Objective: To investigate the damage of rat alveolar type II epithelial cells(RLE-6 TN) 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-6 TN cells were exposed to different concentrations(25, 50, 100, 200, 400 μg/mL) of particulate matter suspensions for 24 h. The morphological changes of RLE-6 TN 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(T-SOD), 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-6 TN 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 ≥50 μg/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 ≥50 μg/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 ≥25 μg/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 ≥50 μg/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 ≥50 μg/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 ≥100 μg/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 PM_(2.5) can induce oxidative stress of rat alveolar type II epithelial cells, reduce the membrane potential, and eventually lead to cell apoptosis.
Keywords: PM_(2.5); alveolar type Ⅱ epithelial cells; apoptosis; oxidative stress.