Protective Effects of Fucoxanthin on High Glucose- and 4-Hydroxynonenal (4-HNE)-Induced Injury in Human Retinal Pigment Epithelial Cells

Abstract

The incidence of diabetes mellitus is increasing due to the eating and living habits of modern people. As the disease progresses, the long-term effects of diabetes can cause microvascular disease, causing dysfunction in different parts of the body, which, in turn, leads to different complications, such as diabetic neuropathy, diabetic nephropathy, and diabetic retinopathy (DR). DR is the main cause of vision loss and blindness in diabetic patients. Persistent hyperglycemia may cause damage to the retina, induce the accumulation of inflammatory factors, and destroy the blood-retinal barrier function. Fucoxanthin (Fx) is a marine carotenoid extracted from seaweed. It accounts for more than 10% of the total carotenoids in nature. Fx is mainly found in brown algae and has strong antioxidant properties, due to its unique biologically active structure. This carotenoid also has the effects of reducing lipid peroxidation, reducing DNA damage, and preventing cardiovascular diseases as well as anti-inflammatory and anti-tumor effects. However, there is no relevant research on the protective effect of Fx in DR. Therefore, in this study, we explore the protective effect of Fx on the retina. Human retinal epithelial cells (ARPE-19) are used to investigate the protective effect of Fx on high glucose stress- (glucose 75 mM) and high lipid peroxidation stress (4-hydroxynonenal, 4-HNE (30 μM))-induced DR. The cell viability test shows that Fx recovered the cell damage, and Western blotting shows that Fx reduced the inflammation response and maintained the integrity of the blood-retinal barrier by reducing its apoptosis and cell adhesion factor protein expression. Using an antioxidant enzyme assay kit, we find that the protective effect of Fx may be related to the strong antioxidant properties of Fx, which increases catalase and reduces oxidative stress to produce a protective effect on the retina.

Keywords: antioxidant; fucoxanthin; retinopathy.

Figure 1

The effects of high glucose, fucoxanthin, and the combination on ARPE19 cell viability. ARPE19 cells were cultured in a 96-well plate (3000 cell/well) after starvation with serum free Dulbecco’s Modified Eagle’s Medium (DMEM) F12 for 18 h. The cells were treated with different concentrations of (A) fucoxanthin for 24, 48, or 72 h; combined with fucoxanthin induced by 30 µM 4-hydroxynonenal (4-HNE) for 24 h or 75 mM glucose for 72 H (n = 4). Used (B) MTT assay and (C) cell counting for analysis of the proliferation change. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with the non-treated group. ##, p < 0.01 compared with the induced group (glucose 75 mM/4-HNE 30 µM).

Figure 2

Effects of fucoxanthin (Fx) on 4-HNE- and high glucose-induced ARPE19 cell propidium iodide (PI) staining and morphology changes. ARPE19 cells were cultured in DMEM F12 for 24 h after serum free starvation for 18 h. This was combined with fucoxanthin induced by (A) 30 µM 4-hydroxynonenal (4-HNE) for 24 h or (B) 75 mM glucose for 72 h. We used PI staining and photographed with a microscope at 20× magnification.

Figure 3

Effects of fucoxanthin on 4-HNE- and high glucose-induced ARPE19 apoptosis-related protein expression. We used Western blotting to analyze the (A,B) PARP; (C,D) Nrf2 protein; and (E,F) apoptosis-related protein expression (n = 4). *, p < 0.05; and **, p < 0.01 compared with the non-treated group. #, p < 0.05; ##, p < 0.01; and ###, p < 0.001 compared with the induced group (glucose 75 mM/4-HNE 30 µM). T-PARP, total PARP; C-PARP, cleaved PARP.

Figure 4

Effects of fucoxanthin on 4-HNE- and high glucose-induced ARPE19 blood–retinal barrier protein expression. ARPE19 cells were cultured in DMEM F12 for 24 h after serum free starvation for 18 h. This was combined with fucoxanthin induced by 30 µM 4-hydroxynonenal (4-HNE) for 24 h or 75 mM glucose for 72 h. We used Western blotting to analyze the (A,B) ICAM-1 and (C,D) occludin protein expression (n = 3). **, p < 0.01; and ***, p < 0.001 compared with the non-treated group. #, p < 0.05; and ##, p < 0.01 compared with the induced group (glucose 75 mM/4-HNE 30 µM).

Figure 5

Effects of fucoxanthin on 4-HNE-induced ARPE19 tight junction-related protein expression. ARPE19 cells were cultured in DMEM F12 for 24 h after serum free starvation for 18 h. This was combined with fucoxanthin induced by 30 µM 4-hydroxynonenal (4-HNE) for 24 h. We used (A) immunofluorescences, (B) the single cell structure completeness change, and (C) Western blotting to analyze the ZO-1 protein expression (n = 4). *, p < 0.05 compared with the non-treated group. ##, p < 0.01; and ###, p < 0.001 compared with the induced group (4-HNE 30 µM). We used a microscope at 40× magnification.

Figure 6

Effects of fucoxanthin on high glucose-induced ARPE19 tight junction-related protein expression. ARPE19 cells were cultured in DMEM F12 for 24 h after serum free starvation for 18 h. This was combined with fucoxanthin induced by 75 mM glucose for 72 h. We used (A) immunofluorescences, (B) the single cell structure completeness change, and (C) Western blotting to analyze the ZO-1 protein expression (n = 4). ***, p < 0.001 compared with the non-treated group. #, p < 0.05 compared with the induced group (glucose 75 mM). We used a microscope at 40× magnification.

Figure 7

Effects of fucoxanthin (Fx) on 4-HNE- and high glucose-induced ARPE19 antioxidant ability change. (A) We used a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to evaluate the antioxidant ability of fucoxanthin. Treated with 4-HNE to measure the DCFHA fluorescence (B) image, (C) level, and (D) antioxidant enzyme activity or high glucose-induced oxidative DCFHA fluorescence (E) image, (F) level, and (G) antioxidant enzyme activity change. ARPE19 cells were cultured in DMEM F12 for 24 h after serum free starvation for 18 h. This was combined with fucoxanthin induced by 30 µM 4-hydroxynonenal (4-HNE) for 24 h or 75 mM glucose for 72 h (n = 3–5). *, p < 0.05; and **, p < 0.01 compared with the non-treated group. #, p < 0.05; and ##, p < 0.01 compared with the induced group (4-HNE 30 µM/glucose 75 mM). We used a microscope at 10× magnification.

Figure 8

Schematic representation of the protective effect of fucoxanthin on the 4-HNE- and high glucose-induced ARPE19 cell damage. The reduction of the ROS and the completeness of the blood–retinal barrier provides structural integrity and indicate a protective ability of fucoxanthin in diabetes retinopathy.