Cardioprotective Effects Between SK&F96365 And LOE908 To Ischemia/ Reperfusion Injury In Vivo Rabbits

Myocardial ischemia reperfusion injury (MIRI) is an usual and common pathophysiological procedure. The vascular recanalization, which provides oxygen and nutrients necessary for survival of the ischemic area, paradoxically leads to additional damage to the myocardium as well. This process is named ischemia reperfusion injury. It’s a very complicated procedure with multi-factors, multi-mechanisms interaction, mutual acceleration and mutual development. Its major physiopathologic mechanisms consist of oxidative stress response, oxygen free radical injury, cell apoptosis and mitochondrial or intracellular calcium overload. Additionally, inflammatory response, insulin resistance and endothelial disfunction also importantly contributed to the myocardial ischemia reperfusion injury. Clinically, The mortality rate now as a consequence of acute myocardial infarction(MI) has dropped rapidly thanks to the introduction of reperfusion therapy by means of timely thrombolysis or primary percutaneous coronary intervention (pPCI), which largely improve patients’ prognosis. However, no reflow phenomenon or slow reflow phenomenon which aggravate myocardial injury have gotten more and more attentions of scholars nowadays. Currently, two major treatments of myocardial ischemia-reperfusion injury are methods and drugs. Ischemia preconditioning(IPC) and ischemia postconditioning(IPO) are the most familiar method to us and be most commonly used, of which differ each other with treatment time point.No-reflow phenomenon or slow reflow phenomenon have disturbed definitely most the cardiologists after percutaneous coronary intervention in some patients clinically. These phenomenon, so called "no-reflow (NR) or slow reflow", leaves myocardial tissue hypoperfusion despite the restoration of epicardial coronary artery patency following primary percutaneous coronary intervention. Although the mechanism of no-reflow or slow reflow hasn’t been clearly known, it can lead to microcirculation disfuntion and structural damage. In the very early stages, studies found the level of plasma ET-1 has slowly increased in some patients with acute myocardial infarction, which is a potent vasoconstrictor and aggravates coronary spasm and no-reflow or slow reflow. Moreover, the constriction of coronary artery is based on inward flow of calcium ion. Evidences show that the constriction resulting from low concentration of ET-1(<0.1 nmol/L) could be blocked by LOE908 or SK&F96365. And the constriction resulting from high constriction of ET-1(>1 nmol/L) could be weakened by LOE908 or SK&F96365 respectively largely, but could not be blocked totally. The blocking effect of LOE908 is 10% and the blocking effect of SK&F96365 is 45%, which may result from the sensitivity to difference calcium channels with these two drugs. Nonselective cation channel type 1(NSCC-1) is not sensitive to SK&F96365, however, sensitive to LOE908. Store-operated calcium channel(SOCC) is sensitive to SK&F96365, but not sensitive to LOE908. Therefore, the cardioprotective effect enhanced by blocking the high concentration of endothelin-1 by SK&F96365 is viable and keep the vessel tension to reduce no-reflow or reflow. And LOE908 could not achieve this effect as its weak blocking effect(10%).Base on these theories, we build up a model of cardiac ischemia reperfusion in vivo New Zealand rabbits to determine the effect of SK&F96365 and LOE908 to the level of blood plasma ET-1 in the plasma and to compare protective effects between SK&F96365 and LOE908 to ischemia/reperfusion injury.Cardioprotective effects between SK&F96365 and LOE908 to ischemia/reperfusion injury in vivo rabbitsObjectiveThe aim of this study was to determine the effect of SK&F 96365 and LOE908 to ET-1 and whether they had a cardioprotective effect against ischemia/reperfusion injury in vivo New Zealand rabbits and compare these effects in different time point after reperfusion.Methods1. AnimalsA total of 24 animals were used in our experiments here. Male New Zealand healthy rabbits (adult, weight 2.0-2.5kg) were obtained from Laboratory Animals Center of Southern Medical University. The animal use and care protocols were all approved through the Committee on Ethical Use of Animals at the Guangdong Academy of Medical Sciences. During the experiments, all efforts were taken to lessen animal suffering and the number of animals used.2. Ischemia and reperfusion model in vivo and groupsThe New Zealand rabbits were divided at random into 4 group(6 rabbits in each one group):(1)Sham group; (2)ischemia/reperfusion(I/R) group; (3)SK&F96365 group; (4) LOE908 group. All the rabbits were anaesthetized with pentobarbital sodium (30 mg/kg). During the surgical procedure, rabbits were artificially ventilated (35 cycles/kg, tidal volume 10 mL air/kg) with a animal ventilator and a venous cannula was inserted into right internal jugular venous after exposing the right internal jugular venous to administrate drugs and collect blood samples. Intercostals thoracotomy was performed at the left side of sternum between third rib and fourth rib to expose the heart. After 10min adaptive phase for the heart,4-0 silk thread was passed through up 1/3 of left