| IntroductionIncidence of cardiovascular disease in old - aged phase is significantly increasing. Cardiac myocyte apoptosis is programmed cell death similar to the other tissue cells in body, playing the important role in the course of normal myocar-dial development and pathogenesis of many cardiovascular diseases. Aging is a kind of degenerative change of body during vital process. There have been some unclear fields and arguments on the topic of aging. In recent years, a lot of studies have indicated that oxidative stress involved in regulating cardiac apoptosis, and the nature of aging cell death was cell apoptosis. Discussing the relationship between oxidative stress and myocardial apoptosis or aging is very important to study pathogenesis of aging and cardiovascular diseases.In this paper, we established the cardiac ischemia - reperfusion injury model of young and aged Wistar rats to investigate changes of ROS activity, Mn - SOD content and the expression of apoptotic factor Bax and Bcl - 2 protein in various groups, and further discuss their differences and relationship, providing theory basis of pathogenesis of cardiovascular diseases and aging for clinic application.Materials and Method1. Animal groupsTotal 48 Wistar rats including 24 rats aged 10 weeks old as young group, and 24 aged 18 months old as aged group, were provided by Animal department of China Medical University, and divided into young control group ( YCG, n =12) , young model group ( YMG, n = 12) , aged control group ( ACG, n = 12) , and aged model group ( AMG, n = 12 ). The ischemia - reperfusion models were made through ischemia for 45min, followed by reperfusion for 90min.2. Specimen disposingAll the cardiac tissues were obtained from the controls and model groups, among which ischemia - reperfusion regions were dissected. Each case of tissue was cut into two portion: one was put into 4% paraformaldehyde, fixed for 24 hours and embedded with paraffin, applying to HE stain, immunohistochemistry (IHC) detection of Bax and Bel - 2 proteins and apoptotic detection via TUNEL; the other was put immediately into fluid nitrogen and then transferred to - 70 t refrigerator for determining MDA content and Mn - SOD activity.3. Determination of MDA content and Mn - SOD activity of cardiac myo-cyteCardiac tissues preserved in fluid nitrogen were grinded into power, homog-enated in ice -cold saline, and then centrifuged at 3000rpm for 10min, finally kept the supernatant for determination of MDA content and SOD activity. Quantities of malomdialdehyde (MDA) and activities of superoxide dismutase (SOD) were determined with thiobarbituric acid reaction (TBA) and nitriteform method, respectively.4. Detection of apoptotic factor Bax , Bcl -2 proteinsThe expression of Bax and Bel - 2 proteins was determinated with PV -9000 immunohistochemistry method, and the detailed procedure referred to the instruction of PV - 9000 kit ( Maixin Corp. China). Simultaneously, PBS as negative control substituted primary antibody. Five high - power fields of each section were obtain to determinate optical density of Bax or Bel - 2 protein via computer image analysis system, that is, every group with 12 sections has total 60 fields5. Detection of myocardial apoptosis by TUNEL methodTUNEL method was subjected to detecting the myocardial apoptosis. The detailed procedure refers to the instruction of TUNEL kit ( Boster Corp. China). PBS as negative control substituted primary antibody. Four random high - power fields of each section were obtained to determinate the apoptosis index ( AI).6. Statistic analysisAll data was express as Mean ± SD ( x ± s ). SPSS window 11.0 software was used to analyze variance,P less than 0.05 indicates statistical correlation,P less than 0.01 indicates significantly statistic correlation.Results1. MDA content and Mn - SOD activities in various groupsThe myocardial SOD activities in ACG were significantly lower than those in YCG (P<0.01); the activities of myocardial SOD in AMG were significantly lower comparing to YMG ( P < 0.01). Model group s SOD activities were significantly higher than those in control group ( P < 0. 01 ). The myocardial MDA contents in AMG were significantly higher comparing to ACG, but there was no statistic difference in the MDA contents between YMG and YCG ( P > 0. 05 ). The ratio of MDA to SOD in AMG was significantly higher than that in ACG (P <0.05).2. Expression of Bax and Bel -2 proteins in various groupsBel - 2 and Bax protein were both stained with brown particles, and located in myocardial cytoplasm, compared with young YCG, Bel - 2 protein expression in YMG obviously decreased,P <0.01. In addition, Bel -2 expression in ACG and AMG were higher than that in YCG,P <0.01 o Bax expression in YCG was significantly lower than that in ACG and AMG,P <0.01. Compared with ACG, protein expression of Bax increased, P <0.05.Comparison of apoptosis in various groups3. Apoptotic myocytes was stained brown in nucleus by TUNEL companied with karyopyknosis, and could be detected in YMG, AMG and ACG. The apoptosis index( AI) in YMG, AMG and ACG was higher than YCG,P <0.01,and there was significant difference in AI among YMG, AMG and ACG, P < 0. 05, among which AI in AMG was the highest. |
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