| Background:Reperfusion after a period of ischemia did not reduce damage to ischemic tissues and organs but rather caused more serious ischemia-reperfusion (I/R) injury. With development of new procedures and technologies for revascularizationin clinical practice,I/R injury has become a more and more important clinical topic which requires imminent and effective solutions.Jennis introduced the concept of I/R injury in 1960, uncovering that,the necrosis pathological process in myocardial ultrastructure continued, even with irreversible damage, after blood reperfusion. This published view has been gradually concerned and further confirmed by the medical profession. The pathophysiological processes after myocardial ischemia and reperfusion bring the damage to ultrastructure, metabolic, function and electrophysiology in ischemic myocardium tissue cells increased further, resulting in the irreversiblely worsened condition, named ischemia-reperfusion injury. After revascularization of occluded coronary, and reperfusion of infarcted myocardial tissue for some time, hypotension, shock, pump failure, malignant arrhythmias,sudden death and other complications emerge, resulting in deteriorating condition. Thus, the mechanism and related measures for the prevention from ischemia-reperfusion injury have been extensively concerned by the medical profession, and it is committed to explore effective means and measures for prevention from the occurrence and progression of I/RI.Primary percutaneouscoronary intervention(PPCI) is the most effective treatment of acute myocardial infarction. In recent years, with more and more application of PPCI, I/RI has become a important factor of hemodynamic abnormalities,pump failure,malignant arrhythmia and even death of after revascularization. According to statistics, more than 60% of patients with acute myocardial infarction suffered from perioperative reversible pump failure after PCI. therefore, it is explored in cardiovascular profession as a hot spot,how to prevent 1/ RI.In myocardium occur these following pathophysiological changes during reperfusionrl. Myocardial Stunning. Myocardial dysfunction has occurred after once or several brief ischemia in cardiac tissue without ccurrence of myocardial necrosis after re perfusion. Although the blood perfusion in myocardial tissue was normal or nearly normal, but the pathological phenomena due to ischemic myocardial dysfunction still exists within a certain time. Cardiac dysfunction caused by I/RI sustains a period of time (approximately 3-24h), and recovers gradually,which is temporary and reversible. Impressively the duration of myocardial contractile dysfunction often exceeds that of ischemia time after reperfusion.For instance, ischemia time in the heart of a dog was only 15min, but myocardial pump dysfunction would remain more than one day after reperfusion.Clinically, the pathological changes are not uncommon in recurrent angina and revascularization after acute myocardial infarction. It was found that myocardial abnormal movement has been detected for several days,in spite of disappearance of pain and discomfort in patients with angina after drug therapy. Such as after the thrombolytic therapy in patients with acute myocardial infarction, the cardiac systolic function did not immediately restore to its original level after reperfusion in blocked coronary artery. Therefore, the evaluation of the effect of PCI or thrombolysis should be appropriate in about 2 weeks after reperfusion. Studies on the mechanism of this phenomenon have pointed out that, myocardial blunt may be caused by increasing oxygen free radicals, activated inflammation such as the aggregation of inflammatory cells,and calcium overload,due to mitochondrial dysfunction after ischemia,reduced synthesis of ATP, and increasing anaerobic glycolysis.2.Hibernating myocardium. After myocardial ischemic injury, although nreperfusion did not lead to myocardial necrosis, but like myocardial hibernation,due to metabolic decrease, ultrastructure remodeling, decreased ATP synthesis, and decreased oxygenation demand,myocardial function and perfusion were redistributed,in oder to maintain new balance between blood flow and function. At this time by the way of self-protection regulation,such as reducing myocardial oxygen consumption and decreased contractile force, the progression to myocardial necrosis was avoided, and more viable cardiomyocytes were preserved.This is also an adaptive, self-regulating mechanism of protection from myocardial