| Objective: To study the protective effects and mechanisms of Caspase-3-siRNA preconditioning on myocardial ischemia-reperfusion injury(MIRI) in rats.Methods: 78 male SD rats were randomly divided into 4 groups: Sham group, Control group, Scrambled-siRNA group and Caspase-3-siRNA group. In Caspase-3-siRNA group and the Scrambled-siRNA group: We made rat MIRI model through left anterior decending coronary artery ligation reversibly. Caspase-3-siRNA or Scrambled Caspase-3-siRNA solution was injected intravenously into rats at 24h, 12h and immidiately before myocardial ischemia. In Control group and Sham group:The method to make rat MIRI model is the same as above, but the left anterior descending coronary artery (LAD) was false ligated in Sham group. PBS was injected intravenously into rats with the same way as above. Reperfusion took place after the left anterior descending coronary artery occlusion 30 min. Collecting the blood from the abdominal aortic to detect the activity of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) after reperfusion 2h, 6h, 12h, 24h. Rats were killed for detecting the apoptosis index (AI) of myocardial cells at the same time points by TUNEL. The desuperoxide dismutase (SOD) activity and malondialdehyde (MDA) content from the cardiac myocytes were detected after reperfusion 24h. The expression of Caspase-3 mRNA was assayed by the methods of reverse transcription polymerase chain reaction (RT-PCR) and its protein in cardiac myocytes were assayed by the methods of Western blot. Optical microscopy and transmission electron microscopy were used to observe the pathological changes of myocardial tissue and cells respectively. The rats in Sham group were killed to detect the same targets as part of an overall control at the only time point of 24h.Result:(1) With the time of reperfusion, except the Sham group, the activity of CK-MB and LDH in serum gradually increased. CK-MB activity reached the highest point at 6h after reperfusion and declined during the period of reperfusion 12h to 24h. LDH activity reached the highest point at 12h and remained high level at 24h after reperfusion. The levels of CK-MB and LDH activity in the Caspase-3-siRNA group were significantly lower than Control group or the Scrambled-siRNA group (P<0.05), but there was no significant difference between Scrambled-siRNA group and Control group (P> 0.05). (2) The results of TUNEL showed that cardiomyocyte apoptosis index (AI) had an increasing trend during the period of reperfusion and reached the highest point at 12h after reperfusion, and also remained high level at 24h after reperfusion. AI in the Control group and Scrambled-siRNA group were significantly higher than Caspase-3-siRNA group (P <0.05 or 0.01), but there was no significant difference between Scrambled-siRNA group and Control group (P> 0.05). (3) SOD activity in Caspase-3-siRNA group was significantly higher than the Control and Scrambled-siRNA group (P<0.05), while the MDA content was significantly lower than the Control and the Scrambled-siRNA group (P<0.05), but there was no significant difference between Scrambled-siRNA group and Control group (P> 0.05). (4) The results of RT-PCR showed that the expression of caspase-3 mRNA in Caspase-3-siRNA group was significantly decreased than the Control and the Scrambled-siRNA group (P<0.05), but there was no significant difference between Scrambled-siRNA group and Control group (P> 0.05). (5) The results of Westem blot showed that the expression of Caspase-3 protein can be found in all the groups. In the Caspase-3-siRNA group, protein expression of Caspase-3 became obviously lower than that of the Control group and the Scrambled-siRNA group at the 12h time point(P<0.05), and there was no significant difference between Scrambled-siRNA group and Control group (P>0.05). The results suggested that the Caspase-3-siRNA successfully inhibited the caspase-3 gene. (6) The change of myocardium under a light microscope: Sham group: Myocardial fibre arranged in order, the structural integrity of the nucleus, myocardial was normal. Control group: myocardial fiber arranging disorderly, irregular nucleus, cytoplasm staining strongly, apparent interstitial edema, and infiltration of inflammatory cells. The pathological change of myocardial tissue in Scrambled-siRNA group was similar to the Control group. The myocardial injury in Caspase-3-siRNA group was milder than Control and Scrambled-siRNA group. Myocardial fibre relatively arranged in order. Interstitial edema was not obvious, we can see a small number of inflammatory cell. (7) Transmission electron microscopy demonstrated that the myocardial ultrastructure in Sham group was normal with parallel myofibril, clear sarcomere , consistent patterns of mitochondrial and structural integrity of the nucleus, while in the Control group and the Scrambled-siRNA group we can serious injury with myofibrils disorder and fracture , shrinkage of mitochondria, cristae dissolution, nuclear chromatin margination and so on. The structural damage in Caspase-3-siRNA group was relatively less than Control and Scrambled-siRNA group. We can see more clear sarcomeres, no significant dissolution, no fracture, almost normal mitochondria and clear nucleolus.Conclusion: (1) Myocardial ischemia-reperfusion injury induced the cardiomyocyte apoptosis, Caspase-3 involved in the process of apoptosis and played an important role. (2) Caspase-3-siRNA preconditioning can protect cardiomyocyte from ischemia reperfusion injure. The mechanism may be associated with that the Caspase-3-siRNA can specially suppress expression of caspase-3 gene and reduce the occurrence of cardiomyocyte apoptosis. |
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