大气细颗粒物PM_(2.5)诱导肺上皮MLE-12细胞的氧化应激和自噬

PM_(2.5) induces oxidative stress and autophagy in lung epithelial MLE-12 cells

目的研究大气细颗粒物PM2.5对小鼠肺泡Ⅱ型上皮细胞MLE-12细胞的氧化应激和自噬的影响。方法用采集和处理的2009年北京市细颗粒物PM2.525,50,100和200 mg.L-1暴露处理MLE-12细胞24和48 h,用MTT比色法测定细胞存活率,双乙酰基二氯荧光素(DCFH-DA)荧光探针检测细胞内活性氧类(ROS)自由基生成,Western蛋白质印迹法检测微管相关蛋白1轻链3Ⅰ蛋白(LC3Ⅰ)和LC3Ⅱ表达,激光共聚焦显微镜观察细胞自噬体的形成。结果与细胞对照组比较,PM2.5100和200 mg.L-1处理24 h组细胞存活率明显降低,分别降低28%和33%(P<0.01);暴露处理48 h,PM2.525～200 mg.L-1组细胞存活率均明显下降(P<0.01),并呈浓度效应关系(R2=0.4940,P<0.01)。PM2.5100和200 mg.L-1处理MLE-12细胞3 h,胞内ROS水平较细胞对照组显著升高,分别升高27.6%和60.7%(P<0.01)。此外,PM2.5100 mg.L-1处理细胞12,24和48 h及PM2.550,100和200 mg.L-1处理细胞24 h细胞自噬标志物LC3Ⅱ蛋白表达均明显增强,呈现明显的时间效应(R2=0.9150,P<0.01)和浓度效应(R2=0.6338,P<0.01)关系。PM2.5100 mg.L-1处理24 h细胞内自噬体荧光强度明显升高(P<0.05),细胞核周边有明显的自噬泡环绕。结论 PM2.5诱导肺泡Ⅱ型上皮MLE-12细胞的氧化应激,并引发细胞自噬。

OBJECTIVE To investigate the cytotoxic effects of inhaled particulate matter（PM） with an aerodynamic diameter of less than 2.5 μm（PM2.5） on oxidative stress and autophagy in typeⅡ alveolar epithelial cells（MLE-12）.METHODS MLE-12 cells were exposed to PM2.5 collected from an urban area of Beijing in 2009 at the final concentrations of 0,25,50,100 and 200 mg·L-1 for 24 or 48 h,respectively.Cell viability was measured by MTT assay and intracellular reactive oxygen species（ROS） generation was detected by fluorometric assay using DCFH-DA as a probe.Moreover,autophagy in cells was determined by confocal microscopy.The level of microtubule-associated protein 1 light chain 3（LC3）Ⅱ conversion was detected by Western blotting.RESULTS The viability of MLE-12 cells was decreased by 28% and 33%（P0.01）respectively after 24 h of exposure to PM2.5 at the final concentrations of 100 and 200 mg·L-1.After 48 h of exposure to PM2.5 at the final concentrations of 25-200 mg·L-1,the viability of MLE-12 cells was also significantly decreased（R2=0.4940,P0.01）.However,no significant difference was found in the viability of MLE-12 cells when exposed to PM2.5 at the same concentration between 24 and 48 h.Compared with control group,ROS level increased by as much as 27.6% and 60.7% in the cells exposed to PM2.5 at the final concentrations of 100 and 200 mg·L-1 for 3 h（P0.01）.Moreover,the expression of microtubule-associated protein 1 light chain 3Ⅱ in the cells significantly increased when exposed to PM2.5 100 mg·L-1 for 12,24 and 48 h or to PM2.5 50,100 and 200 mg·L-1 for 24 h（P0.05） in both a time-（R2=0.9150,P0.01） and concentration-（R2=0.6338,P0.01） dependent manner.After treated with PM2.5 100 mg·L-1,the intracellular of fluorescence intensity of autophagy significantly increased（P0.05）,and autophageosomes were observed around uncleus in MLE-12 cells.CONCLUSION PM2.5 induces oxidative stress and autophagy in typeⅡ alveolar epithelial MLE-12 cells.