| Background:Although the early restoration of blood flow to the ischemiac myocardium after coronary artery occlusion is critical to salvage myocytes from eventual death,accumulating clinical and experimental studies have demonstrated that reperfusion has additional deleterious injury on the ischemic myocardium that are not expressed during ischemia,which was referred to as ischemic reperfusion injury.It is the universal phenomenon in the higher animals when tissue and organ are subjected to ischemia-reperfusion.It often occurred in cardiac surgery,coronary bypass. infarct recanalization,organ transplantation and so on.The mechanisms responsible for ischemia reperfusion injury include increasing of oxygen free radicals,overloading of intracellular calcium,acute inflammatory response and damage of vascular endothelial cell auto-homeostasis, etc.Murry,et al.first discovered that repetitions of brief ischemia-reperfusion led to a pronounced protection against subsequent lethal ischemia.This phenomenon was known as ischemia preconditioning.Ischemic preconditioning can significantly reduce myocardial ischemia-reperfusion injury,but the clinical application is limited.In 2003,Zhao,et al.witnessed for the first time that repetitions short-term coronary ischemia and reperfusion at early period of reperfusion significantly reduced the infarct size and alleviated ischemia reperfusion injury.This phenomenon was called ischemic postconditioning.Postconditioning possesses a comparable protective effect as preconditioning and provides operation spaces for clinical application.After ischemic postconditioning phenomenon was recognized,the possible mechanisms of its protective effect were explored gradually.We were encouraged to testify the hypothesis that the administration of drug intervention at the early period of reperfusion can simulate the protective effect of postconditioning.The protective and remedial effect of this drug intervention will have not only profound significance of theoretical study,but also broad prospects of clinical application.The process of drug intervention before ischemia was known as pharmacological preconditioning.Therefore,the process of drug intervention before reperfusion was accordingly called pharmacological postconditioning.Genistein,one of the isoflavones was first isolated from soybeans in 1931.It has diverse bioactivity on target tissue via different mechanisms.Genistein is beneficial to the prevention and treatment of cardiovascular diseases by anti-oxidation,scavenging reactive oxygen species, inhibiting myocardial calcium overload,hypolipidemic,affecting vascular reactivity,inhibiting smooth muscle cell proliferation,prohibiting cardiomyocyte apoptosis,anti-thrombosis formation,and so on.Reactive oxygen species(ROS)and calcium overloading generated right after reperfusion are two most important triggers to ischemia reperfusion injury.Genistein can resist oxidant,scavenge reactive oxygen species and inhibit myocardial calcium overload. Therefore,we choose Genistein as our drug candidate.We will utilize rat myocardial ischemia reperfusion in vivo and in vitro model to testify the potential pharmacological postconditioning protective effect of genistein to myocardium and its mechanisms.Aim:1.To establish the rat heart ischemia-reperfusion in vivo model,compare the differences in myocardial infarction,myocardial enzyme,heart function indexes,heart pathomorphology and ultrastructure between Genistein given and Genistein not given in early stage of reperfusion,and identify the pharmacological postconditioning protective effect of Genistein on rat's myocardial ischemia-reperfusion injury.2.To observe the effect of Genistein on myocardial NOS activity,serumal NO content and cardiomyocyte apoptosis in rats' ischemia-reperfusion in vivo hearts,and study the interventional effects of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on cardioprotection of Genistein against heart ischemia-reperfusion injury in an in vitro model, we analyze the potential mechanisms of Genistein pharmacological postconditioning.Materials and methods:1.The establishment of the rat heart ischemia-reperfusion in vivo model and the definement of the drug injection methods in Genistein pharmacological postconditioningHealthy male adult Wistar rats weighed 240-260 g were selected to establish the ischemia reperfusion model according to the method published in literature.After blocking the left anterior descending coronary artery reversibly,when electrocardiogram showed ST-segment elevation＞0.15 mV,we thought ischemia model was finished.The experiment was divided into such seven groups as the sham group,ischemia-reperfusion group,Genistein 0.1 mg/kg group, Genistein 0.3 mg/kg group,Genistein 1.0 mg/kg group,Genistein 2.0 mg/kg group and Genistein 5.0 mg/kg group.In the early stage of reperfusion,different