Nrf2-ARE通路在心肌缺血后处理和吡那地尔后处理中的心肌保护作用机制

The activation mechanism of Nrf2-ARE pathway in protective effect of ischemia and pinacidil postconditioning on isolated rat hearts

目的探讨核因子-E2相关因子2（Nrf2）-ARE通路在心肌缺血后处理和吡那地尔后处理中的心肌保护作用机制。方法建立大鼠心肌缺血再灌注损伤模型，随机分为6组（n=8）：正常（N）组、缺血再灌注（Con）组、缺血后处理（IPO）组、吡那地尔后处理（50μmol／L，P50）组、N-（2-巯基丙酰）-甘氨酸（MPG，2mmo]／L）＋IPO（M＋IPO）组、MPG＋P50（M＋PS0）组。K—H液灌注平衡20min后，N组续灌100min；Con组停跳缺血40min，再灌注60min；IPO组于再灌注即刻行10s再灌注和10s缺血，循环6次，再续灌58min；P50组于再灌注即刻给予P50处理2min，续灌58min；M＋IPO组和M＋P50组，分别于再灌注即刻予含MPG的K—H液灌注3min，再IPO或P50处理2min，再续灌55min。记录各组平衡末及再灌注末的左心室发展压（LVDP）、心率（HR）、左心室舒张末压（LVEDP）和左心室内压最大上升速率（＋dp／dtmax）；电镜观察心肌细胞超微结构和线粒体Flameng评分；分别采用RT—PCR和Westemblot法检测灌注末各组心肌组织中醌氧化还原酶1（NQ01）、血红素加氧酶1（HO-1）、超氧化物歧化酶1（SOD-1）、Nrf2基因及蛋白表达。结果平衡末各组间LVDP、HR、LVEDP、＋dp／dtmax值差异均无统计学意义（P〉0．05）。再灌末HR、LVDP及＋dp／dtmax：与N组比较，其余各组均有下降；与Con组比较，IPO和P50组均显著升高（P〈0．05）；与IPO组比较，M＋IPO组较之明显下降（P〈0．05）；与P50组比较，M＋P50组显著降低（P〈0．05）。再灌末LVEDP：Con组较其余各组升高明显（P〈0．05）；M＋IPO组较IPO组显著升高（P〈0．05）；M＋P50组较P50组显著升高（P〈0．05）。电镜观察结果示N组心肌细胞超微结构形态基本正常，Con组超微结构损伤最严重；IPO组和PSO组优于Con组，M＋IPO组较IPO组损伤严重，M＋P50组较P50组损伤严重。线粒体Flameng评分，与N组相比，其余各组分数显著升高（P〈0．05）；IPO组和P50组评分明显低于Con组和应用MPG的组（P〈0．05）。HO-1、NQ01、SODI及Nrf2基因和蛋白表达量，N组表达量最高（P〈0．05）；与Con组比较，IPO和P50组表达显著增高（P〈0．05）；M＋IPO和M＋P50组的表达量比与之对应的IPO和P50组明显降低（P〈0．05）。结论缺血后处理和吡那地尔后处理可能通过再灌注时产生的活性氧类物质激活Nrf2-ARE通路，调控其下游的抗氧化蛋白和Ⅱ相解毒酶，改善缺血后心肌结构和整体功能，达到抗心肌缺血再灌注损伤的作用。

