磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白自噬通路在支气管肺发育不良中的作用

The role of phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin autophagy pathway in bronchopulmonary dysplasia

目的探讨磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)自噬通路在支气管肺发育不良中的作用。方法将A549细胞(腺癌人类肺泡基底上皮细胞)一定条件培养后分成对照组、支气管肺发育不良组、雷帕霉素干预组。支气管肺发育不良组、雷帕霉素干预组高氧环境培养,置于37℃、85%氧气孵化箱中培养48 h,前者不加药剂、后者加10μmol/L的雷帕霉素干预;对照组常规环境培养,置于37℃、5%二氧化碳孵化箱中培养48 h,不加药剂。利用高氧构建支气管肺发育不良的细胞模型,利用蛋白质印迹实验和实时荧光定量聚合酶链反应(RT-qPCR)实验验证自噬对高氧处理后A549细胞肺泡表面标志物合成蛋白复合物(SPC)、水通道蛋白5(AQP5)的影响,利用5-乙炔基-2′-脱氧尿苷(EdU)实验检测自噬对高氧处理后A549细胞增殖的影响,利用蛋白质印迹实验验证PI3K/Akt/mTOR自噬通路在支气管肺发育不良中的作用。结果蛋白质印迹实验和RT-qPCR实验表明自噬激活剂雷帕霉素挽救了高氧处理后A549细胞SPC、AQP5表达的下调。EdU实验表明雷帕霉素显著促进了高氧处理后A549细胞的增殖,EdU阳性细胞百分比高于支气管肺发育不良组[(0.48±0.06)比(0.36±0.02)](P<0.05)。蛋白质印迹实验显示雷帕霉素干预后的A549细胞中的磷酸化-PI3K、磷酸化-Akt、磷酸化-mTOR、自噬选择性底物P62的表达水平显著降低,微管相关蛋白1轻链3的表达水平显著上升(均P<0.05)。结论PI3K/Akt/mTOR自噬通路在支气管肺发育不良中发挥着重要作用。

Objective To discuss the role of phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)autophagy pathway in bronchopulmonary dysplasia.Methods A549 cells(adenocarcinoma human alveolar basal epithelial cells)were cultured under certain conditions and divided into control group,bronchopulmonary dysplasia group and rapamycin intervention group.The bronchopulmonary dysplasia group and the rapamycin intervention group were cultured in a hyperoxic environment and incubated at 37℃in an 85%oxygen incubator for 48 h,and the former was not treated with drugs,while the latter was treated with 10μmol/L of rapamycin intervention.The control group was cultured in a conventional environment and incubated at 37℃in a 5%carbon dioxide incubator for 48 h without drugs.Hyperoxia was used to construct a cell model of bronchopulmonary dysplasia.Western Blotting test and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)test were used to verify the effect of autophagy on the synthesis of protein complexes(SPC)and aquaporin 5(AQP5)on alveolar surface markers of A549 cells after hyperoxia treatment.The effect of autophagy on the proliferation of A549 cells after hyperoxia treatment was detected by 5-ethynyl-2′-deoxyuridine(EdU)test.Western Blotting test was used to verify the role of PI3K/Akt/mTOR autophagy pathway in bronchopulmonary dysplasia.Results Western Blotting test and RT-qPCR test showed that autophagy activator rapamycin rescued the down-regulation of SPC and AQP5 expression in A549 cells after hyperoxia treatment.EdU test showed that rapamycin significantly promoted the proliferation of A549 cells after hyperoxia treatment,and the percentage of EdU positive cells was higher than that in the bronchopulmonary dysplasia group[(0.48±0.06)vs(0.36±0.02)](P<0.05).Western Blotting test showed that the expression levels of phosphorylated PI3K,phosphorylated Akt,phosphorylated mTOR and autophagy selective substrate P62 in A549 cells after rapamycin intervention decreased significantly,and the expression level of microtubule associated protein 1 light chain 3 significantly increased(all P<0.05).Conclusions PI3K/Akt/mTOR autophagy pathway plays an important role in bronchopulmonary dysplasia.