Role Of PINK1/parkin-mediated Mitophagy In Diaphragm Dysfunction Induced By Mechanical Ventilation

Objective:To study the effects of mechanical ventilation?MV?on diaphragm function and atrophy at different duration,and to explore PTEN induced putative kinase 1?PINK1?/parkin-mediated mitophagy in diaphragm dysfunction caused by mechanical ventilation?Ventilator-induced diaphragm dysfunction,VIDD?.Methods:Twenty-four SPF-grade 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?.The rats in the Control group were without any treatment;The rats in the MV-6h group were mechanically ventilated for 6 h;The rats in the MV-12h group were mechanically ventilated for 12 h;The rats in the MV-24h group were mechanically ventilated for 24 h.The following indicators were detected at the corresponding time points after the ending of mechanical ventilation in each group of rats:1.Diaphragm function:Using RM6240 multi-channel physiology signal collection system to measure the compound action potential?CMAP?of the diaphragm;2.Diaphragm atrophy:Hematoxylin-eosin staining method to measure the cross-sectional area?CSA?of the diaphragm,Western blotting?WB?to detect the levels of muscle atrophy F-box?MAFbx?and muscle specific ring finger 1?MURF1?;3.Ultrastructure and membrane potential???m?of diaphragm mitochondria:The changes in mitochondrial ultrastructure of diaphragm was observed by transmission electron microscopy?TEM?;The mitochondrial membrane potential???m?was detected by JC-1 Immunofluorescence Staining;4.Oxidative stress level:The levels of superoxide dismutase?SOD?,glutathione peroxidase?GSH?,and catalase?H2O2?in the diaphragm were detected by WST-1,colorimetric,and visible light methods,respectively.5.Autophagy-related indicators:WB detected the level of mitochondrial fission protein?drp1?and fusion protein?mitofusin-2,mfn2?,autophagy-related protein PINK1,parkin,P62 and microtubule-associated protein 1 light chain 3?LC3?;By labeling the mitochondrial membrane protein tom20 and the autophagy-related proteins PINK1 to observe the position and co-localization,respectively.Results:?1?The amplitude of CMAP of diaphragm in the MV-24h group was significantly decreased compared to the Control group and MV-6h group,these differences were statistically significant?P<0.05?;The duration of CMAP of diaphragm in the MV-24h group was significantly increased compared to the Control group,MV-6h group and MV-12h group,these differences were statistically significant?P<0.05?.?2?Compared with the Control group,the CSA of diaphragm were significantly decreased in the MV-6h group,MV-12h group and MV-24h group,these differences were statistically significant?P<0.05?.Compared with the Control group,the MAFbx and MURF1 were significantly increased in the MV-12h group and MV-24h group,these differences were statistically significant?P<0.05?.?3?In the Control group,the mitochondrial structures of diaphragm were generally intact;After mechanical ventilation for 6 h,the mitochondria began to swell and cristae disappeared;After mechanical ventilation for 12 h,the mitochondria were obviously swollen,and a small number of mitophagy were seen between myofibrils;After mechanical ventilation for 24 h,in addition to mitophagy,a large number of lipid droplets were also found;Compared with the Control group,the??m were significantly decreased in the MV-12h group and MV-24h group,these differences were statistically significant?P<0.05?.?4?Compared with the Control group,the SOD activity was significantly decreased in the MV-12h group and MV-24h group,the GSH activity was significantly decreased in the MV-6h group,MV-12h group and MV-24h group,the H₂O₂ activity was significantly increased in the MV-12h group and MV-24h group,these differences were statistically significant?P<0.05?.?5?Compared with the Control group,drp1 increased significantly in MV-24h group,mfn2 decreased significantly in MV-12h group and MV-24h group,these differences were statistically significant?P<0.05?;Compared with the Control group,PINK1 increased significantly in MV-12h group and MV-24h group,parkin increased significantly in MV-24h group,P62 increased significantly in MV-12h group and MV-24h group,LC3II/I ratio increased significantly in MV-24h group,these differences were statistically significant?P<0.05?;Compared with the Control group,large of the co-localizations of PINK1 and tom20 were found in the MV-24h group.Conclusions:?1?Mechanical ventilation for 6 h will cause diaphragm dysfunction in rats,mechanical ventilation for 12 h will cause diaphragm atrophy in rats,and the diaphragm dysfunction and atrophy of rats will further increase with the prolong of mechanical ventilation time.?2?MV can cause the changes of diaphragm mitochondrial structure,generation of a large number of ROS,imbalance of mitochondrial dynamics,and cause the changes of mitophagy mediated by PINK1/parkin,mitophagy is closely related to the occurrence of VIDD.