谷胱甘肽预处理可改善暖肺缺血再灌注损伤过程中的线粒体功能障碍。
Glutathione preconditioning ameliorates mitochondria dysfunction during warm pulmonary ischemia-reperfusion injury.
机构信息
Department of Cardiothoracic and Thoracic Vascular Surgery, University Hospital Würzburg, and Insitute of Hygiene and Microbiology, University of Würzburg, Würzburg, Germany.
出版信息
Eur J Cardiothorac Surg. 2012 Jan;41(1):140-8; discussion 148. doi: 10.1016/j.ejcts.2011.02.081.
OBJECTIVES
Reduced glutathione (GSH) has been shown to improve pulmonary graft preservation. Mitochondrial dysfunction is regarded to be the motor of ischemia-reperfusion injury (IR) in solid organs. We have shown previously that IR induces pulmonary mitochondrial damage. This study elucidates the impact of GSH preconditioning on the integrity and function of pulmonary mitochondria in the setting of warm pulmonary IR.
METHODS
Wistar rats were subjected to control, sham, and to two-study-group conditions (IR30/60 and GSH-IR30/60) receiving IR with or without GSH preconditioning. Rats were anesthetized and received mechanical ventilation. Pulmonary in situ clamping followed by reperfusion generated IR. Mitochondria were isolated from pulmonary tissue. Respiratory chain complexes activities (I-IV) were analyzed by polarography. Mitochondrial viability (Ca2+-induced swelling) and membrane integrity (citrate synthase assay) were determined. Subcellular-fractional cytochrome C-content (Cyt C) was quantified by enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was analyzed by fluorescence-activated cell sorting (FACS) after energizing and uncoupling. Inflammatory activation was determined by myeloperoxidase activity (MPO), matrix-metalloproteinase 9 (MMP-9) activity by gel zymography.
RESULTS
Pulmonary IR significantly reduced mitochondrial viability in combination with ΔΨm hyper-polarization. GSH preconditioning improved mitochondrial viability and normalized ΔΨm. Cyt C was reduced after IR; GSH protected from Cyt C liberation. Respiratory chain complex activities (I, II, III) declined during IR; GSH protected complex II function. GSH also protected from MMP-9 and neutrophil sequestration (P>.05).
CONCLUSIONS
GSH preconditioning is effective to prevent mitochondrial death and improves complex II function during IR, but not mitochondrial membrane stability. GSH-mediated amelioration of ΔΨm hyper-polarization appears to be the key factor of mitochondrial protection.
目的
还原型谷胱甘肽（GSH）已被证明可改善肺移植物保存。线粒体功能障碍被认为是实体器官缺血再灌注损伤（IR）的动力。我们之前已经表明，IR 会导致肺线粒体损伤。本研究阐明了 GSH 预处理对热肺 IR 情况下肺线粒体完整性和功能的影响。
方法
Wistar 大鼠分为对照组、假手术组和两个研究组（IR30/60 和 GSH-IR30/60），分别接受 IR 和 GSH 预处理。大鼠接受麻醉和机械通气。肺原位夹闭后再灌注产生 IR。从肺组织中分离线粒体。通过极谱法分析呼吸链复合物活性（I-IV）。测定线粒体活力（Ca2+诱导肿胀）和膜完整性（柠檬酸合酶测定）。通过酶联免疫吸附试验（ELISA）定量亚细胞细胞色素 C 含量（Cyt C）。通过荧光激活细胞分选（FACS）在加能和去耦后分析线粒体膜电位（ΔΨm）。通过髓过氧化物酶活性（MPO）和凝胶电泳法基质金属蛋白酶 9（MMP-9）活性来确定炎症激活。
结果
肺 IR 显著降低了线粒体活力，同时伴有 ΔΨm 超极化。GSH 预处理可改善线粒体活力并使 ΔΨm 正常化。IR 后 Cyt C 减少；GSH 可防止 Cyt C 释放。呼吸链复合物活性（I、II、III）在 IR 期间下降；GSH 保护复合物 II 功能。GSH 还可防止 MMP-9 和中性粒细胞隔离（P>.05）。
结论
GSH 预处理可有效防止线粒体死亡，并在 IR 期间改善复合物 II 功能，但不能改善线粒体膜稳定性。GSH 介导的 ΔΨm 超极化缓解似乎是线粒体保护的关键因素。
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