失血复合缺氧缺水缺食诱导心肌细胞凋亡及相关机制研究

Blood loss,hypoxia,water and diet depletion induce apoptosis in cardiomyocytes in rats and its molecular mechanism

目的定量研究失血复合缺氧缺水缺食情况下心肌细胞的凋亡及其机制。方法二级WISTAR大鼠160只,随机分为正常对照、单纯失血、缺氧缺水缺食、失血复合缺氧缺水缺食4组,每组40只,其中失血模型为剪尾法制备,失血量30%;缺氧组为将动物置于常压低氧舱内,氧浓度10%。所有动物于造模后1 d、3 d、5 d、7 d活杀取材,各时间点每组均为10只,雌雄各半;采用TUNEL(原位末端标记法)法检测心肌细胞凋亡,免疫组化SP法检查心肌组织中凋亡相关因子Bax、Bcl-2、caspase-3及核转录因子CREB的表达并进行积分光密度(IOD)和平均光密度(MOD)定量检测。结果单纯失血组、缺氧缺水缺食(三缺)组和失血并三缺组心肌细胞凋亡指数于入低氧舱1 d、3 d、5 d、7 d均显著高于正常对照组(P<0.01),其中失血并三缺组于早期较其他两组尤为明显(P<0.01);各时间点Bax和caspase-3表达均呈增强、MOD值增高趋势,其中早期失血并三缺组较失血组和三缺组更为明显(P<0.05);Bcl-2均呈表达减弱、MOD值降低趋势,尤其5 d～7 d时较明显(P<0.01);CREB于3 d、7 d呈现不同程度的表达增强(P<0.05),上述凋亡相关因子表达变化呈现失血并三缺组依次较三缺组、单纯失血组加重趋势。结论失血或三缺均导致大鼠心肌细胞凋亡指数增加,失血复合三缺可明显加重其上述变化;凋亡相关因子Bax、Bcl-2、caspase-3和CREB均参与其损伤过程。

Objective To investigate the effects of blood loss,hypoxia,water and diet depletion on apop-tosis in cardiomyocytes in rats and its molecular mechanism.Methods 160 healthy adult WISTAR rats（male ： female = 1∶ 1） were divided into 4 groups： control group,blood loss group,hypoxia ＋ water depletion ＋ diet depletion group（three lack group） and blood loss ＋ hypoxia ＋ water depletion ＋ diet depletion group（blood loss ＋ three lack group）.The blood loss model was established by cutting tail.Normobaric hypoxia chamber designed and manufactured by our studying team was used to make the oxy-gen concentration to 10%.The experiment data were recorded on days 1,3,5 and 7.The apoptosis in-dex of cardiomyocytes and expression of apoptotic factors（Bax,Bcl-2,caspase-3 and CREB） were ana-lyzed by immunohistochemistry.The IOD and MOD value of apoptotic factors were detected quantiatly.Results The apoptosis index of cardiomyocytes was significantly higher in the experimental groups than that in the control group（P 0.01）.The expression of Bax and caspase-3 was increased at day 1 to 7,especially in the blood loss ＋ three lack group,and the values of MOD and IOD increased.However,that of Bcl-2 was decreased.Varying increase of CREB（cAMP response element-binding） and Caspase-3 protein were observed,and the values of MOD and IOD increased,especially significantly at early time in three lack group and blood loss combined with three lack group.Conclusions Under the condition of blood loss combined with three lack group,the apoptosis of myocardial cells correlated with the increase of promoting apoptotic factor（Bax） and the decrease of anti-apoptotic factor（Bcl-2）.Furthermore,it might release cytochrome C by mitochondria pathway,activate Caspase-3 and initiate apoptosis of myocardial cells.The increase of p-CREB took part in the process of apoptosis of myocardial cells.