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Abstract
Purpose
To evaluate the effects of simvastatin on the catalase expression in human retinal pigment epithelium.
Methods
Retinal pigment epithelial (RPE) cells were incubated for 6 hours and 24 hours with various concentrations of simvastatin. In addition, RPE cells were incubated with 200 μM of H2O2 and various concentrations of simvastatin. After incubation, real-time polymerase chain reaction (PCR) was performed to examine the catalase messenger ribonucleic acid (mRNA) expression and a catalase assay was performed to examine the catalase activity in RPE. Intracellular reactive oxygen species (ROS) was measured using a fluorescence activated cell sorter (FACS).
Results
Simvastatin increased the amount of catalase mRNA and catalase activity at 10 μM in RPE cells. Under oxidative stress (200 μM of H2O2), 2.5 μM of simvastatin increased the catalase mRNA expression and 5 μM of simvastatin increased catalase activity in RPE cells. In addition, simvastatin reduced free radical formation but this effect was diminished in the presence of an irreversible catalase inhibitor, 3-amino-1,2,4-triazole (3-AT).
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Figure 1.
Expression of catalase mRNA in human retinal pigment epithelium increased with increasing concentrations of simvastatin. Human retinal pigment epithelium was cultured in various concentrations of simvastatin for 6 hours. Expression of catalase messenger ribonucleic acid (mRNA) was calibrated using glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The experiments were performed 3 times independently and the data are shown as means ± SD. *p < 0.05 versus the control.
Figure 2.
Catalase activity increased with increasing concentrations of simvastatin in human retinal pigment epithelium. Human retinal pigment epithelium was cultured with various concentrations of simvastatin for 24 hours. The experiments were performed 3 times independently and the data are shown as means ± SD. *p < 0.05 versus the control.
Figure 3.
Expression of catalase increased as exposure time with simvastatin increased in human retinal pigment epithelium. (A) Expression of catalase mRNA increased as exposure time with simvastatin increased in human retinal pigment epithelium. Human retinal pigment epithelium was cultured in 10 μM of simvastatin for various amounts of time. The highest level of catalase mRNA was expressed following 12 hours of culture and the effect lasted for at least 48 hours. (B) Catalase activity increased as exposure time with simvastatin increased in human retinal pigment epithelium. Human retinal pigment epithelium was cultured in 10 μM of simvastatin for various amounts of time. Catalase activity increased significantly after 6 hours. Catalase activity reached highest levels at 24 hours and the increase lasted for at least 48 hours. The experiments were performed 3 times independently and the data are shown as means ± SD.
Figure 4.
Expression of catalase increased as the concentration of simvastatin increased under oxidative stress (H2O2) in human retinal pigment epithelium. (A) Expression of catalase messenger ribonucleic acid (mRNA) increased as the concentration of simvastatin increased under oxidative stress in human retinal pigment epithelium. Human retinal pigment epithelium was cultured in 200 μM of H2O2 and various concentrations of simvastatin for 6 hours. The highest level of catalase mRNA was expressed with 2.5 μM of simvastatin and the effect lasted by 10 μM of simvastatin. Expression of catalase mRNA was calibrated using glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) Human retinal pigment epithelium was cultured in 200 μM of H2O2 and various concentrations of simvastatin for 24 hours. Catalase activity reached the highest levels with 5 μM of simvastatin. The effects of catalase activity induced by simvastatin diminished with 10 μM of simvastatin. The experiments were performed 5 times independently and the data are shown as means ± SD.
Figure 5.
The effects of simvastatin on reactive oxygen species (ROS) formation in human retinal pigment epithelium. (A) Simvastatin inhibited ROS formation in human retinal pigment epithelial cells. (B) The anti-oxidative effect of simvastatin diminished in the presence of the catalase inhibitor, 3-AT. (C) ROS formation increased following exposure to H2O2 in human retinal pigment epithelial cells. (D) Simvastatin inhibited ROS formation in hum an retinal pigment epithelial cells under oxidative stress. (E) The anti-oxidative effect of simvastatin was diminished in the presence of the catalase inhibitor, 3-AT, under oxidative stress. The data are reported as means ± SD of 3 experiments performed independently.
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