Department of Medicine, National Jewish Health, Denver, CO 80206, USA.
Cell Death Dis. 2013 Apr 4;4(4):e573. doi: 10.1038/cddis.2013.96.
Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)(-/-) mice. We determined human primary ATII cell injury by CS in vitro and analyzed ATII cells isolated from smoker and non-smoker lung donors ex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2(-/-) mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential antioxidant prevention strategy against oxidative injury of ATII cells in CS-related lung diseases.
香烟烟雾（CS）是慢性阻塞性肺疾病（COPD）的主要危险因素。CS 引起的氧化应激导致 DNA 和肺部损伤。吸烟者和 COPD 患者的远端气腔中发生氧化应激/抗氧化失衡。我们研究了 CS 产生的氧化应激对核红细胞 2 相关因子 2（Nrf2）（-/-）小鼠分离的原代肺泡 II 型（ATII）细胞的体内和体外影响。我们在体外研究了 CS 对人原代 ATII 细胞的损伤，并分析了来自吸烟者和非吸烟者肺供体的分离的 ATII 细胞。我们还研究了 Trolox（维生素 E 的水溶性衍生物）是否可以保护鼠和人 ATII 细胞免受 CS 诱导的 DNA 损伤和/或减少损伤。我们通过 4-羟壬烯醛表达分析氧化应激，通过 Amplex Red 过氧化氢测定法分析活性氧（ROS）生成，通过免疫印迹分析 Nrf2、血红素加氧酶 1、p53 和 P53 结合蛋白 1（53BP1）表达，Nrf2 核易位，Nrf2 和 p53 DNA 结合活性，末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记测定法分析细胞凋亡，以及通过 ELISA 分析细胞因子产生。我们发现，与野生型小鼠相比，从 Nrf2（-/-）小鼠中分离的 ATII 细胞对 p53/53BP1 介导的 CS 诱导的氧化 DNA 损伤更为敏感，无论是在体内还是在体外。因此，Nrf2 激活是保护 ATII 细胞免受 CS 损伤的关键因素。此外，Trolox 可消除体外 CS 诱导的人 ATII 细胞损伤并减少 DNA 损伤。此外，我们发现与非吸烟者相比，从吸烟者肺供体中分离的 ATII 细胞的炎症和 p53 mRNA 表达更高。我们的结果表明，ATII 细胞中的 Nrf2 和 p53 串扰会影响这些细胞对 CS 损伤的敏感性。Trolox 可通过清除 ROS 机制保护 ATII 细胞免受 CS 诱导的氧化应激、遗传毒性和炎症，并作为一种潜在的抗氧化预防策略，用于 CS 相关肺部疾病中 ATII 细胞的氧化损伤。
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