| Objectives:1. To observe the myocardial apoptosis in neonatal rats after asphyxia by establishing the model of neonatal rats with normobaric asphyxia.2.To observe the expression of myocardial calpain-1 and Bcl-2 in neonatal rats after asphyxiation.3.To study the effect of Leptin on myocardial injury and the expression of myocardial calpain-1 and Bcl-2 in neonatal rats after asphyxia,and to explore its anti-apoptotic mechanism. Methods:1. Group and Model establishment:Total 48 newborn Sprague-Dawley(SD) rats were randomly divided into following groups:normal control group,asphyxia group,leptin treatment groups(group L20,group L80 and group L160) and calpain inhibitor-1 group(group CAI-1). establishing asphyxia of neonatal rats model except the normal control group, the leptin treatment groups were given intraperitoneal injection with different does of leptin(20μg/kg,80μg/kg,160μg/kg)and the CAI-1 group was given intraperitoneal injection with does of calpain inhibitor-1 10mg/kg immediately after establishing the model,Then the myocardial tissue samples were obtained after reoxygenation 120 min.2.Observation:to observe calpain-1 and Bcl-2 expression changes in myocadial tissue, to use TUNEL analysis method to detect myocardial cell apoptosis and apoptosis index(AI) and myocadial tissue pathology to investigate the influence and mechanism of leptin on myocardial cell apoptosis of asphyxial neonatal rats. Results:1. The HE staining of myocadial tissue: myocadial cells of normal control group were arranged in rules and neat, the cytoplasm in uniform light dye, While compared to asphyxia group,myocadial cells were irregular arranged, a portion of cell nucleus fracture or disappear, obviously a lot of inflammatory cells infiltration,interstitial hyperemia and edema, the above changes were significantly reduced in leptin treatment groups(group L20, group L80 and group L160) and group CAI-1.2. Calpain-1 expression in myocadial tissue: compared to the normal control group,the calpain-1 levels in asphyxia group, leptin treatment groups(group L20, group L80 and group L160) and group CAI-1 were significantly rise(P<0.05),IOD(×103) were respectively(31.18 ± 3.56),(99.70 ± 9.56),(84.95 ± 8.66),(65.07 ±10.15),(50.34±6.74),(43.56±5.34);While compared to asphyxia group, the calpain-1 levels were reduced in leptin treatment groups(group L20, group L80 and group L160) and group CAI-1(P<0.05);Under 20μg/kg-160μg/kg leptin treatment, the myocardial tissue of asphyxial neonatal rats calpain-1 levels were shown decreased by concentration dependent;the calpain-1 levels shown no significant differences between leptin group(group L160) and group CAI-1(P>0.05).3. Bcl-2 expression in myocadial tissue: compared to the normal control group, the Bcl-2 levels in asphyxia group, leptin treatment groups(group L20, group L80 and group L160) and group CAI-1 Were significantly rise(P<0.05),IOD(×103) were respectively(5.95 ± 2.39),(17.35 ± 5.19),(30.35 ± 3.92),(48.71 ± 4.35),(57.76 ±5.35),(62.34 ± 5.63);While compared to asphyxia group, the Bcl-2 levels were enhanced in leptin treatment groups(group L20, group L80 and group L160) and group CAI-1(P<0.05);Under 20μg/kg-160μg/kg leptin treatment, the Bcl-2 levels were shown increased by concentration dependent(P<0.05);the Bcl-2 levels shown no significant differences between leptin group(group L160) and group CAI-1(P>0.05).4. Results of myocardial cell apoptosis index(AI): compared to the normal control group, the AI levels were increased significantly in the rest groups(P<0.05),AI(%) were respectively(2.73±0.47),(25.04±5.18),(19.97±1.70),(14.55±1.24),(8.27±3.37),(6.60±1.72);While compared to asphyxia group, the AI levels were dropped between leptin treatment groups(group L20, group L80 and group L160) and group CAI-1(P<0.05);Under 20μg/kg-160μg/kg leptin treatment, the AI levels were shown decreased by concentration dependent(P<0.05);however, the AI levels shown no significant differences between leptin group(group L160) and group CAI-1(P>0.05).5. Correlation analysis:1)The expression of calpain-1 was positively related to AI in asphyxia group,leptin treatment groups(group L20, group L80 and group L160) and group CAI-1,(r=0.73,0.77,0.79,0.81,0.87, P<0.05)2)The expression of Bcl-2 was negatively related to AI in asphyxia group,leptin treatment groups(group L20,group L80 and group L160) and group CAI-1,(r=-0.84,-0.78,-0.82,-0.73,-0.83,P<0.05)3)The expression of calpain-1 was negatively related to the Bcl-2 in asphyxia group,leptin treatment groups(group L20, group L80 and group L160) and group CAI-1,(r=-0.71,-0.80,-0.81,-0.86,-0.89,P<0.05) Conclusion:1.When neonatal rats caused by asphyxia, the expression of calpain-1 and Bcl-2 in myocadial tissue increased and myocardial cell apoptosis enhanced.2.Leptin could inhibit the activation of calpain-1 and elevate Bcl-2 expression, thereby reducing myocardial cell apoptosis of asphyxial neonatal rats and this kind of action presents a certain concentration dependence under 20μg/kg-160μg/kg leptin treatment. |
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