Protective Effect of Astaxanthin on Blue Light Light-Emitting Diode

marine drugs

Article Protective Eﬀect of Astaxanthin on Blue Light Light-Emitting Diode-Induced Retinal Cell Damage via Free Radical Scavenging and Activation of PI3K/Akt/Nrf2 Pathway in 661W Cell Model

Chao-Wen Lin 1,2, Chung-May Yang 1,3 and Chang-Hao Yang 1,3,*

1 Department of Ophthalmology, National Taiwan University Hospital, Taipei 100, Taiwan; [email protected] (C.-W.L.); [email protected] (C.-M.Y.) 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan 3 College of Medicine, National Taiwan University, Taipei 100, Taiwan * Correspondence: [email protected]; Tel.: +886-2-23123456 (ext. 63193)

Received: 6 July 2020; Accepted: 23 July 2020; Published: 25 July 2020

Abstract: Light-emitting diodes (LEDs) are widely used and energy-efficient light sources in modern life that emit higher levels of short-wavelength blue light. Excessive blue light exposure may damage the photoreceptor cells in our eyes. Astaxanthin, a xanthophyll that is abundantly available in seafood, is a potent free radical scavenger and anti-inflammatory agent. We used a 661W photoreceptor cell line to investigate the protective effect of astaxanthin on blue light LED-induced retinal injury. The cells were treated with various concentrations of astaxanthin and then exposed to blue light LED. Our results showed that pretreatment with astaxanthin inhibited blue light LED-induced cell apoptosis and prevented cell death. Moreover, the protective effect was concentration dependent. Astaxanthin suppressed the production of reactive oxygen species and oxidative stress biomarkers and diminished mitochondrial damage induced by blue light exposure. Western blot analysis confirmed that astaxanthin activated the PI3K/Akt pathway, induced the nuclear translocation of Nrf2, and increased the expression of phase II antioxidant enzymes. The expression of antioxidant enzymes and the suppression of apoptosis-related proteins eventually protected the 661W cells against blue light LED-induced cell damage. Thus, our results demonstrated that astaxanthin exerted a dose-dependent protective effect on photoreceptor cells against damage mediated by blue light LED exposure.

Keywords: astaxanthin; blue light; light-emitting diode (LED); photoreceptor; reactive oxygen species (ROS); photo-injury; PI3K; Akt; Nrf2

1. Introduction Artiﬁcial lighting is indispensable in our daily life. Light-emitting diodes (LEDs) are energy-eﬃcient light sources that are widely used in consumer electronics, such as smartphones, digital displays, and computer screens. However, LEDs emit higher levels of blue light compared to conventional light sources. Blue light has a wavelength between 400–495 nm, which can penetrate the lens to reach the retina and induce greater retinal damage compared to light sources with longer wavelengths [1]. The main sites for light absorption in the eye are retinal photoreceptors and retinal pigment epithelial (RPE) cells [2]. Previous studies have demonstrated LED-induced photoreceptor and RPE cell damage [3,4]. The association between blue light exposure and age-related macular degeneration (AMD) was also disclosed in epidemiological studies [5].