doses of Genistein were given by an intravenous microinjection.2.The determination of myocardial infarct sizeWhen the experiment was over,through the side hole of the pressure pipe,2%Even's blue was injected rapidly into left ventricular by carotid.After rats' lips and foot became blue,the heart was taken out quickly from thorax.The atrium and right ventricular was removed.The left ventricular was averagely cut into six annular slices perpendicular to the longitudinal axis direction of heart from the bottom to the cusp,and the slices were incubated at 37℃in 1% TTC for 15 min,and then were fixed 24 to 48 h with 10%formaldehyde solution.The images of the heart were scanned by scanner,and then the dangerous area and infarct size were determined by image-pro plus image analysis software.The date was analyzed by the one-way ANOVA of the multi-groups.3.The observation of the pathomorphological and ultrastructural changes of myocardial tissueWhen the experiment was over,the heart was taken out quickly from thorax,and was washed with the saline solution at 0℃.The myocardial tissue of left ventricular anterior wall under the ligation thread of anterior descending coronary artery about 2 mm was scissored to the size of about 0.2×0.2 cm~2.After being fixed for 24 to 48 h in 10%formaldehyde solution, followed conventional paraffin-embedded,it was sectioned to 4μm sections.Followed conventional HE staining,the sections were observed by the optical microscope(400×)for pathomorphology.The myocardial tissue of left ventricular anterior wall was fixed in 2.5% glutaraldehyde for 2 to 4 h and in osmium tetroxide acid for 2 h one by one.After acetone dehydration,618 epoxy-embedded,it was sectioned to ultrathin sections.Followed uranium-lead double staining,the ultrathin sections were observed by the TEM(6000～20000×)for ultrastructure.4.The determination of myocardial and serumal enzyme indexesThe experiment was over,through the side hole of the pressure pipe,1.5 ml artery blood was drawn out.After the blood was centrifugated for 10 min at 2000 rpm,the supernatant was collected,and the serumal enzyme indexes were measured.10%myocardial tissue homogenate was prepared with the myocardial tissue of left ventricular anterior wall.The homogenate was centrifugated for 10 min at 10000 rpm.The supernatant was collected,and then the myocardial enzyme indexes were measured.By sandwich enzyme-linked immunosorbent assay test(quantitative),the serumal cardiac troponin I(Tn-I)content was detected with the goat anti-rat cardiac troponin I kit,which was purchased from Bionewtrans Pharmaciutical Biotechnology Co.,Ltd.The myocardial MDA content was detected by TAB test with the maleic dialdehyde detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The myocardial SOD activity was detected by xanthine oxidase method with the superoxide dismutase detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The date was analyzed by the one-way ANOVA of the multi-groups.5.The monitoring of the heart function indexesDuring the experiment,the ECG and left ventricular pressure waveform were entirely recorded using RM6240B biological signal acquisition and processing system 2.0e from before ischemia beginning to after ischemia-reperfusion being finished for 3 h.The data of HR,LVSP, LVDP,+dp/dtmax and -dp/dtmax in 30 min after the ischemia and in 10 minutes,30 min,60 min,120 min,180 min after the reperfusion were compared with before ischemia,then the recovery rate of the HR,LVSP-LVDP,+dp/dtmax and -dp/dtmax at each time point were calculated.The date of recovery rate was analyzed by a variance analysis model of repeated measures.6.The determination of serumal NO content and myocardial NOS activityThe preparation of serum and myocardial specimens for NO and NOS detections were defined as method 4.The serumal NO content was detected by chemic method with the nitric oxide detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The activities of each type NOS in myocardial tissue were detected with the nitric oxide synthase detection kit(typing),which was purchased from Nanjing Jiancheng Bioengineering Institute. The data was analyzed by the one-way ANOVA of the multi-groups.7.The observation of the cardiomyocyte apoptosisThe cardiomyocyte apoptosis was detected with in situ cell death detection kit,which was purchased from Germany Roche Diagnostics Mannheim GmbH Company.Myocardial tissue specimens were prepared to TUNEL paraffin sections strictly in accordance with the manual method.Apoptotic cells in TUNEL paraffin sections were counted by a fluorescence microscopy, and apoptosis index was calculated.The data was analyzed by the one-way ANOVA of the multi-groups.The preparation of myocardial detection specimens was defined as method 4.The myocardial Caspase-3 activity was detected with Caspase-3 activity detection kit,which was purchased from