PINK1/parkin介导的线粒体自噬在机械通气导致的膈肌功能障碍中的作用

Role of PINK1/parkin-mediated mitophagy in diaphragmdysfunction caused by mechanical ventilation

摘要:

目的：研究PTEN诱导激酶1（PTEN induced putative kinase 1，PINK1）/parkin介导的线粒体自噬在机械通气（mechani－cal ventilation，MV）导致的膈肌功能障碍和萎缩中的作用。方法：将24只成年雄性SD大鼠随机分成Control组（n=6）、MV-6h组（n=6）、MV-12h组（n=6）和MV-24h组（n=6）。Control组大鼠未经任何处理，MV-6h组大鼠机械通气6 h，MV-12h组大鼠机械通气12 h，MV-24h组大鼠机械通气24 h。采用RM6240生物信息采集系统测量膈肌复合肌动作电位（compound muscle action potential，CMAP），HE染色测量膈肌肌纤维横截面积（cross-sectional area，CSA），Western blot检测膈肌肌肉萎缩蛋白（muscle atrophy F-box，MAFbx）、肌肉特异性环指蛋白1（muscle specific ring finger 1，MURF1）、线粒体裂解蛋白1（dynamin-related pro－tein 1，drp1）、线粒体融合蛋白2（mitofusin-2，mfn2）、线粒体自噬相关蛋白PINK1、parkin、P62及微管相关蛋白轻链3（micro－tubule-associated protein 1 light chain 3，LC3）水平。结果：MV-12h组[（3.15±0.52） mV]和MV-24h组[（2.44±1.26） mV]膈肌的CMAP幅值明显下降（P=0.000）；MV-24h组[（7.05±0.61） ms]膈肌的CMAP时程明显延长（P=0.000）；与Control组[（5 308.67±228.18） μm2]相比，MV-6h组[（3 378.33±393.40） μm2]、MV-12h组[（2 969.67±35.16） μm2]和MV-24h组[（2 115.33±130.23） μm2]膈肌的CSA均明显减少（P=0.000）；与Control组相比，MV-12h组（2.59±0.19）、MV-24h组（4.11±0.19）的MAFbx明显升高（P=0.000），MV-12h组（1.96±0.26）、MV-24h组（2.88±0.29）的MURF1明显升高（P=0.000）；与Control组相比，drp1在MV-24h组（5.55±0.36）明显上升（P=0.000），mfn2在MV-12h组（0.47±0.13）、 MV-24h组（0.35±0.10）明显降低（P=0.002）；与Control组相比，PINK1在MV-12h组（2.53±0.25）和MV-24h组（4.78±0.31）明显上升（P=0.000），parkin在MV-24h组（3.73±0.16）明显上升（P=0.000），P62在MV-12h组（2.28±0.06）和MV-24h组（3.69±0.17）明显上升（P=0.000），LC3Ⅱ/Ⅰ比值在MV-24h组（1.57±1.32）明显上升（P=0.002）。结论：MV可以引起PINK1/parkin介导的线粒体自噬发生改变，并引起膈肌功能障碍和萎缩。

Abstract:

Objective：To investigate the role of PTEN induced putative kinase 1（PINK1）/parkin-mediated mitophagy in diaphragm dysfunction caused by mechanical ventilation（MV）. Methods：Twenty-four adult male SD rats were randomly divided into the Control group（n=6），the MV-6h group（n=6），the MV-12h group（n=6），and the MV-24h group（n=6）. Rats in the Control group were without any treatment，in the MV-6h group were mechanically ventilated for 6 h，in the MV-12h group were mechanically ventilated for 12 h and in the MV-24h group were mechanically ventilated for 24 h. Using RM6240 multi-channel physiology signal collection and pro－cessing system was used to detect diaphragm compound action potential（CMAP），hematoxylin and eosin staining method was used to measure the cross-sectional area（CSA） of the diaphragm，and Western blot was used to detect levels of muscle atrophy F-box（MAFbx），muscle specific ring finger 1（MURF1），mitochondrial fission protein（drp1），mitofusin-2（mfn2），autophagy-related protein of PINK1，parkin and P62，and microtubule-associated protein 1 light chain 3（LC3）. Results：The amplitude of CMAP in the MV-12h group[（3.15±0.52） mV] and the MV-24 group[（2.44±1.26） mV] was significantly decreased（P=0.000）. The duration of CMAP in the MV-24h group[（7.05±0.61） ms] was significantly longer（P=0.000）. Compared with the Control group[（5 308.67±228.18） μm2]，the CSA of diaphragm in the MV-6h group[（3 378.33±393.40） μm2]，the MV-12h group[（2 969.67±35.16） μm2] and the MV-24h group[（2 115.33±130.23） μm2] was significantly de－creased（P=0.000）. Compared with the Control group，the level of MAFbx in the MV-12h group[（2.59±0.19）] and the MV-24h group[（4.11±0.19）] was significantly increased（P=0.000），the level of MURF1 in the MV-12h group[（1.96±0.26）] and MV-24h group[（2.88±0.29）] was significantly increased（P=0.000）. drp1 in the MV-24h group（5.55±0.36）（P=0.000） was significantly increased and mfn2 in the MV-12h group（0.47±0.13） and the MV-24h group（0.35±0.10） was significantly decreased（P=0.002）. Compared with the Control group，PINK1 in the MV-12h group（2.53±0.25） and the MV-24h group（4.78±0.31） was significantly increased（P=0.000），parkin in MV-24h group（3.73±0.16） was significantly increased（P=0.000），P62 in MV-12h group（2.28±0.06） and MV-24h group（3.69±0.17） was significantly increased（P=0.000），and LC3Ⅱ/Ⅰ ratio in MV-24h group（1.57±1.32） was increased significantly（P=0.002）. Conclusion：MV can change the PINK1/parkin-mediated mitophagy，leading to diaphragm dysfunction and atrophy.