2类多巴胺受体通过促进PKC-ε转位参与心肌缺血后适应抑制细胞凋亡

Involvement of dopamine receptor-2 in myocardial ischemic postconditioning inhibited apoptosis by promoting translocation of PKC-ε

目的以蛋白激酶C-ε(PKC-ε)转位为切入点,探讨2类多巴胺受体(DR2)在心肌缺血后适应抑制细胞凋亡中的作用及可能机制。方法复制原代培养乳鼠心肌细胞缺氧/复氧和缺血后适应模型。MTT检测心肌细胞的存活率;Hoechst 33342染色观察细胞凋亡;Western blotting检测Bcl-2、caspase-3、caspase-9、PKC-ε蛋白的表达和细胞色素C(Cyt c)的释放;免疫共沉淀检测PKC-ε和DR2的相互作用。结果与正常组比较,缺氧/复氧组细胞存活率降低,细胞凋亡增加,促凋亡因子(Cyt c、caspase-3、caspase-9)表达增加,抑凋亡因子(Bcl-2)表达亦增加,PKC-ε没有转位到细胞膜,PKC-ε和DR2不存在相互作用。与缺氧/复氧组比较,缺血后适应明显升高细胞存活率,降低细胞凋亡,抑制促凋亡因子(Cyt c、caspase-3、caspase-9)表达,促进抑凋亡因子(Bcl-2)表达,PKC-ε转位到细胞膜,PKC-ε和DR2存在相互作用。与缺血后适应组比较,DR2激动剂进一步增加缺血后适应的保护作用,而DR2抑制剂则取消了DR2激动剂的作用。结论DR2参与心肌缺血后适应抑制细胞凋亡,其机制与DR2促进PKC-ε转位及DR2和PKC-ε存在相互作用有关。

Objective To investigate the effect and possible mechanism of dopamine receptor-2 （DR2） activation in ischemic postconditioning （ PC） inhibited cardiomyocytes apoptosis through translocation of PKC-ε. Method The hypoxia/reoxygenation （ H/R） injury and PC models was established using primarily cultured neonatal rat cardio-myocytes. The cell survival rate was detected by MTT. The cell apoptosis was observed using Hoechst 33342 sta-ning. The protein expression of Bcl-2, caspase-3, caspase-9, the release of cytochrome c（ Cyt c） and translocation of PKC-εwere analyzed using Western blotting. The interaction of DR2 and PKC-εwas tested by co-immunoprecipi-tation. Result Compared with Control group, the cell survival rate was decreased, cardiomyocytes apoptosis was heightened, the expression of Bcl-2,caspase-3,caspase-9 and the release of Cyt c were increased, PKC-ε were not translocated to the cell membrane and the interaction of DR2 and PKC-ε was not exist in H/R group. Compared with H/R group, PC increased the cell survival rate, decreased cardiomyocytes apoptosis, down-regulated the ex-pression of caspase-3, caspase-9 and the release of Cyt c, up-regulated the expression of Bcl-2, promoted transloca-tion of PKC-ε and the interaction of DR2 and PKC-ε. Compared with PC group, the agonist of DR2 further in-creased cardioprotection of PC, however, the antagonist of DR2 canceled this effect of DR2 agonist. Conclusion DR2 activation is involved in PC inhibited apoptosis by promoting translocation of PKC-ε.