Protective effect of TBN, a free radical scavenger, against excessive light-induced retinal damage in vivo and vitro

Light-induced retinal damage is a common cause of retina degeneration. Here, we use in vivo and in vitro model to examine projective effects of 2-[[(1,1-dimethylethyl) oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a radical scavenger with neuro protective properties, on light-induced retinal damage. White cool light (3500 +/- 200 Lux) was used to induce retinal damage of SD rats for 48 hour, and then the animals were randomly assigned to different groups either injected with vitamin C or TBN. The electro retina gram (ERG) had been evaluated of different groups and the number and thickness of outer nuclear layer (ONL) had been measured. The tert-butyl hydro peroxide (t-BHP) was used to induce apoptosis of ARPE-19 cells, and then the pre-protection was given at different time points and concentrations of vitamin C and TBN. The activity of different group of cells was detected by CCK8. Intracellular mitochondrial membrane potential (Delta psi m), ROS and Ca2+ level was detected with flow cytometry. The oxidative stress related proteins expression was detected by western analysis. After intense exposure of SD rats to light, a- and b-wave of ERG almost disappears, number of ONL significantly reduced. After TBN treatment, the a- and b-wave amplitude of ERG was obviously increased. Number of ONL significantly increased. ARPE-19 cells treated with t-BHP exhibited slow cell activity and Delta psi m loss, increased ROS and Ca2+ level, and upregulation of caspase-3, Bax, CHOP, Bip and CaMKII protein as well as downregulation of Bcl-2 protein. Vitamin C and TBN treatment can significantly decrease ROS and Ca2+ level, increase cell activity and Delta psi m in ARPE-19 cells treated with t-BHP and ameliorate the expression of Bcl-2 protein caspase-3, Bax, CHOP, Bip and CaMKII. These findings suggest that the TBN has protective effects on excess light-induced retinal damage and t-BHP induced apoptosis of ARPE-19 cells, via inhibition of mitochondria and endoplasmic reticulum (ER) oxidative stress.