American Biological Company.The ratios of Caspase-3 activity in each group were calculated.The data was analyzed by the one-way ANOVA of the multi-groups.8.The establishment of the rat heart ischemia-reperfusion in vitro model and the definement of the drug intervention methods in Genistein pharmacological postconditioningHealthy Wistar male rats weighed 240-260 g were selected to establish the ischemia reperfusion in vitro model by Langendorff isolated heart perfusion technology according to the method in literature.After blocking the left anterior descending coronary artery reversibly,when electrocardiogram showed ST-segment elevation≥0.15 mV,we thought ischemia model finished. Using estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate as tools drug,the area of myocardial infarction,activities of myocardial enzyme and heart function indexes in isolated rat myocardium were observed.The model rats was divided into such seven groups as sham group,ischemia-reperfusion group,Genistein group,ICI 182780 group,vanadate group,Genistein + ICI 182780 group and Genistein + vanadate groups.The fusion fluid containing drug was used to intervene at 1 min before reperfusion until 9 min after reperfusion,a total of 10 min intervention,and the different drug intervention groups were established.Result:1.The damage effect of regional ischemia-reperfusion on rat hearts in vivoThe percentage of myocardial infarct size to the ischemic area at risk,the cardiac troponin I levels of serumal samples,the MDA levels of myocardial tissue samples and the activity of SOD of myocardial tissue samples of the ischemia-reperfusion group were 47.25±1.89%,0.72±0.06 ng/ml,2.42±0.15 nmol/mgprot and 23.38±1.67 U/mgprot,respectively,and the dates of the sham group were 4.36±0.77%,0.22±0.01 ng/ml,1.27±0.10 nmol/mgprot and 44.21±1.38 U/mgprot.The ischemia-reperfusion group significantly increased the area of myocardial infarction,cardiac troponin I and MDA levels,and reduced SOD activity than the sham group. These results indicated that ischemia-reperfusion had obvious injury effect on rat in vivo heart, and it also proved that we were successful in establishing the myocardial ischemia-reperfusion in vivo model.2.The effect of Genistein pharmacological postconditioning on myocardial infarction sizeThe percentage of myocardial infarct size to the ischemic area at risk of Genistein 0.1 mg/kg group,0.3 mg/kg group,1.0 mg/kg group,2.0 mg/kg group and 5.0 mg/kg group were respectively 20.37±2.05%,16.93±2.26%,8.76±2.17%,28.46±2.34%and 40.75±1.90%.They were significantly reduced myocardial infarction area than the ischemia-reperfusion group, which was 47.25±1.89%.In 0.1～1.0 mg/kg group,there were a dose-dependent decrease of myocardial infarction area,but in 1.0～5.0 mg/kg group,it showed a dose-dependent increase.3.The effect of Genistein pharmacological postconditioning on the pathomorphological and ultrastructural changes of myocardial tissueBy the optical microscopy and TEM,the pathomorphology and ultrastructure of the myocardial tissue after ischemia or reperfusion for 30 minutes and 3 hours were observed.By the optical microscopy,the myocardial tissue showed extensive necrosis and severe muscle fiber fracture,myocardial cells dissolution and disappearance,neutrophils exudation,and so on.Under the TEM,it showed myofibrils disarrangement,large areas of myofibrils fracture,amalgamation, disappearance,sarcomere structure unclear,myocardial nuclear swelling,the nuclear membrane rupture,nuclear chromatin asymmetry,condensation,margination,mitochondrial morphological abnormalities and swelling,the ridge disarrangement,fracture,disappearance,forming cavity, glycogen granules between the mitochondrial vanishment,some mitochondria parceled by double membrane,and completely desquamated from the mother cells,forming apoptotic bodies. In the different dosage Genistein groups,with the difference of doses,myocardial pathomorphological and ultrastructural changes were discriminating,it was showed that the injury became gradually lighter in 0.1～1.0 mg/kg group,however,gradually more serious in 1.0～5.0 mg/kg group.4.The effect of Genistein pharmacological postconditioning on myocardial and serumal enzyme indexesIn 0.1～5.0mg/kg dose range,the cardiac troponin I levels and MDA levels of the different dosage Genistein groups were significantly less than ischemia-reperfusion group,and the activity of SOD of myocardial tissue samples were significantly higher than ischemia-reperfusion group. In 0.1～1.0 mg/kg group,there were a dose-dependent decrease of the cardiac troponin I levels and MDA levels,and a dose-dependent increase of the SOD activity,but in 1.0-5.0 mg/kg group, it was the opposite.5.The effect of Genistein pharmacological postconditioning on the heart function indexesThe recovery rates of the HR,LVSP-LVDP,+dp/dtmax and -dp/dtmax in Genistein 