Pulmonary Vascular Endothelial Cell Injury And The Protective Effect Of HSP In Lung Ischemia-reperfusion Of Rabbit

Objective: Studies have indicated that reperfusion may trigger a series of injury responses, and cause the corresponding organs lethality damage. Pulmonary reperfusion injury could occur after lung transplantation, extracorporeal circulation surgery, pulmonary artery thromboendarterectomy, pulmonary embolism thrombolysis and so on, but few therapies have been demonstrated for preventing such injury. The mark changes of ischemia-reperfusion lung injury is pulmonary capillary endothelial cell injury which could increase pulmonary vasopermeability, pulmonary vascular resistance and pulmonary artery pressure. The mechanism of lung reperfusion injury is still not clearly elucidated, which might possibly be involved with many kinds of factors such as the inflammation medium, the oxygen free radicals, the alveolar epithelium and the lung blood vessel endothelium damage. Heat or other damage factors such as oxygen deficiency, heavy metals, could induce cell secrete heat shock protein (HSP). HSP play an important role in resisting surrounding injury, maintaining homeostasis and adapting environmental changes. Many studies indicated HSP70 protected liver, heart, brain from ischemia and reperfusion injury. But the role of HSP70 in the lung ischemia reperfusion process is not clear. The article is for the purpose of discussing the changes of factors released by the damaged endothelia cell, including endothelin-1 (ET-1), nitric oxide (NO), von Willebrand factor (vWF) and the expression of HSP-70 in pulmonary tissue, investigating the possible mechanism of ischemia-reperfusion lung injury and the role of HSP-70 in the lung ischemia-reperfusion process.Method: Thirty healthy New Zealand rabbits of either gender and weighing 2.5~3.5kg were studied, which the left lower lung artery were obstructed by inflating gas of 5F Berman sacculus catheter to set up rabbit pulmonary embolism model, then the gas of sacculus was put out to result in blood reperfusion. The animals were randomly divided into five groups: sham operation group (n=6), embolism 1h group (n=6), embolism 2h group (n=6), embolism 2h and reperfusion 1h group (n=6), embolism 2h and reperfusion 2h group (n=6). Drawing venous blood of all group rabbit at before, embolism 1h, embolism 2h, embolism 2h reperfusion 1h, embolism 2h reperfusion 2h, anticoagulated and centrifuged, then extracted the supernatant. All the plasmas and supernatants were preserved in the -20℃refrigerator. We detected the content of ET in plasma by radio-immunoassay (RIA) method , calculated vWF by enzyme linked immunosorbent assay (EILSA), and determined NO by nitrate reductase method. At the corresponding time, we puted the rabbit to death, the left lung tissue specimen was fixed by 4% polyformaldehyde and 4% glutaraldehyde respectively, then immunohistochemistry was carried to determine the expression of HSP70 and histopathologic analysis was carried to observe the ultrastructural change in the lung tissue.Results: (1)The changes of ET-1, NO and NO/ET-1 ratio in plasma: Except sham group, the content of the ET-1, NO in other four groups was higher than before embolism (P <0.05). Compared with sham group, the content of the ET-1, NO in plasma significantly increased 1h, 2h after lung embolism, further increased after reperfusion, and the increase was most obvious at reperfusion 2h. The NO/ET-1 ratio in embolism 1h, 2h and reperfusion 1h, 2h were obviously lower than pre-embolization and sham group. (2)The content changes of vWF in plasma: Except sham group, the content of the vWF in other four groups was higher than before embolism (P <0.05). Compared with sham group, the content of the vWF in plasma significantly increased 1h, 2h after lung embolism, futher increased at 1h after reperfusion, and reached its peak value 2h after reperfusion. (3)After the lung ischemia/reperfusion, immunohistochemistry demonstrated that the massive expressions of HSP70 positive cell in rabbit lung tissue were mainly located in pulmonary alveolus epithelial cells, bronchial epithelium. Compared with sham group, the positive area of HSP70 in rabbit lung tissue was remarkably increased at embolism 1h, 2h, and reperfusion 1h, 2h (P<0.05). HSP70 expression in reperfusion 1h, 2h groups were higher than embolism 1h, 2h groups, HSP70 expression in reperfusion 2h group was higher than reperfusion 1h group (P<0.05). (4)Transmission electron microscope showed cell swelling, vacuolization, partial plasmolysis, and dense nuclear chromatin in capillary endothelium after lung reperfusion.Conclusion: (1)In our experiment, we successfully established lung ischemia/reperfusion animal model by using Berman balloon catheter. The experimental animal model of lung ischemia/reperfusion was ideal animal model in elementary research because it owned the advantages in convenient controllability and easy reproducibility. (2)Transmission electron microscope showed in the lung ischemia-reperfusion process existed capillary endothelium injury. (3)ET-1, NO, vWF may be the marker for endothelial cell damage. The result of study demonstrated endothelial cell injury and functional disturbance participatied in the lung eischemia-reperfusion injury. (4)HSP70 overexpressed in the process of the lung ischemia-reperfusion, we presumed HSP70 participatied in the protection of the lung injury.