ischemia-reperfusion injury in ischemic myocardium. Thus, as the characteristic features of hibernating myocardium,in the state of low perfusion can myocardium readjust the balance between energy metabolism and synthesis, reduce the amount of myocardial perfusion, and decrease myocardial oxygen consumption. By similar compensatory mechanism achieve the still normal myocardial cells all the functions.The mechanism is associated with a series of changes caused by the reduced ability of mitochondrial synthesize,such as reduced fatty acid synthesis, increased conversion and utilization of sugar,and decreased reserve capacity. Hibernating myocardial cells show degenerative.If adaptive myocardial structural changes or myocardial fibrosis sustained for a long time, the recovery ability will be losed.Thus, these adaptive changes are limited and incomplete. CCB (calcium channel antagonist), β-blockers and ACEI (angiotensin-converting enzyme inhibitors) can promote the gradual improvement of metabolism and function in the hibernating myocardium.3. No-reflow phenomenon. Ischemia myocardial regional caused by the ligation of coronary artery in dogs,after relief of the ligation of arterial blood in some time,can not be effectively primed, known as no-reflow or no reperfusion. As a unique ischemia-reperfusion injury, it is the continuation of hypoperfusion in ischemic tissue and organ. Noneffective reperfusion in ischemic tissue,and persisting ischemia may seriously hamper the recovery of the function of ischemic myocardium tissue. No-reflow phenomenon occures in endomyocardium and endocardial coronary microvascular branch,due to smaller diameter of terminal branches, higher withstanded tension in subendocardial,the dual bearing roles of myocardial contractility and high filling pressure in ventricular lumen,the maximum oxygen demand,and the worst tolerance of hypoperfusion.4.Cell apoptosis. As a characteristic pathological changes of IRI, apoptosis show the morphological changes,such as decreased cell volume, condensed cytoplasm,and nuclear condensation. Cell membrane ultimately fault and surrounded cytoplasm and chromatin segmentation,leading to the formation of dense granules,named as apoptotic bodies. Membranes is still intact, and organelles remain relatively intact, without inflammation and spillover of cellular contents. Necrosis is the result of pathological attack, showed as the destroy of the cell membrane and structure, cell disintegration, the release of cell contents,and the emergence of progressive damage and inflammation. Within 6 hours after the occurence of myocardial ischemia, apoptosis is the main form of cell death.But with continued existence of ischemia,myocardial necrosis will be significant and will reach a peak within 24 hours.In spite of limited tolerance to myocardial ischemic, myocardial necrosis will gradually appear,when the maximum tolerated interval is exceeded. Myocardial apoptosis occurs in the early stages of myocardial ischemia, otherwise, in the late stage of myocardial ischemia a large number of myocardial necrosis appears. The manner of death depends on the storage capacity of intracellular ATP. When ATP synthesized in mitochondria was depleted, especially when severe myocardial ischemia sustained, apoptosis could be transformed into necrosis. Apoptosis is the most important pathological change of IRI. Effective protection from the occurrence and development of myocardial apoptosis, has become a new direction and a means to prevent IRI.Myocardial ischemia for a short period can increase the tolerance to myocardial ischemia and attenuate myocardial IRI. Thus, as a stimulation of injury,the pre-condition can improve resistance or resilience capabilities of myocardial cell. Transient ischemia for 3 times in a row length of 5 minutes and 10 times in a row length of 2 minutes can induce ischemic preconditioning (IP), but hypoperfusion for only once or twice, in about 5 minutes can not promote the emergence of ischemic preconditioning. The duration and frequency of hypoperfusion may play a decisive role in terms of successful induction.Only appropriate stimulation can produce ischemic preconditioning. Preconditioning is difficult to occur due to too short ischemia time,otherwise, cardiac damage become irreversible when ischemia time is too long. In case of hypoperfusion exceeding 30min, preconditioning effect was weakened,and if hypoperfusion exceed more than 90-120min,ischemic preconditioning can nearly not be induced.Generally citrate acts as a blood