Mar. Drugs 2020, 18, 387; doi:10.3390/md18080387 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, x 2 of 16 Mar. Drugs 2020, 18, 387 2 of 16 Oxidative stress and inflammatory reactions play major roles in the mechanism of blue light- induced retinal injury [6–8]. AMD is the leading cause of blindness worldwide [9]. Although Oxidative stress and inflammatory reactions play major roles in the mechanism of blue intravitreal injections of anti-vascular endothelial growth factor are the standard treatment for AMD light-induced retinal injury [6–8]. AMD is the leading cause of blindness worldwide [9]. [10], their effectiveness remains limited. Nutritional supplements for the improvement of eye health Although intravitreal injections of anti-vascular endothelial growth factor are the standard treatment have received considerable research attention. Sulforaphane, which is abundant in cruciferous for AMD [10], their effectiveness remains limited. Nutritional supplements for the improvement of eye vegetables, exerts anti-oxidative effects and protects RPE cells from photo-oxidative damage [11]. health have received considerable research attention. Sulforaphane, which is abundant in cruciferous Our research group previously demonstrated the protective effect of chitosan oligosaccharides on vegetables, exerts anti-oxidative effects and protects RPE cells from photo-oxidative damage [11]. blue light LED-induced RPE cell damage [12]. Bilberry and lingonberry have also been proven to Our research group previously demonstrated the protective effect of chitosan oligosaccharides on blue protect retinal photoreceptor cells from blue light LED-induced damage [13]. light LED-induced RPE cell damage [12]. Bilberry and lingonberry have also been proven to protect Astaxanthin is a keto-carotenoid [14]. It belongs to xanthophyll family, which includes retinal photoreceptor cells from blue light LED-induced damage [13]. carotenoid compounds containing oxygen molecules in hydroxyl, epoxy or ketone groups. Astaxanthin is a keto-carotenoid [14]. It belongs to xanthophyll family, which includes carotenoid Astaxanthin is also a metabolite of zeaxanthin and canthaxanthin. It contains several double bonds, compounds containing oxygen molecules in hydroxyl, epoxy or ketone groups. Astaxanthin is also a which results in a region of decentralized electrons that can help scavenge reactive oxygen species metabolite of zeaxanthin and canthaxanthin. It contains several double bonds, which results in a region (ROS). Therefore, astaxanthin is a very powerful antioxidant and anti-inflammatory agent [15–17]. of decentralized electrons that can help scavenge reactive oxygen species (ROS). Therefore, astaxanthin Astaxanthin is produced naturally by microalgae Haematococcus pluvialis and yeast Xanthophyllomyces is a very powerful antioxidant and anti-inflammatory agent [15–17]. Astaxanthin is produced naturally dendrorhous [18], especially when faced by a stressful environment, such as starvation or high salinity. by microalgae Haematococcus pluvialis and yeast Xanthophyllomyces dendrorhous [18], especially when Astaxanthin is also present in animals who feed on the algae, such as salmon, trout, sea bream, faced by a stressful environment, such as starvation or high salinity. Astaxanthin is also present in flamingos, and crustaceans. The safety of astaxanthin as a dietary supplement intended for humans animals who feed on the algae, such as salmon, trout, sea bream, flamingos, and crustaceans. The safety and animals has been proven [18,19]. However, the effects of astaxanthin on blue light LED-induced of astaxanthin as a dietary supplement intended for humans and animals has been proven [18,19]. retinal damage have not been reported. In the present study, we used 661W photoreceptor cells as a However, the effects of astaxanthin on blue light LED-induced retinal damage have not been reported. retinal cell model system to investigate the potential protective effects of astaxanthin on blue light- In the present study, we used 661W photoreceptor cells as a retinal cell model system to investigate the induced retinal injury. Furthermore, we aimed to elucidate the detailed mechanisms underlying these potential protective effects of astaxanthin on blue light-induced retinal injury. Furthermore, we aimed protective effects. to elucidate the detailed mechanisms underlying these protective effects. 2. Results 2. Results

2.1.2.1. Astaxanthin Astaxanthin Prevented Prevented Blue Blue Light Light LED-Induced LED-Induced 661W 661W Cell Cell Death Death TheThe viability viability of of 661W 661W cells, cells, determined determined using using Cell Cell Counting Counting Kit-8 Kit-8 (CCK-8) (CCK-8) assay, assay, decreased decreased significantlysignificantly after after exposure exposure to to blue blue light light LED, LED, whic whichh validated validated the theblue blue light light LED-induced LED-induced retinal retinal cell damage.cell damage. Cell Cellviability viability was wassignif significantlyicantly higher higher in inthe the high-dose high-dose astaxanthin astaxanthin treatment treatment group group comparedcompared to that in untreated cells (Figure1 1).). Moreover,Moreover, astaxanthin astaxanthin treatment treatment reduced reduced cell cell death death in in a adose-dependent dose-dependent manner. manner. Thus, Thus, the the protective protective effects effects of astaxanthin of astaxanthin on blue on lightblue LED-inducedlight LED-induced 661W 661Wcell death cell death was concentration was concentration dependent. dependent.

Figure 1. Astaxanthin (AST) suppressed blue light light-emitting diode (LED)-induced cell death. Figure 1. Astaxanthin (AST) suppressed blue light light-emitting diode (LED)-induced cell death. Cell Cell viability was analyzed by Cell Counting Kit-8 (CCK-8) assays. Cells were exposed to 2000 lx viability was analyzed by Cell Counting Kit-8 (CCK-8) assays. Cells were exposed to 2000 lx blue light blue light LED for 24 h after treatment with various concentrations of AST for 1 h. (C: control group, LED for 24 h after treatment with various concentrations of AST for 1 h. (C: control group, no AST or no AST or blue light LED exposure. All data represent mean SD. *p < 0.05, **p < 0.01 compared to blue light LED exposure. All data represent mean ± SD. *p < ±0.05, **p < 0.01 compared to the AST- the AST-untreated blue light LED exposed group; ANOVA with Dunnett’s multiple comparisons test; untreated blue light LED exposed group; ANOVA with Dunnett’s multiple comparisons test; N = 5 N = 5 per group). per group).

