硫氧还蛋白通过阻断过氧亚硝酸阴离子导致的蛋白硝基化减少缺血后心肌细胞凋亡

摘要

目的:本研究旨在阐明硫氧还蛋白(Trx)心肌保护作用的机制.方法:采用成年雄性小鼠心肌30 min缺血/3 h再灌注模型,再灌注前10 min随机给予磷酸缓冲液(PBS,对照)或重组人Trx (hTrx,2 mg/kg) 腹腔注射.在再灌注终末,摘取实验动物的心脏,分别检测心肌细胞凋亡和心肌组织中硝基酪氨酸的含量.结果:再灌注前给予Trx可以显著减少缺血/再灌注导致的心肌细胞凋亡(P＜0.01);与对照组相比,Trx组心肌组织中硝基酪氨酸的含量显著减少 (4.8±0.53 比 9.5±0.86 pmol/mg蛋白质,P＜0.01), 提示Trx可能通过阻断蛋白硝基化和随后的细胞凋亡而发挥心脏保护作用.为了寻找Trx可以阻断蛋白硝基化的直接证据,在培养的成年心肌细胞给予SIN-1 (过氧亚硝酸阴离子供体) 50 μmol处理、或SIN-1 和hTrx(500 nmol)共同处理后,观察Trx 在过氧亚硝酸阴离子诱导的细胞凋亡中的作用.结果表明SIN-1 处理导致心肌细胞凋亡显著增加 (P＜0.01),Trx可以显著减少SIN-1导致的心肌细胞凋亡(P＜0.01) .结论:硫氧还蛋白,一种新的抗凋亡和心肌保护分子,通过阻断过氧亚硝酸阴离子导致的蛋白硝基化和随后的细胞凋亡而发挥心脏保护作用.%To determine the mechanism responsible for thioredoxin(Trx )cardioprotective effect. Method: Adult male mice were subjected to 30 min of myocardial ischemia (MI) followed by 3 h reperfusion(R). Mice were randomized to receive vehicle (PBS)or human Trx(hTrx,2 mg/kg, ip.) 10 min before R. At the end of the reperfusion period, the animal heart was quickly excised, and the ischemic/reperfused cardiac tissue was isolated. The levels of myocardial apoptosis and nitrotyrosine were determined 3 h after R. Results: Treatment with hTrx before R decreased significantly post-ischemic myocardial apoptosis (p ＜0.01). Moreover, the level of cardiomyocyte protein nitration represented by nitrotyrosine reduced markedly in animals treated with Trx (4.8±0.53 vs. 9.5±0.86 pmol/mg protein, p ＜0.01), which suggests that Trx may exert its cardioprotective effect by blocking protein nitration and subsequent apoptosis. To further demonstrate a causative-link between the anti-nitrative and cardioprotective effect of Trx, peroxynitrite was added in cultured adult cardiomyocytes and the effect of Trx on peroxynitrite-induced apoptosis were determined. Incubation of cardiomyocytes with SIN-1 (50 μmol, a peroxynitrite donor) for 3 h caused significant cardiomyocyte apoptosis (P＜0.01). Treatment with Trx decreased markedly apoptosis induced by SIN-1 (P＜0.01). Conclusion: It is demonstrated that Trx is a novel anti-apoptotic and cardioprotective molecule that exerts its cardioprotective effects by blocking peroxynitrite-induced protein nitration and subsequent apoptosis.