| 1,BackgroundCoronary heart disease(CHD)is a kind of common and frequently-occurring disease. CHD is the single-most important cause of death worldwhile, and about 1.1 million people die of the disease each year in china. After an acute myocardial infarction,with the use of thrombolytic therapy or primary percutaneous coronary intervention(PCI) or coronary artery bypass graft(CABG) is the most effective strategy reducing the size of myocardial infarction and improving clinical outcome.But in some animals or patients,we can find that the myocardial cell dysbolism and structural damage became more serious after ischemia-reperfusion.The phenomenon was called myocardial ischemia- reperfusion injury(MIRI). In clinical situations, reperfusion following ischemia result in a sharp drop in blood pressure,heart failure,arrhythmia and even sudden death which worsening pathogenetic condition. Therefore, people show greater concern for mechanisms and control of myocardial ischemia-reperfusion injury, and always try to find out the drugs which have exactly protective effects to myocardial ischemia-reperfusion injury. The causing mechanisms of myocardial ischemia-reperfusion injury are very complicated, and cell apoptosis is one of the important mechanisms of myocardial ischemia-reperfusion injury. Apoptosis is distinguished from necrosis,apoptosis is the process of programmed cell death in which nucleated cells occur the process of natural cell death on condition that through activation of an internally suicide program,mainly through activation of endogeneity DNA endonuclease.Apoptosis occurs in ischemia-reperfusion injury, which may be concerned with the following factors:①lots of reactive oxygen species generation:a burst of Oxygen free radicals generation occur during myocardial reperfusion, Oxygen free radicals generation results in membrane lipid peroxidation,inactivation to kinds of apoenzymes and damage to DNA.②intracellular calcium overload:lots of calcium concentrates in mitochondrial membrane during the process of myocardial ischemia-reperfusion injury, mitochondrial calcium overload results in the opening of mitochondrial permeability transition pore, releases of cytochrome c, and caspase activation leading to myocardial apoptosis.③mitochondria damage:mitochondria still can maintain normal ultrastructure during the process of apoptosis, but its function has been changed significantly, such as increase of inner mitochondrial membrane permeability, decrease of mitochondrial transmembrane potential and the levels of energy production decreased significantly. But these factors can not induce cell apoptosis dircetly,they activate survival or death gene by signal transmission,then the signal transmit to the incision enzyme intranuclear to perform the function of death.To block the signal transduction of cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion may inhibit cardiomyocyte apoptosis, Prevent or cure myocardial ischemia-reperfusion injury.Semen Ziziphi Spinosae is the dried ripe seed of Ziziphu spsinosa Hu, It has the efficacies of constraining sweat, protecting liver, quieting the heart, inducing sleep,and so on.Semen Ziziphi Spinosae mainly contains kinds of jujubosides.Jujubosides are water-soluble constituents of Semen Ziziphi Spinosae,and jujuboside A is one of main effective components. Studies had found that Spine Date Seed PE could regulate blood lipid, lower blood pressure,anti-atherosclerosis, anti-arrhythmia,anti-myocardial ischemia,and,relieve cardiomyocyte following hypoxia/reoxy-genation injury.The others studies had confirmed jujuboside A could anti-atherosclerosis by means of inhibiting overproliferation of vascular smooth muscle cells, and it also could inhibit cell apotosis significantly during the processof cerebral ischemia-reperfusion injury, but now there is no Study on protective effects and mechanisms of jujuboside A on myocardial ischemia reperfusion injury.2,Methods:Objective:In vivo we set up myocardial ischemia-reperfusion injury models in rats simulate the pathophysiologic changes in myocardial ischemia-reperfusion injury, and jujuboside A was administrated by intraperitonea injection before myocardial ischemia. The aim of this study is to demonstrate if jujuboside A can inhibit apoptosis and protect myocardium from ischemia-reperfusion injury, discuss the mechanism involved,chiefly find out the effects of preconditioning with jujuboside A on apoptosis related protein Bax,Bcl-2,Caspase-3 and CytC; In cellular level we set up oxidative injury models by damage to neonate rat myocardial cell with hydrogen peroxide, The aim of this study is to observe the effects of preconditioning with jujuboside A on cardiomyocytes morphology and apoptosis, discuss the mechanism involved, chiefly find out the relation between the protection and PKCs