Objective To observe the activation mechanism of Nrf2-ARE pathway in protective effect of ischemia and pinacidil postconditioning on isolated rat hearts. Methods The hearts of adult male Sprague Dawley rats were established ischemia-reperfusion injury model, and devided into six groups（ n = 8, each group） , i.e. Normal group （Group N ） , ischemia- reperfusion group（ Group Con, I/R）, ischemic postconditioning group （Group IPO）, pinacidil postconditioning group （Group P50 ）, N- （2-mercaptopropionyl） -giycine （ MPG, 2mmoL/L ） ＋ IPO group （ Group M ＋ IPO ）, MPG ＋ P50 group （ Group M ＋ PS0）. Rat hearts were perfused with Krebs-Henseleit（K-H） buffer for 20 minutes for equilibration. Subsequently, Group N was perfused with K-H buffer for 100 minutes after equilibration, Group Con was perfused with 4℃ ST. Thomas solution to stop the heart beating after equilibration, then the hearts were underwent 40 minutes global isehemia under 32℃, and followed by the K-H solution for 60 minutes. Group IPO after global ischemia period, the hearts were subjected to six 10-seconds cycles of ischemia/reperfusion at the beginning of reperfusion, then were reperfused for 58 minutes. Group P50 after global ischemia, rat hearts were perfused with K-H buffer containing pinacidil（50 μmol/L） for 2 minutes before reperfusion. Group M ＋ IPO after global ischemia, the hearts were subjected to perfuse with K-H buffer containing MPG（2 mmol/L） for 3 minutes, and then underwent six 10-seconds cycles of ischemia/reperfusion before reperfusion. Group M ＋ P50 after global ischemia, the hearts were perfused with K-H buffer containing MPG （2 mmol/L） for 3 minutes, and then subjected to perfuse with K-H buffer containing pinacidil（50 μmol/L） for 2 minutes before reperfusion. Cardiac function indexes（ such as HR, LVDP, LVEDP, and the Max dp/dt） at the end point of equilibration and reperfusion were observed and recorded. The uhrastructure of myocardial tissue was observed by electron microscopy and the mitochondrial Flameng score was calculated. RT-PCR and western-blot were applied to detect the gene transcription and protein expression of HO-1, NQO1, SOD1, and Nrf2 in left ventricular myocardial tissue after reperfusion. Results The HR, LVDP and ＋ dp/dtmax at the end of reperfusion ： the cardiac function indexes are lower among each group compared with group N, group IPO and group P50 are better than group Con （ P 〈 0.05）. Compared with group IPO, there is no significant difference in group group P50, but group M ＋ IPO is obviously decreased （P 〈 0.05 ）. Compared with group P50, group M ＋ 1750 index is decreased significantly（ P 〈 0.05 ）. The LVEDP at the end of reperfusion is lower than that among each group as compared with group Con, which is significantly increased in group Con（ P 〈 0.05 ）. Compared with group IPO, there is no significant difference in group P50, but group M ＋ IPO is significantly increased （ P 〈 0.05 ）. Compared with group P50, the group M ＋ 1750 is obviously decreased（ P 〈 0.05 ）. The uhrastructure of myocardial tissue in group N is mostly normal, group Con presence serious damage. The ultrastructure damage of myocardial tissue is improved in group IPO and group P50 as compared with that in group Con, while group M ＋ IPO is more serious than group IPO, group M ＋ P50 is more serious group 1750. The mitochondrial Flameng score is higher among each group as compared with group N （ P 〈 0.05 ）, the score is lower in group IPO and group P50 as compared with group Con and corresponding nonblocking group （ M ＋ IPO, M ＋ P50, P 〈 0.05 ）. The mRNA and the protein expressions of HO-1, NQO1, SOD1 and Nrf2 among each group are lower as compared with group N （P 〈 0.05 ）. Compared with those in group Con, the mRNA and the protein expressions in group IPO and group P50 are obviously increased（ P 〈 0.05 ）, group IPO and group P50 are higher than those in group adding active oxygen scavenger（MPG） （ P 〈 0.05 ）. Conclusion Ischemic postconditioning and pinacidil postconditioning have protective effect of myocardial tissue from ischemia reperfusion injury, while improve the cardiac function index. The cardiac protective effect of Ischemic and Pinacidil postconditioning methods may be involved the ROS in early reperfusion,which activate the Nrf2- ARE pathway, and up-regulate the expression downstream antioxidant protein and phase H detoxifying enzyme, ultimately im- prove the cardiac function index during the reperfusion period. [Key words] NF-E2 related factor 2 Myocardial ischemia Pinacidil Cardioprotective effects