| PerfaceATP-sensitive potassium channel(KATP channel) is affected by the concentration of intracellular ATP and many other factors that regulate potassium(K+) channel,KATP channel can be divided into two categories:one is the cell member ATP-sensitive K+ channel; the other is mitochondrial ATP-sensitive K+(mitoKATp)channel. In recent years, a large number of researches suggest that mitoKATP channel has a protective effect in myocardial ischemia-reperfusion injury[1][2]. nicorandil,as a mitoKATP channel opener, could open mitochondrial membrane ATP-sensitive potassium channel, has a protective effect on the heart.Na+/Ca2+exchanger protein(NCX),as a kind of sugar protein, presents in the cell membrane, the NCX1 subtype of Na+/Ca2+exchanger is a non-ATP-dependent bi-directional transport protein, plays an important role in the concentration of intracellular Ca2+regulation[3]. In pathological conditions such as ischemia-reperfusion injury, sodium-calcium exchange has led to intracellular calcium overload, resulting in abnormal cardiac function.KB-R7943 is a specific NCX1 inhibitor[4][5][6]. nicorandil and KB-R7943 combination may be synergistic, which could reduce the myocardial infarct size, but the two combined with how it works is still unclear.Materials and Methods1. Established the Langendorff perfusion model of isolated rat hearts:Rat heart was linked to Langendorff perfused device with the full perfusion 15min, we started the experiment when left ventricular filling pressure and flow rate stability, selecting 45min incomplete ischemia (about left and right coronary perfusion flow reduced irrigation flow of the original 5%) and 2h reperfusion time. 2. Detected SOD activity and MDA content in coronary perfusion fluid by spectrophotometer:Selecting the end of reperfusion time point in each group, as the observation point, 1ml coronary effluent were collected in 1minute,a spectrophotometer and ancillary reagents measured SOD activity, MDA content.3. Analyzed the myocardial infarct size using BI-2000 image analytical system: Removing the heart into the-20℃refrigerator freezed 1h the end of the experiment, and then the heart was cutting to lmm intervals cross-sectional slices, placed in 37℃TTC staining in 30 min, and then placed in 4% paraformaldehyde fixed 24h, go to our school facilities Department to conduct physical image acquisition, infarct area (TTC unstained white area) and non-infarct zone (TTC stained pink area), then BI-2000 image analysis system to calculate myocardial infarct size, using statistical analysis software SPSS 16.0 in each group.4. Observed the ultra structure of rat myocardium by transmission electron microscope:Removing the heart the end of the experiment, the left ventricle before the lower part of the apex along the filaments towards taking lmm×lmm×lmm size of the heart into the middle of a 2.5% glutaraldehyde fixation,1% osmium tetroxide post-fixed, The series of acetone dehydration, epoxy resin embedding, ultrathin sections,1% uranyl acetate and lead citrate double staining,,with JEOL transmission electron microscope (JEM-1200EX) under observation, radiography.Result1. SOD activity in coronary perfusion fluid was 5.298±2.968 (U/ml) in control group; 7.025±3.725 (U/ml) in nicorandil group; 5.567±3.146 (U/ml) in KB-R7943 group; and 10.969±3.503 (U/ml) in combination of nicorandil and KB-R7943 group.2. MDA content in coronary perfusion fluid was 0.449±0.123(nmol/ml) in control group; 0.329±0.078(nmol/ml) in nicorandil group; 0.282±0.150 (nmol/ml) in KB-R7943 group; and 0.109±0.081 (nmol/ml) in combination of nicorandil and KB-R7943 group.3. The myocardial infarct size was 34.31%in control group; 26.35%in nicorandil group; 28.74% in KB-R7943 group; and 19.23% in combination of nicorandil and KB-R7943 group.4. Myocardial electron microscopy:myocardial focal sarcoplasmic coagulation, focal necrosis, etc., dissolved filaments, the majority of mitochondrial swelling, vacuolization, mitochondrial cristae broken cristae reduced; nuclear chromatin condensation; glycogen granules decreased; muscle cells swelling significantly in control group; the normal structure of muscle cells, the sarcomere is clear, a small number of filaments focal dissolved; mitochondria slightly swollen, cristae clear; a small number of sarcoplasmic reticulum dilation in combination drug group. Nicorandil group, KB-R7943 group of electron micrographs of the results between the control group and between the combination drug group.Combination drug group compared with the control group, myocardial ultrastructural injury significantly reduced.ConclusionThe isolated rat heart perfused by modified Langendorff device, after 15-minute balanced perfusion,45-minute ischemia (about left and right coronary perfusion flow reduced to 5% of the original irrigation flow), and 2-hour reperfusion were performed. SOD activity in coronary perfusion fluid is the highest in the combination of nicorandil and KB-R7943 group, and MDA content is the lowest, and myocardial infarct size is least. Combination drug group compared with the control group, myocardial ultrastructural injury significantly reduced. Hence, the combination of nicorandil and KB-R7943 significantly reduced myocardial infarct size, significantly reduced myocardial ultrastructural damage, can increase coronary perfusion fluid SOD activity, reduced MDA levels.
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