心电图检查并记录Ⅱ导联心电图，对心电图异常的大鼠予以剔除。选用浓度为30g/L戊巴比妥钠腹腔注射对大鼠进行麻醉，用量为1.5ml/kg，大鼠麻醉后进行固定，连接ALC-V型号动物呼吸机，同时记录心电图。随后沿大鼠左锁骨中线纵行剪开胸部皮肤约2厘米，选用5号针线结扎大鼠左冠状动脉的前降支，大鼠心肌梗死模型制备成功的心电图为ST段弓背向上抬高。当每组大鼠在心肌梗死30分钟后，松开结扎线，进行再灌注3小时。假手术组中大鼠只做穿线而不结扎左前降支处理。
2.2.2实验取材
2.2.2.1摘取大鼠的心脏，使用冰生理盐水进行清洗，摘除血管、脂肪等多余的组织。应用4%的多聚甲醛对大鼠心肌进行固定4小时，再用30%的蔗糖对心肌组织进行脱水（4摄氏度过夜）。
2.2.2.2剪下结扎线以下的左心室心肌组织，剪碎后置于玻璃研磨器中，加入蛋白裂解液后进行研磨，静置30分钟。将左心室匀浆于4摄氏度的离心机中，进行时速3000转/分钟，连续离心10分钟，抽取上清液后，再次进行离心，选用12000转/分钟，连续离心30分钟，再次取其上清液，每20微升分装，并在-80摄氏度的冰箱中保存备用。
2.2.3观察指标
2.2.3.1心肌细胞凋亡指数的检测
在每一个标本中各取已制备的切片1张，应用末端脱氧核苷酸转移酶所介导dUTP缺口末端标记法进行标记心肌凋亡细胞核中DNA3-OH的末端，并采用原位荧光法对凋亡细胞进行标记与显示。在荧光显微镜高倍（×200）视野下观察每张切片标本，在心肌梗死区域随机地选择4个视野，并计算凋亡的细胞指数（心肌细胞的凋亡指数%=镜下凋亡心肌细胞总数/镜下总细胞数×100%）。
2.2.3.2Western Blot检测Bcl-2、Bax基因蛋白的表达
从-80摄氏度的冰箱中将心肌匀浆蛋白取出后，对浆液进行复温，蛋白上清液经过SDS-聚丙烯酰胺凝聚电泳作用下转移至硝酸纤维素膜上，用10%的脱脂奶粉TBST液进行封闭1小时，加入一抗（比例：1:1000）（兔抗鼠bcl-2,bax），在室温下进行孵育2小时后，应用TBST液进行3次洗涤，加入二抗在室温条件下孵育2小时，3次洗涤，经过ECL试剂盒后显色，再对其采用凝聚成像系统进行扫描处理，显色后的结果采用Quality one软件对灰度进行分析，观察并计算蛋白相对的表达水平。
结果：
1.心肌细胞凋亡指数
在200倍的荧光显微镜显示下，在大鼠心肌梗死区域呈现绿色荧光的地带为心肌凋亡细胞主要的分布区域，其中药物干预组大鼠的凋亡细胞数目显著少于模型组，二者统计学具有显著的统计学差异（p<0.01）。
2.基因蛋白Bcl-2与Bax的表达水平
与假手术组大鼠比较，模型组大鼠基因蛋白Bax表达增强；基因蛋白Bcl-2表达明显减弱，二者具有显著的统计学差异(P
机体组织出现缺血后，对缺血组织进行再灌注治疗后，多种因素导致缺血的组织细胞出现代谢功能障碍及结构破坏，从而使组织损害程度进一步加重。人体的许多组织都存在缺血再灌注损伤的现象，尤其是在大脑及心脏。心肌缺血再灌注损伤是多种因素参加的极其复杂的环节，就目前研究表明较为认同的再灌注损伤机制为：氧自由基产生、钙离子超载、心肌细胞能量代谢障碍、炎症细胞激活、内皮细胞功能障碍以及心肌细胞发生凋亡等。心肌缺血再灌注损伤发生过程中心肌细胞的过度凋亡是再一次导致心肌损伤的重要原因，心肌细胞发生凋亡作为缺血再灌注损伤的早期事件，贯穿于缺血再灌注损伤的病理生理过程。
细胞凋亡是受基因调控的一种程序性的细胞死亡，参与心肌细胞凋亡的主要调控基因有Bc1-2、p21、Fas、p53、热休克蛋白及caspase家族蛋白酶。根据调控基因的不同功能可以分为三个种类： Bc1-2、lAP、EIB等为具有抑制凋亡功能的基因；Fas、p53、Bax等为具有促凋亡功能的基因；C-myc、Bcl家族等为具有双向调控功能的基因。目前研究中，受关注程度最多、功能较明确的具有抑制凋亡功能的基因为Bc1-2，促进细胞凋亡的基因为Bax。基因蛋白Bcl-2及Bax是Bcl-2基因蛋白家族中作用互为对立的重要的调控细胞凋亡基因，Bcl-2对细胞凋亡起抑制作用，Bcl-2高度表达对于线粒体内钙离子稳态起到了维持作用，对线粒体膜电位下降起到阻止作用，并抑制线粒体膜通透性转换孔开放，抑制促进凋亡蛋白质如凋亡诱导因子、细胞色素C的释放，从而防止细胞发生凋亡。Bcl-2还能够与Apaf-1及Caspase9进行结合，并维持Bcl-2的非活化状态，阻断Caspase的级联反应，从而抑制细胞的凋亡。基因蛋白Bax对细胞的凋亡起到促进作用。部分研究结果尚提示，调节心肌细胞凋亡的基因水平不仅仅取决于基因蛋白Bcl-2及Bax自身水平的表达高低，还与基因蛋白Bcl-2及Bax水平比值有关，基因蛋白Bcl-2及Bax的比值反映了细胞出现凋亡的程度。
冠心舒通胶囊以广枣、丹参、丁香、冰片、天竺黄等中药药材以一定的比例所组成，是在现代中医药理论与蒙医蒙药理论相互贯通结合的基础上开发研制所成，冠心舒通胶囊有活血化瘀、通经活络、行气止痛的功效。中药广枣及丹参的药理作用均有多种，例如对实验动物的外周微循环具有改善作用、对炎症反应起到抑制作用以及抗氧化作用等。本实验研究主要通过用药后制备心肌缺血再灌注损伤的模型后，检测大鼠心肌细胞的凋亡指数与基因蛋白Bcl-2及Bax的表达水平，实验结果表明，与假手术组大鼠相比较，模型组大鼠基因蛋白Bax的表达增强，基因蛋白Bcl-2的表达明显降低；与模型组大鼠相比，冠心舒通胶囊组大鼠，心肌细胞凋亡指数、基因蛋白Bax的表达明显下降，基因蛋白Bcl-2的表达明显上调。本实验研究最终证实，冠心舒通胶囊能够明显降低缺血再灌注损伤的心肌细胞发生凋亡，从而表明其具有抗心肌缺血再灌注损伤的作用。
结论：
冠心舒通胶囊可以通过抑制心肌细胞的凋亡，下调基因蛋白Bax表达，上调基因蛋白Bcl-2表达，在心肌缺血再灌注损伤时起到保护心肌细胞的作用。
Background：
With the progress of science and technology,and the ascension of diagnosisand treatment,advanced medical treatment method of coronary arteryrecanalization (such as percutaneous coronary intervention, thrombolysis,coronary artery bypass grafting) is being applicated in clinical perfectly,whichcan supply the blood to the ischemic damaged heart promptly. This technologycan lessen the morbidity and mortality remarkablely of acute cardiovascularadverse events. The benefit of reperfusion therapy for the ischemic