Effects Of Short Durations Hyperoxia On Mitochondrial Calcium And Ros Of Rat TypeⅡAlveolar Epithelial Cells

Objective It has been proved that long-term hyperoxia can damage lung tissue and cells.General anesthesia is associated with a very high incidence of hyperoxemia,despite it usually lasts for a relatively short period of time.It is not clear whether this short-term hyperoxia has adverse effects on lung tissue and cells.This experiment was aimed to investigate whether short time hyperoxia causes rats typeⅡAlveolar Epithelial Cells（AECⅡ）injury,and to detect the effects of short durations hyperoxia exposure on mitochondrial calcium and ROS production of AECⅡ,to explore the related mechanism of lung injury with hyperoxia.Methods Rats AECⅡcell lines（RLE-6TN）were randomly divided into three groups:a nomoxia group（C,21%O₂）,a middle oxygen fraction group（M,60%O₂）,a high oxygen fraction group（H,90%O₂）.Each group was exposed for 4h.According to the exposure duration,the three groups were divided into 1h（T1）,2h（T2）,3h（T3）and 4h（T4）groups,named group C1,C2,C3 and C4,group M1,M2,M3,M4 and group H1,H2,H3 and H4.The calcium concentration in mitochondria of all groups was measured with the method Rhod-2-AM detection kit and ROS level was detected by DCFH-DA kit,AECⅡcells and mitochondria ultrastructure changes were observed by inverted fluorescent contrast phase microscope and transmission electron microscope.Results 1.Effects of oxygen concentration on ROS and mitochondrial Ca²⁺concentration of AECⅡcells:Compared with group C,the ROS level in group H and group M were significantly increased（P<0.05）;compared with group M,the ROS level in group H were significantly increased（P<0.05）;Compared with group C,the mitochondrial calcium concentration in group H2,H3,H4 were significantly increased（P<0.05）;compared with group M,the mitochondrial calcium concentration in group H2,H3,H4 were significantly increased（P<0.05）;2.Effects of oxygen exposure durations on ROS and mitochondrial Ca²⁺concentration of AECⅡcells:In group M,the ROS level were in the order M4>M3>M2>M1（P<0.05）;in group H,the ROS level were in the order H4>H3>H2>H1（P<0.05）;in group H,the mitochondrial calcium concentration in group H2,H3 and H4 were significantly higher than that in group H1（P<0.05）,and in group H4 were significantly higher than that in group H2 and group H3（P<0.05）;the mitochondrial calcium concentration were no significant in group H2and group H3（P>0.05）;the mitochondrial calcium concentration at each time point were no significant of group C and M（P>0.05）.3.Microscopic observation:in group C,the cells were showed an island-like adherent growth with a variety of forms,such as cube or polygon.In group M4,the cells were gradually reduced and almost rounded in shape,with intercellular junctions were partial lossed.In group H4,the cells volume were reduced,the morphology of the cells were mostly rounded,and the intercellular connections were missing.Transmission electron microscope observation:in group C,mitochondrial membrane of the cells were integrated,mitochondrial cristae were normal,the basic structure of cells also normal;in group M,basic substructure of the mitochondria were normal;in group H4,the mitochondrial were swollen,and there were no obvious vacuoles.Conclusion Rats AECⅡcells exposed to hyperoxia for 4h,mitochondria Ca²⁺concentration and ROS were increased with the oxygen exposed concentration and durations.