Protective Effect Of Procyanidin On Myocardial Ischemia Reperfusion Injury By The Medium Of PI3K/Akt/GSK-3βand Mitochondrial ATP-sensitive Potassium Channel

BackgroupWith the wide application of thrombolysis、percutaneous coronary intervention and coronary bypass grafting in clinical medicine, more and more doctors and scientists realize the importance of myocardial ischemia reperfusion injury. Early、 continuously and fully restoration of blood flow through the occluded coronary artery is currently the most effective therapy to limit infarct size and preserve cardiac function after acute myocardial infarction.Nevertheless, reperfusion is considered as a double edged sword as it can lead to worsening of tissue injury, cause myocardial dysfunction and cardiac arrhythmias and increase the myocardial infarct size. Therefore, it is extremely important for us to find feasible ways to protect myocardium and avoid ischemia reperfusion injury. Some studies have proved that ischemic preconditioning and Pharmacological preconditioning are effective means of protecting heart from ischemia reperfusion injury, which can release some endogenous active substances,and activate signaling pathways such as protein kinase C(PKC), phosphoinositide kinase-3(P13K), and opened mitochondrial ATP sensitive K+channels(mitoKATP).Myocardial ischemia reperfusion injury is an extremely complicated pathophysiological process, the main pathogenic mechanism recognized at present includes: oxidative stress injury, intracellular calcium overload, cells apoptosis, cells energy loss, activation of neutrophil inflammatory reaction, etc. Meanwhile, ischemia reperfusion injury involves in many regulatory mechanisms in organism and activation and inactivation of signaling pathways, reactive oxygen species generats in large quantities trough ischemia and reperfusion. ROS has a very strong chemical activity, active electron can easily makes cell membrane lipid peroxidation enhanced and restrains the function of protein and damages nucleic acid and chromosome, thus damages the structure and function of cells.Cell apoptosis is consecutive programmed cell death. it involves a series of special gene expression during the whole process, characteristic morphological and biochemical changes in cells. Apoptosis plays an important role in the pathological process of myocardial ischemia-reperfusion injury. Inhibiting myocardial apoptosis can significantly reduces the area of myocardial infarction.Phosphoinositide3-kinases (PI3Ks), and their downstream target serine/threonine kinase (Akt) are a conserved family of signal transduction enzymes which are involved in regulating cellular activation, in flammatory responses,and apoptosis. Activation of PI3K/Akt-dependent signaling has been shown to prevent cardiac myocyte apoptosis and protect the myocardium from I/R injury Mitochondrial ATP-sensitive K+channels (mitoKATP) have been extensively shown to participate in ischemic preconditioning. Events triggered by ischemic preconditioning can be mimicked by mitoKATP agonists, while antagonists abrogate the protective effects of ischemic preconditioning. mitoKATP channels are present in the inner mitochondrial membrane that transport K+into mitochondria, leading to increased matrix volumes, The cardioprotective results of opening mitoKATP channels include improved energy metabolism, decreased mitochon drial Ca2=uptake during ischemia, and prevention of oxidative stress during reperfusion.The procyanidin(PC)from the grape seeds is an effective antioxidant and possesses extensive pharmacological activities. Studies have found that PC could inhibit oxidative stress, regulate blood lipid, relieve arteriosclerosis, attenuate inflammation.If PC preconditioning can protect heart against ischemia reperfusion injury and the endogenous mechanism are not clear. The present study established ischemia reperfusion rats heart model and anoxia/reoxygenation cardiomyocytes model to study the effects of PC preconditioning on ischemia reperfusion injury from the level of organ、cell and molecule. the first Part Procyanidin Relieve Myocardial Ischemia Reperfusion InjuryObjective:To observe the effects of PC on the size of myocardial infarction、systolic and diastolic function、serum concentration of CK-MB and CTnI、via establishing the ischemia reperfusion model of rats in vivo. PI3K/Akt/GSK-3βsignal pathway blocker LY294002was given to testify the role of PI3K/Akt/GSK-3βsignal pathway in myocardial ischemia reperfusion.Methods:Spragre—Dawley rats were randomly divided into following four groups, each group included10rats:sham operation group(SO); ischemic/reperfusion group (I/R); PC preconditioning+A/R group(PC); LY294002blocking group (LY294002).the rats in PC and LY294002groups were fed with PC(200mg/kg/d for4weeks, intragastricly)while the other rats were given0.9%NaCI intragastricly only for4weeks. The hearts of all animals except for SO group were subjected to30min ischemia and120min reperfusion. The parameters of cardiac function including left ventricular