Abstract
Exposure to fine ambient particulate matter (PM2.5) is known to be associated with cardiovascular disease. To uncover the molecular mechanisms involved in cardiovascular toxicity of PM2.5, we investigated alterations in the protein profile of human umbilical vein endothelial cells (HUVECs) treated with PM2.5 using two-dimensional electrophoresis in conjunction with mass spectrometry (MS). A total of 31 protein spots were selected as differentially expressed proteins and identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. The results demonstrated that DNA damage and cell apoptosis are important factors contributing to PM2.5-mediated toxicity in HUVECs. It is further proposed that PM2.5 can inhibit superoxide dismutase (SOD) activity and increase reactive oxygen species (ROS) and malonaldehyde (MDA) production in a concentration-dependent manner. Induction of apoptosis and DNA damage through oxidative stress pathways may be one of the key toxicological events occurring in HUVECs under PM2.5 stress. These results indicated that the toxic mechanisms of PM2.5 on cardiovascular disease are related to endothelial dysfunction.
摘要
目的
应用双向电泳及质谱技术分析PM2.5 对人脐静脉 内皮细胞(HUVEC)蛋白质组的影响,探讨PM2.5 引起心血管毒性的潜在机制。
创新点
多项流行病学调查表明,PM2.5 与人类心血管疾病 有密切关系,而血管内皮细胞是血管的第一道防 线。本项目应用蛋白质组技术研究PM2.5 对血管 内皮细胞损伤的作用,并从DNA 损伤的角度探 讨了PM2.5 心血管毒性机制,具有较好的创新性。 研究结果可为PM2.5 的危害分析提供基础数据, 同时为PM2.5 的防治提供新的依据及思路。
方法
培养HUVEC 细胞,分为正常组(正常培养的 HUVEC 细胞)、PM2.5 处理组(50、100 μg/ml PM2.5 处理HUVEC 细胞24 h)。双向电泳技术建立各 组细胞蛋白质组图谱,质谱技术鉴定差异表达的 蛋白质,流式细胞术分析细胞凋亡情况,酶联免 疫吸附法(ELISA)检测DNA 损伤产物8-OHdG 的水平,荧光标记技术分析DNA 双链断裂的形 成,并用免疫印迹法(Western blotting)检测DNA 损伤相关蛋白的表达。
结论
经PM2.5 处理HUVEC 后,31 个蛋白表达发生了 显著性的变化(图2,表1),其中8 个蛋白质 参与了DNA 的损伤与RNA 的编缉,7 个蛋白质 与细胞凋亡有关(表2)。进一步实验表明:PM2.5 能够促细胞凋亡(图5),提高DNA 损伤产物 8-OHdG 的含量(图6),促进DNA 双链断裂位 点的形成(图7),调节损伤修复相关蛋白 (Mer11A、Rad50 和Rad51)的表达(图8), 抑制超氧化物歧化酶(SOD)的活性,增加 HUVEC 细胞内活性氧(ROS)和丙二醛(MDA) 的水平(图9)。综上所述,PM2.5 能够通过调节 一系列蛋白的表达,加重HUVEC 细胞氧化应激 水平,增加DNA 损伤,促进细胞凋亡,造成内 皮细胞损伤,从而导致心脑血管事件的发生。
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Project supported by the Medical and Health Science and Technology Fund of Zhejiang Province (No. 2016KYB224) and the Scientific Research Fund of Zhejiang Chinese Medicine University (No. 2015ZG17), China
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Zhu, J., Zhu, Lws., Yang, Jh. et al. Proteomic analysis of human umbilical vein endothelial cells exposed to PM2.5. J. Zhejiang Univ. Sci. B 19, 458–470 (2018). https://doi.org/10.1631/jzus.B1700103
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DOI: https://doi.org/10.1631/jzus.B1700103
Key words
- Fine ambient particulate matter (PM2.5)
- Human umbilical vein endothelial cell (HUVEC)
- Proteomics
- Toxic mechanism