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

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 mL/L O₂ 和50 mL/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10 μmol/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。

Abstract:

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, Pin1, 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, Pin1, 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 Pin1, 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 LY333531 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 PKCβ in alveolar epithelial cells and production of mitochondrial ROS and decrease mitochondrial membrane potential. PKCβ inhibitor LY333531 can partially disrupt these changes and thus alleviate the hyperoxia-induced alveolar epithelial cell injury.