sistolic pressure(LVSP), left ventricular end—dastolic pressure(LVEDP), maximal differentials of LVDP(±LVdp/dtmax) were recorded respectively by multileads physiologic recorder at preischemia (To)、ischemia30min(Tl)、 postreperfusion30min(T2)、postreperfusion1h(T3) and postreperfusion2h(T4). Plasma CK-MB、CTnl were measured after2h reperfusion. Infarct size were defined by Evan’S and TTC staining.Results:1.Comparison of the systolic and diastolic function of heart:There was no significant difference of LVSP、LVEDP、±dp/dtmax at preischemia in four groups. The indicators has no difference at five time in SO group, while LVSP、±dp/dtmax decreased and LVEDP increased at2h reperfusion compared with at preischemia in I/R、PC、LY294002group, the difference is significant (p<O.05, respectively).Compared with group SO, LVSP、±dp/dtmax decreased and LVEDP increased at2h reperfusion in group I/R, the difference is significant (p<O.01, respectively). Compared with group I/R, LVSP、=dp/dtmax increased and LVEDP decreased at2h reperfusion in group PC, the difference is significant (p<O.05, respectively). Compared with group PC, LVSP、±dp/dtmax decreased and LVEDP increased at2h reperfusion in group LY294002, the difference is significant (p<O.05, respectively). The indicators has no difference at2h reperfusion in I/R group and LY294002group.2. Comparison of activities of serum CK—MB and CTnI concentrations between Groups:The activities of serum CK—MB and CTnI were significantly higher in group I/R than in group SO(p<O.01, respectively), while lower in group PC than in group I/R(p<O.01, respectively). The activities of serum CK-MB and CTnI were significantly higher in LY294002group(p<O.01, respectively).There was no significant difference between group I/R and LY294002.3. Comparison of the myocardial infarction size in rats between groups:the myocardial infarction size of I/R group (38.6±2.7%)significantly enlarged when compared with that of SO group(0), The infarct size was significantly reduced in group PC group (15.2±2.2%)compared with I/R group(p<O.01). The infarct size was significantly enlarged in group LY294002(30.3±2.4%) compared with group PC(p<O.01). There was no significant difference between group I/R and LY294002. There was no significant difference in the area of at risk between I/R、PC、 LY294002groups.Conclusion:1、Myocardial ischemia/reperfusion injury in rats can cause severe damage to myocardial systolic and diastolic function and myocardial tissues.2、PC can significantly improve the impairedmyocardial systolic and diastolic function、reduce myocardial infarction size and decrease serum concentrations of CK—MB and CTnI.It is to say that PC preconditioning can protect tissue injury caused by myocardial ischemia/reperfusion in rats.3、LY294002, a specific PI3K inhibitor, can lessen the protective effects of PC on myocardial ischemia/reperfusion injury.Suggesting that the protective effects of PC may be related to the activation of P13/Akt signaling pathways. the second part The Mechanism of PI3K/Akt/GSK-3p and Mitochondrial ATP-sensitive Potassium Channel on The Cardiomyocyte Anoxia/reoxygenation Injury ModelObjective:Cardiomyocytes were isolated from the ventricles of1-3days Spmgue-Dawley neonatal rats and primarily cultured for experiment. Ischemia reperfusion injury was mimicked by anoxia/reoxygenation model. to study the effect of PC in rat cardiomyocytes injured by anoxia/reoxygenation by measure cell viability、ROS、apoptosis. PI3K/Akt/GSK-3βsignal pathway blocker LY294002and mitochondrial ATP sensitive potassium channel blocker5-HD were given to testify the role of PI3K/Akt/GSK-3βsignal pathway and mitochondrial ATP sensitive potassium channel in PC cardioprotection.Methods:Cultured myocardial cells of SD neonatal rats(1-3d) by the differential attachment technique, and then randomly divided into five groups after cultured48hours.control group; anoxia/reoxygenation group(A/R group):anoxia for3h and reoxygeonation for3h; PC preconditionging+A/R group(PC group):PC with final concentration100ug/ml was given30min later,then processed with anoxia and reoxygenation; LY294002+PC+A/R group(LY294002group):culture medium was added LY294002with concentration of15umol/1, added PC30min later, then anoxia and reoxygenation were conducted30min later;5-HD+PC+A/R group (5-HD group):5-HD with final concentration100umol/1was given, culture medium was added5-HD with concentration of100umol/1, added PC30min later, then anoxia and reoxygenation were conducted30min later.At the end of experiment, measured cell viability and the amount of ROS.Apoptosis of myocardial cells was detected by Tunel and Armexin-V/PI assays with flow eytometry.The expression of Caspase-3-. t-Akt、p-Akt、t-GSK-3β、p-GSK—3β protein in cytoplasm were measured by western blotting.Results:1.Cell morphology by inverted microscope:the cardiomyocytes are radial and spiral, in sheet arranged.The nucleus is oval and in the middle of cell. 2. The cell viability:the cell viability of A/R group(41.51±3.86%)significantly decreased when compared with that