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Objective
To observe the influences of hyperoxia on the migration of human lens epithelial cells (HLECs) and the inhibitory effects of glutathione 2 (Grx2) on excessive migration of HLECs induced by hyperoxia.
Methods
The HLECs were divided into the hyperoxic model group and normoxic control group.HLECs were cultured in 80% oxygen mixture for 2 days to establish the oxidative damage model of HLECs induced by hyperoxia in vitro (hyperoxia model group), and cultured in normoxia mixture as the normoxic control group.Cell migration was observed by scratch test, cytoskeleton morphology was observed by phalloidin staining, and Grx2 mRNA level was detected by real-time quantitative PCR.HLECs in the hyperoxic model group and the normoxic control group were subdivided into empty plasmid transfection group and Grx2 plasmid transfection group, respectively.The migration behavior and cytoskeleton changes of hyperoxic model cells in different transfection groups were observed.MitoSOX staining was used to detect reactive oxygen species (ROS) content of mitochondria under flow cytometry, and JC-1 staining was used to observe the changes of mitochondrial membrane potential under laser-scanning confocal microscopy.
Results
In the process of cell culture, the cells grew well.Twenty-four hours and 48 hours after scratching, the scratch distance of the hyperoxic model group was smaller than that of the normoxic control group, with significant differences between them (both at P<0.05). The cell circumference of the hyperoxic model group was increased compared with that of the normoxic group, with a significant difference between them (t= -2.254, P<0.05). After 2 days culture, the relative expression level of Grx2 mRNA in the hyperoxic model group was 0.54±0.11, which was significantly lower than that in the normoxic control group (t=7.128, P<0.01). The transfection efficiency of Grx2 was higher than 90%.At 24 hours and 48 hours after scratching, the scratch distance in the Grx2 transfected hyperoxic model group was wider than that in the empty plasmid transfected hyperoxic model group, with significant differences between them (both at P<0.05). The relative expression level of Grx2 mRNA in Grx2 transfected cells was up-regulated compared with that in empty plasmid transfected cells, with a significant difference between them (P<0.01). Compared with the normoxic control group, the cell circumference of the hyperoxic model group was decreased, with a significant difference between them (P<0.05). The relative mean fluorescence intensity of mitochondrial ROS in the Grx2 transfected hyperoxic model group was lower than that in the empty plasmid transfected hyperoxic model group, with a significant difference between them (P<0.05). Under the hyperoxic condition, the red/green fluorescence intensity of Grx2 transfected group was higher than that of the empty plasmid transfected group, with a significant difference between them (P<0.01).
Conclusions
Grx2 may inhibit the excessive migration of HLECs under hyperoxia by regulating oxidative stress in mitochondria.