细胞外信号调节激酶通路在七氟醚后处理对抗大鼠离体心脏缺血再灌注损伤中的作用

The role of extracellular signal-regulated kinase 1/2 signaling pathway in the cardio-protection induced by sevoflurane postconditioning in isolated rat hearts exposed to ischemia-reperfusion injury

目的研究活性氧(ROS)、细胞外信号调节激酶(ERK1/2)及线粒体通透性转换孔(mPTP)在七氟醚缺血后处理减轻离体大鼠心脏缺血-再灌注损伤中的作用。方法以K-H缓冲液灌注离体大鼠心脏,全心缺血30min后复灌60min建立缺血-再灌注损伤模型。七氟醚缺血后处理的心脏于缺血后复灌最初15min以3%七氟醚饱和的K-H缓冲液灌注。分别单独给予或与七氟醚同时给予ROS清除剂NAC(4mM)或ERK1/2阻断剂PD98059(20μM),用以评价ROS及ERK1/2在七氟醚缺血后处理中的作用。比较各组间血流动力学、心肌梗死面积、冠脉流出液中乳酸脱氢酶(LDH)及肌酸肌酶-MB(CK-MB)水平。同时,测定各组缺血30min复灌60min后心肌丙二醛(MDA)含量以反映氧化应激损伤程度。Western blotting测定ERK1/2的磷酸化情况。测定心肌烟酰胺腺嘌呤二核苷酸(NAD+)含量以反映mPTP的开放情况。结果与对照组相比,复灌之初给予3%七氟醚可显著改善心功能(增加左室发展压力、左室最大收缩/舒张速率、冠脉流量、心率,并降低左室舒张末期压力)、降低心肌梗死面积及减少LDH及CK-MB释放(P<0.05)。七氟醚的心肌保护作用同样表现在降低缺血-再灌注损伤后心肌的MDA含量(P<0.05)。然而,给予NAC或PD98059不仅可消除上述保护作用,而且可以抑制七氟醚增强ERK1/2磷酸化及抑制mPTP开放的保护作用(P<0.05)。结论 3%七氟醚缺血后处理通过ROS-ERK1/2-mPTP信号通路可为健康大鼠离体心脏的缺血-再灌注损伤提供保护。

Objective To investigate the roles of reactive oxygen species （ROS）, extracellular signal regulated kinase 1/2 （ERK 1/2） and mitochondrial permeability transition pore （mPTP）, and their possible linkages in sevoflurane postconditioning （SpostC） in isolated healthy rat hearts exposed to myocardial ischemia-reperfusion injury （MIRI）. Methods Isolated rat hearts were subjected to 30 min of global ischemia, followed by 1 h of reperfusion with Krebs-Henseleit （K-H） buffer. SpostC was induced by perfusing the hearts with K-H buffer saturated with 3% sevoflurane during the first 15 min of reperfusion. To evaluate the role of ROS and ERK 1/2 in SpostC, ROS scavenger NAC （4 mM） or ERK 1/2 inhibitor PD98059 （20 μM） was administered alone or together with sevoflurane during the first 15 min of reperfusion. Hemodynamics, infarct size, lactate dehydrogenase （LDH） and creatine kinase-MB （CK-MB） were compared among groups. Additionally, myocardial malondialdehyde （MDA） content, an indicator of oxidative injury was also determined after 60 min of reperfusion. ERK 1/2 phosphorylation was measured by Western blotting analysis. The status of mPTP opening was determined by analyzing the nicotinamide adenine dinucleotide （NAD＋） content in myocardium. Results When compared with unprotected ISCH hearts, exposure of 3% sevoflurane during early reperfusion significantly improved functional recovery （improved LVDP, ±dp/dt, CF, HR and reduced LVEDP）, decreased myocardial infarct size and reduced LDH and CK-MB release （P〈0.05）. The protective effect of sevoflurane postconditioning was also manifested by reduced MDA content in myocardium after ischemia-reperfusion. However, these protective effects were all abolished in the presence of either NAC or PD98059, which was accompanied by prevention of ERK 1/2 phosphorylation and reduction of myocardial NAD＋ content. Conclusion Sevoflurane postconditioning protects isolated rat hearts against ischemia reperfusion injury via the recruitment of the ROS-ERK 1/2-mPTP signaling cascade.