pathway, and find new evidence to protect myocardium from ischemia-reperfusion injury. Our study includes three parts:The first part is to find out if jujuboside A can protect myocardium from ischemia-reperfusion injury when it was administrated by intraperitonea injection before myocardial ischemia,especially to explore the effect of jujuboside A on the change of myocardial tissue,the marker of myocardial damage,the use of antioxidatio,the occurrence of arrhythmia after reperfusion and heart function. The second part is to demonstrate the effects of preconditioning with jujuboside A on cardiomyocytes apoptosis in protein,discuss the mechanism involved;The third part is to demonstrate the effects of preconditioning with jujuboside A on neonate rat myocardial cell apoptosis induced by hydrogen peroxide and the experssion of PKCs,identify the mechanism involved.Methods:The first part of this study:The left anterior descending (LAD) coronary artery was occluded for 15min followed by reperfusion for 30min to establish myocardial ischemia-reperfusion injury model.44 androgenic wistar rats were randomly divided into four groups:the Sham group (n=8), the I/R control group(n= 12), the jujuboside A group (n=12)and the EGCG group (n=12). Rats of the jujuboside A group and the EGCG group were given drug at a dose of 20mg/kg for three consecutive days(intraperitoneal injection), rats of the Sham group and the I/R control group were given physiological saline of the same volume for three consecutive days (intraperitoneal injection),the operation was conducted 30 min after the last intraperitoneal injection in rats of each group. We observed the occurrence of arrhythmias in electrocardiogram of lead II during myocardial reperfusion for 30min, and scored the ventricular arrhythmias (VA)acceording to the method of Ravingerova T. Through the medlab 6.0 biological signal collecting and processing system, the left ventricle systolic pressure(LVSP),end-diastolic (LVEDP) pressure and the max rate of rise and decline(±dp/dtmax) were measured at the time of before ischemia, ischemia 15min, reperfusion 15min and reperfusion 30min respectivly. Specimen collection:After the experiment had been done, we drew blood from the abdominal aortic vascular of rats, mutilated the great vessels and tissues from the cardiac base, took out of the heart, removed the atriums and the right ventricles, washed the hearts by cold physiological saline for three times, cut the heart to take the damaged cardiae muscles according to the need of detection, divided the left ventricle in three parts.the first part was weighed by electronic precision balance,enoughly cut and then homogenized about8 stroke with a glass Dounce homogenizer, measured the contents of MDA and SOD in heart;the second part was fixed with 4% polysorbate; the third part was keptin icebox at 80℃below zero. The blood was stood for 30min at room temperature, then centrifugated at 4℃.the serum was kept in icebox at 80℃below zero, measured the levels of CK and LDH.The second part of this study:40 androgenic wistar rats were randomly divided into four groups:the Sham group (n=10), the I/R control group(n=10),the jujuboside A group (n=10)and the EGCG group (n=10), The rest is the same as the first part. The apoptotic cardiomyocytes were detected by TUNEL staining, counted apoptotic index.The expression of Bcl-2 and Bax protein were measured by western blotting,cytochrome C in the cytoplasm were detected by western blotting, the activity of caspase-3 was measured by spectrophotometry in each group.The results were compared among the groups.The third part of this study:Primary cultures of cardiac myocytes were prepared from ventricles of 1～3 day new born witar rats and cultured for 72 hours. we set up oxidative injury models by damage to neonate rat myocardial cell with hydrogen peroxide.the neonate rat myocardial cells were divided as follows:①Control group:There was not any treatment in the cultural course of neonate rat myocardial cells.②Model group:The model group was treatment with the final concentration of 500μmol/L hydrogen peroxide in the cell culture fluid in 4 hours. ③Jujuboside A treatment group:The treatment was similar to that of the model group,but dealed with the final concentration of 20mg/L jujuboside A in the cell culture fluid in 24 hours before adding hydrogen peroxide.