Trx-ASK1在多柔比星诱导的乳鼠心肌细胞凋亡中的作用

Role of thioredoxin-ASK1 in doxorubicin-induced apoptosis of neonatal rat cardiac myocytes

目的:探讨硫氧还蛋白-凋亡信号调节激酶1(Trx-ASK1)在多柔比星(doxorubicin,DOX)诱导的乳鼠心肌细胞凋亡中的作用。方法:体外分离原代新生SD大鼠心肌细胞,待细胞可见搏动率大于95%、预孵育抗氧化剂ebselen(Ebs)后,给予DOX(1μmol/L)作用24 h。MTT检测细胞存活率;活性氧簇(ROS)敏感的荧光探针2',7'-二氯二氢荧光素二乙酯(H2DCFDA)检测细胞内ROS含量;细胞凋亡荧光Hoechst 33258试剂盒进行凋亡检测;凋亡试剂盒检测caspase-3的活性;Western blotting法检测聚腺苷酸二磷酸核糖聚合酶1[poly(ADP-ri-bose)polymerase 1,PARP1]、ASK1、p-ASK1、p38和p-p38;免疫沉淀法检测Trx-ASK1是否发生分离。结果:乳鼠心肌细胞给予DOX后细胞内ROS量明显增多,荧光染色可以观察到明显凋亡,给予抗氧化剂Ebs后凋亡得到明显改善。与正常组相比,凋亡组caspase-3活性明显升高(P<0.01);PARP1、ASK1和p38等蛋白活化形式表达增加(P<0.01);Trx-ASK1的分离显著增加(P<0.01)。Ebs保护组与凋亡组相比,caspase-3活性明显降低(P<0.05);PARP1、ASK1和p38等蛋白活化形式表达减少(P<0.01);Trx-ASK1分离明显减少(P<0.05)。结论:给予DOX后心肌细胞可发生明显的凋亡,Trx与ASK1发生了一定程度的分离,从而使得ASK1介导的凋亡信号通路参与了该凋亡过程,给予Ebs后凋亡得到明显改善。本研究充分说明Trx-ASK1在DOX诱导心肌细胞凋亡过程中发挥着重要的作用,这将有助于进一步研究DOX诱导心肌细胞凋亡的机制。

AIM： To investigate the role of thioredoxin（Trx）-apoptosis signal-regulating kinase 1（ASK1） in doxorubicin-induced apoptosis of neonatal rat cardiac myocytes （NRCMs）. METHODS： Primary cardiomyocytes were isolated from newborn Sprague-Dawley rats with the purity of NRCMs 〉95%. NRCMs were pretreated with the indicated concentrations of ebselen 2 h prior to the addition of doxorubicin, then treated with doxorubicin at concentration of 1 μmol/L for another 24 h. The viability of the cells was examined by MTT assay.Reactive oxygen species（ROS） levels were measured by a ROS-specific probe 2＇,7＇-dichlorodihydrofluorescein diacetate （H2DCFDA）. Apoptotic cardiomyocytes were determined by Hoechst 33258 nuclear staining. The activity of caspase-3 was detected with a caspase-3 colorimetric assay kit. The protein levels of poly（ADP-ribose） polymerase 1（PARP1）, ASK1, p-ASK1, p38 and p-p38 were determined by Western blotting. Immunoprecipitation and immunoblotting were performed to detect whether the Trx-ASK1 was dissociated. RESULTS： Doxorubicin induced significant apoptosis of NRCMs. The levels of ROS were significantly increased. Ebselen significantly decreased the apoptosis. Compared with control group, increased activity of caspase-3 was showed in doxorubicin group （P〈0.01）. Increased protein levels of PARP1, ASK1 and p38 were observed （P〈0.01）. The increase in the dissociated Trx-ASK1 was also found. Compared with doxorubicin group, ebselen decreased the activity of caspase-3 （P〈0.01）, the levels of PARP1,ASK1 and p38 proteins （P〈0.05）, and the dissociated Trx-ASK1. CONCLUSION： Doxorubicin induces significant apoptosis of NRCMs. ASK1 is partly dissociated from Trx, and starts the ASK1-mediated apoptotic signaling. The process is significantly attenuated by pretreatment with ebselen. Trx-ASK1 plays an important role in doxorubicin-induced apoptosis of cardiomyocytes.