Topical instillation of Metformin Prevents Oxidative Stress-Induced Eye Lens Opacity Through Induction of AMPK-Nrf2-Mediated Antioxidant Pathway

Abstract

Purpose : Oxidative stress amplification due to dysregulation of antioxidant pathway with advancing age is a major cause of aging diseases. Metformin (Met), an antidiabetic, has been found to be a potent inducer of Nrf2 (NFE2-related factor 2) antioxidant pathway. Using C57BL/6 mice as a model system, herein we showed that topical application of Met in the eye internalizes in lenses and prevents lens opacity ex-vivo through activation of AMPK-Nrf2/antioxidant-mediated defense pathway.

Methods : 18-month-old mice (n=18) were used for study and anaesthetized with an oxygen-isoflurane pump before Met topical application. Buffered saline (n=6) or 5mM/5µl (n=6) of Met in buffered saline (pH7.4) or Met blocker, atropine (3.5mM) followed by Met (n=6) was instilled once daily in the eyes for 7 days and lenses were collected. To assess Met penetration, lenses were isolated and lens extract was processed and analyzed by Acquity UPLC with Waters triple-quad Xevo TQS. Also, total RNA and protein were isolated and processed for qPCR and Western analyses to assess levels of Nrf2 and its target genes, like Prdx6 and phaseII enzymes expression using specific probes. In another set of ex-vivo experiments, the isolated lenses were exposed to 0-100µM of H₂O₂ for variable times to assess lens opacity. The lenses were photographed (Nikon SMZ 745T) and lens opacity was determined using densitometry. H2DCF-DA dye measured reactive oxygen species (ROS) levels. Two-tailed Student’s t-test and one–way ANOVA were used for statistical analysis.

Results : Data analyses revealed that Met could internalize in the lenses, and the lenses displayed upregulation and activation of AMPK. These lenses showed significantly increased expression of Nrf2 and its target antioxidant genes mRNA and protein like Prdx6 and PhaseII enzymes (p<0.001) with dramatic reduction in ROS levels compared to untreated lenses. Untreated lenses developed H₂O₂-induced lens opacity ex-vivo, while treated showed significantly less opacity; 81% at 48h and 68% at 120h of H₂O₂ exposure.

Conclusions : All together, our results demonstrate that topically applied Met internalizes in eye lenses and prevents oxidative stress-evoked the lens opacity through activation of AMPK-Nrf2- antioxidant defense pathway, thus raise the possibility of Met-based interventions to delay/prevent aging-related cataract.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.