The Mechanism Of50Hz MF-induced ROS Production And The Relationship With EGF Receptor Clustering In FL Cells

Objective:To explore the mechanism of50-Hz magnetic field (MF)-induced ROS production and the relationship with epidermal growth factor receptor (EGFR) clustering in FL cells.Methods:After human amniotic (FL) cells were exposed to a50-Hz sinusoidal magnetic field (MF) for different durations, ROS levels were detected by reactive oxygen species (ROS) assay kit, NADPH oxidase activity was analysed with chemiluminescence, Mito SOX Red and DHE were used to anylyse the mechanism of MF-induced ROS production, immunochemical fluorescence and laser confocal microscopy were used to explore the role of NADPH oxidase in MF-induced EGFR clustering. Datas were analysed by SAS9.1, using paired t-test, variance analysis and SNK test.Results:The levels of intracellular total ROS increased after exposure to0.4mT MF for5min,15min and30min, the differences were all statistically significant (P<0.05); however, after exposure for60min, the intracellular total ROS level did not increase compared with sham group (P>0.05).0.2mT MF exposure for15min still could increase intracellular total ROS level compared with sham group (P<0.05); but0.1mT MF exposure for15min did not (P>0.05). After0.4mT MF exposure for15min and30min, mitochondrial ROS increased (15min group P<0.01and30min group P<0.05); but exposure for5min and60min did not (P>0.05). After0.4mT MF exposure for5min, 15min and30min, cytoplasmic ROS increased, differences were all statistically significant (P<0.05), but exposure for60min did not (P>0.05). After0.4mT MF exposure for5min,15min and30min, intracellular NADPH oxidase activity elevated(P<0.05), but exposure for60min did not (,P>0.05). In addition, the increased intracellular total ROS and cytoplasmic ROS induced by0.4mT MF exposure could be completely eliminated by pretreatment with1μM DPI (a NOX inhibitor) for15min (P<0.05); But the mitochondrial ROS production induced by0.4mT MF exposure for15min and30min both could not be inhibited by DPI (1μM)(P>0.05). After exposure to0.4mT MF for15min, the percentage of EGFR clustering cell increased significantly (P<0.01), which could be inhibited by pretreatment with DPI (1μM) for15min.Conclusion:Based on the experiments, it is concluded that:1. MF exposure could induce ROS production in FL cells and the intensity threshold was between0.1mT to0.2mT.2. MF exposure induced cytoplasmic ROS production via enhancing NADPH oxidase activity, which mediated EGF receptor clustering.3. MF exposure induced mitochondrial ROS production, which was not related to NADPH oxidase.