Mar. Drugs 2020, 18, 387 3 of 16

2.2. Astaxanthin Inhibited Blue Light LED-Induced 661W Cell Apoptosis Mar. Drugs 2020, 18, x 3 of 16 We analyzed the damage in 661W cells due to 2000 lx blue light LED exposure using the Terminal deoxynucleotidyl2.2. Astaxanthin transferase-mediated Inhibited Blue Light LED-Induced dUTP-biotinide 661W Cell Apoptosis end labeling (TUNEL) assay. The number of apoptoticWe cells analyzed increased the damage after bluein 661W light cells LED due exposureto 2000 lx blue (Figure light 2LEDa,b). exposure However, using the number of TUNEL-positiveTerminal deoxynucleotidyl cells decreased transferase-mediated markedly after dUTP-biotinide astaxanthin end pretreatment, labeling (TUNEL) especially assay. The at higher number of apoptotic cells increased after blue light LED exposure (Figure 2a,b). However, the concentrations (20 and 50 µM). The protective eﬀect was found to be concentration dependent. B-cell number of TUNEL-positive cells decreased markedly after astaxanthin pretreatment, especially at lymphomahigher 2-associated concentrations X protein (20 and 50 (Bax) μM). isThe a protective pro-apoptotic effect was protein, found to while be concentration B-cell lymphoma dependent. 2 (Bcl-2) is an importantB-cell anti-apoptotic lymphoma 2-associated protein. X protein We found (Bax) that is a astaxanthinpro-apoptotic protein, pretreatment while B-cell enhanced lymphoma the 2 expression of Bcl-2 and(Bcl-2) inhibited is an important that of anti-apoptotic Bax; it dose-dependently protein. We found increased that astaxanthin the mRNA pretreatment and enhanced protein expressionthe of expression of Bcl-2 and inhibited that of Bax; it dose-dependently increased the mRNA and protein Bcl-2/Bax (Figure2c,d). expression of Bcl-2/Bax (Figure 2c,d).

Figure 2. Astaxanthin (AST) inhibited blue light light-emitting diode (LED)-induced 661W cell apoptosis.Figure The 2. 661W Astaxanthin cells were (AST) exposed inhibited toblue 2000 light lx light-emitting blue light LEDdiode for(LED)-induced 24 h after being661W cell treated with various concentrationsapoptosis. The 661W of ASTcells were for 1exposed h. (a )to Apoptotic2000 lx blue light cells LED detected for 24 h byafter terminal being treated deoxynucleotidyl with various concentrations of AST for 1 h. (a) Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotinide end labeling (TUNEL). Nuclei were counterstained with transferase-mediated dUTP-biotinide end labeling (TUNEL). Nuclei were counterstained with 4′,6- 40,6-diamidino-2-phenylindolediamidino-2-phenylindole (DAPI). (DAPI). Ctrl: Ctrl: control control group—no group—no AST or blue AST light or LED blue exposure. light LED (b) exposure. (b) NumberNumber of TUNEL-positive of TUNEL-positive cells cells werewere counted counted in inat least at least four fourrandomly randomly chosen chosenviews, represented views, represented as columns.as columns. (C: control (C: control group. group. All All data data represent represent mean mean ± SD. *pSD. < 0.05, *p **

ANOVA with Dunnett’s multiple comparisons test; N = 5 in each group.) (d) Evaluation of the protein expression of Bcl-2 and Bax by Western blot analysis. β-actin was used as the internal control. (e) Relative protein expression of Bcl-2/Bax. (All data represent mean ± SD. * p < 0.05, ** p < 0.01 compared to the AST-untreated blue light LED exposed group by Kruskal–Wallis test with post hoc Dunn test;

Mar. DrugsN 2020= 4 per, 18 ,group.) 387 4 of 16

2.3. Astaxanthin Inhibited ROS Production in 661W Cells 2.3. Astaxanthin Inhibited ROS Production in 661W Cells To detect the anti-oxidative effects of astaxanthin on 661W cells, we used an ROS assay to assess ROSTo production. detect the anti-oxidative ROS production effects increased of astaxanthin substantia onlly 661W after cells, exposure we used to blue an ROS light assay LED. to However, assess ROSastaxanthin production. pretreatment ROS production before increased exposure substantially led to a significant after exposure decline to in blue ROS light levels, LED. especially However, at astaxanthinhigher concentrations pretreatment before(20 and exposure 50 μM) led(Figure to a significant 3). The anti-oxidative decline in ROS effect levels, was especially also concentration at higher concentrationsdependent. (20 and 50 µM) (Figure3). The anti-oxidative e ffect was also concentration dependent.

