| Background-In1960, Jennings[1] first proposed the concept of myocardial ischemia reperfusion injury (ischemia/reperfusion, I/R), myocardial ischemia reperfusion, not only didn’t make tissue and organs function recovery, but make ischemia further aggravated what caused by function metabolic disorder and structural failure.More and more attention on the mechanism of ischemia reperfusion injury has been paid after the appcarance of conception of ischemiareperfusion injury. Intracellular calcium metabolism disorders, the generation of oxygen free radicals and neutrophil infiltration are three main ways that cause myocardial ischemia-reperfusion injury. in1972,Shen and Jennings found dog of heart coronary brief occlusion and reperfusion can accelerate the accumulation of intracellular calcium, and calcium overload theory was first proposed, from this Ca2+in ischemia-reperfusion injury in rats become research hotspot. Calcium overload on myocardial ischemia reperfusion damage effects including:mitochondrial dysfunction, Activate Ca2+dependence phospholipase, promote the phospholipase decomposition; promote the formation of oxygen free radicals; cause arrhythmias. Citric acid, also known as citrate, Citrate ions and the blood calcium ions can generate citrate calcium, this complex is soluble in water, but it is not easy to dissociation, lower serum calcium concentration. Suppose citrate may affect the extracellular calcium ions that involved in myocardial I/R injury, which may produce myocardial protection. In this study, using rats in vivo myocardial ischemia-reperfusion model, to observed citrate on rat myocardial I/R injury influence, to explore whether citrate pretreatment in rat myocardial I/R can lessen the damage effect.Objective---The purpose of this study was to investigate the role of citrate attenuates injury on myocardial ischemia-reperfusion in rats and explore the possible mechanism. Methods-BW of male SD rats was350-400g,The thoracotomy and left anterior descending coronary artery was ligated. The study was carried out in a series of experiments:1. The impact of citrate to ventricular arrhythmias and hemodynamics caused of myocardial ischemia-reperfusion:Animals were randomized to divide into five groups(n=8each). Sham-operated rats(group S)were treated identically except the left coronary artery was not tied;Ischemic-reperfusion group(group I/R)was treated30min of occlusion and120min of reperfusion(intravenous give an equal volume of saline);Different concentrations administered group(0.01mol·L-1,0.05mol·L-1and0.1mol·L-1), lml dose at10min before reperfusion.Then record and analysis changes of ventricular arrhythmias, and systolic blood pressure, diastolic blood pressure, mean arterial pressure, pulse pressure and heart rate within120minutes after reperfusion.2. Citrate on serum Ca2+impact study:Observed in vivo in rats given0.05mol· L-1citrate before drug administration and after5min,10min,15min,30min carotid artery, Observe citrate serum Ca2+, and to explore the possible mechanism of citrate role.3. Citrate on rat myocardial cell injury research:Rat in vivo model of ischemia-reperfusion, reperfusion after120min, Evans blue and TTC staining method for measuring the ischemic area and infarct area; At the end of reperfusion120min left ventricular myocardial tissue protein Capase-3activity as the detection of indicators, Use Caspase-3activity spectrophotometric assay kit to detect its activity.4. Citrate on ischemia-reperfusion rats semi-quantitative analysis of superoxide dismutase (SOD):At the end of reperfusion120min, extracted the left ventricular myocardium tissue protein in each experimental group, using Western blot (western-blot) methods to analyze the content of each experimental group SODResults-1. The impact of citrate to ventricular arrhythmias and hemodynamics caused of myocardial ischemia-reperfusion: 1.1ArrhythmiaReperfusion period:Compared with Sham group, The number of I/R group, ventricular arrhythmia was significantly increased (P<0.01); Compared with the I/R group, the administered group reperfusion120min incidence of ventricular arrhythmias are on a downward trend, Which0.05M group was significantly decreased (P<0.01). From the time distribution, citrate down the role of ventricular arrhythmias peak is mainly concentrated in the early reperfusion (20min within).1.2HemodynamicCompared with Sham group, IR group of systolic blood pressure, diastolic blood pressure, average pressure in reperfusion first30min were significantly lower (P <0.05); Compared with the I/R group, Citrate pretreatment group of systolic blood pressure, diastolic blood pressure, and average arterial pressure have shown a downward trend; Pulse pressure increased slightly; Changes in heart rate slightly smaller, But were not statistically different.2. Citrate on serum Ca2+impact study:After5min of serum calcium ion concentration and before using this drug is reduced by7.64%(P<0.001);10min after treatment was significantly decreased by20.52%(P<0.05);15min after treatment decreased by18.70%, But this was not statistically significant; Medication after30min, Serum calcium picks up4%. Prompted that citrate’s ability in medication10min chelated serum calcium achieved peak.3. Citrate on rat myocardial cell injury research:3.1Compared with the I/R group:Citrate pretreatment group AAR/LV has reduced after ischemic myocardium reperfusion120min,0.05M groups ischemic area decreased34%(n=6, P<0.05); IS/AAR of citrate pretreatment group after reperfusion significantly reduced by more than40%(n=6, P<0.05); IS/LV has reduced in citrate pretreatment group after reperfusion, the0.05M group significantly reduced68%(n=6, P<0.01);3.2Compared with Sham group, IR group obviously activity increased (P<0.01); Compared with the I/R group, Caspase-3activity has a downward trend in the administration group, Which0.05M was reduced most significantly (P <0.01);Compared with0.05M group, they have a statistically significant difference in the dose group (0.1M group of P<0.01,0.01M group, P<0.01), But no dose-dependent manner.4. SOD-Western Blot results:Compared with Sham group (0.78±0.04), IR group (0.85±0.08) of SOD activity decreased; Compared with the I/R group, The administration group0.01M group (1.20±0.12),0.05M (1.30±0.12),0.1M group (1.16±0.12) significantly increased the SOD released, Improve the oxygen free radical removal force, It has statistically significant (P<0.05).Conclusion-Citrate pretreatment can remarkably enhance cardial-protective effect against myocardial ischemic-reperfusion injury in rats.And the effect of Citrate reduce incident rate of arrhythmia and myocardial infact size were the more effective when the dose of management was0.05mol·L-1. The effectiveness of anti-apoptotic caused by citrate,which was by down-regulation with the protein expression of Caspase-3. Preliminary evidence that citrate pretreatment can reduce myocardial ischemia reperfusion injury, with myocardial protection. |
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