④PKC inhibitor group(JA+Chelerythrine), The treatment was similar to that of the jujuboside A treatment group,but dealed with the final concentration of 5umol/L chelerythrine in the cell culture fluid in 5min before adding hydrogen peroxide. Morphology and the beating rate change of primary cultured neonatal rat myocardial cell were observed under the inverted microscope; viability of myocardial cell was assayed by MTT; Apoptosis rate was determined by flow cytometric analysis. The activity of caspase-3 in myocardial cell was measured by spectrophotometry,and Western blotting was used to analyze expression of cytochrome C of the cytoplasm and expression of PKCs of the cytomembrane in each group.The results were compared among the groups.Statistiealanalysis:The data was analyzed by SPSS13.0.measurement data was presented as the means±SEM.Test of homogeneity of variance was done in each group.If datas accorded with homogeneity of variance statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Games-Howell. Heart function parameters in different time points was analyzed by repetitive measurement and analysis of varianee.Level of signifieance a=0.05.Results:The first part of this study:1,Histopathologic observation of the myocardium in rats:Pathologic changes of myocardial tissue were observed under hematoxylin-eosine staining(HE).In sham group there was no detectable histopathological change. Rats in the I/R control group displayed a serious degree of myocardial neutrophilic infiltrate, necrosis,hemorrhage, and spindle-shaped interstitial cells as compared with rats in the sham group. pathologic changes of cardiac tissue in the JA and EGCG group were significantly milder than that of the I/R group.2,The effects of myocardial ischemia-reperfusion on the levels of LDH,CK,SOD and MDA in rats:Compared with Sham group,I/R group,JA group and EGCG group markedly increased the activity of LDH and CK in serum,the content of MDA in myocardium,but significantly decreased the activity of SOD in myocardium (P< 0.01). The results indicated that ischemia-reperfusion could result in serious injury of myocardium, and confirmed that myocardial ischemia-reperfusion injury model was established successfuly.3,The effects of JA on the levels of LDH,CK,SOD and MDA of myocardial ischemia-reperfusion in rats:Compared with I/R group, JA group and EGCG group markedly decreased the activity of LDH,CK in serum and the content of MDA in myocardium,but significantly increased the activity of SOD in myocardium (P<0.01).The values of MDA,CK,LDH and SOD in JA and EGCG groups had no significant difference(P>0.05).The results indicated that effects of JA and EGCG antioxidation decreased the injury of ischemia/reperfusion of myocardium.4,Reperfusion arrhythmia:Twelve rats were excluded from entry to this study,arrhythmia was found in 26 rats,arrhythmia occurred mainly at the early period of reperfusion. The arrhythmia appeared in rats inclding ventricular premature beat,ventricular tachycardia(VT),ventricular fibrillation(VF),and so on.Only accidental ventricular premature beat appeared in rats of Sham group. The serious arrhythmia appeared in rats of I/R group,remained frequent ventricular tachycardia(VT),frequent ventricular fibrillation(VF),and accompanied a small quantity of atrioventricular block,there were 2 dead rats in I/R group,most of arrhythmia occurred in reperfusion. Arrhythmia score in I/R group was more than that in sham group (P=0.000).The arrhythmia also appeared in rats of JA group mainly inclding ventricular premature beat and ventricular tachycardia(VT), and Arrhythmia score in the group was lower than that in I/R group (P=0.005).The arrhythmia appeared in rats of EGCG group mainly inclding ventricular premature beat and paroxysmal ventricular tachycardia(VT),Compared with JA group,There was no difference between them (P=0.080).5,Index of cardiac function:No significant difference of LVSP,LVEDP and±dp/dtmax were found in all groups of preischemic (P>0.05). LVSP was significantly decreased in the sequence of Sham group, EGCG group, JA group and I/R group,±dp/dtmax was significantly decreased in the sequence of Sham group, JA group, EGCG group and I/R group, while LVEDP was significantly increased in the sequence of Sham group, EGCG group, JA group and I/R group.The results indicated that ischemia-reperfusion could lead toheart malfunction,and pretreatmenting with jujuboside A can improve the heart function.The second part of this study:1,Myocardial TUNEL staining:The apoptotic cardiomyocytes were detected by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) assay.The normal cardiomyocytic nucleus is blue, The nucleus of apoptotic cardiomyocyte is brown.AI of I/R group was (18.14±2.22)%,after pretreatmenting with jujuboside A and EGCG, AI was significantly lower than that of I/R group (P=0.000).Compared with sham group, AI of JA group and EGCG group were significantly raised (P<0.01)2,Bcl-2,Bax and CytC protein expression:According to the densitometry of bands under the CCD imagine system, a certain amount of Bcl-2 and Bax were expressed in myocardium of sham group, the cytochrome C expression in cytoplasm was little. Compared with the sham group,the expression of Bcl-2 and the rate of Bcl-2/Bax decreased in the I/R group, the expression