1.0 mg/kg group were respectively 100.40%,109.69%,133.28%and 116.03%,and they were significantly higher than in ischemia-reperfusion group,which were respectively 78.51%, 77.96%,80.66%and 79.32%.6.The effect of Genistein pharmacological postconditioning on the serumal NO content and myocardial NOS activityThe NO contents detected in different doses of Genistein groups were significantly higher than in ischemia-reperfusion group,the NO content detected in Genistein 1.0 mg/kg group was the highest.The activities of T-NOS and cNOS of myocardial tissue samples detected in the different doses of Genistein groups were significantly higher than in ischemia-reperfusion group, the activity of T-NOS and cNOS detected in Genistein 1.0 mg/kg group was the highest.There were no differences in activity of iNOS detected among the groups,and the activity of iNOS was much lower than cNOS.7.The effect of Genistein pharmacological postconditioning on the cardiomyocyte apoptosisIschemia-reperfusion injury of ischemia-reperfusion group was more serious,in which the apoptosis index went up to 28.58±2.37%.Apoptosis index in 0.1,1.0 and 5.0 mg/kg genistein treated groups were respectively 19.48±2.13%,10.55±1.93%and 21.49±2.34%,and the 1.0 mg/kg genistein groups treated showed the lowest apoptosis index.Apoptosis index in the different dose of Genistein treated groups were significantly lower than in ischemia-reperfusion group.After the ratio of Caspase-3 activity in sham group was set at 1.00,the ratios of Caspase-3 activity in other groups were calculated,which increased to 2.75±0.25 in ischemia-reperfusion group and which decreased tol.51±0.15,1.28±0.10 and 1.71±0.13 in 0.1,1.0,5.0 mg/kg groups.The ratios of Caspase-3 activity in Genistein given groups were significantly lower than in ischemia-reperfusion group.8.The intervention effect of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on cardioprotection against ischemia-reperfusion injury by Genistein pharmacological postconditioningAfter ischemia-reperfusion,the percentage of myocardial infarct size to the ischemic area at risk of isolated rat heart in ICI 182780,vanadate group and Genistein + ICI 182780 group were respectively 48.92±1.63%,46.07±1.81%and 29.02±1.65%,and they were significantly higher than in Genistein group,which was 10.35±1.74%.It was significantly lower in Genistein + vanadate group than in Genistein given group,which only was 7.45±1.40%.The varying tendency of SOD activity in heart perfusate in each group was completely opposite to the varying tendency of myocardial infarction area.There was no significant difference in the recovery rates of the HR between the groups after reperfusion for 180 min.The recovery rates of LVSP-LVDP,+dp/dtmax and -dp/dtmax in ICI 182780 group,vanadate group and Genistein + ICI 182780 group were significantly lower than in Genistein group.The recovery rates in Genistein+ICI 182780 group was almost as high as in ischemia-reperfusion group.The recovery rates in Genistein + vanadate group was almost as high as in Genistein group.Most of the recovery rates in ICI 182780 group and vanadate group did not differ from in ischemia-reperfusion group.Conclusion:1.Using the heart ischemia-reperfusion model of rat in vivo,it was identified that genistein could effectively reduce myocardial ischemia-reperfusion injury by intravenous injection in early reperfusion,which was mainly manifested in the reduction of the myocardial infarction size, lower activity of cardiac troponin I and MDA,higher activity of SOD,the improvement of the recovery of the heart function,and the marked attenuation of destruction of myocardial ultrastructure,indicating genistein has cardioprotection effect against ischemia-reperfusion injury by the pharmacological postconditioning.2.Generally,genistein showed protective effects on myocardial ischemia-reperfusion injury in 0.1～5.0 mg/kg dose range.However,this protective effect showed two opposite trends in different dose range:a dose-dependent increasing in 0.1～1.0 mg/kg dose range,while a dose-dependent weakening in 1.0～5.0 mg/kg dose range.3.The effects of genistein on the myocardial NOS activity,serumal NO content, cardiomyocyte apoptosis index and Caspase-3 activity in ischemia-reperfusion hearts of rat, associating with application of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on ischemia-reperfusion heart of rat in vitro showed that the protective effect of Genistein pharmacological postconditioning related to the enhancement of myocardial NOS activity,the inhibition of cardiomyocyte apoptosis and the excitement of estrogen receptor.Its protein tyrosine kinase inhibitor property was detrimental to myocardial protection,and this may be the major reasons for a dose-dependent decrease of the protective effect in the higher dose range. |
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