Abstract
Purpose :
Oxidative stress is a significant factor involved in many retinal diseases such as age-related macular degeneration, diabetic retinopathy, etc. We have recently shown that caffeine (Caf) prevents reactive oxygen species (ROS)-induced metabolic aberrations and decrease in level of glutathione in the neural retina; the effect is attributed to its reactions neutralizing ROS, esp. hydroxyl radical. The main goal here was to determine if Caf protects the retina by modulating activities of endogenous antioxidant enzymes and their expression.
Methods :
Initially, activity of catalase, a peroxisomal enzyme decomposing H2O2, was determined. Bovine neural retinas were incubated in Medium 199 under 5% CO2 for 4 hours in 3 groups: Control (Medium 199 +0.5mM xanthine (XA)), Experimental (Medium 199+ 0.5mM XA + 10U xanthine oxidase (XO)), and Caffeine (Medium 199+ 0.5mM XA + 10U XO + 5mM caf). XA-XO reaction was a source of ROS. Retinas were then processed for protein and catalase activity assays. Separate incubations under above conditions were conducted for gene expression studies. Total RNA was purified and RT-PCR was done with appropriate primers to determine the expression of catalase and superoxide dismutase (SOD).
Results :
Catalase activity was 65±16.5 μM/mg protein/minute in controls, 115±31.5 μM/mg protein/minute in experimentals, and 69±18.5 μM/mg protein/minute in the caffeine group. Exposure to ROS increased the activity of catalase in the experimental group by 1.7 times of the controls. Caffeine maintained catalase activity close to the controls. Catalase expression in both, experimental and caffeine groups, decreased to ~50% of the controls. SOD expression in experimental and caffeine groups was ~200% and ~187% of the controls, respectively.
Conclusions :
The increase in catalase activity by ROS could be due to its substrate-induced activation and a compensatory response to short-term oxidative stress. Caffeine prevented this increase by neutralizing ROS (as shown before), thereby not necessitating increase in catalase activity. These findings agree with our previous studies demonstrating the neuroprotection offered by caf in the ROS-exposed retina reflected in maintenance of glutathione and glycolysis. Interestingly, expression of catalase decreased significantly, while SOD expression almost doubled, in both experimental and caffeine groups. Reasons for these changes are being investigated.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.