Objective: To investigate the role of cisatracurium in diaphragm atrophy in mechanically ventilated (MV) rats and its possible mechanism.
Methods: 30 adult male Sprague-Dawley (SD) rats were randomly assigned to 5 groups: Rats in the control (CON) group ( n=6) were fasted for 30 h without any other intervention; rats in the MV group ( n=6) were fasted for 6 h, and then mechanically ventilated for 24 h while receiving continuous infusion of sodium pentobarbital and 0.9% NaCl; rats in the MV+cisatracurium (MVC) group ( n=6) were fasted for 6 h, and then mechanically ventilated for 24 h while receiving continuous infusion of sodium pentobarbital and cisatracurium; rats in the MV+chloroquine (QMV) group ( n=6) and rats in the MV+cisatracurium+chloroquine (QMVC) group ( n=6) received intraperitoneal injection of chloroquine (30 mg/kg), an autophagy inhibitor, at 24 h and 30 min prior to MV in addition to the treatments given to the MV group and the MVC group, respectively. The rats in each group were sacrificed 30 hours later, and costal diaphragm muscle specimens were collected. The cross-sectional area (CSA) of the diaphragm fibers was observed through HE staining, and the colocalizations of TOM20 and LC3 were assessed by immunofluorescence staining. The expression levels of PINK1, Parkin, P62 and LC3, the mitophagy-related proteins, and the expression levels of MAFbx and MURF-1, muscular-atrophy-related proteins, were evaluated by Western blot.
Results: Respective comparisons of the MV group with the CON group and the MVC group with the MV group showed that the CSA decreased ( P<0.05), the expression of MURF-1, MAFbx, PINK1, Parkin and LC3Ⅱ/Ⅰproteins increased ( P<0.05), the number of co-expressed mitochondria of TOM20 and LC3 and the expression of LC3 increased and the expression of P62 protein decreased ( P<0.05) in the MV and MVC groups. Respective comparisons of the QMV group with the MV group and the QMVC group with the MVC group showed that the CSA increased ( P<0.05), the expression of MURF-1, MAFbx, PINK1, Parkin and LC3Ⅱ/Ⅰ proteins increased ( P<0.05), the number of co-expressed mitochondria of TOM20 and LC3 and the expression of LC3 decreased and the expression of P62 protein decreased ( P<0.05) in the QMV and QMVC group.
Conclusion: Mechanical ventilation for 24 h caused diaphragm atrophy in SD rats. Cisatracurium may aggravate diaphragm atrophy in mechanically ventilated rats through the autophagy-lysosome (AL) pathway, a process that may be related to the PINK1/Parkin-mediated mitophagy, and chloroquine may reduce diaphragmatic atrophy induced by cisatracurium by blocking the AL pathway.
目的: 研究神经肌肉阻断剂顺式阿曲库铵在机械通气SD大鼠膈肌萎缩中的作用及可能的机制。
方法: 将30只成年雄性SD大鼠随机分为5组：①对照组（CON组，n=6），禁食30 h；②机械通气组（MV组，n=6），禁食6 h后机械通气24 h，同时持续泵入戊巴比妥钠+0.9%氯化钠；③机械通气+顺式阿曲库铵组（MVC组，n=6），禁食6 h后机械通气24 h，同时持续泵入戊巴比妥钠+顺式阿曲库铵；④机械通气+氯喹（chloroquine, CQ）组（QMV组，n=6），机械通气前24 h和前30 min腹腔注射自噬抑制剂CQ 30 mg/kg，其余操作同MV组；⑤机械通气+顺式阿曲库铵+CQ组（QMVC组，n=6），机械通气前24 h和前30 min腹腔注射自噬抑制剂CQ 30 mg/kg，其余操作同MVC组。各组大鼠于30 h后处死，取肋膈肌标本。HE染色观察膈肌纤维横截面积（cross-sectional area, CSA），免疫荧光染色观察线粒体外膜转移酶蛋白-20（TOM20）和微管相关蛋白1-轻链3（LC3）的共定位，Western blot检测肋膈肌中肌肉萎缩相关蛋白MAFbx、MURF-1，线粒体自噬相关蛋白PINK1、Parkin、P62、LC3的表达水平。
结果: MV组和CON组相比，MVC组和MV组相比，CSA减小（P<0.05），MURF-1、MAFbx蛋白的表达量增加（P<0.05），TOM20和LC3的共表达的线粒体数量增加，LC3表达增加（P<0.05），PINK1、Parkin、LC3Ⅱ/Ⅰ蛋白的表达量增加（P<0.05），P62蛋白的表达量下降（P<0.05）；QMV组和MV组相比、QMVC组和MVC组相比，CSA增大（P<0.05），MURF-1、MAFbx蛋白的表达量减少（P<0.05），TOM20和LC3的共表达的线粒体数量减少，LC3表达减少（P<0.05），PINK1、Parkin、LCⅡ/Ⅰ蛋白的表达量减少（P<0.05），P62蛋白的表达量增加（P<0.05）。
结论: 机械通气24 h，引起SD大鼠膈肌萎缩；顺式阿曲库铵通过自噬-溶酶体（autophagy-lysosome, AL）途径加重机械通气大鼠的膈肌萎缩，此过程可能与PINK1-Parkin介导的线粒体自噬途径有关；氯喹通过阻断AL途径可改善顺式阿曲库铵引起的机械通气大鼠膈肌萎缩。
Keywords: Chloroquine; Cisatracurium; Diaphragm atrophy; Mechanical ventilation; Mitophagy.
Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).