硫氧还蛋白硝基化在多柔比星诱导的乳鼠心肌细胞凋亡中的作用

Role of thioredoxin nitration in doxorubicin-induced apoptosis of neonatal rat cardiomyocytes

目的:探讨硫氧还蛋白(thioredoxin,Trx)硝基化在多柔比星诱导的乳鼠心肌细胞凋亡过程中的作用。方法:体外分离培养新生SD大鼠心肌细胞,直接给予多柔比星刺激和预孵育过氧亚硝基阴离子(ONOO-)清除剂锰(III)四(1-甲基-4-吡啶基)卟啉(MnTMPyP)后再给予多柔比星刺激。MTT检测细胞存活率,细胞凋亡荧光Hoechst 33258试剂盒检测细胞凋亡情况,分光光度法检测caspase-3的活性,Western blotting法检测聚腺苷酸二磷酸核糖聚合酶1剪切片段[cleaved poly(ADP-ribose)polymerase-1,cleaved PARP-1]、凋亡信号调节激酶1(apoptosis signal-regulating kinase 1,ASK1)、磷酸化ASK1(p-ASK1)、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)和磷酸化p38 MAPK(p-p38 MAPK)的表达,免疫沉淀法检测Trx-NT和Trx-硝基酪氨酸的形成。结果:给予多柔比星后,心肌细胞Hoechst荧光染色观察到明显凋亡,MnTMPyP预保护后,凋亡情况减轻。多柔比星组与正常组相比,Trx硝基化水平升高,caspase-3活性、cleaved PARP-1和p-p38 MAPK表达增加(P<0.05),TrxASK1和p-ASK1表达减少(P<0.05)。MnTMPyP预保护组与多柔比星组相比,Trx硝基化水平降低(P<0.05),caspase-3活性、cleaved PARP-1和p-p38 MAPK表达降低(P<0.05),Trx-ASK1和p-ASK1表达增加(P<0.05)。结论:多柔比星刺激心肌细胞后,Trx硝基化水平和细胞凋亡明显增加;给予ONOO-清除剂MnTMPyP后,Trx硝基化程度和凋亡情况得到明显改善。Trx硝基化可能是多柔比星诱导心肌细胞凋亡的机制之一。

AIM： To investigate the role of thioredoxin nitration in the apoptosis of neonatal rat eardiomyocytes （NRCMs） induced by doxorubicin （DOX）. METHODS： Cardiomyoeytes treated with DOX were isolated from newborn Sprague-Dawley rats and cultured in vitro. NRCMs were treated with DOX alone （DOX group）, pretreated with Mn （III） tetrakis（1-methyl-4-pyridyl） porphyrin （ MnTMPyP）, a peroxynitrite （ ONO0- ） scavenger, and then treated with DOX （ MnTMPyP ＋ DOX group）, or treated with MnTMPyP alone （ MnTMPyP group）. NRCMs without any treatment served as a normal control （ control group）. The viability of the ceils was examined by MTI？ assay, and the apoptosis was measured by Hoechst 33258 nuclear staining kit. The activity of caspase-3 was detected by spectrophotometry. The expression of cleaved poly（ADP-ribose） polymerase 1 （ PARP-1 ）, apoptosis sigual-regulating kinase 1 （ ASK1 ）, phosphorylated ASK1 （p-ASK1）, p38 mitogen-activated protein kinase （p38 MAPK） and phosphorylated p38 MAPK （p-p38 MAPK） was meas- ured by Western blotting. Immunoprecipitation and immunoblotting were performed to detect the formation of Trx-ASK1 and Trx-nitrotyrosine. RESULTS： DOX induced significant apoptosis of NRCMs. MnTMPyP could significantly attenuate the apoptosis induced by DOX. Compared with control group, Trx nitration in DOX group increased obviously. The increases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were also observed, besides the expression of Trx- ASK1 compound and p-ASK1 decreased significantly （P 〈 0. 05）. MnTMPyP could decrease the nitration of Trx. The decreases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were detected in MnTMPyP ＋ DOX group, while the expression of Trx-ASK1 compound and p-ASK1 increased significantly （ P 〈 0. 05 ）. CONCLUSION： DOX could induce significant apoptosis of NRCMs and increase Trx nitration. The process was significantly attenuated by pretreatment with MnTMPyP. Therefore, Trx nitration may play an important role in doxorubicin-induced apoptosis of cardiomyocytes. [