Investigation Of The Effects Of Autophagy In Ventilator-Induced Diaphragm Dysfunction Of Rat

Background Mechanical Ventilation(MV)is an important tool of life support in clinical,the objective of using MV is to fight for the time to cure the patient and find the etiology.However,prolonged mechanical ventilation(PMV)can cause diaphragm dysfunction(DD),characterized by rapid decrease muscle strength and muscle atrophy,which termed ventilator-induced diaphragmatic dysfunction(VIDD).VIDD was significantly associated with difficulty in weaning,prolonged mechanically ventilatory time and length of hospital stay,and increased complications and mortality.Mitochondrial oxidative stress-induced proteolytic enzyme system activation is one of the important mechanisms of VIDD.Reactive oxygen species(ROS)have been shown to mediate autophagy activation,but autophagy is considered to have a "double-edged sword" effect.Therefore,this study aimed to investigate the role of autophagy in prolonged mechanical ventilation and the interaction between ROS and autophagy on the regulation of proteolytic enzyme systems.Objectives(1)To investigate whether prolonged mechanical ventilation increases the level of autophagy in the diaphragm and the possible role of autophagy in the development of VIDD;(2)To investigate the relationship between autophagy activation and mitochondrial ROS formation during prolonged mechanical ventilation;to investigate the effect of PMV on autophagic flux.Methods(1)Prolonged mechanical ventilation induced diaphragm autophagy and VIDD: Adult male Wistar rats were randomly assigned to 4 groups:(1)short-term spontaneous breathing group(SSB,spontaneous breathing for 4 h after sham surgery,n = 5);(2)short-term mechanical ventilation group(SMV,mechanical ventilation for4 h,n = 5);(3)prolonged spontaneous breathing group(PSB,spontaneous breathing for 12 h after sham surgery,n = 5);(4)prolonged mechanical ventilation group(PMV,mechanical ventilation for 12 h,n = 5).The heart rate,blood pressure,and rectal temperature were monitored throughout the experiment.Arterial blood gas and blood routine analysis in the short-term group and the prolonged group were every 2 h and 4h,respectively.The strength of the diaphragm muscle strip was measured by electrical stimulation.The autophagic structures was observed by transmission electron microscopy.The cross-sectional area(CSA)was observed by HE and immunofluorescence staining.The expression of autophagy-related genes was detected by real-time quantitative PCR.Western-blot was used to detect microtubule-associated protein LC3B-II,LC3B-II/I ratio,Caspase-3 and calpain activity,and protein ubiquitin E3 ligase Atrogin-1 and MuRF1 expression;immunochemical assay was used for assessing diaphragmatic ROS products.(2)Relationship between autophagy activation and mitochondrial ROS production and their roles in the development of VIDD: Adult male Wistar rats were randomly assigned to 3 groups(n = 10/group):(1)prolonged spontaneous breathing control group(CON,spontaneous breathing for 12 h after sham surgery,n = 5);(2)prolonged mechanical ventilation(12 h)group(CMV,n = 5);(3)prolonged mechanical ventilation plus mitochondrial oxygen free radical scavenger Mito-Tempol group(CMV + Mito-T,n = 5);another 5 rats in each group were given the autophagic tool medicine colchicine before intervened.Heart rate,blood pressure,and rectal temperature were monitored throughout the experiment.Arterial blood gas and blood routine were analysed every 4 hours;Contractile ability of the diaphragmatic muscle was measured in vitro;autophagy lysosome-related structures were observed by transmission electron microscopy;HE and immunofluorescence staining were performed to assess cross-sectional area;real-time quantitative PCR was used to detect the relative expression of autophagy-related genes;Western-blot was used to detect the ratio of microtubule-associated protein LC3B-II/I,Caspase-3,calcium,and rotein ubiquitin E3 ligase Atrogin-1 and MuRF1;immunochemical assay for diaphragmatic ROS products.Results(1)Prolonged mechanical ventilation induced diaphragm autophagy: There was no significant changes in body weight and vital signs(heart rate,blood pressure,and rectal temperature)before and after the experiment.The in vitro diaphragm muscle contractile test showed that the maximal twitch force,maximumtetanic force,frequency-force effect curve and anti-fatigue ability of the diaphragm muscle of the PMV group were significantly lower than those of the SSB,SMV and PSB groups.Transmission electron microscopy showed that the number of autophagy-related structures was significantly increased as compared to SSB,SMV and PSB groups,and lipid droplet and disordered myofilament structure were observed.Immunofluorescence staining showed that the myofilament cross-sectional area of the