胰岛素对新生大鼠缺氧/复氧诱导心肌细胞凋亡的影响

Protective effect of insulin on anoxia/reoxygenation-induced apoptosis in cultured cardiomyocyte of neonatal rats

目的探讨胰岛素对缺氧/复氧所诱导心肌细胞凋亡的影响及其机制。方法通过给原代培养乳鼠心室肌细胞行缺氧2h/复氧4h,建立缺氧/复氧(anoxia/reoxygenation)心肌细胞损伤模型。于复氧期开始随机给予0.9%生理盐水(VC组)、胰岛素(INS组)、LY294002(LY组)、胰岛素+LY294002(INS+LY组)干预。于复氧4h后,利用2,4二硝基苯肼显色法检测乳酸脱氢酶(LDH)活性,硫代巴比妥酸显色法检测丙二醛(MDA)含量,原位末端标记法(TUNEL)和DNA梯带法(DNALadder)标测细胞凋亡,免疫印迹法(Westernblotting)检测磷酸化Akt表达,并比较各组间差异。结果与VC组相比,INS组中LDH活性、MDA含量、凋亡指数(AI)显著降低(P<0.01),磷酸化Akt表达明显增加(P<0.01);但上述指标变化可被LY294002(PI3K抑制剂)所抑制。结论在复氧早期给予胰岛素干预可显著地减少缺氧/复氧所诱导的心肌细胞凋亡,其保护机制与PI3K/Akt所介导的抗细胞凋亡作用有关。

Objective To explore the protective effects and mechanisms of insulin on anoxia/reoxygenation-induced cardiac injury. Methods The model of anoxia/reoxygenation （A/R） injury was extablished through anoxia for 2 hours and reoxygenation for 4 hours in cultured cardiomyocytes of neonatal rats, which were randomized to receive 0.9% Nacl, insulin, LY294002, insulin plus LY294002 at the beginning of reoxygenation. At the end of reoxygenation, activities of lactate dehydrogenase （LDH） and contents of malondialdehyde （MDA） were measured through spectrophotometric procedures. Apoptosis of cardiomyocytes was assessed through TUNEL and DNA Ladder. Western blotting was used to analyse expression of phosphorylated Akt in all groups. Results Compared with Nad group, activities of LDH, contents of MDA, apoptosis index （AI） were significantly decreased, and expression of phosphorylated Akt was markedly increased in insulin-treated group. However those changes of LDH, MDA, AI and phosphorylated Akt were inhibited by LY294002 （PI3K inhibitor）. Conclusion The early administration of insulin at reoxygenation could protect cardiomyocytes from anoxia/reoxygenationinduced apoptosis through PI3K/Akt signaling pathway.