On Myocardial Ischemia Reperfusion Injury In Experimental Studies Guanxinshu Cells Through Capsules

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 husbandry1.1GroupingDivided average averagely the30SD grade rats into3groups randomly.Theyare the sham group, the model group and the drug group.1.2BreedingThe 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 determination2.1Preparating the model of myocardial ischemia reperfusionAll 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 materials2.2.1When 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 target2.3.1Myocardial apoptosis indexOnly1section 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 indexUnder 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 BaxCompared 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).DiscussionThe 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 medicationThe 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.