摘要：
Objective To investigate the preventive effect and mechanism of erythropoietin (EPO) on apoptosis induced by hyperoxia in human alveolar epithelial cells (A549 cells).Methods A549 cells were divided into the observation groups 1,2,3,and 4 as well as the model control group and blank control group.The cells in the observation groups 1,2,3,and 4 were pretreated with 10,20,50,and 100 IU/mL EPO for 24 h,and then were placed in a hyperoxia incubator with an oxygen concentration of ＞ 90％ for 24 h.The cells in the model control group were directly cultured in a high-oxygen incubator with an oxygen concentration of ＞ 90％ for 24 h.The cells in the blank control group were cultured for 24 h under normal conditions.Then,CCK-8 method was used to observe the proliferation of cells in each group (indicated by OD450).Flow cytometry was used to measure the apoptosis rate in each group.Inverted phase contrast microscopy was used to observe the cell morphology in each group.Scanning electron microscopy was used to observe mitochondrial morphology in each group.Immunohistochemical SP method was used to observe the protein of optic atrophy 1 (OPA1) in the observation group 4,the model control group and the blank control group [Results were expressed by the integrated optical density (IOD) value].Tetrachloro-tetraethyl-imidacarbocyanineiodide (JC-1) method was used to detect the mitochondrial membrane potentials of the above six groups (indicated by the ratio of red-green fluorescence intensity ratio).Results ①The OD450 of observation groups 1,2,3,4,model control group,and blank control group were 0.21 ± 0.03,0.28 ± 0.02,0.32 ± 0.03,0.34 ± 0.04,0.16 ± 0.04,and 0.37 ± 0.06 respectively.The OD450 of the model control group was lower than that of the control group (P ＜0.05).The OD450 of the observation groups 1,2,3,and 4 were higher than that of the model control group (all P ＜ 0.05).The observation groups 1,2 were lower than the blank control group (all P ＜ 0.05).②The apoptosis rates of the observation groups 1,2,3,and 4,the model control group,and the blank control group were 3.70 ± 0.66,2.31 ± 0.54,1.08 ± 0.49,1.13 ± 0.51,5.24 ± 0.93,and 0.95 ± 0.37,respectively.Significant difference was found in the apoptosis rate between the model control group and the blank control group,between the observation groups 1,2,3,and 4 and the model control group,and between the observation groups 1,2 and the blank control group (all P ＜ 0.05).③Under the inverted phase contrast microscope,the cells in the model control group were significantly damaged,became round or oval,and the intercellular space increased,the number of living cells decreased significantly,and the suspended cells increased significantly.As the EPO concentration increased,the cell damage of the observation groups 1,2,3,and 4 was significantly improved as compared with that of the model control group.Cell morphology of the observation groups 3 and 4 was similar to the that of the blank control group.④Transmission electron microscopy showed that a large number of mitochondria in the model control group were swollen or ruptured,the number of mitochondria increased,and the apoptosis characteristics such as decreased or disappeared mitochondrial cristae,chromatin condensation,and lysis of nuclei into fragments were observed.However,the recovery of mitochondrial rupture was observed in the observation groups 1,2,3,and 4.The mitochondrial rupture of the observation groups 3 and 4 was similar to that of the blank control group.⑤The IOD values of OPAl in the observation group 4,model control group,and blank control group were 23.01 ± 2.01,7.53 ± 1.16,and 38.25 ± 6.67,respectively.The observation group 4 was higher than the blank control group,and was lower than the model control group (all P ＜ 0.05).⑥The red-green fluorescence intensity ratio in the observation group 4,the model control group,and the blank control group were 1.73 ±0.06,1.32 ±0.07,and 1.96 ± 0.84,respectively.The observation group 4 was lower than the blank control group,and was higher than the model control group (all P ＜ 0.05).Conclusions EPO can prevent the apoptosis of A549 cells induced by hyperoxia.The mechanism may be that EPO pretreatment can increase OPA1 expression and stabilize mitochondrial membrane potential.%目的 观察促红细胞生成素(EPO)对高氧诱导的人肺泡上皮细胞A549凋亡的预防作用,并探讨其机制.方法 将体外培养的A549细胞分为观察1、2、3、4组及模型对照组、空白对照组.观察1、2、3、4组细胞分别加入10、20、50、100 IU/mL的EPO预处理24h,然后置于氧浓度＞90％的高氧培养箱内培养24 h.模型对照组细胞直接置于氧浓度＞90％的高氧培养箱内培养24h.空白对照组细胞常规条件下培养24 h.采用CCK-8法观察各组细胞增殖情况(以OD450表示),采用流式细胞术测算各组细胞凋亡率,采用倒置相差显微镜观察各组细胞形态,采用扫描电镜观察各组线粒体形态;采用免疫组化SP法检测观察4组、模型对照组和空白对照组神经萎缩症蛋白1(OPA1)蛋白[结果以IOD值表示],采用四氯四乙基苯并咪唑基羰花青碘化物(JC-1)法检测上述三组线粒体膜电位(结果用红绿荧光强度比值表示).结果 ①观察1、2、3、4组及模型对照组、空白对照组OD450分别为0.21 ±0.03、0.28 ±0.02、0.32 ±0.03、0.34±0.04和0.16 ±0.04、0.37 ±0.06,模型对照组0D450低于空白对照组,观察1、2、3、4组OD450高于模型对照组(P均＜0.05),观察1、2组OD450低于空白对照组(P均＜0.05).②观察1、2、3、4组及模型对照组、空白对照组细胞凋亡率分别为3.70±0.66、2.31 ±0.54、1.08 ±0.49、1.13 ±0.51和5.24±0.93、0.95 ±0.37,模型对照组细胞凋亡率高于空白对照组,观察1、2、3、4组细胞凋亡率低于模型对照组,观察1、2组细胞凋亡率低于空白对照组(P均＜0.05).③倒置相差显微镜下,模型对照组细胞受损明显,变为圆形或椭圆形,细胞间空隙加大,活细胞数量明显降低,悬浮细胞明显增多;观察1、2、3、4组随EPO浓度增加,细胞损伤状况与模型对照组相比明显改善;观察3、4组细胞形态与空白对照组相似.④透射电子显微镜下,模型对照组细胞大量线粒体肿胀或破裂,线粒体的数量增多,并可伴线粒体嵴数减少或消失、染色质浓缩、细胞核裂解为碎块等细胞凋亡特征.观察1、2、3、4组线粒体断裂情况有所恢复,观察3、4组细胞线粒体断裂情况与空白对照组相似.⑤观察4组、模型对照组、空白对照组OPA1的IOD值分别为23.01±2.01、7.53 ±1.16、38.25±6.67.观察4组OPA1的IOD值高于空白对照组,低于模型对照组(P均＜0.05).⑥观察4组、模型对照组、空白对照组红绿荧光强度比值分别为1.73 ±0.06、1.32 ±0.07、1.96±0.84.观察4组红绿荧光强度比值低于空白对照组,高于模型对照组(P均＜0.05).结论 EPO可以预防高氧诱导的A549细胞凋亡,其机制可能是EPO预处理可以增加OPA1表达,稳定线粒体膜电位.
