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 control 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 apoptosis detection kit was used to determine the apoptosis and necrosis rates of BEAS-2B and A549 cells after GEE treatment. The expression levels of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase1 (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 exposure flow (P < 0.01). GEE exposure resulted in decreased expression of Nrf2 and HO-1 proteins in BEAS-2B cells and increased expression 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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