**3. Discussion**

This work used a polarized RPE cell culture model exhibiting similar morphological and physiological characteristics to the intact RPE monolayer, including the apical microvilli, well-defined tight junctions, membrane transport capability, and melanocytic pigmentation [25]. In our previous studies, when unpolarized RPE cells were exposed to 3500 lux visible light plus 25.0 μmol/L DHA for 12 h, the phagocytic index remained at only approximately 50% [1]. However, a phagocytic index with 81.49 ± 4.36% was observed when the differentiated RPE cell monolayer was exposed to the same visible light and lipid environment, suggesting that the differentiated RPE cells exhibited greater tolerance to visible light exposure. Therefore, it was hypothesized that, compared with the conventional in vitro cell model, differentiated RPE cells would more effectively reflect the true physiological conditions of retinal photodamage.

The RPE is considered a major target for retinal photodamage, which has long been attributed to the intracellular accumulation of lipofuscin particles during the phagocytizing of POSs [1,26]. Upon irradiation with visible light, N-retinylidene-N-retinylethanol-amine (A2E), one of the bis-retinoids derived from lipofuscin, produces singlet oxygen and other ROS, thus becoming a source of oxidative stress in RPE cells [27,28]. Due to their abundance of PUFAs, lipid peroxidation of POSs occurs before being swallowed in the presence of light exposure [29]. Given the close contact between RPE and POSs, this lipid peroxidation appears to be an important cause of visible light-induced damage to RPE cells. The results of this present study further confirmed that under the condition of visible light plus DHA

exposure, obvious oxidative injury occurred in the RPE cells. Numerous studies have indicated that excessive light exposure can not only lead to oxidative stress by triggering ROS generation but also induce retinal cell dysfunction. When Wistar rats were illuminated by white light-emitting diode light, disruption to the outer blood-retinal barrier, which is constructed by RPE cells, was observed [30]. Furthermore, excessive light exposure could result in increased vascular endothelial growth factor (VEGF) secretion in cultured RPE cells, thus increasing the risk of neovascularization and edema [31]. In our previous studies, cellular senescence was also discovered when RPE cells were subjected to visible light exposure [1], while, in this work, it was further found that intense visible light radiation could lead to the degradation of phagocytic functioning in differentiated RPE cells. It is, thus, apparent that the protection of RPE cells against visible light-induced oxidative damage and phagocytosis disorder within a PUFA-rich environment should be an important pathway for vision-protecting dietary nutrients.

Antioxidant activity has been shown to be one of the important ways in which dietary nutrients perform their vision-protective functions. For example, dietary flavonoids (such as fisetin, luteolin, and quercetin) and vitamins C and E are capable of protecting RPE cells against hydrogen peroxide (H2O2)- or *t*-butyl hydroperoxide (*t*-BOOH)-induced death by scavenging intracellular ROS [32]. Curcumin pretreatment was shown to display a protective effect on light-induced retinal degeneration in a rat model through antioxidant pathways [33], while malvidin-3-galactoside/glucoside, the characteristic component of blueberry anthocyanins, could reduce oxidative stress in RPE cells by decreasing the levels of ROS and MDA [34]. It was also reported that madecassoside, a major bioactive triterpenoid saponin, could attenuate H2O2-induced ROS and MDA production in RPE cells [35]. As mentioned above, RPE cells are highly susceptible to cell damage induced by lipid peroxidation in the body; however, as shown via the results in this work, fucoxanthin can effectively inhibit oxidative stress and inflammation induced by visible light plus DHA in RPE cells.

There is increasing evidence that the activation of endogenous cellular antioxidant systems is an important way in which dietary functional ingredients exert their antioxidant physiological activities. As a nuclear transcription factor, Nrf2 controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins, which are critically important mechanisms for cellular protection and cell survival [36]. Resveratrol pretreatment reportedly significantly restored the SOD activity and upregulated the protein and mRNA expressions of Nrf2 and HO-1 in rat brain [37] and, as a carotenoid, lycopene pretreatment has also been reported to be effective in enhancing the expressions of Nrf2 and HO-1 in rat brain, thereby exerting a neurocytoprotective function [38]. Furthermore, when RPE cells were pretreated with hesperetin for between 1 and 6 h, the effect on Nucl-Nrf2 activation was discovered to be treatment time-dependent [20]. Based on our present results, when RPE cells were pretreated with fucoxanthin for 6–24 h, the expressions of Nucl-Nrf2 and its regulated downstream antioxidant proteins (such as GCLC, GPx, TrxR, HO-1, and NQO1) increased progressively. In addition, the addition of Nrf2 inhibitor ML385 during the whole experiment was found to effectively inhibit the expressions of Nucl-Nrf2, NQO1, and HO-1, and no protective effect from fucoxanthin on the RPE cells was observed. Thus, it was apparent that fucoxanthin performed an ameliorative effect on the visible light-induced phagocytic disorder of the RPE cells via the Nrf2 signaling pathway.

#### **4. Materials and Methods**

#### *4.1. Materials and Chemical Reagents*

The fucoxanthin and lutein standards were purchased from Dexter Biotechnology Co., Ltd. (Chengdu, China). Dimethyl sulfoxide, Dulbecco's modified Eagle's/Ham's F12 media, DHA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2,7 - dichlorofluorescin diacetate (DCFH-DA), blue fluorescent amine-modified microspheres (0.05 μm) and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (MO, USA).

Commercial test kits, including LDH (CAS: A020-2-2), MDA (CAS: A003-4-1), SOD (CAS: A001-3-1), and HO-1 (CAS: H246-1), were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Penicillin, streptomycin, and Hanks' balanced salt solution (HBSS) were obtained from Gibco Life Technologies (Grand Island, NY, USA). The IL-6 (CAS: SEKM-0007) and TNFα (CAS: SEKM-0034)kits were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The nuclear extraction kit (CAS: ab113474), Nrf2 transcription factor assay kit (CAS: ab207223), GCLC (CAS: ab233632), NQO1 (CAS: ab184867) were purchased from Abcam Shanghai Trading Co., Ltd. (Shanghai, China). The antioxidant protein test kits, including GPx (CAS: BC1195) and TrxR (CAS: BC1155), were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The Nrf2 inhibitor ML385 was provided by Sigma-Aldrich (MO). All other reagents were analytical reagent-grade and purchased from the China National Pharmaceutical Industry Corporation Ltd. (Shanghai, China).