fast muscle fibers and slow muscle fibers in PMV group was significantly decreased than that of SSB,SMV,and PSB group;western-blot results showed that LC3B-II level,LC3B-II/LC3B-I ratio,Caspase-3,calpain activity,and E3 ligase Atrogin-1 and MuRF1 protein expression were significantly higher in the PMV group than those in the SSB group.The ROS producttion in the PMV group was significantly higher than those of the SSB,SMV,and PSB groups.However,there was no significant difference between the SSB,SMV and PSB groups.These results suggest that prolonged mechanical ventilation leads to decreased contractile force of the diaphragm,accompanied by activation of the proteolytic enzyme system and activation of the diaphragm autophagy,suggesting that the proteolytic enzyme system and autophagy play an important role in the development of VIDD.(2)Relationship between autophagy and mitochondrial ROS production and their roles and mechanisms in the development of VIDD: animal experiments showed that the characters of animals(body weight,rectal temperature,heart rate,blood pressure,blood gas,and blood routine)was insignificant different between the groups after the experiment.The level of TNF-α was significant in the CMV group as compared to the other three groups.Both Colchicine and Mito-Tempol significantly increased diaphragm contractility and diaphragm muscle fatigue resistance,improved diaphragmatic atrophy,and reduced the number of autophagosome lysosomal-related structures.The inhibition of autophagosome degradation by Colchicine can effectively reduce the production of mitochondrial ROS production induced by PMV,indicating that the mitochondrial ROS formation in diaphragmatic muscle during mechanical ventilation is dependent at least in part on the activation ofautophagosomes.In addition,the use of mitochondria-targeted superoxide scavenger Mito-Tempol significantly reduced mitochondrial ROS production and reduced the number of autophagosomes.At the same time,the activities of calpain and Caspase-3in PMV + Mito-T group were significantly decreased than those in PMV group,while the activities of calpain and Caspase-3 in PMV + COL group were significantly higher than those in PMV group and PMV + Mito-Group T,and the differences were statistically significant(p < 0.05).In addition,the ratio of autophagy-related proteins LC3B-II and LC3B-II/I in the PMV + COL group was significantly higher than that in the PMV group,whereas the ratio of autophagy-related proteins LC3B-II and LC3B-II/I in the PMV + Mito-T group was significantly decreased than those of the PMV group;prolonged CMV significantly increased the protein level of Atrogin-1and MuRF1 in the diaphragm muscle,while in the PMV + COL and PMV + Mito-T group ubiquitin ligases Atrogin-1 and MuRF1 Protein expression was significantly lower than that of the PMV group.Morphological analysis showed that the CSA in the PMV + COL group and the PMV + Mito-T group was significantly higher than that in the PMV group,and the difference was statistically significant(p < 0.05).These results indicate that there is a positive regulation between autophagic activity and mitochondrial ROS production,which is an important mechanism of PMV-induced diaphragmatic atrophy and contractile dysfunction;inhibition of autophagosome degradation,diaphragmatic compensatory increase in calpain and Caspase-3 protease activity to degrade oxidatively damaged cytoplasmic proteins and organelles.Conclusions(1)Prolonged mechanical ventilation can induce ventilator-associated diaphragma dysfunction in the healthy rats,which is characterized by the decrease of maximal twitch force,maximal tetanic force,anti-fatigue ability and force-frequency effect curve of diaphragm muscle;The cross-sectional area of the fast and slow muscle fibers of the diaphragm is reduced;the molecular level shows the activity of four proteolytic enzyme systems(calpain,Caspase-3,ubiquitin protease,and autophagy)is increased.The proteolytic enzyme system is the main mechanism ofVIDD,and the activation of the proteolytic enzyme system may be related to the oxidative stress in the diaphragm.(2)The effect of prolonged CMV on autophagic flow is: increase the synthesis of autophagosomes and inhibit the degradation of autophagosomes;CMV-induced autophagy activation and positive feedback between skeletal muscle ROS production mediates the progression of VIDD;when autophagosome degradation is inhibited,calpain and Caspase-3 degrade the accumulated cytoplasmic proteins and dysfunctional organelles in a compensatory manner;mitochondria-targeted free radical scavengers can alleviate VIDD development process.It indicates that autophagy plays an important role in the development of VIDD,suggesting that inhibition of autophagy and the use of mitochondria-targeted antioxidants can alleviate the development of VIDD.