anterior descending coronary artery to make an occlusion. The reperfusion was achieved through removing the silk thread. For the Sham group:after 10min stabilization for the heart,4-0 silk thread was just passed through LAD but not ligate for 30min, after that the silk thread was removed. We collected blood sample from the right internal jugular venous to detect LDH, CKMB, ET-1 after reperfusing 30min,60min and 120min. The total time was 160min for the heart. For the I/R group, after 10min stabilization, all rabbits were subjected to 30min ischemia and then followed by 120min reperfusion. We collected blood sample from the right internal jugular venous to detect LDH, CKMB, ET-1 after reperfusing 30min, 60min and 120min. For SK&F96365 group, based on I/R group, SK&F96365 was injected into vein at the onset of reperfusion and collected blood sample from the right internal jugular venous to detect LDH, CKMB, ET-1 after reperfusing 30min, 60min and 120min. For LOE908 group, LOE908 was administrated the same as SK&F96365 group.3. Index observationsAfter reperfusion, the levels of LDH、CK-MB and ET-1 were tested in plasma after reperfusing 30min,60min and 120min. The heart was used to examed the infarct size in TTC at the end of experiment.4. Statistical analysisAll data were statistically analyzed by the software of SPSS 19.0 and showed as mean±SD(x±s). One way ANOVA followed by the LSD-test (homogeneity of variance) or Dunnett-T3 test (heterogeneity of variance) was used for multiple comparisons between groups. The significance level was set at 0.05.Results1. The effect to the myocardial enzymeAfter 30min reperfusion, the level of plasma LDH (1961.5±59.94 U/L) and the level of plasma CK-MB(1069.5±48.62 U/L) were siginificantly increased (P=0.000) in the I/R group when compared with Sham group(P=0.000 and P=0.000 respectively). It showed a successful operation about building the schemia reperfusion injury model as the myocardial enzymes rised up as expectant. Compared with I/R group, the level of plasma LDH (1458.33±65.53 U/L and 1468.83±36.30 U/L) and the level of plasma CK-MB (724.83±22.60 U/L and 732.67±11.20 U/L) in the SK&F96365 and LOE908 group decreased by administration of SK&F 96365 and LOE908, it was statistically significant (P=0.000 and P=0.000 respectively). However, there was no difference between SK&F96365 group and LOE908 group about the level of plasma LDH and the level of plasma CK-MB (P= 0.71 and P=0.77, respectively).After 60min reperfusion, compared with I/R group, the level of plasma LDH (1535.83±68.99 U/L and 1486.33±41.05 U/L) and the level of plasma CK-MB (1151.17±55.62U/L and 1166.67±49.06 U/L) in the SK&F 96365 and LOE908 group decreased by administration of SK&F 96365 and LOE908, it was statistically significant (P=0.000 and P=0.000 respectively). However, the difference between SK&F96365 group and LOE908 group about the level of plasma LDH and the level of plasma CK-MB was not statistically significant (P= 0.13 and P=0.54, respectively).After 120min reperfusion, compared with I/R group, the level of plasma LDH (1594.83±41.18 U/L and 1871.33±30.61 U/L) and the level of plasma CK-MB (1472.33±137.24 U/L and 1732.17±38.18 U/L) in the SK&F 96365 and LOE908 group decreased by administration of SK&F 96365 and LOE908, it was statistically significant (P=0.000 and P=0.000 respectively). Moreover, the difference between SK&F96365 group and LOE908 group about the level of plasma LDH and the level of plasma CK-MB was statistically significant (P=0.000 and P=0.000 respectively).2. The increase of the level of ET-1 in plasmaAfter 30min reperfusion, compared with Sham group (425.15±9.22 pg/ml), the levels of plasma ET-1 in the other three groups were siginificantly increased (P=0.000 among all). After 60min reperfusion, compared with Sham group (424.63± 5.46 pg/ml), the levels of plasma ET-1 in the other three groups also increased and statistically significant (P=0.000 among all). After 120min reperfusion, compared with Sham group (426.33±6.40 pg/ml), the levels of plasma ET-1 in the other three groups also increased and statistically significant (P=0.000 among all).3. The effect to the infarct sizeComparing to Sham group, the myocardial infarct size (63.55±3.53%) increased after inschemia and reperfusion. And it decreased after the use of SK&F 96365 (33.02±3.24%) and LOE908 (43.58±1.49%). It was statistically significant (P=0.000 and P=0.000 respectively). Moreover, the difference between SK&F96365 group and LOE908 group about the infarct size was statistically significant (P=0.000). The results were consistent with that in myocardial enzyme.Conclusions for this paper1.The level of plasma endothelin-1 increased markedly after myocardial ischemia reperfusion in New Zealand healthy rabbits.2.Two different calcium antagonists SK&F96365 and LOE908 both provided a protective effect against ischaemia reperfusion injury in some degrees in this study.3.There was no difference between these two different calcium antagonists against ischaemia/reperfusion injury at the time point after reperfusing 30min and 60min. After 120min reperfusion, the cardioprotection effect of SK&F96365 was better than LOE908. The mechanism of cardioprotection from SK&F96365 and LOE908 was not clearly known, Continue in-depth studies were needed.