摘要
目的探讨抑制自噬后对大鼠乳鼠心肌细胞缺氧诱导的促凋亡蛋白Bim、caspase-3表达的影响及低氧诱导因子1(HIF-1)的调控作用。方法体外培养1~3 d龄SD大鼠心肌细胞建立缺氧模型,检测缺氧0、2、4、8、14和24 h时自噬情况,取自噬显著升高的时间点细胞作为单纯缺氧组(anoxia组),缺氧前用10 mmol/L3-甲基腺嘌呤(3MA)预处理心肌细胞1h作为缺氧+3-甲基腺嘌呤组(anoxia+3MA组),设立正常对照组(control组)。倒置显微镜下观察各组心肌细胞的搏动频率和异常节律;全自动血液生化分析仪检测乳酸脱氢酶LDH的活性;Western blot检测各组中LC3-Ⅱ/Ⅰ、Bim、caspase-3和HIF-1蛋白表达情况。结果抑制自噬后心肌细胞搏动频率明显减弱并可见异常节律、LDH释放增加(P<0.05),同时Bim和caspase-3表达明显升高(P<0.05);缺氧+3MA组中HIF-1蛋白表达明显低于单纯缺氧组(P<0.05)。结论自噬抑制剂抑制自噬后导致缺氧诱导的大鼠乳鼠心肌细胞凋亡增加,HIF-1正调控缺氧诱导的自噬抵抗凋亡发挥心肌保护作用。
Objective To investigate the potential roles of autophagy induced by anoxia in effecting on expression of Bim and caspase-3 and tries to clarify the regulati anoxia of neonatal rat ventricular myocytes as an on of HIF-I in this process. Methods We employed simulated in vitro model of ischemia injury to the heart. Cardiac myocytes were exposed to 0,2,4,8,14 and 24 h anoxia. Cardiac myocytes were pretreated with 10 mmol/L 3-Methylade- nine (3 MA) to inhibit autophagy. The beating rate and arrhythm of myocardial cells were detected by inverted microscope. The activity of lactate dehydrogenase was determined by automatic biochemistry analyzer. Western blot analysis was used to examine variation in the expression of LC3- ]I / I ( a marker of autophagy), Bim, caspase-3 and HIF-1. Results As the beating rate of myocardial cells was slightly decreased, and the cell ar- rhythm was increased (P 〈0. 05 ) after inhibition autophagy; the activity of lactate dehydrogenase was increased (P 〈 0. 05 ) when inhibiting autophagy. Moreover, significant autophagy was reduced by pretreating with 3-Methy- ladenine (3 MA) in anoxia, and the expression of Bim and caspase-3 was significantly increased (P 〈 0. 05). The expression of HIF-1 was significantly increased in anoxia group and reduced in anoxia +3MA group (P 〈 0. 05).Conclusions Inhibition of autophagy constitutes a powerful and previously uncharacterized apopotosis induced by anoxia in cardiac myocytes via upregulating the pro-apopototic protein Bim and caspase-3. And HIF-1 positively regulated the process of autophagy induced by anoxia.
出处
《基础医学与临床》
CSCD
北大核心
2014年第2期185-189,共5页
Basic and Clinical Medicine
基金
国家十二五科技支撑计划(2013BAI05B10)