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Bis-allylic Deuterated DHA Alleviates Oxidative Stress in Retinal Epithelial Cells.

ABSTRACT: Oxidative stress plays a crucial role in developing and accelerating retinal diseases including age-related macular degeneration (AMD). Docosahexaenoic acid (DHA, C22:6, n-3), the main lipid constituent of retinal epithelial cell membranes, is highly prone to radical and enzymatic oxidation leading to deleterious or beneficial metabolites for retinal tissue. To inhibit radical oxidation while preserving enzymatic metabolism, deuterium was incorporated at specific positions of DHA, resulting in D2-DHA when incorporated at position 6 and D4-DHA when incorporated at the 6,9 bis-allylic positions. Both derivatives were able to decrease DHAs' toxicity and free radical processes involved in lipid peroxidation, in ARPE-19 cells (Adult Retinal Pigment Epithelial cell line), under pro-oxidant conditions. Our positive results encouraged us to prepare lipophenolic-deuterated-DHA conjugates as possible drug candidates for AMD treatment. These novel derivatives proved efficient in limiting lipid peroxidation in ARPE-19 cells. Finally, we evaluated the underlying mechanisms and the enzymatic conversion of both deuterated DHA. While radical abstraction was affected at the deuterium incorporation sites, enzymatic conversion by the lipoxygenase 15s-LOX was not impacted. Our results suggest that site-specifically deuterated DHA could be used in the development of DHA conjugates for treatment of oxidative stress driven diseases, or as biological tools to study the roles, activities and mechanisms of DHA metabolites.

SUBMITTER: Rosell M

PROVIDER: S-EPMC6826779 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Bis-allylic Deuterated DHA Alleviates Oxidative Stress in Retinal Epithelial Cells.

Rosell Mélissa M Giera Martin M Brabet Philippe P Shchepinov Mikhail S MS Guichardant Michel M Durand Thierry T Vercauteren Joseph J Galano Jean-Marie JM Crauste Céline C

Antioxidants (Basel, Switzerland) 20191001 10

Oxidative stress plays a crucial role in developing and accelerating retinal diseases including age-related macular degeneration (AMD). Docosahexaenoic acid (DHA, C22:6, n-3), the main lipid constituent of retinal epithelial cell membranes, is highly prone to radical and enzymatic oxidation leading to deleterious or beneficial metabolites for retinal tissue. To inhibit radical oxidation while preserving enzymatic metabolism, deuterium was incorporated at specific positions of DHA, resulting in D ...[more]

PMID: 31581525

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Project description:PurposeIn this work, we investigated the ability of pinosylvin (PS), 3,5-dihydroxy-trans-stilbene, to modulate oxidative stress in human RPE cells. PS, a stilbenoid polyphenol, occurs in high concentrations in bark byproducts and therefore represents an attractive bioactive compound for health-promoting applications.MethodsFirst, we evaluated the toxicity range of PS by exposing ARPE-19 cells to 0.1-200 µM concentrations of PS for 24 h followed by the cell viability test. In the next stage, the ARPE-19 cells were preincubated in PS for 24 h followed by hydroquinone (HQ) exposure without PS for another 24 h. The cell viability test was conducted after HQ exposure. To elucidate the potential mechanisms behind PS-mediated protection against oxidative stress, the ARPE-19 cells were treated with 5 µM PS for 6 h, and mRNA was extracted at four time points (2 h, 6 h, 12 h, 24 h) to determine changes in the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), sequestosome 1 (p62/SQSTM1), heme oxygenase-1 (HO-1), and glutathione S-transferase pi 1 (GSTP1) genes. To clarify the molecular mechanism behind PS-mediated protection further, the ARPE-19 cells were transfected with p62 and Nrf2 siRNAs for 24 h, and the roles of p62, Nrf2, and its target gene HO-1 in conferring protection against oxidative stress were studied with quantitative real-time PCR (qRT-PCR) and the cell viability test.ResultsPS treatment at concentrations of 5 and 10 µM significantly enhanced cell survival from oxidative stress. The expression levels of an enzyme with antioxidative, anti-inflammatory, and immunomodulatory properties, HO-1, were increased by PS treatment and correlated strongly with cell survival. PS treatment did not elevate the expression levels of Nrf2 or its target genes, p62 or GSTP1, even though it had a clear effect on the expression of HO-1, another gene controlled by Nrf2. RNA interference analysis further confirmed the important role of Nrf2 and HO-1 in PS-mediated protection against oxidative stress whereas the role of p62 seemed to be insignificant at the gene expression and cell viability levels.ConclusionsOur results suggest that PS treatment conferred protection against oxidative stress through the induction of HO-1 in human RPE cells. Consequently, PS-stilbene compounds, which can be isolated in significant amounts from bark waste, may possess health-promoting properties against aging-related diseases associated with oxidative stress such as age-related macular degeneration (AMD) and Alzheimer's disease. These natural compounds may offer opportunities for high-value use of bark waste in diverse health-related applications.

Project description:Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNF? in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.

Dataset Information

Bis-allylic Deuterated DHA Alleviates Oxidative Stress in Retinal Epithelial Cells.

Publications

Bis-allylic Deuterated DHA Alleviates Oxidative Stress in Retinal Epithelial Cells.

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