Abstract
Purpose :
Among elderly people in developed countries age-related macular degeneration (AMD) is the leading cause of progressive vision loss. Hallmark features of AMD are an accumulation of drusen in the Bruch’s membrane and dysfunction of retinal pigment epithelial (RPE) cells. Oxidative stress is an important factor contributing to the damaged RPE cells. Multiple studies provide evidence that melanin is an important antioxidant factor which leads to reduction of the reactive oxygen species (ROS). Melatonin also has anti-inflammatory, antitumor, and antiangiogenic properties.
Evaluate melatonin protection in Non-AMD cybrids and in AMD cybrids.
Methods :
AMD patients and age-matched non-AMD individuals provided platelets that were combined with mitochondria-deficient (Rho0) ARPE-19 cells. The resultant Cybrid cell lines have identical nuclei but mitochondria from different individuals. Cybrids were cultured in 96-well plates (10,000 cells/well) and treated with Melatonin 200 µM for 24 hours. Ethanol was used as vehicle-control. ROS levels and cellular metabolism were measured by the H2DCFDA and MTT assays, respectively. After adding 100 µl H2DCFDA solution/well, plates were read via the fluorescent plate reader with the excitation (EX, 492nm) and emission (EM, 520nm) wavelengths. For cell metabolism, 10 µl of MTT assay reagent was added per well and plates were incubated in 37°C for 3 hours. After adding 100 μl/well of DMSO, plates were analyzed (signal at 570 nm and reference at 630 nm) with the absorbance plate reader. – Statistics were calculated using Student T-test.
Results :
ROS levels of non-AMD and AMD cybrids with melatonin declined to 90.50 ± 3.79 (P< 0.0001, n=4) and 84.50 ± 1.55 (P< 0.0001, n=4) versus Ethanol groups (100.8 ± 0.478, n=4) and (99.75 ± 1.75, n=4), respectively.
MTT Assay levels of non-AMD and AMD cybrids with Melatonin improved to 129 ± 2.97 (P< 0.0001, n=4) and 132.3 ± 2.78 (P< 0.0001, n=4) versus Ethanol groups (100.8 ±0.478, n=4) and (99.75 ±1.75, n=4), respectively.
Conclusions :
Non-AMD cybrids and AMD cybrids treated with melatonin had significantly increased cellular metabolism and lower levels of ROS, demonstrating that melatonin could protect cells against oxidative stress.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.