空气细颗粒物诱导大鼠肺泡Ⅱ型上皮细胞损伤

Air fine particulate matter induced alveolar type Ⅱ epithelial cell injury in rats

目的探讨空气细颗粒物(fine particulate matter,PM_(2.5))导致大鼠肺泡Ⅱ型上皮细胞(RLE-6TN)损伤及相关机制。方法采集潍坊市2020年大气中的PM_(2.5),用细胞培养液配成颗粒物混悬液。RLE-6TN细胞暴露于不同浓度(25、50、100、200和400μg/mL)颗粒物混悬液24 h,倒置显微镜观察细胞形态变化,用噻唑蓝法测定细胞活力;微板法测定细胞上清液中乳酸脱氢酶(lactate dehydrogenase,LDH)浓度;采用DCFH-DA、Annexin V-FITC/PI、JC-1探针法结合激光共聚焦检测荧光强度以确定细胞活性氧(reactive oxygen species,ROS)、细胞凋亡、线粒体膜电位水平;比色法测定细胞内总超氧化物歧化酶(total superoxide dismutase,T-SOD)、谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)含量和活性;Caspase-3和Caspase-9试剂盒结合酶标仪检测细胞凋亡蛋白相对表达活力。采用单因素方差分析和皮尔逊相关分析比较各结果差异和相关性。结果PM_(2.5)可导致RLE-6TN细胞形态发生改变,细胞间隙变大,细胞活力降低,与对照组相比,各剂量组差异均有统计学意义(P<0.05)。≥50μg/mL染毒组上清液中LDH浓度升高,其LDH浓度≥(377.82±29.84),与对照组(278.51±23.76)相比差异均有统计学意义(P<0.05)。激光共聚焦检测细胞活性氧结果显示,≥50μg/mL染毒组细胞内绿色荧光逐渐增强,其相对荧光强度≥(2.77±0.18),与对照组相比差异均有统计学意义(P<0.05);细胞凋亡水平升高,与对照组相比差异有统计学意义(P<0.05);细胞线粒体膜电位水平逐渐降低,≥25μg/mL染毒组线粒体膜电位水平≤(4.22±0.45),与对照组(6.16±0.49)相比差异有统计学意义(P<0.05)。PM_(2.5)可导致细胞T-SOD和GSH水平降低,≥50μg/mL染毒组细胞T-SOD和GSH水平分别≤(14.67±0.49)和≤(433.29±39.24),与对照组[(16.58±0.60)和(542.90±45.06)]相比显著降低(P<0.05),MDA水平随着PM_(2.5)浓度升高而上升,与对照组(1.15±0.19)相比,≥50μg/mL染毒组MDA水平≥(1.72±0.13),差异均有统计学意义(P<0.05)。≥100μg/mL染毒组细胞Caspase-3和Caspase-9活性水平升高,其活性水平分别≥(1.62±0.27)和≥(1.23±0.06),与对照组相比差异均有统计学意义(P<0.05)。结论一定浓度PM_(2.5)暴露可诱导大鼠肺泡Ⅱ型上皮细胞发生氧化应激,膜电位降低,最终导致细胞凋亡。

OBJECTIVE To investigate the damage of rat alveolar type II epithelial cells(RLE-6TN)caused by air fine particulate matter(PM_(2.5))and its related mechanism.METHODS PM_(2.5) in the atmosphere of Weifang City in 2020 was collected and cell culture medium was used to prepare particulate suspension.RLE-6TN cells were exposed to different concentrations(25,50,100,200,400μg/mL)of particulate matter suspensions for 24 h.The morphological changes of RLE-6TN cells were observed under inverted microscope,and the cell viability was determined by MTT method.The concentration of lactate dehydrogenase(LDH)in cell supernatant was determined by microplate method.DCFH-DA,Annexin V-FITC/PI,JC-1 probe and laser confocal fluorescence intensity were used to determine the levels of reactive oxygen species(ROS),apoptosis and mitochondrial membrane potential.Total superoxide dismutase(TSOD),glutathione(GSH)and malondialdehyde(MDA)contents and activity levels in cells were determined by colorimetric method.Caspase-3 and Caspase-9 kit were used to detect the relative expression activity of apoptosis proteins.RESULTS PM_(2.5) could lead to morphological changes of RLE-6TN cells,enlarged cell space and decreased cell viability.Compared with the control group,there were statistically significant differences in each dose group(P<0.05).LDH concentration in the supernatant of≥50μg/mL infected group increased,and LDH concentration was≥(377.82±29.84),which was significantly different from that of the control group(278.51±23.76)(P<0.05).The result of laser confocal detection of ROS showed that the intracellular green fluorescence increased gradually in the≥50μg/mL group,and the relative fluorescence intensity was≥(2.77±0.18),which was statistically significant compared with the control group(P<0.05).The level of apoptosis was significantly increased compared with the control group(P<0.05).The level of mitochondrial membrane potential decreased gradually,and the level of mitochondrial membrane potential in the≥25μg/mL group was≤(4.22±0.45),which was statistically different from that in the control group(6.16±0.49)(P<0.05).PM_(2.5) could reduce the levels of T-SOD and GSH,and the levels of T-SOD and GSH in≥50μg/mL exposed group were≤(14.67±0.49)and≤(433.29±39.24),respectively,significantly lower than those in control group((16.58±0.60)and(542.90±45.06))(P<0.05).MDA level increased with the increase of PM_(2.5) concentration.Compared with the control group(1.15±0.19),MDA level in≥50μg/mL exposed group was≥(1.72±0.13),with statistical significance(P<0.05).The activity levels of Caspase-3 and Caspase-9 increased in≥100μg/mL group,and the activity levels were≥(1.62±0.27)and≥(1.23±0.06),respectively,compared with the control group,the differences were statistically significant(P<0.05).CONCLUSION Exposure to a certain concentration of PM2. 5 can induce oxidative stressof rat alveolar type II epithelial cells,reduce the membrane potential,and eventually leadto cell apoptosis.