二氧化氮(NO_2)吸入暴露对大鼠心肌线粒体的损伤作用

The damage of NO_2 inhalation exposure to myocardial cell mitochondria in rats

除呼吸系统外心脏可能是二氧化氮(NO2)作用的重要靶器官之一,而线粒体是心脏运作高度依赖的细胞器,故通过研究线粒体相关指标的变化,探究空气NO2污染与心血管系统疾病的相关性对有效预防这类污染物暴露和干预由此引发的疾病治疗具有重要意义.建立Wistar大鼠NO2动式吸入染毒模型,考察慢性(对照、1×10-6和2.5×10-6)和急性(对照、2.5×10-6、5×10-6和10×10-6)暴露条件下大鼠心肌细胞线粒体超微结构变化、线粒体膜电位和活性功能水平变化,以及调控线粒体呼吸链组分相关因子PGC-1α、NRF1和TFAM蛋白表达变化.结果显示,长期低浓度NO2暴露致使心肌细胞线粒体数量减少并伴有肿胀;活性均显著降低,分别为对照组的0.79倍(P<0.05)和0.56倍(P<0.05);膜电位较对照组均显著下降,分别为0.89倍(P<0.05)和0.79倍(P<0.05);调控线粒体呼吸链组分相关因子PGC-1α、NRF1和TFAM蛋白表达较对照组下调,并在2.5×10-6时均显现出显著性差距,分别为对照组的0.76倍(P<0.05)、0.85倍(P<0.001)和0.52倍(P<0.05).而急性较高浓度NO2暴露时结果不同,大鼠心肌细胞线粒体数量增加;活性在5×10-6和10×10-6时显著性上升,分别为对照组的1.52倍(P<0.001)和2.12倍(P<0.001);膜电位在各浓度均显著增加,分别为对照组的1.50倍(P<0.05)、1.62倍(P<0.05)和2.25倍(P<0.001);调控线粒体呼吸链组分相关因子PGC-1α、N R F1和TFA M表达依赖性上调,同时在10×10-6下呈现显著增高趋势,分别为对照组的1.51倍(P<0.001)、1.47倍(P<0.05)和1.60倍(P<0.001).本研究表明,长期低浓度NO2暴露会引起心肌细胞线粒体氧化磷酸化功能减弱,进而导致线粒体功能退化;然而,急性较高浓度NO2暴露则引发相反的生物学效应,大鼠心肌细胞线粒体增强相关功能以适应外界刺激,这与线粒体功能代偿作用有关.由于线粒体功能和结构的完整性对于心血管系统疾病的生理机能至关重要,其损伤很可能是空气NO2污染与心血管系统疾病发生发展相关的重要分子机制.

The heart is suggested to be one of the important target organs of nitrogen dioxide （NO2） besides the respiratory system. Since effective operation of the heart is based on mitochondria, it is of great significance to study the correlation between NO2 pollution and mitochondrial structure and function for prevention and intervention of cardiovascular diseases （CVD） after NO2 exposure. In the present study, we established NO2 inhalation exposure model using Wistar rats with chronic （control, 1 × 10^-6, 2.5 × 10^-6） and acute （control, 2.5× 10^-6, 5 × 10^-6, 10 × 10^-6） exposure, to investigate the mitochondrial ultrastructure of myocardial cell. The mitochondrial function was detected by membrane potential and activity. The protein levels of mitochondrial respiratory chain transcription factors PGC-lct, NRF1, and TFAM were assayed by Western blot. The results showed a decrease of the number of mitochondria and swelling of myocardial cells after chronic and low concentration NO2 exposure. The mitochondria activity declined significantly, being 0.79 times （P 〈 0.05） and 0.56 times （P 〈 0.05） respectively of the control. The membrane potential was significantly lower, being 0.89 times （P 〈 0.05） and 0.79 times （P 〈 0.05） respectively of the control. Expressions of PGC-1α, NRF1 and TFAM declined significantly to 76% （P 〈 0.05）, 85% （P 〈 0.001） and 52% （P 〈 0.05） respectively in myocardial cell after 2.5 x 10-6 chronic and low concentration NO2 exposure. However, after acute and high concentration NO2 stimulation the number of myocardial cell mitochondria increased. The mitochondria activity increased to 1.52 （P 〈 0.001） and 2.12 （P 〈 0.001） times respectively with 5 × 10^-6 and 10 × 10^-6NO2. The membrane potential increased significantly to 1.50 （P 〈 0.05）, 1.62 （P 〈 0.05） and 2.25 （P 〈 0.001） times respectively with 2.5 × 10^-6, 5× 10^-6 and 10 × 10^6 NO2. The expression levels ofPGC-lct, NRF1 and TFAM all increased significantly in myocardial cell of rats, to 1.51 （P 〈 0.001）, 1.47 （P 〈 0.05） and 1.60 （P 〈 0.001） times respectively under 10× 10^-6 exposure. The results indicated that chronic and low concentration NO2 exposure causes mitochondrial dysfunction by myocardial mitochondria oxidative phosphorylation weakening. Meanwhile, the opposite biological effect after acute and high concentration NO2 stimulation could be due to the compensative effect. The mitochondrial structure and function damage might be one of the molecular mechanisms of CVD induced by NO2 pollution.