This work was supported by the National Natural Science Foundation of China (No. 30571782 ) and the Foundation of Educational Committee of Jiangsu Province(No. 05KJD32023 ).

^*Corresponding author. E-mail: jnyuanli@163.com

Ro5-4864——外周苯二氮卓受体激动剂抑制大鼠心肌线粒体通透性转换

李敬远^1,2 王俊科¹ 曾因明^2,*

¹中国医科大学附属第一医院麻醉科，沈阳 110001；²江苏省麻醉医学研究所，徐州医学院江苏省麻醉学重点实验室，徐州 221002

摘 要：线粒体通透性转换（mitochondrial permeability transition，MPT）导致线粒体氧化应激性损伤。近年研究认为位于线粒体外膜的外周苯二氮卓受体(peripheral benzodiazepine receptor，PBR)参与了线粒体的重要生理功能。本研究在心肌线粒体水平探讨激动外周苯二氮卓受体是否能抑制Ca²⁺诱发的MPT。分离SD大鼠心肌线粒体，将外周苯二氮卓受体激动剂Ro5-4864（50,100,200μmol/L）和线粒体孵育，利用150μmol/L Ca²⁺诱发MPT ，部分线粒体和100μmol/L Ro5-4864孵育之前5min加入线粒体通透性转换孔道(mitochondrial permeability transition pore, mPTP)开放剂苍术苷(ATR)。采用分光光度法观察线粒体膨胀情况，Western Blot法检测线粒体细胞色素C(CytoC)释放，利用荧光探针JC-1在激光共聚集显微镜下观察线粒体膜电位的变化。50,100,200μmol/L Ro5-4864均显著抑制Ca²⁺诱发的线粒体在520nm处吸光度的下降，而且Ro5-4864抑制Ca²⁺引起的线粒体CytoC释放和线粒体膜电位下降，但ATR可取消Ro5-4864的上述作用。结果提示PBR激动剂可抑制大鼠心肌MPT，保持线粒体CytoC含量和稳定线粒体膜电位，减轻心肌线粒体损伤，PBR的激活可能成为减轻心肌细胞应激性损伤及心肌保护的新方法。

关键词：外周苯二氮卓受体；Ro-54864；线粒体通透性转换

中图分类号：Q463

Ro5-4864— peripheral benzodiazepine receptor agonist inhibits mitochondria permeability transition of rat heart

LI Jing-yuan^1,2 , WANG Jun-ke ¹, ZENG Yin-ming^2,*

¹ Department of Anesthesiology, the First Affiliated Hospital, China Medical University, Shenyang 110001,China; ² Jiangsu Institute of Anesthesiology, Jiangsu Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221002,China

Abstract: Opening of mitochondrial permeability transition (MPT) pores leads to mitochondrial injury during oxidative stress. The peripheral benzodiazepine receptor (PBR), located at mitochondrial outer-membrane, has been shown to be involved in several mitochondrial functions. In the current study, we use Ro5-4864, a PBR agonist, to test if activation of PBR can prevent mitochondrial permeability transition opening during Ca²⁺ overloading. Cardiac mitochondria were isolated from Sprague-Dawley hearts. The isolated mitochondria were treated by 150 μmol/L Ca²⁺ to induce MPT. Ro5-4864 (50, 100, 200 μmol/L) was added into incubation buffer before adding 150 umol/L calcium, respectively. In additional group (ATR+Ro group), Atractyloside (ATR, 20μmol/L), an opener of MPT was added 5 minutes before the addition of 100μmol/L Ro5-4864. The change of absorbance at 520 nm (Abs520 nm) was monitored with a spectrophotometer at 30℃ for 10 min. Western Blotting was used to detect cytochrome c loss. The mitochondrial membrane potential were monitored with the fluorescence dye JC-1. Ro5-4864 inhibited the decrease of absorbance at 520 nm compared to the untreated Ca²⁺ group（P<0.01, P<0.05）. In the presence of ATR, Ro5-4864 was not able to prevent MPT anymore. Opening of MPT by Ca²⁺ decreases the content of cytochrome c in mitochondria, but increases cytochrome c content in cytosol. Ro5-4864 preserved cytochrome c content in mitochondria and led to less cytochrome c release to cytosol. ATR treatment reverses the protective effect of Ro5-4864 on cytochrome c content. Opening of MPT with calcium led to mitochondrial depolarization, whereas RO5-4864 treatment maintains mitochondrial membrane potential in the presence of 100 μmol/L calcium. Thus, prevention of MPT by activation of PBR during calcium overloading maintains mitochondrial cytochrome c content and membrane potential. Activation of PBR during cardiac ischemia and reperfusion may be an alternative way for cardioprotection.

Key words: peripheral benzodiazepine receptor; Ro5-4864; mitochondria permeability transition