myocardialcells is above rubies,which not only can make the ischemic myocardial cellsacquire vitality once again,but also can reduce infarction area of the heart.Whencoronary artery occlusion of blood vessels was reperfused after some time inthe clinical, myocardial celles suffer the reperfusion injury a certain extent.Refer to the evidence-based medicine,repassing the occluded coronary arterycan not harvest the desired effect after4hours of myocardial infarction. Thetreatment promotes the myocardial cell to death.In recent years, many medicinalscholars have observed the reperfusion injury when myocardial ischemia happenswhich can arouse a series of bad waterfall chain event.Myocardial ischemia-reperfusion injury is one of the complications after coronary arteryrecanalizated,and it is easy to cause myocardial stunning, NO-reflow,reperfusion arrhythmia, cardiac cell death and so on in the process of myocardialischemia reperfusion. With the development of cardiovascular progress in thestudy of molecular biology,we have already step by step confirmed that thereis a close relationship between myocardial Ischemia-reperfusion injury andmyocardial apoptosis at present.Myocardial apoptosis is in the process ofmyocardial Ischemia-reperfusion injury which can accelerate irreversible cell apoptosis,and the number of myocardial apoptosis cells is correlated with thelength of duration of ischemia reperfusion injury.Therefore myocardial Ischemia-reperfusion is the adverse event of affect prognosis of acute coronary arterylesions.Clinical drug intervening myocardial apoptosis is the focus of theresearch。
Traditional Chinese Medicine plays an irreplaceable role in continuationthousands years civilization of the Chinese nation.TCM not only guarantees themasses of working people’s health and longevity,but also plays an indeliblecontribution to the breakthroughs in medical science.Traditional Chinesemedicine which is from all natural can healing disease pathogenesis,and thereis a small side effect.The TCM which is suitable for people's physicalfitness,life cultivation and health preservation, curing diseases and improvinghealth is the crystallization of the working people for thousands of years ofour country.Many clinical diseases donot have therapeutic method of westernmedicine and western medicine.Medical scholars translate their focus on Chinesemateria medica preparation.The process of molecular biology promotes theresearch of Chinese native medicine ingredient and pharmacological mechanism.Single herb is limited on sudocrem.Chinese herbal compound’s chemical componentis very complex and pharmacologic action has the characteristic of multilevel,multiple target point, multiway and comprehensive.Facing the clinicalcomplicated diseases, Chinese herbal compound embodies enormous worth ofcurative effect.
