中文摘要：

目的探讨胰岛素（INS）在抑制缺血再灌注损伤（IRI）诱导的心肌微血管内皮细胞（CMECs）凋亡中的作用及其分子机制。方法分离和培养大鼠CMECs,建立模拟缺血再灌注（SI/R）模型,分别选用INS、磷脂酰肌醇-3-激酶（PI3K）特异性抑制剂渥曼青霉素（Wort）和Fox O1/3a干扰RNA处理细胞。采用随机数字表法对大鼠CMECs进行分组：SI/R组（5只大鼠的CMECs）,SI/R＋INS组（5只大鼠的CMECs）,SI/R＋INS＋Wort组（5只大鼠的CMECs）,SI/R＋INS＋Fox O1siRNA组（6只大鼠的CMECs）,SI/R＋INS＋Fox O3asiRNA组（4只大鼠的CMECs）。采用末端转移酶标记技术（TUNEL）检测各组CMECs凋亡率,ELISA法检测各组Caspase-3活性,Western blotting法检测各组p-Akt（Ser473）、p-Fox O1（Ser256）、p-Fox O3a（Thr32）、生存素（SVV）蛋白表达水平。结果与SI/R组比较,SI/R＋INS组、SI/R＋INS＋Fox O1siRNA组、SI/R＋INS＋Fox O3asiRNA组CMECs凋亡率及Caspase-3活性均降低（P〈0.01）;而SI/R＋INS＋Wort组CMECs凋亡率及Caspase-3活性与SI/R组比较,差异无统计学意义（P〉0.05）;与SI/R＋INS组比较,SI/R＋INS＋Fox O1siRNA组、SI/R＋INS＋Fox O3asiRNA组CMECs凋亡率及Caspase-3活性升高（P〈0.01）;且SI/R＋INS＋Fox O1siRNA组CMECs凋亡率高于SI/R＋INS＋Fox O3asiRNA组（P〈0.01）。Western blotting检测结果显示：SI/R＋INS组p-Akt、p-Fox O1、p-Fox O3a、SVV蛋白表达水平均高于SI/R、SI/R＋INS＋Wort组、SI/R＋INS＋Fox O1siRNA组以及SI/R＋INS＋Fox O3asiRNA组（P〈0.01）;且SI/R＋INS＋Fox O3asiRNA组SVV蛋白表达水平低于SI/R＋INS＋Fox O1siRNA组（P〈0.01）。结论 NIS可显著抑制IRI诱导的CMECs凋亡,其机制可能与激活Akt/Fox O/SVV信号通路相关,且主要通过Fox O3a的激活上调SVV蛋白表达水平,从而抑制CMECs凋亡。

英文摘要：

Objective To explore the effect and mechanisms of insulin（ INS） inhibition of apoptosis of microvascular endothelial cells（ CMECs） induced by ischemia reperfusion injury（ IRI）. Methods CMECs were isolated from the hearts of adult rats to establish IRI model. INS,Wortmannin（ Wort） and Fox O1 /3asiRNA were used to process the cells. The CMECs were randomly divided into SI / R group（ CMECs of 5 rats）, SI / R ＋ INS group（ CMECs of 5 rats）, SI / R ＋ INS ＋ Wort group（ CMECs of 5 rats）,SI/R ＋ INS ＋ Fox O1 siRNA group（ CMECs of 6 rats） and SI / R ＋ INS ＋ Fox O3 asiRNA group（ CMECs of 4rats）. TUNEL was used to detect the apoptosis rate of CMECs in each group. ELISA was used to detect the activity of Caspase- 3in each group. Western blotting method was used to detect the levels of p- Akt（ Ser473）,p- Fox O1（ Ser256） and p- Fox O3a（ Thr32） and the protein expression of SVV. Results Compared with SI/R group,apoptotic index and Caspase- 3 activity were significantly lower in groups of SI / R ＋ INS, SI / R ＋ INS ＋ Fox O1 siRNA, SI / R ＋ INS ＋ Fox O3asiRNA（ P〈0. 01）; but the apoptotic index and Caspase- 3 activity in SI / R ＋ INS ＋ Wort group showed no significant difference compared with SI / R group（ P〈0. 05）; compared with SI/R ＋ INS group,apoptotic index and Caspase- 3 activity was significantly increased in groups ofSI / R ＋ INS ＋ Fox O1 siRNA and SI / R ＋ INS ＋ Fox O3 asiRNA,and the apoptotic index in SI / R ＋ INS ＋ Fox O1 siRNA group was significantly higher than that of the SI / R ＋ INS ＋ Fox O3 asiRNA group（ P〈0. 01）. Western blotting analysis showed that p- Akt level,p- Fox O1 level,p- Fox O3 alevel and SVV protein expression in SI / R ＋ INS group were significantly higher than groups of SI / R,SI / R ＋ INS ＋ Wort,SI / R ＋ INS ＋ Fox O1 siRNA and SI / R ＋ INS ＋ Fox O3asiRNA（ P〈0. 01）; and the SVV protein expression in SI / R ＋ INS ＋ Fox O3 asiRNA group was significantly lower than that