AhR/CYP1B1介导香烟烟雾诱导人永生化支气管上皮细胞损伤的机制
Cigarette smoke-induced human bronchial epithelial cell injury mediated by AhR/CYP1B1
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摘要:
背景 研究表明，吸烟与数十种病理状况有关。香烟烟雾中包含近5 000种化学物质，包括致癌物和芳香烃受体（AhR）配体等。
目的 探究AhR信号通路中细胞色素P450（CYP）1B1介导香烟烟雾诱导人永生化支气管上皮细胞损伤的机制。
方法 从基因表达数据库（GEO）中获取吸烟相关基因表达芯片数据集GSE994、GSE17913和GSE10072，从临床蛋白质组肿瘤分析协会（CPTAC）获取肺癌患者正常肺组织蛋白表达数据，根据吸烟史将四个数据集中的样本进行分组，分析各组之间CYP1B1的表达差异。采用细胞烟雾动式染毒装置，以体积分数0%、20%和40%香烟烟雾对人永生化支气管上皮细胞（BEAS-2B）进行染毒；用AhR拮抗剂CH223191（CH22，10 μmol·L-1）预处理细胞。采用荧光定量PCR和Western blotting检测CYP1B1的mRNA和蛋白表达水平，用流式细胞术分析各组细胞活性氧（ROS）水平和凋亡率，用EdU法检测各组细胞的增殖能力变化。
结果 基于四个数据集分析结果，发现目前吸烟者的肺内气道上皮细胞、口腔上皮细胞和正常肺组织中CYP1B1的mRNA和蛋白表达均高于从未吸烟和已戒烟人群（均
P < 0.05）。20%和40%香烟烟雾暴露组BEAS-2B细胞CYP1B1表达水平明显上调（均P < 0.05）。AhR拮抗剂CH22预处理后，香烟烟雾暴露组的细胞CYP1B1表达与未经CH22预处理组相比较出现下调（均P < 0.05）。20%和40%香烟烟雾暴露组ROS水平和细胞凋亡率明显上调（P < 0.05），CH22预处理后香烟烟雾暴露组的细胞ROS水平和凋亡率明显下调（均P < 0.05）。同时，20%和40%香烟烟雾暴露组细胞增殖能力下调（均P < 0.05），CH22预处理后香烟烟雾暴露组增殖能力上调（均P < 0.05）。结论 CYP1B1在吸烟者肺组织中上调，香烟烟雾诱导的人支气管上皮细胞的损伤作用与AhR/CYP1B1信号通路激活相关。
Abstract:Background Studies have shown that smoking is associated with dozens of pathological conditions. Cigarette smoke contains nearly 5000 chemicals, including carcinogens and aryl hydrocarbon receptor (AhR) ligands.
Objective This study is designed to explore potential mediating mechanism of AhR/CYP1B1 in cigarette smoke-induced injury of human bronchial epithelial cells.
Methods Three smoking-related datasets, viz. GSE994, GSE17913, and GSE10072, were downloaded from the Gene Expression Omnibus (GEO), and protein expression data of normal tissues of lung cancer patients were obtained from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The samples from the four datasets were categorized by smoking history and compared for CYP1B1 expressions. Human immortalized lung epithelial (BEAS-2B) cells were exposed to 0%, 20% and 40% (v/v) cigarette smoke in a cell-smoke dynamic exposure system, and pretreated with 10 μmol·L-1 AhR antagonist CH223191 (CH22). The expression levels of CYP1B1 mRNA and protein were assessed by quantitative PCR and Western blotting; the reactive oxygen species (ROS) level and apoptosis rate were analyzed by flow cytometry; the proliferative ability was measured by EdU assay.
Results In all four datasets, the expression levels of CYP1B1 mRNA and protein in airway epithelial cell, oral epithelial cell, and lung tissues of smoking patients were significantly higher than that of never and former smokers (all
Ps < 0.05). The mRNA and protein expressions of CYP1B1 in the 20% and 40% cigarette smoke exposure groups were significantly up-regulated (allPs < 0.05), while the expressions in the cigarette smoke exposure groups pretreated with CH22 were significantly down-regulated than those in the CH22-unpretreated groups (allPs < 0.05). Similarly, the ROS levels and apoptosis rates in the 20% and 40% cigarette smoke exposure groups were increased (allPs < 0.05), while the two indicators were decreased after the pretreatment with CH22 (allPs < 0.05). Furthermore, the 20% and 40% cigarette smoke exposure groups showed significant down-regulation of cell proliferative ability (allPs < 0.05), while the CH22 pretreatment groups showed increased proliferative ability (allPs < 0.05).Conclusion CYP1B1 is up-regulated in the lung tissues of smoking patients. The cigarette smoke-induced damage in human bronchial epithelial cells is mediated by the activation of AhR/CYP1B1 signaling pathway.
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