Released Mitochondrial Dna By Mitophagy Contributes To Ventilator Induced Lung Inflammation And Injury

Objective: To explore whether mechanical ventilation trigger mitophagy and its released mt DNA contributes to ventilator induced lung inflammation and injury.Methods: 1.Eighty specific pathogen free SD rats were randomly divided into five groups(n = 16),who underwent spontaneous breathing and mechanical ventilation with VT of 10,20,30 and 40 m L/kg.All rats in each group were then divided into four subgroups with duration of 1.0,2.0,3.0 and 4.0 hours(n = 4).Autophagic protein expression was assessed by RT-q PCR and WB,and further to observe autophagosomes by transmission electrion microscope in accordance to autophagy level.Mitochondria damage was evaluated by ATP level,ROS production and ??m variation during mitophagy.2.Thirty-six SD rats were randomly divided into three groups(n=12):(1)spontaneous breathing group(CON),normal VT group(NTV,VT= 8 m L/kg)and high VT group(HTV,VT= 40 m L/kg).All rats received endotracheal tube by tracheostomy.These rats in CON group were maintained spontaneous breathing,while rats in NTV and HTV group received mechanical ventilation.After four hours,the blood serum,BALF and lung tissues were collected.The lung wet/dry(W/D)ratio was assessed,and morphology changes were observed by light microscopy and electron microscopy.The infiltrated cells in BALF were counted and total protein in BALF was measured by BCA methods.ELISA was executed to measure the concentration of IL-1?,IL-6 and TNF-? in the serum and BALF.The m RNA expressions and protein levels of LC3 B,COX IV and NF-?B in lung tissues were assayed by immunoblotting and RT-q PCR.3.Another forty-eight SD rats were randomly divided into following four groups(n=12):(1)spontaneous breathing group(CON),high VT group(HTV),enhanced autophagy group(Am A)and inhibited autophagy group(Cs A).The rats except for these in CON groups were both received endotracheal tube by tracheostomy and mechanical ventilation with VT of 40 m L/kg.After four hours,the blood serum,BALF and lung tissues were collected.The lung W/D ratio was assessed,and morphology changes were observed by light microscopy and electron microscopy.The infiltrated cells in BALF were counted and total protein in BALF was measured by BCA methods.The concentration of IL-1?,IL-6,TNF-?,MPO and MIP-2 in serum and BALF were assayed by ELISA.Furthermore,the expression of mt DNA and DNase-II were assessed,and whether mt DNA released by mitophagy was proved by IF tracking.Results: Mechanical ventilation with VT greater than 30 m L/kg and duration more than 2.0 hours spontaneously would activate mitophagy,which induced mitochondria damage with dysfunction of ATP synthesis,accumulation of ROS and loss of ??m.Mechanical ventilation with high VT also induces acute inflammation and injury in lung tissues.The condition of lung edema,morphology injury and inflammation were both positively associated with autophagic level(both P < 0.05).In the enhanced autophagy group,theexpression of DNase-II in serum was decreased while mt DNA and inflammation in cytoplasm were severer compared with other groups(both P < 0.05).Conclusions: Inappropriate mechanical ventilation induces mitochondrial damage and activation of mitophagy.The mt DNA then is released from damaged mitochondrial to cytoplasm and contributed to ventilator induced lung inflammation and injury.