Yu Ganggang, Zeng Xiangjun, Wang Hongxia, Hou Qi, Tan Chunting, Xu Qiufen, Wang Haoyan
The Department of Respiratory Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
Cell Physiol Biochem. 2015;36(2):474-86. doi: 10.1159/000430113. Epub 2015 May 11.
BACKGROUND/AIMS: Epoxyeicosatrienoic acids (EETs), a type of lipid mediators produced by cytochrome P450 epoxygenases, exert anti-inflammatory, angiogenic, anti-oxidative and anti-apoptotic effects. However, the role of EETs in cigarette smoke-induced lung injury and the underlying mechanisms are not fully known. The aim of this study was to explore the effects of CYP2J2-EETs on cigarette smoke extracts (CSE)-induced apoptosis in human bronchial epithelial cell line (Beas-2B) and the possible mechanisms involved.
Cytochrome P450 epoxygenase 2J2 (CYP2J2) and its metabolites EETs were assessed by western blotting or LC-MS-MS. Cell viability and apoptosis were determined by MTT assay and AnnexinV-PI staining. Reactive oxygen species (ROS) were assessed by measuring H2DCFDA. Caspase-3, HO-1, MAPK and endoplasmic reticulum (ER) stress-related markers GRP78, p-elF2a, and CHOP were evaluated by western blotting.
CSE suppressed expression of both CYP2J2 and EET by Beas-2B cells. CSE also induced apoptosis, the generation of ROS and the ER stress in Beas-2B cells. These changes were abolished by pretreatment with exogenous 14,15-EET while pretreatment with 14,15-EEZE, a selective EET antagonist, abolished the protective effects of 14,15-EET. In addition, EETs increased the expression of antioxidant enzyme HO-1. Furthermore, 14,15-EET reduced CSE-induced activation of p38 and JNK.
The data suggest that CYP2J2-derived EETs protect against CSE-induced lung injury possibly through attenuating ER stress.
背景/目的：环氧二十碳三烯酸（EETs）是细胞色素P450环氧化酶产生的一类脂质介质，具有抗炎、促血管生成、抗氧化和抗凋亡作用。然而，EETs在香烟烟雾诱导的肺损伤中的作用及其潜在机制尚不完全清楚。本研究旨在探讨CYP2J2-EETs对香烟烟雾提取物（CSE）诱导的人支气管上皮细胞系（Beas-2B）凋亡的影响及其可能的机制。
采用蛋白质免疫印迹法或液相色谱-质谱联用（LC-MS-MS）检测细胞色素P450环氧化酶2J2（CYP2J2）及其代谢产物EETs。通过MTT法和AnnexinV-PI染色检测细胞活力和凋亡情况。通过检测H2DCFDA评估活性氧（ROS）水平。采用蛋白质免疫印迹法评估半胱天冬酶-3、血红素加氧酶-1（HO-1）、丝裂原活化蛋白激酶（MAPK）以及内质网（ER）应激相关标志物葡萄糖调节蛋白78（GRP78）、磷酸化真核细胞起始因子2α（p-elF2α）和C/EBP同源蛋白（CHOP）。
CSE抑制Beas-2B细胞中CYP2J2和EET的表达。CSE还诱导Beas-2B细胞凋亡、ROS生成和ER应激。外源性14,15-EET预处理可消除这些变化，而选择性EET拮抗剂14,15-EEZE预处理则消除了14,15-EET的保护作用。此外，EETs增加抗氧化酶HO-1的表达。此外，14,15-EET降低CSE诱导的p38和JNK激活。
数据表明，CYP2J2衍生的EETs可能通过减轻ER应激来预防CSE诱导的肺损伤。