anticoagulant In coagulation process, calcium ions can facilitate the formation of fibrin, thrombin, clotting factor HI, and promote platelet activation. Citric acid can be binded with the free Ca2+to form calcium citrate, a soluble complexes, thereby inhibiting coagulation and preventing blood clotting,by the way of decreasing the concentration of plasma Ca2+.Objective:Currently there is little published research about the protecting effect and its underlying mechanisms of citrate pre-treatment for I/R. We aim to investigate the effect of citrate pre-treatment on the MI and cardiac function of I/R rats, based on the conventional I/R preparation model. We also aim to study the impact of citrate on the apoptosis and signaling pathway inI/R myocardial cells.Methods:1. I/R model preparation and experimental groups:the conventional preparation I/R model. Healthy male SD rats were randomly divided into three groups, each containing 15 rats. I/R group:ischemic 30min/reperfusion 120min, in the period of reperfusion with infusion of normal saline through the femoral vein; I/R+citrate group: same as I/R group with addition of citron acid (0.05mol/L) through femoral vein; Sham group:no other procedure other than open chest.2. Monitor and record arrhythmias using ECG during the period of 30 mins ischemic and 120 mins of reperfusion.3. Measurement of cardiac MI:TTC staining method for measuring MI, which are EF, LVSP, LVEDP,+dp/dtmax and -dp/dtmax.4. Detection of LDH, CK, SOD, MDA and GSH-PX.5. Detection of myocardial apoptosisrelate proteins:TUNEL method to detect myocardial apoptosis, western blot method to detect apoptosis-related proteins.6. The signaling molecule PI3K/Akt detection:application Western blot to detect the expression p-PI3K, p-Akt.Results:1. A marked increasdoccurrenceof ventricular arrhythmiasin I/R group than Sham group.15 minutes of reperfusion inducedpremature ventricular contractions, ventricular tachycardia, ventricular fibrillation and other malignant arrhythmias. When pre-treating withcitric acid 0.05mol/L before reperfusion, frequencies of all above mentioned ventricular arrhythmias decreased siginificantly. (P<0.01)2.There was statistically significant change in levels of EF, LVSP, LVEDP, +dp/txmax and -dt/txmax in the I/R group compared to sham group. LDH and CK increased significantly in I/R group compared to sham group. There was significant concentration decrease in the two after pre-treatment with citrate (p<0.05).3.The Mean[SD] infarct area proportion in the I/R group was 28.8%[6.4%], which decreased significantly in I/R+citrate group (p<0.01).4.1n comparison to sham group, SOD and GSH-PX decreased in I/R group and MDA increased in I/R group. This change was reversed after pre-treatment with citrate (p<0.05).5.The Mean[SD] TUNEL staining positive ratio was 10.37%[2.04%] in the I/R group whereas the I/R+citrate group had 3.20%[0.96%]. This indicates citrate pre-treatment introduced a decrease of cell apoptosis.6. We observed an increased expression of caspase-9, caspase-7, caspase-3and Bax proteins in the I/R group, in comparison to a decrease expression in I/R+citrate group (p<0.001). Bcl-2 protein expression was decreased in I/R group but increasedin I/R+citrate group. (p<0.001)7.Citric acid can activate PI3K/Akt signaling pathway. There was a significant increase of p-PI3K and p-AKT in citrate pre-treated group than I/R group. Citrate whether further inhibit myocardial cell apoptosis by upregulating PI3K/Akt signaling pathway study, we used the PI3K inhibitor LY294002 (20μmol/L) on myocardial cells were treated detect apoptosis related factors found I/R+Citrate+LY294002 group each apoptosis-related factor protein levels and I/R+Citrate group, there is a statistically significant difference.Conclusions:1. Citric acid pretreatment reduced ischemia-reperfusion arrhythmia severity and incidence, protected cardiac function2. Citrate pre-treatment decreased I/R. myocardial cell apoptosis, decreased area size of myocardial infarct and improved cardiac muscle function.3. Citrate pretreatment significantly increased the p-Akt levels in myocardial cells. PI3K specific inhibitor LY294002 inhibited the increase of p-Akt levels, which inhibited the protection of citrate pre-treatment on I/R myocardial cells.4. Citrate pretreatment activated PI3K/Akt signaling pathway,reduced apoptosis, reduced area size of myocardial infact,protectedmyocardial cellsreduccedthe severity and incidence of ventricular arrhythmia. |
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