PURPOSE: Ischemia-reperfusion injury (I/R injury) is known not only to induce hypoxic and oxidative stress, but also to cause retinal degeneration in rats. Crystallins, known to inhibit the formation of reactive oxygen species, reduce apoptotic cell death. Our goal was to clarify not only the role of I/R injury-mediated crystallins, but also to evaluate the correlation of these compounds to anti-inflammation in the vitreous body. METHODS: Twenty-four Sprague-Dawley rats were used in this study. We induced I/R injury by clamping the optic nerve for 30 minutes and then releasing it. The vitreous bodies were obtained from the experimental and control subjects 24, 48, and 72 hours after I/R injury. Two-dimensional electrophoresis was performed, and the targeted spots were further investigated using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, spectrophotometry, Western blotting, and histological examination. RESULTS: After I/R injury, 23 spots were identified as crystallins. The betaB2 crystallins were transcriptionally and post-translationally regulated, whereas the alphaB crystallins were controlled by post-translational modifications in the vitreous bodies of the rats. The total amounts of alphaA and beta crystallins (including isotypes of beta crystalline) had increased 48 hours after injury. The phosphorylation of alphaB crystallin (at serine residues 19, 45, and 59) was significantly increased 48 hours later, whereas phosphorylation of ERK1/2 showed the greatest decrease. CONCLUSIONS: During hypoxic and oxidation stress, our results suggest that phosphorylated alphaB crystalline inhibits RAS, resulting in the inactivation of ERK1/2. The phosphorylation of alphaB crystallin may be associated with the inflammatory suppression in the vitreous body via the I/R injury model system.
Animals ; Blotting, Western ; Oxidative Stress ; Phosphorylation ; Protein Processing, Post-Translational ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/*metabolism/pathology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Vitreous Body/*metabolism/pathology ; beta-Crystallins/*metabolism