Fig. 6

The effect of oxidative stress in cultured primary human corneal endothelial cells (HCECs) senescence in vitro a Oxidative stress on HCECs shows elevated ROS Levels. Intracellular ROS and mitochondria ROS accumulation by DCFH-DA staining after treatment of H₂O₂ using confocal microscopy. At 60 minutes after treatment with 100 μM H₂O₂, ROS generation was observed in HCECs (Fig. 6a-i and -ii). SA-β-Gal positivity was also observed in HCECs after 60 minutes of treatment with 100 μM H₂O₂ (Fig. 6a-iii and iv) compared with no H₂O₂-treatment. b Localization of MitoTracker Green FM with MitoSOX red in HCECs. Because of the localization of Mito Tracker Green (Fig. 6b-ii and -v), H₂O₂ treated cells showed red fluorescence in mitochondria, indicating increased mitochondrial ROS production (Fig. 6b-iv), compared with control HCECs (Fig. 6b-i). c and d Oxidative stress on HCECs shows up-regulated Levels of CDK inhibitors. At 2 hours after 100 μM H₂O₂ treatment, a brief up-regulation of p16^INK4A, p21^Cip1, and p27^kip1 mRNA was found in HCECs, whereas after 50 μM H₂O₂ treatment, the mRNA expressions of p16^INK4A, p21^cip1, and p27^kip1 had no statistically difference between H₂O₂ treatment and no H₂O₂ treatment (Fig. 6c). When HCECs were exposed to 100 μM H₂O₂, the level of p16^INK4A, p21^cip1, and p27^kip1 protein expression was further elevated, and this up-regulation persisted from 2 to 6 hours post H₂O₂ treatment (Fig. 6d). The HCECs with no H₂O₂ treatment was used as control. Three more additional experiments achieved equivalent results. Data are means ± SD (n = 3). All means marked with * (P < 0.05) are significantly different from the control