冬季北京城区PM2.5诱导人肺上皮细胞系A549中多环芳烃受体(AhR)通路的激活

Activation of aryl hydrocarbon receptor(AhR)pathway in human lung epithelial cell line A549 exposed to PM2.5 from the urban area of Beijing in winter

使用冬季北京市城区的细颗粒物(PM2.5)评估其对人肺上皮细胞系A549中多环芳烃受体(AhR)通路的激活作用.利用CCK-8法检测细胞暴露PM2.5后的存活率,选取50%抑制浓度(IC50)作为细胞暴露剂量,采用Western blot和免疫荧光检测PM2.5暴露诱导AhR定位变化,采用荧光定量PCR和Western blot检测AhR的靶基因CYP1A1的转录和翻译水平改变,应用双荧光素酶报告基因法测定AhR与靶基因结合位点(XRE)的活性,利用酶标仪测定CYP1A1的酶活性改变.结果显示,随着暴露时间的增加,AhR逐渐从细胞质易位至细胞核;CYP1A1 mRNA和蛋白质的表达水平也呈时间依赖性显著增加;AhR与XRE结合活性的增加表明了PM2.5诱导CYP1A1增加的调节机制;最后引起细胞内CYP1A1酶活性显著增加.研究表明,来自冬季北京城区的PM2.5样品可激活A549细胞的AhR通路,提示AhR介导的信号途径可能与PM2.5的暴露毒性有关.

Fine particulate matter(PM2.5)of Beijing urban area in winter was used to evaluate its effects on the aryl hydrocarbon receptor(AhR)pathway in human lung epithelial cell line A549.Cell viability was measured by CCK8 assay to detect the dose of 50%inhibitory concentration(IC50).The intracellular localization of AhR triggered by exposure of PM2.5 was detected by Western blot and immunofluorescence.The transcription and translation levels of target gene CYP1A1 were measured by q⁃PCR and Western blot.The dual luciferase report assay was used to determine XRE⁃binding form activation.The enzyme activity of CYP1A1 was measured by microplate reader.The results presented that with the increase of exposure time,AhR gradually translocated from the cytoplasm to the nucleus.The expression of CYP1A1 mRNA and protein significantly enhanced in a time⁃dependent manner.The increase of XRE⁃binding activity of AhR indicated the mechanism of PM2.5⁃driven up⁃regulation of CYP1A1.Ultimately,exposure to PM2.5 caused an enhancement of the enzymatic activity of CYP1A1.These data support the activation of AhR induced by PM2.5 from the urban area of Beijing in winter,suggesting that the toxicity of PM2.5 may be related to the AhR⁃mediated pathway.