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目的探讨磷脂酰肌醇3激酶(PI3-K)/蛋白激酶B(Akt)信号通路在DIDS(4,4′-diisothiocyanostilbene-2,2′-disulfonic acid)减弱缺血/再灌注损伤(I/RI)诱导心肌细胞凋亡中的作用。方法以I/RI诱导心肌细胞凋亡,然后用PI3-K特异性的抑制剂LY294002[22(42吗啉基)282苯基24氢212苯并吡喃242酮]进行干预。实验分为正常对照组、I/RI组、I/RI+DIDS组和I/RI+DIDS+LY294002组。通过噻唑蓝(MTT)比色法、Hoechest-33258染色和半胱天冬蛋白酶(Apo-ONETMHomogeneous Caspase)-3试剂盒以及Western blot分别检测:心肌细胞的存活率(%)、细胞核的形态变化和caspase-3活性、Akt的磷酸化。结果①DIDS能够显著抑制I/RI诱导的细胞存活率的下降,抑制凋亡小体的出现,抑制caspase-3的活性的增加(P<0.01)。②用LY294002预处理后,DIDS保护I/RI心肌细胞存活的作用减弱、凋亡小体增加和caspase-3活性明显升高(P<0.01)。③I/RI组与正常对照组Akt磷酸化无明显差异,DIDS能够显著增加I/RI组中Akt蛋白的磷酸化(P<0.01)。用LY294002预处理后,DIDS对I/RI组Akt蛋白磷酸化的影响明显减弱(P<0.01)。结论DIDS可通过激活PI3-K/Akt信号通路减弱I/RI诱导的心肌细胞的凋亡。
Objective To investigate the effects of PI3-K / Akt signaling pathway on I / R injury induced by DIDS (4,4’-diisothiocyanostilbene-2,2’-disulfonic acid) RI) induces cardiomyocyte apoptosis. Methods Cardiomyocyte apoptosis was induced by I / RI and then interfered with PI3-K specific inhibitor LY294002 [22 (42 morpholino) 282 phenyl 24hydrogen 212 benzopyran 242 ketone]. The experiment was divided into normal control group, I / RI group, I / RI + DIDS group and I / RI + DIDS + LY294002 group. Hoechest-33258 staining and Apo-ONETMHomogeneous Caspase-3 kit and Western blot were used to detect the viability of cardiomyocytes (%), morphological changes of the nucleus and caspase-3 activity, phosphorylation of Akt. Results (1) DIDS could significantly inhibit the decrease of cell viability induced by I / RI, inhibit the appearance of apoptotic bodies and inhibit the activity of caspase-3 (P <0.01). ② Pretreatment with LY294002 attenuated the survival of cardiomyocytes in DID-protected I / RI rats, increased apoptotic bodies and increased caspase-3 activity (P <0.01). There was no significant difference in Akt phosphorylation between I / RI group and normal control group. DIDS could significantly increase Akt phosphorylation in I / RI group (P <0.01). After LY294002 pretreatment, the effect of DIDS on Akt phosphorylation in I / RI group was significantly weaker (P <0.01). Conclusion DIDS attenuates I / RI-induced apoptosis in cardiomyocytes by activating the PI3-K / Akt signaling pathway.