**2. Results**

#### *2.1. Cytotoxicity of Fucoxanthin to RPE Cells in a Lipid-Rich Environment*

The cytotoxicity of fucoxanthin and lutein to RPE cells in a high-lipid environment was evaluated, with the results shown in Figure 1. When the medium contained 25.0 μmol/L of DHA, a slight proliferative effect was observed on the RPE cells after 24 h incubation, with no statistically significant difference between the fucoxanthin and lutein. It was therefore clear that under the condition of 25.0 μmol/L DHA, no obvious (*p* < 0.05) neither fucoxanthin nor lutein exerted a significant inhibitory effect on RPE cells' proliferation when their dosages ≤50.0 μg/mL. Compared with the control group, there was also no significant difference (*p* < 0.05) in the lactic dehydrogenase (LDH) expression in all treatment groups. The LDH analysis further confirmed that 25.0 μmol/L DHA plus ≤50.0 μg/mL fucoxanthin or lutein exerted no cytotoxicity on the RPE cells.

#### *2.2. Effects of Visible Light Exposure on Oxidative Damage in Differentiated RPE Cells*

The effects of different light exposure conditions on the oxidative damage to and phagocytic function of RPE cells were investigated, the results of which are presented in Figure 2. When the differentiated RPE cells were irradiated with 1500 lux visible light for 6–24 h, no obvious (*p* < 0.05) oxidative damage, inflammation, or phagocytic dysfunction were observed. However, when the light intensity was increased to 3500 or 5000 lux, visible light-induced injury was induced with prolonged light exposure. After 24 h light exposure under 3500 lux, the intracellular ROS and malondialdehyde (MDA) level had increased by approximately 1.46 and 1.05 times, respectively, while superoxide dismutase (SOD) activity had decreased by 29.85%. Compared with the control group, the inflammatory cytokines interleukin (IL)-6 (354.13 ± 25.44 pg/mL of control) and tumor necrosis factor-α (TNF-α) (114.13 ± 5.39 pg/mL of control) in the media were found to have increased to 632.36 ± 22.39 pg/mL and 432.36 ± 21.35 pg/mL, respectively, and a 37.64% loss was simultaneously observed in the phagocytic index. It is clear that under the more intense light exposure conditions and consequently more extensive oxidative damage, the phagocytic function of the RPE cells was further degraded. For example, when the RPE cells were exposed to light irradiation at 5000 lux for 24 h, their phagocytic index remained only 45.34 ± 5.68%. Considering the degree of oxidative damage and degradation of the phagocytic function of RPE cells, light exposure at 3500 lux for 24 h was employed in the subsequent experiments.

**Figure 2.** The effects of visible light exposure on oxidative damage, inflammation and phagocytosis in differentiated RPE cells: (**a**) intracellular ROS levels; (**b**) intracellular MDA levels; (**c**) SOD activity; (**d**) inflammatory factor IL-6; (**e**) inflammatory factor TNF-α; (**f**) phagocytic index. (\* *p* < 0.05 and \*\* *p* < 0.01 vs. control; ΔΔ *p* < 0.01 means 1500 vs. 3500 lux; -- *p* < 0.01 means 3500 vs. 5000 lux).

#### *2.3. Fucoxanthin Pretreatment Activated the Nrf2 Signal Pathway in RPE Cells*

In this study, the results showed that adequate pretreatment time with fucoxanthin was beneficial in activating the antioxidant system of the RPE cells. As shown in Figure 3, after RPE cells were pretreated by fucoxanthin for 6–24 h, the expressions of nuclear Nrf2 (Nucl-Nrf2) protein and its regulated downstream antioxidant proteins or detoxification enzymes were investigated. When the RPE cells were co-incubated with 20.0 μmol/L fucoxanthin for 6 and 12 h (Figure 3a), it was found that, compared with the control group, Nucl-Nrf2 activity was increased by approximately 1.28 and 1.48 times, respectively. However, there was no further significant (*p* < 0.05) increase in Nucl-Nrf2 expression when the fucoxanthin pretreatment time was extended to 24 h. Interestingly, lutein, which is also a xanthophyll, performed worse (*p* < 0.01) than fucoxanthin in activating Nucl-Nrf2. After 12 h pretreatment, the Nucl-Nrf2 level in the lutein group had only increased 1.21 times. As shown in Figure 3b–e, similar phenomena were observed in the antioxidant proteins, including glutamate-cysteine ligase catalytic subunit (GCLC), glutathione peroxidase (GPx), thioredoxin reductase (TrxR), and HO-1, as well as the detoxification enzyme NQO1. The above results, thus, indicate that 12 h fucoxanthin pretreatment was sufficient to effectively activate the Nrf2 signaling pathway in the differentiated RPE cells.