Guan Xin Shu Tong capsule which is prepared, developed, rolled out andpopularized is the three kinds of new Mongolian drug of China in the world.GuanXin Shu Tong capsule is united Traditional Chinese Medicine theory, clinicalpractice and characteristic theory of Traditional Mongolian Medicine.Guan XinShu Tong capsule is a compound Chinese medicine which is made up of fructuschoerospondiatis,the root of red-rooted salvia, clove,borneol andtabasheer.Guan Xin Shu Tong capsule possesses the effects of promoting bloodcirculation to remove blood stasis, clearing and activating the channels and collaterals, promoting qi circulation to relieve pain,which is used to cure thesyndrome of blood stasis in heart.The syndrome contains stethalgia, suppressionin the chest, be nervous, be short of breathof coronary heart disease andangina.Fructus choerospondiatis is the monarch herb which has the effect ofpromoting qi to activate blood, nourishing heart and soothing the nerves,andcan cure the symptom of qi depression to blood stasis,obstruction of pain,heartpalpitations shortness of breath and malaise.The root of red-rooted salviais the ministerial herb which has the effect of promoting blood circulation torestore menstrual flow,removing stasis pain,healing carbuncle, clearing awaythe heart-fire and relieving restlessness and nourishing the blood andtranquilization, and can cure the symptom of abnormal menstruation,amenorrheadysmenorrhea, concretions and gatherings, ulcers sore, hot bi pain,chest pain,upset and nosleeping, angina and so on.Clove is the adjuvant herb which has theeffect of warming middle energizer descend adverse-rising, reinforcing kidneyto strengthen yang,and can cure the symptom of trusted subordinate crymodynia,deficiency-cold in spleen and stomach,hiccups vomiting, eat less and vomitingand diarrhoea,kidney deficiency impotence.Borneol is the adjuvant herb whichhas the effect of faint scent scattering, inducing resuscitation, clearing heatand diffusing poison and removing nebula, and can cure the symptom of high fevercoma, stroke sputum jue chronic ulcerated,dusting confusion by summer-heatand wet, pharyngitis and deaf, aphtha and tooth swollening, carbuncle,sore redswollen eyes, film covering eyes.Tabasheer is the conductant herb which has theeffect of clearing heat and eliminating phlegm,cold heart to calm the frightened,and can cure the symptom of god faint delirium,stroke sputum fan not language,pediatric epilepsy,epilepsia, sputum and heat cough.In the process of many yearsclinical application,we found that Guan Xin Shu Tong capsule can restrainapoptosis in myocardial ischemia-reperfusion injury.Scholars have researchedthe Guan Xin Shu Tong capsule only on clinical curative effcet observationempirical using summary in previous studies which are short of researching ofcurative effect mechanism system.For the sake of expliciting the mechanism of action of Guan Xin Shu Tong capsule restraining apoptosis in myocardial ischemia-reperfusion injury, reducing the clinical morbidity and mortality,enhancingthe level of patients’life,we proceed this research to provide reliable theorybasis and empirical data.
