促红细胞生成素对缺氧复氧心肌细胞 caspase-3表达及Omi/HtrA2转位变化和 Omi/HtrA2沉默时对其的影响

摘要

Objective To observe the expression of caspase-3 and transposition of Omi/HtrA2 in H9C2 by erythropoietin and/or Omi/HtrA2 silencing to explore the related anti-apoptotic mechanisms.Methods The cultured H9C2 cardiomyocytes were divided into control group,H/R group (anoxia 2 h and re-oxygenation 24 h), and different concentrations of EPO treatment groups.The release rate of lactate dehydrogenase (LDH)in cell supernatant was measured in each group.Expressions of cleaved caspase-3 and Omi/HtrA2 were measured by Western blot;then the transposition of Omi/HtrA2 between cytoplasm and mitochondria was observed.Specific siRNA interfering fragment was transfected into H9C2 cardiomyocytes by liposome method.Its silencing effect on Omi/HtrA2 was measured by RT-PCR and Western blot.The survival rate,release rate and expression of cleaved caspase-3 were measured.And the expression of Omi/HtrA2 was measured in cytoplasm and mitochondria in H9C2 (transposition of Omi/HtrA2 ).Results Compared with H/R group,the release of LDH and expression of cleaved caspase-3 were decreased; the transposition of Omi/HtrA2 from mitochondria to cytoplasm in H/R treatment groups was increased compared with control group,while that in EPO (20 IU/mL)group decreased.si-HtrA2 group transfected with siRNA showed a decreased release of LDH and expression of cleaved caspase-3 with all significant variances (P <0.05).Conclusion EPO exerts a cytoprotective effect by inhibiting the transposition of Omi/HtrA2 and hence the activation of caspases-3 pathway.%目的：观察促红细胞生成素(EPO)及/或进一步沉默 Omi/HtrA2表达时对缺氧复氧(H/R)心肌细胞的影响,探索相关抗细胞凋亡机制。方法培养乳鼠心肌细胞株(H9C2细胞),分组(对照组、H/R 组及各浓度的 EPO 干预组)处理后,酶标仪检测各组细胞上清液中乳酸脱氢酶(LDH)释放率,Western blot 检测细胞内 cleaved caspase-3、Omi/HtrA2蛋白表达变化；并观察 Omi/HtrA2在胞质和线粒体的表达变化(转位情况)。再经脂质体法将 Omi/HtrA2特异性 siRNA 干扰片段转染至 H9C2细胞中,RT-PCR 和 Western blot 验证 siRNA 对 Omi/HtrA2表达的沉默效应后,分组同前干预,分别检测各组细胞 LDH 释放率及 cleaved caspase-3表达变化。结果与 H/R 组相比, EPO 干预组细胞上清液 LDH 释放减少,cleaved caspase-3表达减弱；H/R 组 Omi/HtrA2蛋白表达在胞质中表达较对照组增多(向胞质发生转位),而 EPO(20 IU/mL)组其胞质转位减少。经 siRNA 干扰后,与 H/R 组相比,LDH 释放降低,cleaved caspase-3表达减弱,差异均有统计学意义(P <0.05)。结论EPO 可能通过减少 Omi/HtrA2蛋白的转位,抑制 caspases-3通路激活,而发挥细胞保护作用。