#### *2.4. Fucoxanthin Attenuated Visible Light-Induced Oxidative Stress and Phagocytosis Disorder in RPE Cells*

Following 12 h pretreatment with fucoxanthin, the differentiated RPE cells were subjected to visible light exposure at 3500 lux for 24 h. As shown in Figure 4, when the concentration of fucoxanthin was 5.0 μmol/L, both the intracellular ROS and MDA levels began to be effectively (*p* < 0.01) inhibited, although no obvious (*p* < 0.05) ameliorative effects on inflammation or phagocytosis were evident. However, when the fucoxanthin concentrations were further increased to 10.0 or 20.0 μmol/L, the oxidative stress and inflammatory response in the RPE cells were significantly (*p* < 0.01) ameliorated. Under the condition of 20.0 μmol/L fucoxanthin, the intracellular ROS level decreased from 208.24 ± 8.56% (model group) to 118.56 ± 7.68%, and, compared with the model group, the levels of MDA, IL-6 and TNF-α were reduced by 44.87, 39.16, and 63.89%, respectively. Simultaneously, the phagocytic index of RPE cells recovered from 62.36 ± 4.15% to 89.56 ± 6.36%.

#### *2.5. Fucoxanthin Protected against Phagocytosis Disorder of RPE Cells via the Nrf2-Mediated Pathway*

In order to further elucidate the mechanism by which fucoxanthin provides protection against visible light-induced phagocytosis disorder of RPE cells, the Nrf2-mediated signaling pathway was investigated. As shown in Figure 5a, when the RPE cells were irradiated with visible light, the expression levels of Nucl-Nrf2 and its regulated NQO1 and HO-1 were slightly increased compared with those of the control group. Interestingly, when the RPE cells were administered with 20.0 μmol/L fucoxanthin (12 h pretreatment time plus 24 h light exposure time), the levels of Nucl-Nrf2, NQO1, and HO-1 were increased by approximately 1.64, 1.54, and 1.78, respectively, compared with those of the control group. Correspondingly, the intracellular ROS level was close to that of the control, while the TNFα was reduced by 64.21%, and the phagocytic index recovered to approximately 86.04%. However, when the Nrf2 inhibitor ML385 was added at the same time as the fucoxanthin pretreatment of the RPE cells, the levels of Nucl-Nrf2, NQO1, and HO-1 after the light exposure were only 24.56, 26.21, and 21.16%, respectively, of those of the control group. Moreover, under the same ML385 pretreatment condition, the fucoxanthin did not exhibit ameliorative effects on the oxidative stress, inflammatory response, or phagocytosis disruption in the RPE cells. As shown in Figure 5b–d, there were obvious (*p* < 0.05) differences between the fucoxanthin +ML385 group and the model group at the intracellular ROS level, inflammatory factor TNFα level, and the phagocytic index. Thus, it is clear that ML385 can effectively block activation of the Nrf2 signaling pathway, resulting in the inability of fucoxanthin to improve the phagocytosis of RPE cells.

**Figure 3.** The effect of pretreatment time on Nrf2 signaling pathway activated by fucoxanthin in differentiated RPE cells. (**a**) Nucl-Nrf2 activity; (**b**) GCLC expression level; (**c**) GPx expression level; (**d**) TrxR expression level; (**e**) HO-1 expression level; (**f**) NQO1 expression level. (\*\* *p* < 0.01 vs. control; -- *p* < 0.01 vs. lutein).

**Figure 4.** Inhibitory effects of fucoxanthin on oxidative damage and phagocytosis disorder in RPE cells induced by visible light: (**a**) intracellular ROS levels; (**b**) intracellular MDA levels; (**c**) inflammatory factor IL-6; (**d**) inflammatory factor TNF-α; (**e**) phagocytic indexes. (\*\* *p* < 0.01 vs. control; ## *p* < 0.01 vs. light exposure).

**Figure 5.** Ameliorative effects of fucoxanthin on phagocytosis disorder in RPE cells via the Nrf2 signal pathway: (**a**) the expressions of Nucl-Nrf2, NQO1, and HO-1 when RPE cells were treated with fucoxanthin or fucoxanthin +ML385; (**b**) ROS production when RPE cells were treated with fucoxanthin or fucoxanthin + ML385; (**c**) TNF-α levels when RPE cells were treated with fucoxanthin or fucoxanthin +ML385; (**d**) phagocytic indexes when RPE cells were treated with fucoxanthin or fucoxanthin +ML385. (\* *p* < 0.05 and \*\* *p* < 0.01 vs. control; ## *p* < 0.01 vs. light exposure).
