Effects of different exposure flow rates in air-liquid interface exposure systems on the expression of oxidative stress-related proteins in lung cells induced by gasoline engine exhaust
Objective To investigate the effect of gasoline engine exhaust(GEE)from two-wheel motorcycles on the expression of oxidative stress-related proteins in human lung cells using an air-liquid interface(ALI)culture and exposure system.Methods There were three groups in the experiment,including blank control group,clean air group,and GEE group.Except for the blank con-trol group,the in vitro ALI exposure technology was used in the other groups.Human bronchial epithelial cells BEAS-2B and human lung cancer cells A549 cultured on porous membranes in the ALI were exposed to clean air or GEE at the exposure flow rate of 10,15,and 25 mL/min for 60 min in a 37 ℃ water bath,respectively.The cell relative viability was evaluated using the Cell Counting Kit-8.The level of reactive oxygen species(ROS)was measured by a 2',7'-dichloro-dihydro-fluoresceindiacetate probe.Annexin V-FITC ap-optosis detection kit was used to determine the apoptosis and necrosis rates of BEAS-2B and A549 cells after GEE treatment.The ex-pression levels of nuclear factor erythroid-2-related factor 2(Nrf2)and heme oxygenasel(HO-1)proteins in whole cell lysates were determined by Western blotting.Results Under the three flow rates of 10,15,and 25 mL/min,the cell relative viability of BEAS-2B and A549 cells in the GEE group decreased gradually,and the intracellular ROS generation and apoptosis rate increased gradually with the increasing flow rate;there were significant differences compared with the clean air group(P＜0.05).There were main effects of GEE exposure and exposure flow on the cell relative viability,intracellular ROS generation,early apoptosis rate,late apoptosis and necrosis rate,and total apoptosis rate of BEAS-2B and A549 cells,and there was an interaction effect between GEE exposure and expo-sure flow(P＜0.01).GEE exposure resulted in decreased expression of Nrf2 and HO-1 proteins in BEAS-2B cells and increased ex-pression of Nrf2 and HO-1 proteins in A549 cells.The expression of Nrf2 protein in BEAS-2B cells was significantly different only in the main effect of exposure flow(P＜0.01).The expression of HO-1 protein in BEAS-2B cells was statistically significant only in the main effect of GEE(P＜0.01).There were significant differences in Nrf2 protein expression in A549 cells in the main effect of GEE,the main effect of exposure flow,and the interaction effect(P＜0.05).There was no significant difference in the expression of HO-1 protein in A549 cells in the main effect of GEE,the main effect of exposure flow,and the interaction effect(P＞0.05).Conclusion GEE has a strong acute toxic effect on lung cells.GEE-induced alterations in cell viability,intracellular ROS generation,and apoptosis rate are significantly affected by exposure flow.The exposure flow has a significant effect on the Nrf2 protein expression in BEAS-2B and A549 cells induced by GEE,but not on HO-1 protein expression.
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