of Bax and the cytochrome C expression in cytoplasm increased with significant difference (P<0.01). Compared with the I/R group,the expression of Bcl-2 and the rate of Bcl-2/Bax increased in the JA group and EGCG, the expression of Bax and the cytochrome C expression in cytoplasm decreased with significant difference (P<0.01).The expression of Bcl-2,Bax,cytochrome C and the rate of Bcl-2/Bax were no significant difference between JA group and EGCG group(P>0.05), and there was no a significant difference of the Bax expression between the two groups and sham group (P>0.05), but The expression of Bcl-2,cytochrome C and the rate of Bcl-2/Bax were significant difference between the two groups and sham group(P<0.01).3,The results of Caspase-3 Activity:The levels ofcaspase-3 activity in normal cardiomyocyte were very low, the levels of caspase-3 activity in I/R group were increased significantly, which was decreased significantly in JA and EGCG groups(P <0.01). The caspase-3 activity was no significant difference between JA group and EGCG group (P=0.817).The third part of this study:1,Morphological characters of cellular:Cell morphology was observed under reverse microscope.From 72 hours on,the normal cardiomyocytes grew in clusters,the reflected light of nucleus was good,and the pseudopodia was plump, connected reciprocally. In addition, cell beating intension of cardiac cells was apparent,the rhythm was 60-80 beats per minute.Most of cells in H2O2 group were crenulated, the nucleus were dim, the pseudopodia were became thin significantly, the rhythm of cell beating was decreased significantly, the.frequency was 20～30 beats per minute. Compared with the I/R group, the morphological characters of JA group was improved significantly.the morphological characters of PKC inhibitor group was worse that of JA group. 2,The results of viability and apoptosis rate in myocardial cell:Compared with the control group, the viability in myocardial cell of model group was decreased significantly,and the apoptosis rate was increased significantly (P<0.01).Compared with the model group, the viability in myocardial cell of JA group was increased significantly, and the apoptosis rate was decreased significantly (P<0.01). Compared with the JA group, the viability in myocardial cell of PKC inhibitor group was decreased significantly, and the apoptosis rate was increased significantly (P<0.01,).3,Protein expression of CytC and PKCs were measured by Western blot analysis: Compared with the control group, the expression of cytochrome C in the cytosol of model group was increased significantly (P=0.000), and the expression of PKCεin the membrane was no significant difference between them (P=0.235).Treatments with jujuboside A (20 mg/L) significantly decreased the expression of cytochrome C in the cytosol, and significantly increased the expression of PKCεin the membrane(P <0.01). Compared with the jujuboside A group, the expression of cytochrome c in the cytosol of PKC inhibitor group was increased significantly, and the expression of PKCεin the membrane was decreased significantly (P< 0.01).4,The results of Caspase-3 Activity in cardiomyocyte:Compared with the control group,the Caspase-3 Activity of model group was increased significantly (P=0.000). Compared with the model group,the caspase-3 activity of jujuboside A group was decreased significantly (P=0.000).Treatments with Chelerythrin significantly increased the caspase-3 activity (P=0.000).Conclusion:1,The experiment has proved that myocardial ischemia/reperfusion could lead to myocardial injury by an in vivo myocardial ischemic reperfusion model in rats. Pretreatment with jujuboside A could decrease the injury of ischemia/reperfusion of myocardium,decrease the levels of LDH,CK and MDA, increase the activity of SOD, decrease the reperfusion arrhythmia and improve cardiac function.2,Myocardial ischemia/reperfusion and hydrogen peroxide injury could result in cardiomyocyte apoptosis,pretreatment with jujuboside A could decrease cardiomyocyte apoptosis.Pretreatment with jujuboside A can relieve the ischemic reperfusion injury,the mechanism of which may be related with the inhibited myocardial apoptosis.3,The protective effect of preconditioning with jujuboside A was probably achieved through decreasing myocardium apoptosis,and modulating expression of Bcl-2 and Bax,increasing the ratios of Bcl-2/Bax,inhibiting the release of cytochrome C and decreasing the activity of caspase-3.4,Protein kinase C is the important link of preconditioning, pretreatment with jujuboside A can strengthen the expression of PKCεin the membrane,but the PKC inhibitor can significantly attenuate the effects of jujuboside A, and so the PKCεsignaling pathway may be a key point in the anti-apoptosis of jujuboside A. |
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