Purpose：
To function the effect of Guan Xin Shu Tong capsule on apoptosis in myocardialischemia-reperfusion injury in rats.
Material and method：
1.Laboratory animal breeding husbandry
1.1Grouping
Divided average averagely the30SD grade rats into3groups randomly.Theyare the sham group, the model group and the drug group.
1.2Breeding
The rats were breed under the condition of daily.The indoor temperature wasmaintained between18and22centigrade.The humidity was dominated at（55±2）percent.All the rats can drink water with freedom.All the rats were taken inthe solar radiation12hours.The ten rats in the sham group were offered thenormal saline by gavage,2times a day, which was continuous for5days byirrigation.The ten rats in the model group were offered the normal saline bygavage,2times a day, which was continuous for5days by irrigation,before themodel of myocardial ischemia reperfusion was established.The ten rats in thedrug group which was controled by Guan Xin Shu Tong capsule were offered GuanXin Shu Tong capsules by gavage, before the model of myocardial ischemiareperfusion was established.The Guan Xin Shu Tong capsule dose was1.5gram perkilogram which was dissolved in normal saline, every time to be6ml per kilogramby lavage,2times a day, drenched for5days consecutively.
2. Experimental target determination
2.1Preparating the model of myocardial ischemia reperfusion
All the rats were fasted for12hours before the preparation of model,but the rats can be free to drink.Preoperative ECG examination of all the rats andrecording the electrocardiogram of lead II.Eliminate the rats which the Ecg wereabnormal.All the rats were anesthetized by pentobarbital sodium whoseconcentration was30gram per litre.All the rats were used medicinebyintraperitoneal injection.The dose was1.5milliliter per kilogram.Fixed therats which were the state after anesthesia,and were for endotrachealintubation.Animal Ventilator model number is ALC-V which was used to providethe rats to breath.The rats’s breathing rate was set to50to60times per minute,continuing to record electrocardiogram in rats at the same time.The rats’ leftmid-clavicular line were cut open the chest skin about2centimeter.The rats’left anterior descending coronary artery were ligated by surgery line.The modelof myocardial infarction were success when ST segment of electro cardiogramarchupward.When the rats in each group were at the state of myocardial infarctionfor30minutes,the rats were loosen the ligature and cured by reperfusion therapyfor3hours.The rats in the sham group were given the same treatment,but therats were only did the thread without ligation of left anterior descendingbranch.
2.2Draw materials
2.2.1When the rats in each group were at the state of myocardial infarctionfor30minutes,the rats were cured by reperfusion therapy for3hours.the rats’hearts were taken which were washed by ice normal saline and removed blood vessels,fat and other redundant organization.The myocardium in rats was fixed on4hoursby application of4%parafor maldehyde,and the myocardium in rats was sucrosedin the application of30%on myocardial tissue dehydration (4centigrade forthe night).
2.2.2The left ventricular myocardial tissue was cut out following ligation,and put in glass grinding machine after cuting up.The tissue was grinded afterAdding protein pyrolysis liquid,and standed for30minutes calmly.Leftventricular homogenate was put in the centrifuge in4degrees Celsius,and wasto be carried on the speed of3000round per minute for10minutes continuously.The supernatant fluid was extracted and was centrifugal again onthe speed of12000round per minute for30minutes continuously.Then thesupernatant fluid was choosed, and shipmented every20microlitre partially.andsaved in the refrigerator in-80degrees Celsius.
