Lutein and zeaxanthin supplementation reduces H2O2-induced oxidative damage in human lens epithelial cells.

PURPOSE: Epidemiological studies suggest that dietary intake of lutein and zeaxanthin is inversely related to the risk for senile cataract. The objectives of this work were to investigate the mechanisms by which these nutrients provide anti-cataract effects. We evaluated their modulation of oxidative damage in human lens epithelial cells (HLEC) and their interaction with intracellular glutathione (GSH). METHODS: Subconfluent HLEC were pre-incubated with or without 5 µM lutein, zeaxanthin, or α-tocopherol for 48 h and then exposed to 100 µM H(2)O(2) for 1 h. Levels of protein carbonyls in the cells were measured by western-blotting analysis following reaction with 2,4-dinitrophenylhydrazine (DNPH). Levels of malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by an HPLC system. DNA damage was assessed using comet assays. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: In the absence of H(2)O(2), HLEC had very low levels of protein carbonyl and MDA. Supplementation with lutein, zeaxanthin, or α-tocopherol to the unstressed HLEC had no detectable effects on levels of oxidized proteins and lipid in the cells. Exposure of HLEC to H(2)O(2) significantly increased levels of oxidized proteins, lipid peroxidation, and DNA damage. Pre-incubation with lutein, zeaxanthin, or α-tocopherol dramatically reduced the levels of H(2)O(2) -induced protein carbonyl, MDA, and DNA damage in HLEC. The protective effects of lutein, zeaxanthin, and α-tocopherol against protein oxidation, lipid peroxidation, and DNA damage were comparable. Supplementation with lutein, zeaxanthin, or α-tocopherol increased GSH levels and GSH:GSSG ratio, particularly in response to oxidative stress. Depletion of GSH resulted in significant increase in susceptibility to H(2)O(2)-induced cell death. Supplementation with α-tocopherol, but not lutein or zeaxanthin, can partially restore the resistance of GSH-depleted cells to H(2)O(2). CONCLUSIONS: These data indicate that lutein or zeaxanthin supplementation protects lens protein, lipid, and DNA from oxidative damage and improves intracellular redox status upon oxidative stress. The protective effects are comparable to that of α-tocopherol, except that lutein and zeaxanthin cannot compensate for GSH depletion. The data imply that sufficient intake of lutein and zeaxanthin may reduce the risk for senile cataract via protecting the lens from oxidative damage.

Upregulation of DR3 expression in CD4⁺ T cells promotes secretion of IL-17 in experimental autoimmune uveitis.

PURPOSE: This study investigated the role of death receptor 3 (DR3) in experimental autoimmune uveitis (EAU). METHODS: EAU was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein (IRBP) 161-180 emulsified with complete Freund's adjuvant and evaluated with clinical and histopathologic observation. Total protein of draining lymph nodes (DLNs) was extracted from the control, EAU, or recovery phase mice. CD4⁺ T cells were separated from lymphocytes with magnetic-assisted cell sorting. At the same time, some of the CD4⁺ T cells were cultured with or without recombinant TL1A (rTL1A, the DR3 ligand) for three days, and the supernatants were collected for the interleukin-17 (IL-17) test. DR3 mRNA and protein levels in CD4⁺ T cells and the endogenous concentration of TL1A in mice DLNs were assessed with real-time PCR or western blotting. Levels of IL-17 in the supernatants were determined with enzyme-linked immunosorbent assay. RESULTS: Histopathological and clinical data revealed severe intraocular inflammation in the immunized mice. The inflammation reached its peak on day 14 in EAU and had resolved in the recovery phase (weeks 4-5 or more after IRBP immunization). CD4⁺ T cells obtained from EAU (day 7 or 14) had higher levels of DR3 mRNA and protein expression compared with the control group treated with complete Freund's adjuvant alone and the recovery group. However, the DR3 mRNA and protein levels on day 21 in EAU were similar to those observed in the control and recovery groups. The endogenous levels of TL1A were upregulated in EAU, and decreased in the recovery phase mice. Adding rTL1A increased the production of IL-17 by CD4⁺ T cells isolated from mice DLNs. Moreover, the increased IL-17 levels in the culture supernatant of CD4⁺ T cells from EAU were much higher than those from the control and recovery phase mice. However, the effects on promoting IL-17 production in TL1A-stimulated CD4⁺ T cells were similar between the controland recovery groups. CONCLUSIONS: Our data suggest that DR3 expression is induced during EAU and may be involved in the development of this disease, possibly by promoting IL-17 secretion.