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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 investigate the effect of insulin on cardiomyocyte apoptosis induced by hypoxia / reoxygenation and its mechanism. Methods Primary cultured neonatal rat ventricular myocytes were subjected to hypoxia 2h / reoxygenation for 4h to establish an anoxia / reoxygenation cardiomyocyte injury model. At the beginning of reoxygenation period, 0.9% saline (VC group), insulin (INS group), LY294002 (LY group) and insulin + LY294002 (INS + LY group) After reoxygenation for 4h, lactate dehydrogenase (LDH) activity was assayed by 2,4-dinitrophenylhydrazine colorimetric assay. Malondialdehyde (MDA) content was detected by thiobarbituric acid chromogenic assay. TUNEL and DNALadder were used to detect apoptosis. The phosphorylation of Akt was detected by Western blotting, and the differences between groups were compared. Results Compared with VC group, the activity of LDH, the content of MDA and the apoptosis index (AI) in INS group were significantly decreased (P <0.01) and the phosphorylated Akt expression was significantly increased (P <0.01). However, the changes of these indexes could be detected by LY294002 PI3K inhibitor). Conclusion Insulin intervention in early reoxygenation can significantly reduce myocardial cell apoptosis induced by anoxia / reoxygenation, and its protective mechanism is related to the anti-apoptotic effect mediated by PI3K / Akt.