2.3Observation target
2.3.1Myocardial apoptosis index
Only1section card was chosen from in each prepared specimen.The terminalof DNA3-OH in the myocardial apoptotic nuclei was signed by the way of terminaldeoxyribonucleotide transferase mediated dUTP Nick end labeling.The apoptoticcells were signed and displayed by fluorescence in situ tag.Apoptotic cell countanalysis: according to Nick end labeling kits related reaction after operationinstructions, the apoptosis of myocardial cells were in the greenfluorescence.All of the nucleated cells present blue fluorescence after colouredby Desktop Application Programming Interface (DAPI).Each biopsy specimens wasobserved under fluorescence microscope (200) at high magnification view.Inmyocardial infarction area four horizons are randomly selected.The number oftotal number of cells and apoptosis cell number were counted in Each themicroscopic view.The index of the apoptotic cells was calculated by formula ofMyocardial cell apoptosis index=Total number of the apoptosis of myocardialcells/Microscopically the total cell number×100%.
2.3.2Detecting the expression of the pretein gene of Bcl-2and Bax by the wayof Western Blot.
Myocardial homogenate protein was taken out from the refrigerator in-80degrees Celsius and was recovered to the room temperature.Protein in thesupernatant was removed to the nitrocellulose blotting membranes by the way ofSDS-condensation of polyacrylamide electrophoresis,and was closed for1hourby10%skimmed milk powder Tris Buffered Saline Tween (TBST) fluid.The firstresistance was Added in the fluid which the proportion was1vs1000. Afterincubation for2hours at room temperature,the protein in the supernatant wadwashed3times by the Tris Buffered Saline Tween fluid.The second resistance was Added in the fluid which was marked by horseradish peroxidase in the roomtemperatureThe fluid was colored after ECL reagent kit,which was in the condensedimaging system adopted to improve the scanning and used Quality one softwareto analyze the gray level,and observatiing and calculating the relativeexpression level of the protein.
Results：
1.Myocardial apoptosis index
Under the fluorescence microscope show that two hundred times，in themyocardial infarction area of rats the primary distribution area of apoptosisof myocardial cells presents green fluorescence strip.The results shows thatthe numbers of the apoptosis cell was significantly reduced of the rats in thegroup intervened by drugs than the rats in model group.There is a obviousdifference between the rats in the group intervened by drugs and the rats inmodel group(p<0.01).
2.Expression level of the gene proteins of Bcl-2and Bax
Compared with the rats in the control group,the enhanced expression of thegene protein of Bax is remarkable, and the expression decreased of the geneprotein of Bcl-2is evident. There is a obvious difference between the rats inthe control group and the rats in model group(p<0.01). Compared with the ratsin the model group,the rats in the group intervened by Guan Xin Shu Tong capsuleshave an enhanced expression of the gene protein of Bax,and there is a obviousdifference between the rats in the model group and the rats in druggroup(p<0.01).Compared with the rats in the model group,the rats in the groupintervened by Guan Xin Shu Tong capsules have a decreased expression of the geneprotein of Bcl-2,which has a difference between the rats in the model group andthe rats in drug group(p<0.05).
Discussion
The body tissues occurred ischemia which were cured by reperfusion turn upmetabolic dysfunction and structural damage of the tissue cells by a number of factors.This pathological process aggravated the extent the tissue damage.Anumber of the organizations in the body exist the phenomenon of the ischemiareperfusion injury. This kind of situation is consist in the brain and heart.Myocardial Ischemia-reperfusion injury is a vital and complicated segment whichis in a variety of factors. The current research suggests that the ischemiareperfusion injury mechanism which most people agree oxygen free radicalsproduced, intracellular calcium overload, myocardial cells energy metabolismdisordered, activation of inflammatory cells, Endothelial cell dysfunction,myocardial cell apoptosis, and so on.In the process of myocardial ischemia-reperfusion injury, the excessive apoptosis of myocardial cells results in thesignificant cause the myocardial cells damage.The cells of cardiac turning upapoptosis is the early event in the course of ischemia reperfusion injuryprogression.This event throughouts the pathology and physiology process ofischemia-reperfusion injury.
