Abstract
Purpose :
Deterioration of antioxidant response with advancing age is a leading cause of age-related pathologies. Therapeutic activation of antioxidant pathway has been suggested as an effective strategy to combat aging pathobiologies. Hydralazine (Hyd), an antihypertensive, has been shown to be a potent activator of Nrf2 (NFE2-related factor 2) pathway. Using C57BL/6 mice as a model system, we showed that topical instillation of Hyd in the eye activates Nrf2/ARE (antioxidant response element) pathway, and these lenses gained resistance against oxidative stress-induced insult.
Methods :
16-month old mice (n=12) were used for study, and anaesthetized with an oxygen-isoflurane before Hyd instillation. Buffered saline (n=6) or 25µM/5µl (n=6) of Hyd in buffered saline (pH7.4) was instilled once daily in the eyes for 5 days and lenses were collected. 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 other phaseII enzymes expression using probes specific to the molecules. In another set of ex-vivo experiments, Hyd-treated or untreated lenses were exposed to 100µM of H2O2 to assess lens opacity. 72h later lenses were photographed (Nikon SMZ 745T) and intensity of 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 :
Hyd instilled lenses displayed significantly increased expression of Nrf2 and its target antioxidant genes mRNA and protein, such as Prdx6, Catalase, SOD1, HO1, Gpx1 and GSTπ (p<0.001) compared to untreated lenses. ROS quantitation showed that Hyd instilled lenses had significantly reduced ROS levels compared to untreated lenses. Untreated lenses developed H2O2-induced lens opacity ex-vivo, while Hyd treated lenses showed significantly less opacity; 82% at 48h and 70% at 72h of H2O2 exposure in comparison to untreated lenses as analyzed by densitometry.
Conclusions :
Our findings, for the first time, reveal that topical instillation of Hyd can activate Nrf2/ARE-pathway in lenses in vivo and these lenses engender resistance against oxidative stress. Our study also provide a foundation for further study of Hyd as a viable therapeutic candidate for the treatment or prevention of age-related diseases, including cataract.
This is a 2021 ARVO Annual Meeting abstract.