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目的:本研究旨在明确过氧亚硝酸阴离子是否可以导致硫氧还蛋白(thioredoxin,TRX,一种新的抗氧化和抗凋亡蛋白质)硝基化修饰失活,从而促进缺血/再灌注心肌细胞凋亡。方法和结果:硫氧还蛋白中加入100μmol/L SIN-1(过氧亚硝酸阴离子供体)离体孵育可导致TRX发生硝基化,提示心肌组织内TRX也对硝基修饰敏感。为进一步确定TRX硝化是否改变其心肌保护作用,采用缺血30 min,再灌注3 h(凋亡检测)或24 h(心肌梗死范围)小鼠缺血/再灌注模型,分别于再灌前10 min给予磷酸盐缓冲液(PBS,对照组),TRX或硝化TRX。TRX治疗可以显著减少缺血/再灌注导致的心肌细胞凋亡和心肌梗死范围(P<0.01);而在体外用SIN-1孵育TRX,则完全阻断了其心肌保护作用;此外,用SIN-1和血红蛋白(清除NO)或SIN-1和超氧化物岐化酶(SOD,清除过氧化物)同时孵育TRX,其心肌保护作用则完全保留。结论:上述结果表明,SIN-1通过产生过氧亚硝酸阴离子使TRX失活(丧失对心脏的保护作用),提示过氧亚硝酸阴离子可能通过使TRX硝基化失活而发挥促凋亡作用。
OBJECTIVE: This study aimed to determine whether peroxynitrite anion can lead to the inactivation of nitration of thioredoxin (TRX, a new anti-oxidant and anti-apoptotic protein) to promote ischemia / reperfusion Cardiomyocyte apoptosis. Methods and Results: In vitro incubation of thioredoxin with 100 μmol / L SIN-1 (peroxynitrite anion donor) resulted in the nitration of TRX, suggesting that TRX in myocardium is also sensitive to nitro-modification. To further determine whether TRX nitrification changed its cardioprotective effect, ischemia / reperfusion models of mice were performed at 30 min after ischemia, 3 h after reperfusion (apoptosis test) or 24 h (myocardial infarction range) min phosphate buffered saline (PBS, control group), TRX or nitrated TRX. TRX treatment could significantly reduce the extent of myocardial apoptosis and myocardial infarction caused by ischemia / reperfusion (P <0.01); while in vitro incubation of TRX with SIN-1 completely blocked its cardioprotection; in addition, -1 and hemoglobin (NO removal) or SIN-1 and superoxide dismutase (SOD, peroxide scavenging) at the same time incubation of TRX, its cardioprotection was completely preserved. CONCLUSIONS: The above results show that SIN-1 inactivates TRX (loss of cardioprotective effect) by generating peroxynitrite anion, suggesting that peroxynitrite anion may exert a pro-apoptotic effect by deactivating TRX nitration .