The cell apoptosis which is regulated by the genes is a kind of programmedcell death.The main control genes which participates in the process of myocardialcell apoptosis include the regulatory gene of Bc1-2, the regulatory gene of p21,the regulatory gene of Fas, the regulatory gene of p53,the heat shock protein,the caspase family of protease,and so on. According to the different functionof regulating the genes can be divided into three categories.The regulatory geneof Bc1-2,the regulatory gene of lAP and the regulatory gene of EIB are providedwith function of inhibition cell apoptosis. The regulatory gene of Fas, theregulatory gene of p53and the regulatory gene of Bax are provided with functionof promoting cell apoptosis.The regulatory gene family of C-myc and theregulatory gene family of Bcl have the function of promoting cell apoptosis andinhibition cell apoptosis.In the present study,the regulatory genes of mostconcern and the functions relatively clear is the gene of Bc1-2,which has thefunction of inhibition cell apoptosis,and the gene of Bax which has the functionof promoting cell apoptosis.The regulatory gene of Bcl-2and the regulatory geneof Bax are the most important regulatory genes in the protein gene family. The regulatory gene of Bcl-2and the regulatory gene of Bax have a relation withmutually opposite effects.The regulatory gene of Bcl-2highly expressed has thefunction of inhibition cell apoptosis,which has the act of maintaining calciumof steady state in the mitochondria,holding back the membrane potential declineof mitochondria, inhibition of mitochondrial membrane permeability transitionpore opening,and inhibition of promoting apoptosis proteins such as the factorapoptosis inducing, the release of cytochrome C, so as to prevent the cellapoptosis.The regulatory gene of Bcl-2also has the fuctions of combining withon the gene of Apaf-1and Caspase9, maintaining the non-antiactive state ofBcl-2and blocking the cascade reaction of the family pretein of Caspase.Generally speaking,the regulatory gene of Bcl-2also has the fuctions of theprocess of inhibit cell apoptosis.The gene protein of Bax has the funtion ofpushing forward myocardial cell apoptosis.Part of the research results indicatethat the gene level of regulation of myocardial cell apoptosis not only dependson the protein gene level expression of Bcl-2and Bax itself,but also the ratioof the levels of gene protein Bcl-2and Bax.The ratio of the levels of geneprotein Bcl-2and Bax reflects the degree of the appearance of cell apoptosis.
Guan Xin Shu Tong capsule is made up of fructus choerospondiatis,the rootof red-rooted salvia, clove,borneol and tabasheer,which is the product ofmutually penetrating combination of the modern theory of Traditional ChineseMedicine and Mongolian medicine and theory.Guan Xin Shu Tong capsule has theeffects of promoting blood circulation to remove blood stasis, clearing andactivating the channels and collaterals, promoting qi circulation to relievepain,which is used to cure the syndrome of blood stasis in heart.The herbalmedicine of fructus choerospondiatis and the root of red-rooted salvia havea variety of pharmacological effects,such as improving the appearance of theperipheral cycle of experimental animals,possessing the funtion of inhibitingthe inflammatory response, having an antioxidant effect.The experiment researchmainly detecte the apoptosis index of rat myocardial cells and the expressionlevel of the protein gene Bcl-2and Bax through preparation of the model of myocardial ischemia-reperfusion injury after the medication
The experimental result shows that compared with the rats in the controlgroup,the enhanced expression of the gene protein of Bax is remarkable, and theexpression decreased of the gene protein of Bcl-2is evident.Compared with therats in the model group,the rats in the group intervened by Guan Xin Shu Tongcapsules have an enhanced expression of the gene protein of Bax and have adecreased expression of the gene protein of Bcl-2.This experimental studyconfirmes that Guan Xin Shu Tong capsule can obviously decrease ischemia-reperfusion injury of myocardial cell apoptosis occurred finally.so thisexperimentalshows that Guan Xin Shu Tong capsule has the role ofwithstandingredients myocardial ischemia-reperfusion injury.
Conclusion：
Guan Xin Shu Tong capsule has the fuction of protecting myocardial cellsby restraining myocardial cell apoptosis, increasing the expression of proteingene Bcl-2and weakening the expression of protein gene Bax.
引文
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