Macular Carotenoids Lutein (L) and Zeaxanthin (Z) Protect Human Retinal Pigment Epithelial (RPE) Cells from Cobalt chloride-Induced Hypoxia Stress.

Abstract

Purpose : Hypoxia-induced oxidative stress is one of the vital factors that activate the pathogenic mechanism of retinal diseases such as diabetic retinopathy and retinopathy of prematurity. Cobalt chloride (CoCl₂) is a commonly used hypoxia mimetic agent that induces oxidative stress-mediated apoptosis by generating reactive oxygen species (ROS). Most retinal diseases are irreversible with no effective treatment; therefore protecting the RPE cells from hypoxia stress is an effective therapeutic way to prevent or slow down the progression of retinal diseases. L and Z are potent antioxidants and can be used as an effective therapy for the treatment of hypoxia-induced oxidative stress. In this study, we evaluate the protective effect of L and Z from CoCl₂-induced hypoxia stress.

Methods : To determine the anti-hypoxia effect of L and Z, ARPE-19 cells were pretreated with 1, 2, and 5 μM of L and Z for 24 h and then incubated with and without 8mM CoCl₂ for 4 h. Cell viability was measured by incubating cells with 0.5 mg/ml MTT solution for 2 h in dark and measured at 550 nm. To determine ROS levels, cells were pretreated with 1, 2, and 5 μM of L and Z for 24 h, and then incubated with 2 μM 5-(and 6)- chloromethyl-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) reagent. Cells were measured at Ex/Em: 485nm/535nm.

Results : ARPE-19 cells treated with CoCl₂ result in reduced cell viability and increased ROS levels compared to the untreated CoCl₂. Cells pretreated with L and Z before CoCl₂ treatment efficiently attenuate CoCl₂ induced cytotoxicity and ROS levels in a dose-dependent manner (1-5 μM). Pretreatment with 5 μM of L significantly increase the cell viability by 1.3 fold and decrease the ROS levels by 1.7 fold. Pretreatment with 5 μM of Z significantly increased the cell viability by 1.5 fold and decreased the ROS levels by 2.2 fold compared to control (without carotenoid treatment).

Conclusions : L and Z protect ARPE-19 cells from CoCl₂-induced hypoxia stress and attenuate cytotoxicity and ROS levels. Z had a comparatively better protective effect relative to L. This may be due to their better antioxidant activity, but a further detailed study is warranted. Macular carotenoids alone or in combination with other nutrients can be used in the prevention of hypoxia-related retinal diseases.

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