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Bhavana Chhunchha, Eri Kubo, Sanjay P Singh, Dhirendra P Singh; Metformin Restores Impaired Antioxidant Defense Response In Aging Lens Epithelial Cells By Reactivating Nrf2/ARE/Prdx6 Axis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1112.
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© ARVO (1962-2015); The Authors (2016-present)
Dysregulation of antioxidant pathway is a leading factor in the etiology of age-related blinding diseases. Metformin (Met), an antidiabetic, has been found to promote antioxidant pathway via Nrf2 (NFE2-related factor 2) activation. Here, using human lens epithelial cells (hLECs)/lenses, we demonstrate that age-related increase in oxidative load and cell death are linked to dysregulation of Nrf2/ARE (antioxidant response element) and protective protein Peroxiredoxin (Prdx) 6, and that the process can be reversed by Met treatment.
hLECs isolated from 14 normal donor eyes were treated with Met (0-1mM) and exposed to H2O2 (0-150 μM) for different times. Subjects were in three age groups: young, 18-24y (n=4), middle, 42-54y (n=5), and old, 62-70y (n=5). We assessed the levels of Nrf2, Prdx6 and senescence markers p16/21 by Western blot and qPCR, and examined the correlation of these parameters with reactive oxygen species (ROS) levels and pathobiology of aging hLECs, if any. ChIP and TransAM Nrf2 activity were used to assess Nrf2/ARE binding (-357/-349) in Prdx6 promoter (-918), and transactivation assay was used to assess functionality of binding. MTS and H2DCF-DA dye assays measured cell viability and ROS. Two-tailed Student’s t-test and one way ANOVA were used for statistical analysis.
hLECs from the middle and old groups showed reduced levels of Nrf2 and Prdx6 and increased oxidative load and senescence markers, and were more susceptible to cell death than younger hLECs (p<0.05). DNA binding assays revealed a progressive reduction in Nrf2/ARE binding in Prdx6 promoter, which was directly linked to a marked reduction of the promoter activity in aged hLECs (p<0.001). Met treatment augmented Prdx6 expression and reduced p16/21 in time-dependent fashion, by reactivating Nrf2/ARE binding and promoter activity and by enhancing expression of Nrf2 and facilitating its translocalization in nucleus. Met rescued cells from H2O2-induced toxicity, which was consistent with Met's activation of Nrf2/ARE and increased Prdx6, and reduction in ROS levels.
Findings reveal a mechanistic insight into the underlying causes of age-related dysregulation of Nrf2/ARE/Prdx6 and its restoration through Metformin, and thus provide a proof of concept that Met or mimetics can be considered as a therapeutic agent.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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