Urocortin Ⅰ后处理对缺氧/复氧大鼠心肌细胞线粒体膜电位及活性氧自由基的影响

The effect of urocortin Ⅰ postconditioning on mitochondrial membrane potential and reactive oxygen species in rat heart during hypoxia/reoxygenation

目的观察Urocortin I后处理对缺氧/复氧大鼠心肌细胞线粒体膜电位及活性氧自由基的影响。方法利用ALC-HP型离体心脏灌注装置分离成年大鼠心肌细胞,将培养1 d后存活状态良好的细胞随机分为正常组(N组)、缺氧/复氧组(HR组)、UrocortinⅠ后处理组(Ucn Ⅰ组)、5-羟葵酸拮抗UrocortinⅠ组(5-HD+UcnⅠ组)。N组:37℃培养箱中持续培养150 min;HR组:缺氧40 min后,复氧110 min;UcnⅠ组:缺氧40 min复氧10 min,然后UcnⅠ处理30 min再复氧70min;5-HD+UcnⅠ组:特异性线粒体ATP敏感性钾通道(mito KATP)拮抗剂5-HD处理10 min后,在含UcnⅠ的培养基中处理30 min,余处理同UcnⅠ组。各组于复氧末加入荧光探针并用倒置相差显微镜观察细胞线粒体膜电位(MMP)及活性氧自由基(ROS)的变化。结果 (1)MMP变化:N组心肌细胞MMP较其余各组高(P<0.01);Ucn Ⅰ组虽低于N组,但高于HR组及5-HD+Ucn Ⅰ组(P<0.05);5-HD+Ucn Ⅰ组与HR组比差异无统计学意义(P>0.05)。(2)ROS变化:N组心肌细胞ROS荧光强度低于其余各组(P<0.01);Ucn Ⅰ组及5-HD+Ucn Ⅰ组较HR组低(P<0.01),但5-HD+Ucn Ⅰ组高于Ucn Ⅰ组(P<0.01)。结论 Urocortin I后处理能抑制缺氧/复氧心肌细胞活性氧自由基的生成,并抑制线粒体膜电位的衰减,其机制与其开放线粒体ATP敏感性钾通道有关。

Objective To explore the effect of urocortin I postconditioning on mitochondrial membrane potential and reac.tive oxygen species in rat heart during hypoxia/reoxygenation. Methods Isolated rat myoeytes by using ALC - HP type isolation heart peffusion device. Myocytes after being continuously cultivated 24 h were randomly divided into four groups ： Nor group, HR group, Ucnl postconditioning group and mitochondrial ATP - sensitive potassium channel blocker 5 - HD ＋ Ucn I group. Nor group： cultivated in a 37 ~C incubator continuously for 150 rain; HR group： suffered from hypoxia for 40 rain, then reoxggenation for 110 min;Ucn I group： reoxygen- ation for 10 min after being hypoxia for 40 min, then cultivated in a nutrient medium containing Ucn 1 for 30 rain, and then reoxygenation for 70 rain. 5 - HD ＋ Ucn I group： cultivated in the nutrient medium containing 5 -HD for 10 min before reoxygenation, the rest managements were the same as Ucn I group. Observing the change of MMP and ROS by using inverted phase contrast microscope at the end of reoxygenation. Results 1 ） Flu- orescence intensity change of MMP：Nor group was higher than other three groups （P 〈 0.01 ） ;Ucn 1 group was lower than Nor group,but higher than groups HR and 5 - HD ＋ Ucn I （P 〈 0.05 ） ; but there was no significantdifference between groups HR and 5 - HD ＋ Ucn I （ P 〉 0.05. 2） Fluorescence intensity change of ROS ： Nor group was lower than other three groups（ P 〈 0.01 ） ; HR group was higher than groups Ucn I and 5 -HD ＋ UcnI （ P 〈 0.01 ） ; while 5 - HD ＋ Ucn I group was higher than Ucn I group（P 〈 0.01 ）. Conclusion Urocortin I post- conditioning can restrain the excessive generation of ROS and attenuation of MMP, these effects relate to the o- pening of mitochondrial ATP - sensitive potassium channel.