PKCβ/P66Shc氧化应激通路在高氧诱导人肺泡上皮细胞活性氧簇产生中的作用  

Roles of PKCβ/P66Shc oxidative stress signal pathway in mediating hyperoxiainduced ROS production in alveolar epithelial cells

作　　者：车忠丽[1] 董文斌[1] 李清平[1] 雷小平[1] 康兰[1] 郭琳[1] 翟雪松[1] 王胜会[1] 陈枫[2] 

机构地区：[1]泸州医学院附属医院新生儿科,四川泸州646000 [2]泸州医学院附属医院医学实验研究中心,四川泸州646000

出　　处：《中国当代儿科杂志》2015年第3期275-280,共6页Chinese Journal of Contemporary Pediatrics

基　　金：中华儿科杂志第二届双鹤珂立苏科研基金;四川省教育厅科研基金(08ZA150);四川省卫生厅科研基金(90191)

摘　　要：目的探讨PKCβ/P66Shc氧化应激通路在高氧诱导的肺泡上皮细胞(A549)活性氧簇(ROS)产生中的作用及其抑制剂对高氧诱导的A549细胞损伤的保护作用。方法体外培养A549细胞系,随机分为对照组、高氧组和LY333531组。对照组细胞置于含5%CO2培养箱中;以3 L/min速度向高氧组细胞中通入950 m L/L O2和50 m L/L CO2的高纯混合气,10 min后密闭培养;LY333531组细胞于高氧诱导前用终浓度为10μmol/L的PKCβ抑制剂LY333531预处理24 h。采用Western blot检测A549细胞中PKCβ、Pin1、P66Shc和P66Shc-Ser36蛋白的表达变化。激光共聚焦显微镜检测P66Shc的线粒体转位率、线粒体ROS产生及线粒体膜电位的变化。结果与对照组相比,高氧组A549细胞内PKCβ、Pin1、P66Shc和P66Shc-Ser36蛋白表达均显著增加(P<0.05);LY333531组Pin1、P66Shc和P66Shc-Ser36的表达明显低于高氧组,但仍高于对照组(P<0.05)。与对照组相比,高氧组细胞P66Shc的线粒体转位率和ROS生成水平明显增加(P<0.05);LY333531组P66Shc转位率和ROS生成水平明显低于高氧组,但仍高于对照组(P<0.05)。与对照组相比,高氧组线粒体膜电位明显下降(P<0.05);LY333531组线粒体膜电位较高氧组升高,但仍低于对照组(P<0.05)。结论高氧能诱导肺泡上皮细胞内PKCβ表达增多,线粒体ROS产生增多,膜电位下降。LY333531能抑制PKCβ蛋白的表达,从而减轻高氧诱导的A549细胞的损伤。Objective To explore the roles of PKCβ/P66Shc oxidative stress signal pathway in mediating hyperoxia-induced reactive oxgen species （ROS） production in alveolar epithelial cells （A549） and the protective effects of PKCβ inhibitor on hyperoxia-induced injuries of alveolar epithelial cells. Methods A549 cells were cultured in vitro and randomly divided into three groups： control, hyperoxia and PKCβ inhibitor LY333531 treatment. The hyperoxia group was exposed to a mixture of O2 （950 mL/L） and CO2 （50 mL/L） for 10 minutes and then cultured in a closed environment. The LY333531 group was treated with PKCβ inhibitor LY333531 of 10 μmol/L for 24 hours before hyperoxia induction. Cells were collected 24 hours after culture and the levels of PKCβ, Pinl, P66Shc and P66Shc- Ser36 were detected by Western blot. The intracellular translocation of P66Shc, the production of ROS and cellular mitochondria membrane potential were measured using the confocal microscopy. Results Compared with the control group, the levels of PKCβ, Pinl, P66Shc and P-P66Shc-Ser36 in A549 cells 24 hours after culture increased significantly in the hyperoxia group. These changes in the hyperoxia group were accompanied with an increased translocation rate of P66Shc from cytoplasm into mitochondria, an increased production of mitochondrial ROS, and a reduced mitochondrial membrane potential. Compared with the hyperoxia group, the levels of Pinl, P66Shc and P66Shc-Ser36 in A549 cells,the translocation rate of P66Shc from cytoplasm into mitochondria and the production of mitochondrial ROS decreased significantly, while the mitochondrial membrane potential increased significantly in the L^333531 treatment group. However, there were significant differences in the above mentioned measurements between the LY333531 treatment and control groups. Conclusions Hyperoxia can increase the expression of PKCI3 in alveolar epithelial cells and production of mitochondrial ROS and decrease mitochondrial membrane potential. PKCβ inhibitor LY33353

关 键 词：PKCβ P66SHC 活性氧簇 氧化应激 肺泡上皮细胞 

分 类 号：R722.1[医药卫生—儿科]