Department Of Thoracic Surgery, Union Hospital Of Huazhong University Of Science And Technology

Background:Lung ischemia-reperfusion (I/R) injury occurs in various clinic events, such as lung transplantation, sleeved lobectomy, pulmonary thromboendarterectomy, aortic surgery, heart surgery, hemorrhagic shock, cardiopulmonary bypass and post-resuscitation for circulatory arrest. For example, Bronchiolitis obliterans syndrome, the main cause of lung allograft dysfunction, remains to be a great troublesome for lung transplant doctors, but appears to be related to lung I/R injury after organ implantation. The peroperative complication and long-time graft function are tightly associated with the initial intensity of I/R injury. Lung I/R injury, which causes elevated morbidity and mortality, is characterized by interstitial edema, inflammatory cells infiltration, disruption of respiratory membrane and confused gas exchange. However, understanding of the integrated mechanism of lung I/R injury remains incomplete.Autophagy, which resides in almost all cells that have mitochondria, is a traditional conserved homeostasis process. On one hand, autophagic double-membrane vesicles, termed autophagosomes, separate and digest dysfunctional organelles, defective proteins and the intracellular pathogen through the lysosomes-dependent degradation pathway. On the other hand, the "self-eating" process can provide anabolic substrates for cells. Virtually, autophagy occurs at low basal levels in cells to execute homeostatic function, such as organelles and protein turnover. It is up-regulated rapidly when intracellular nutrition and energy is deficient, like hypoxia, starvation, and grow factor depletion. Because of autophagic procedure, the cells not only get rid of the impaired mitochondria and endoplasmic reticulum (ER) in stresses, but also get enough substrates for survival. Autophagy also can induce programmed cell death, termed type II programmed cell death, when it is inappropriately activated. Besides that, autophagy can lead to apoptosis because of their crosstalk. So autophagy is considered as an adaptation mechanism for cells in stress.Increasing lines of evidences suggest that the formation of autophagosomes play a considerable role in many pathophysiological states including cancer, infection, immunity and neurodegenerative diseases. Previous studies showed that ischemia and immediate reperfusion can trigger elevated autophagic flux. Chigure Suzuki and coworkers observed an increase of autophagosomes in the renal tubular epithelial cells of I/R-injured mouse models. They also found inhibition of autophagy by an inhibitor significantly inhibited H2O2-induced cell death in the human renal proximal tubular epithelial cell line HK-2. Their results indicated that autophagy participated in renal I/R injury. But Man Jiang’s research presented a converse result in other renal I/R model. They declared autophagy was a renoprotective mechanism during in vitro hypoxia and in vivo I/R injury. As to liver, Kunihito Gotoh detected inhibition of autophagy can attenuate rat liver I/R injury after transplantation. Therefore, autophagy is presumed to have both favorable and damaging effects, and its role in I/R injury appears to be model-depended and/or tissue-depended. Dose autophagy participates in lung I/R injury pathophysiological process? What’s the role of autophagy in lung I/R injury? Here we employed rats to set up lung I/R model in situ to find clues.Methods:The lung ischemia/reperfusion injury mode in rats was developed to explore the level of autophagy in lung I/R injury. LC-3, autophagy labeled protein, was detected by western blot. Autophagosome was observed by electronic microscope. Four group rats which were subjected sham operation,3hours ischemia,1hour ischemia followed2hours reperfusion or rapamycin pretreated five days and sham operation to study the role of autophagy in activating autophagy. Inhibitor (3-methyladenine) of autophagy were injected before ischemia to study the the role of autophagy in lung I/R injury. Lung W/D, MPO, MDA and lung injury score were used to measure the level of injury in lung. LC-3was detected to inform the inhibit action of3-MA. Apoptosis associated protein caspas-3were detected by western blot and TUNEL was was to measure the level of apoptosis in3-MA pretreated lung to explore the mechanism.Result:The results indicated that sham group expressed base LC3-Ⅱ density, which was increased after60min ischemia, and the reperfusion2h and6h groups presented the most labeled protein. After24hours reperfusion, the densitometry of LC3-Ⅱ returned to base level. The autophagic flux was elevated during ischemia period, and was significantly enhanced during reperfusion.30mg/kg3-methyladenine (3-MA) can inhibit autophagy in lung and ameliorated lung I/R injury, as indicated by reduced lung wet/dry ratio (W/D), myeloperoxidase (MPO) activity, malondialdehyde (MDA) concentration and lung injury score.3-MA pretreatment also reduced cleaved caspase-3and apoptosis in lung, as indicated by Terminal dUTP nick end-labeling (TUNEL) assay.Conclusion:Together, the results demonstrated that lung ischemia and the followed reperfusion can activate autophagy. And the elevated autophagy might be a scathing factor in lung I/R injury. It may aggravate lung ischemia-reperfusion injury by activate apoptosis.