| Myocardial ischemia/reperfusion injury (I/RI) is an important pathological and physiological process, which was involved in a variety of common clinical diseases and conditions such as coronary artery spasm, myocardial infarction, thrombolytic therapy, coronary artery bypass surgery, cardiopulmonary bypass heart surgery and cardiac arrest after cardiopulmonary resuscitation. However, the exact role with the mechanism of I/R remains unclear. According to some studies, I/R is also related to the functional regulation of ion channels in the cell membrane except oxygen free radicals, calcium overload, vascular endothelial cells, nitris oxide and the neutrophil. It is necessary to thoroughly explore the heart I/R injury of ion channels mechanisms and effective protection measures for clinical I/R injury-related diseases so as to provide a potential therapeutic strategy.In recent years, many studies have shown that the regulation role of extra or intracellular ions in cell homeostasis is closely related to cell apoptosis. The early cell shrinkage, also named apoptotic volume decrease (AVD) is an important morphological characteristic of apoptosis, which was related to the activated K+, Cl- channels and H2O outflow. The activated Cl- currents were detected in various cell apoptosis by patch clamp. Apoptotic cell death in variety of cell types can be rescued by blocking the Cl- currents. Our previous study testified that the chloride channels participated in cardiomyocyte apoptosis and cardiomyocyte apoptosis induced by STS can be effectively inhibited by chloride channel blockers. However, to the best of our knowledge, there are only few studies about chloride channel inhibition on the level of organs and the whole-animal, and currently only two was reported with opposite conclusions. Therefore, the aims of the present study were to explore a non-selective chloride blocker of DIDS on whole-animal, based on our previous foundation and present issues about chloride channel inhibition on various cell protections. AimThe purpose of this study is to observe whether 4,4'-disothiocyano- stibibene-2,2'-disulfonic acid (DIDS) has protective effects of injury rats myocardium in established myocardial ischemia reperfusion rats model. It will provide a new theory for chloride channel blockers so as to prevent the myocardial I/R injury effectively.Methods1. The animal model of myocardial ischemia-reperfusion: healthy SD rats were randomly divided into four groups. Sham-operated group, the left anterior descending (LAD) coronary artery was exposed without further procedures; I/R (30 min/4 h), the LAD coronary artery was reperfused for 4 hours following 30 minutes occlusion; I/R (30 min/12 h) group, the animal model was the same as that in the I/R(30 min/4 h) group; In I/R(30 min/24 h)group, the animal model was the same as that in the I/R(30 min/4 h) group. Choose the optimal condition of stabilized model of rats ischemia/reperfusion (I/R) for more experiment.2. Experiment groups: healthy SD rats were divided into five groups: A. sham-operated group; B. I/R group; C. I/R+ DIDS (7 mg/kg) group; D. I/R+ DIDS (14 mg/kg) group; E. I/R+ DIDS (28 mg/kg) group. In DIDS group, the rats were administered by program control micro pump injection of DIDS 2 h (4 ml/kg/h), at the beginning of reperfusion.3. Observation index:①The ECG changes were recorded incessantly from myocardial ischemia to reperfusion and was analyzed and given score.②The hemodynamic parameters were determined by multiple channel electrophy siolo graphs before ischemia, after ischemia and after reperfusion.③Measurement of plasma levels of creatine kinase (CK) and lacate dehydrogenase (LDH) reperfusion. Measure the malonaldehyde (MDA) and the activity of superoxide dismatast (SOD) of myocardial .④The apoptotic of cardiomyocytes was investigated by DNA fragmentation (DNA-ladder) and Terminal deoxynucleoside transferase mediated dUTP nick end labeling (TUNEL) method.⑤The ischemia and infarct size of hearts were measured by double staining with 2,3,5-Triphenyl-2H-tetrazoli- um chloride (TTC) and Evan's blue dye.Results1. The stable animal model of myocardial ischemia-reperfusion was successfully produced by reversibly ligating and releasing the coronary artery in rats. At the completion of the reperfusion period, we dyed the myocardium with evans blue dye and observed the normal ventricular myocardium was blue. Then we dyed the area at risk with TTC. TTC stained the viable tissue into brick red, leaving the necrotic area pale white. Ischemia for 30 min with reperfusion for 4 hours was an optimal condition to measure cardiomyocyte apoptosis and ischemia for 30 min with reperfusion for 24 hours was an optimal condition to staining the ischemia and infarct size of hearts. The results serve as a satisfactory foundation for further research.2. DIDS can degrade arrhythmia score. After 30-minute ischemia and 4-hour reperfusion; in sham-operated group, I/R group, DIDS (7 mg/kg) group, DIDS (14 mg/kg) group, DIDS (28 mg/kg) group the arrhythmia score were 1.20±0.45,4.57±1.62,3.97±1.51,3.23±1.13,3.56±1.47。DIDS can degrade arrhythmia in reperfusion phase(n=5,P <0.05), DIDS 14 mg/kg group provide the most significant effect(n=5,P <0.01).3. DIDS can improve haemodynamics index:①The hemodynamic parameters indicated that: with sham-operated group, left ventricular end diastolic pressure (LVEDP) were significantly increased while left ventricular systolic pressure (LVSP), maximal rate of increase of ventricular pressure (dp/dtmax) and maximal rate of decrease of ventricular pressure (dp/dtmin) reduced remarkably in I/R group (n=5,P <0.05);②+dp/dtmax and -dp/dtmax increased remarkably in DIDS group(n=5,P <0.05),LVEDP were decreased significantly and LVSP increased remarkably in DIDS 14 mg/kg group(n=5,P <0.05) when compared with I/R group.4. The levels of plasma CK and LDH in DIDS group decreased significantly (n=5,P <0.05).5. DIDS can significantly reduce myocardial MDA, and increase myocardial SOD activity compared with I/R group(n=5,P <0.05).6. DIDS can reduce myocardial apoptosis. The results of the myocardial DNA-ladder and TUNEL staining revealed that the TUNEL positive cardiomyocytes were increased dramatically after I/R injury but compared with I/R group, they were significantly reduced in DIDS group (n=5,P <0.05), especially in DIDS 14 mg/kg group.7. DIDS can decrease myocardial infarct size. After 30-minute ischemia and 24-hour reperfusion: The results of the myocardium staining showed that the myocardial infarct size was reduced in DIDS group compared with I/R group (n=5,P <0.05).Conclusion1. The rat model of myocardial ischemia-reperfusion is successfully produced, Ischemia for 30 min with reperfusion for 4 hours was an optimal condition to measure cardiomyocyte apoptosis and ischemia for 30 min with reperfusion for 24 hours was an optimal condition to staining the ischemia and infarct size of hearts.2. DIDS can provide an obvious cardioprotective effect on the ischemia/reper- fusion myocardium. The protective mechanism involve DIDS inhibit the chloridion efflux of damaged cadiocyte, inhibit the lipid peroxidation and increase myocardial SOD activity. DIDS can inhibit the apoptosis of cardiomyocytes., promote the recovery of myocardial function during ischemia-reperfusion and reduce myocardial infarct size.3. In our present study, DIDS (14 mg/kg) is optimal in cardioprotections, In details, it obviously inhibited reperfusion arrhythmia and atettenuated cardiomyocyte apoptosis with improved LVSP,LVEDP.
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