of control group(92.36±3.12%),(p<O.01).the cell viability of PC group (75.74±4.69%)significantly increased when compared with that of A/R group,(p<O.01).The viability of LY294002group(47.52±4.71%) significantly decreased when compared with that of PC group(p<O.01,),no significant difference compared with that of A/R group.The viability of5-HD group(48.13±4.68%) significantly decreased when compared with that of PC+A/R group(p<0.01),no significant difference compared with that of A/R group.the difference between LY294002+PC+A/R group and5-HD+PC+A/R group no significance.3. Myocardial cell nuclear changes were tested by Hoechst33258staining:Cells have uniform nuclear and light blue staining in control group.Cells appear chromatin condensation nucle、marginalization and some fragments of cell cleavage to the morphological changes after A/R, irregular and deeply blue staining cells increase in A/R group. Deeply blue staining cells decrease in PC group.Deeply blue staining cells increase in LY294002group and5-HD group,have the same changes with A/R group.4. Cell ROS level:the ROS in A/R group(211.6±38.4) significantly increased compared with that in control group(27.0±5.6),(p<O.01). ROS in PC group(80.1±10.3) significantly decreased compared with that in A/R group,(p<O.01). The ROS of LY294002group(167.2±18.3) significantly increased when compared with that of PC group(p<0.01),no significant difference compared with that of A/R group.The ROS of5-HD group(178.3±22.8) significantly increased when compared with that of PC group(p<0.01),no significant difference compared with that of A/R group.the difference between LY294002group and5-HD no significance.5. The apoptosis of cells by Tunel:the apoptosis in A/R group(25.3%±2.64) significantly increased compared with that in control group(3.1%±0.65),(p<0.01).the apoptosis in PC group(10.2%±1.59) significantly decreased compared with that in A/R group(p<0.01).The apoptosis in LY294002group(19.6+1.79) significantly increased when compared with that of PC group(p<0.01),no significant difference compared with that of A/R group.The apoptosis in5-HD group(18.1%±2.03) significantly increased when compared with that of PC+A/R group(p<O.01),no significant difference compared with that of A/R group.the difference between LY294002group and5-HD group no significance.6. The apoptosis of cells by flow eytometry:the apoptosis in A/R group(30.71%±3.82%) significantly increased compared with that in control group(8.36±1.25%)(p<O.01). the apoptosis in PC group(13.11±1.36%) significantly decreased compared with that in A/R group (p<O.01).The apoptosis in LY294002group(26.12±2.07%) significantly increased when compared with that of PC group(p<O.01),no significant difference compared with that of A/R group.The apoptosis in5-HD group(27.47±2.15%) significantly increased when compared with that of PC group(p<O.01),no significant difference compared with that of A/R group.the difference between LY294002group and5-HD group no significance.7. Western blotting measure t-Akt、p-Akt、t-GSK-30、p-GSK—3β protein: Expression of Caspases-3in cardiomyocyte:the expression of Caspases-3in A/R group increase apparently comparing with control group,while that in PC group reduced obviously comparing With A/R group.The expression of Caspases-3increased obviously in LY294002group and in5-HD group compared with PC+A/R group. The difference between LY294002group and5-HD group no significance.Expression of Akt protein and phosphorylated Akt protein in cardiomyocyte:There was no significant difference among t-Akt protein of all groups. P-Akt in A/R group increase, but no significant difference compared with control group.There was significantly increased in PC+A/R group comparing With A/R group.The expression of P-Akt decreased obviously in LY294002+PC+A/R group. The difference between5-HD+PC+A/R group and PC+A/R group no significance.Expression of GSK3(3protein and phosphorylated GSK3β protein in cardiomyocyte: There was no significant difference among t-GSK3(3protein of all groups. P-GSK3β in A/R group increase, but no significant difference compared with control group.There was significantly increased in PC+A/R group comparing With A/R group.The expression of P-GSK3P decreased obviously in LY294002+PC+A/R group. The difference between5-HD+PC+A/R group and PC+A/R group no significance.Conclusion:1. PC Can protect cardiac cells against hypoxia-reoxygeonation injury,which include improving cell survival rate and reducing apotosis、ROS as well as release of myocardial damaging enzyme.2. The cardioprotection of PC could be blocked obviously by signaling pathway blocker LY294002, so PC preconditioning activating PI3K/Akt/GSK-3psignal pathway.3、The cardioprotection of PC could be blocked obviously by the mitochondrial ATP sensitive potassium channel blocker5-HD, so it can be say that mitochondrial ATP sensitive potassium channel is involved in PC cardioprotection.4、Only the two signal pathways be existed and have the normal function, PC has the cardioprotection in anoxia/reoxygenation injury.