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H. Wu, K. Xing, L.-R. Li, F. Giblin, Y.-S. Ho, M. F. Lou; Glutaredoxin 2 (Grx2) Kockout Increases Celluar Sensitivity to H202-Induced Cell Injury in Mouse Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1594.
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Glutaredoxin 2 (Grx2) is an isozyme of thioltransferase (TTase or Grx1) present in the mitochondria but its function is not well understood. The purpose of this study was to evaluate the roles of Grx2 in anti-oxidative and mitochondrial complex I protective functions in the lens by using Grx2 knockout mouse lens epithelial cells (LECs) as a model.
Primary cultures of LECs were established from the lenses of wild-type (WT) and Grx2-knockout (Grx2 KO) mice. Cells were probed for αA-crystallin, Grx2 by Western blot analysis while cell viability was examined by WST-8 assay. Glutathione (GSH) level, Lactate dehydrogenase (LDH) release, Grx2 activity and Complex I activity were determined by spectrophotometric assays. Reactive oxygen species (ROS) was detected using DCF-DA fluorescein with a cell sorter. Apoptosis was quantified by flow cytometry.
LECs identity was confirmed by positive immunoreactiviy to anti-αA-crystallin antibody. Western blotting showed normal expression of Grx2 in WT cells but not in Grx2 KO cells. KO cells had only trace Grx2 activity (15%) in comparison with the WT. Both cells showed similar morphology and growth pattern, and contained same level of GSH and complex 1 activity in mitochondria. However, KO cells were more sensitive to oxidative stress (100 µM H2O2 for 6 h) and exhibited lower cell viability and more LDH leakage in comparison with the WT cells. In addition, knockdown of Grx2 weakened the cell’s ability to detoxify H2O2 and deteriorated the H2O2-induced complex I activity loss.
Grx2 play a major role in protecting mouse HLE cells against H2O2-induced cell injury. The mechanism of this protection is likely associated with its ability to protect complex I in the electron transport chain and its peroxidase activities.
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