Effect of glutaredoxin 2 gene knockout on lens epithelial cells against oxidative stress

Abstract

Abstract Purpose The mitochondrial glutaredoxin 2 (Grx2) is known to possess both dethiolase and peroxidase activities, and has shown an ability to protect cells from oxidative stress‐induced apoptosis in the human lens epithelial cells. In this study, we further studied the function of Grx2 by using Grx2 knockout mouse lens epithelial (MLE) cells as a model. Methods Primary culture of MLE cells was established from the lenses of wild‐type (WT) and Grx2‐knockout (Grx2 KO) mice. Cells were probed for αA‐crystallin and Grx2 by Western blot analysis while cell viability was examined by WST‐8 assay. Glutathione (GSH) level, Grx2 and Complex I activities, and lactate dehydrogenase (LDH) release were determined by spectrophotometric assays. Reactive oxygen species was detected using DCF‐DA fluorescein with a cell sorter. Apoptosis was quantified by flow cytometry. Results Both primary cell cultures were confirmed to be lens epithelial cells by the presence of αA‐crystallin. Western blotting showed normal expression of Grx2 in WT cells but absent in Grx2 KO cells. Both cell types showed similar morphology and growth rate with same level of GSH pool and complex 1 activity in the mitochondrial fraction. 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 enhanced the H2O2‐induced inactivation of complex I in the electron transport chain. Conclusion Grx2 can protect MLE cells from H2O2‐induced cell injury, and the mechanism of this protection is likely associated with its ability to detoxify H2O2 and its preservation of complex I activity in the mitochondria.