ROS在乳化异氟醚后处理促进心肌损伤大鼠Nrf2／ARE信号通路激活中的作用

Role of ROS in emulsified isoflurane postconditioning-induced promotion of Nrf2/ARE signaling pathway activation in rats with myocardial injury

目的探讨活性氧（ROS）在乳化异氟醚后处理促进心肌损伤大鼠转录因子NF-E2相关因子2（Nrf2）/抗氧化反应原件（ARE）信号通路激活中的作用。 方法健康雄性SD大鼠，体重250～300 g，16～20周龄，采用Langendorff灌注装置建立大鼠离体心脏灌注模型，取模型制备成功的心脏80个，采用随机数字表法分为5组（n＝16）：对照组（C组）、缺血再灌注组（I/R组）、乳化异氟醚后处理组（EIP组）、脂肪乳后处理组（FEP组）和ROS清除剂N-（2-巯基丙酰）-甘氨酸＋乳化异氟醚后处理组（N＋EIP组）。除C组外；其它4组缺血40 min再灌注60 min；EIP组和FEP组于再灌注即刻分别灌注含乳化异氟醚1.68 mmol/L和脂肪乳712 mg/L的K-H液2 min，再灌注58 min；N＋EIP组灌注含N-（2-巯基丙酰）-甘氨酸2 mmol/L的K-H液3 min，然后行乳化异氟醚后处理。分别于平衡灌注末及再灌注末时记录HR、左室发展压（LVDP）、左室舒张末压（LVEDP）和左心室压力最大上升速度（＋dp/dtmax）。分别于再灌注5 min和再灌注末时取心肌组织，测定ROS含量，于再灌注末时取心肌组织，电镜下观察心肌细胞超微结构，并进行线粒体损伤评分，检测心肌组织Nrf2、血红素加氧酶-1（HO-1）、醌氧化还原酶1（NQO1）、超氧化物歧化酶-1（SOD-1）及其mRNA的表达水平。 结果与C组比较，再灌注末时I/R组HR、＋dp/dtmax和LVDP降低，LVEDP、线粒体损伤评分和心肌组织ROS含量升高，心肌组织Nrf2、HO-1、NQO1和SOD-1及其mRNA的表达下调（P〈0.05）；与I/R组比较，再灌注末时EIP组HR、＋dp/dtmax和LVDP升高，LVEDP、线粒体损伤评分、心肌组织ROS含量降低，心肌组织Nrf2、HO-1、NQO1和SOD-1及其mRNA的表达上调（P〈0.05），N＋EIP组上述指标比较差异无统计学意义（P〉0.05）；与EIP组比较，N＋EIP组HR、＋dp/dtmax和LVDP降低，LVEDP和线粒体损伤评分升高，再灌注5 min时心肌组织ROS含量降低，再灌注末时心肌组织ROS含量升高，心肌组织Nrf2、HO-1、NQO1及SOD-1及其mRNA的表达下调（P〈0.05）。EIP组心肌损伤程度轻于I/R组；N＋EIP组心肌损伤程度与I/R组比较无明显差异。 结论乳化异氟醚后处理促进心肌损伤大鼠Nrf2/ARE信号通路激活的机制全部与ROS有关。

Objective To investigate the role of reactive oxygen species （ROS） in emulsified isoflurane postconditioning-indueed promotion of nuclear factor-E2 related factor 2 （Nrf2） /antioxidant re- sponse element （ARE） signaling pathway activation in rats with myocardial injury. Methods Healthy male Sprague-Dawley rats, aged 16-20 weeks, weighing 250-300 g, were heparinized and anesthetized. Their hearts were excised and peffused in a Langendorff apparatus with K-H solution. Eighty isolated rat hearts were divided into 5 groups （n= 16 each） using a random number table： control group （group C） ; ischemia-reperfusion （I/R） group; emulsified isoflurane postconditioning group （group EIP） ; fat emulsion postconditioning group （group FEP）; N-（2-mercaptopropionyl）-glyeine （a ROS scavenger） plus emulsi- fied isoflurane posteonditioning group （group N＋EIP）. Group C was perfused with K-H solution for 100 rain, and the other 4 groups were subjected to 40 min of ischemia followed by 60 min of repeffusion. EIP and FEP groups were peffused for 2 min with K-H solution containing 1.68 mmol/L emulsified isoflurane and 712 mg/L intralipid, respectively, starting from the onset of repeffusion, and then continuously per- fused with K-H solution for 58 rain. Group N＋EIP was peffused for 3 min with K-H solution containing N- （2-mereaptopropionyl）-glyeine 2 mmol/L, and then emulsified isoflurane postconditioning was performed. Heart rate （HR）, left ventrieular developed pressure （LVDP）, left ventricular end-diastolic pressure （LVEDP） and the maximum rate of increase of left ventricular pressure （＋dp/dt ax） were recorded at the end of equilibration and of reperfusion. At 5 min of reperfusion and the end of reperfusion, myocardial specimens were obtained for determination of ROS content. At the end of reperfusion, myocardial specimens were obtained for examination of the ultrastructure of myocardial cells and for determination of the expression of Nff2, heine oxygenase-1 （ HO-1 ） , quinone oxidoreductase 1 （ NQOI ） and superoxide dismutase-1 （SOD-l） protein and mRNA in myocardial tissues. Mitoehondrial injury scores （Flameng scores） were e- valuated. Results Compared with group C, HR, ＋dp/dt and LVDP were significantly decreased, LV- EDP, mitoehondrial Flameng scores and ROS contents were increased, and the expression of Nff2, HO-1, NQO1 and SOD-I protein and mRNA was down-regulated at the end of reperfusion in I/R group （P〈0. 05）. Compared with group I/R, HR, ＋dp/dt and LVDP were significantly increased, LVEDP, mitochondrial Flameng scores and ROS contents were decreased, and the expression of Nrf2, HO-1, NQO1 and SOD-1 protein and mRNA was up-regulated at the end of reperfusion in group EIP （P〈0.05） , and no significant changes in the parameters mentioned above were found in group N＋EIP （P〉0.05）. Compared with group EIP, HR, ＋dp/dtnax and LVDP were significantly decreased, LVEDP and mitoehondrial Flameng scores were increased, ROS contents at 5 min of reperfusion were decreased, ROS contents at the end of reperfu- sion were increased, and the expression of Nrf2, HO-1, NQOI and SOD-1 protein and mRNA was down- regulated in group N＋EIP （P〈0.05）. The degree of myocardial injury was reduced in group EIP as com- pared with group I/R. There was no significant difference in the degree of myocardial injury between group N＋EIP and group I/R. Conclusion The mechanism by which emulsified isoflurane postconditioning promotes Nff2-ARE signaling pathway activation is totally related to ROS in rats with myocardial injury.