Figure 3. Astaxanthin (AST) inhibited the production of reactive oxygen species (ROS). (a) ROS Figure 3. Astaxanthin (AST) inhibited the production of reactive oxygen species (ROS). (a) ROS production was analyzed in cells pretreated with different AST concentrations for 1 h after exposure to production was analyzed in cells pretreated with different AST concentrations for 1 h after exposure 2000 lx blue light light-emitting diode (LED) for 24 h. The left column shows 2 , 7 -dichlorfluorescein to 2000 lx blue light light-emitting diode (LED) for 24 h. The left column shows0 20′, 7′-dichlorfluorescein (DCF) fluorescent cells. Nuclei were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI). (DCF) fluorescent cells. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole0 (DAPI). (Ctrl: (Ctrl: control group—no AST or blue light LED exposure.) (b) Relative DCF fluorescence intensity in control group—no AST or blue light LED exposure.) (b) Relative DCF fluorescence intensity in AST AST pretreated groups compared to that in the control group. (C: control group. All data represent pretreated groups compared to that in the control group. (C: control group. All data represent mean mean SD. * p < 0.05, ** p < 0.01 compared to the AST-untreated blue light LED exposed group; ± SD.± * p < 0.05, ** p < 0.01 compared to the AST-untreated blue light LED exposed group; Kruskal– Kruskal–Wallis test with post hoc Dunn test; N = 3 in each group.). Wallis test with post hoc Dunn test; N = 3 in each group.). 2.4. Astaxanthin Inhibited Oxidative Stress Biomarkers in 661W Cells 2.4. Astaxanthin Inhibited Oxidative Stress Biomarkers in 661W Cells The expression of 8-hydroxy-20-deoxyguanosine (8-OHdG), nitrotyrosine, and acrolein, which are indicatorsThe of expression oxidative of stress 8-hydroxy-2 induced′ by-deoxyguanosine the peroxidation (8-OHdG), of DNA, nitrotyrosine, protein, and lipid,and acrolein, respectively, which increasedare indicators considerably of oxidative in 661W stre cellsss after induced exposure by tothe blue peroxidation light LED. However,of DNA, pretreatmentprotein, and with lipid, astaxanthinrespectively, before increased exposure considerably resulted in a in significant 661W cells decrement after exposure in fluorescence to blue intensity, light LED. especially However, at higherpretreatment concentration with (20astaxanthin and 50 µ M)before (Figure exposure4). These resu e ffltedects in were a significant concentration decrement dependent. in fluorescence intensity, especially at higher concentration (20 and 50 μM) (Figure 4). These effects were concentration dependent.

Mar. Drugs 2020, 18, 387 5 of 16 Mar. Drugs 2020, 18, x 5 of 16

FigureFigure 4.4. AstaxanthinAstaxanthin (AST) (AST) inhibited inhibited the theexpressi expressionon of ofoxidative oxidative stress stress biomarkers. biomarkers. (a) (Immunofluorescencea) Immunofluorescence dete detectionction of 8-hydroxy-2 of 8-hydroxy-2′-deoxyguanosine0-deoxyguanosine (8-OHdG) (8-OHdG),, nitrotyrosine, nitrotyrosine, and andacrolein acrolein in cells in pretreated cells pretreated with different with di ASTfferent conc ASTentrations concentrations for 1 h and for then 1 exposed h and thento 2000 exposed lx blue tolight 2000 light-emitting lx blue light diode light-emitting (LED) for diode 24 h. (LED) Nuclei for were 24 h.counterstained Nuclei were with counterstained 4′,6-diamidino-2- with 4phenylindole0,6-diamidino-2-phenylindole (DAPI). (Ctrl: control (DAPI). group—no (Ctrl: control AST group—no or blue light AST LED or blue exposure.) light LED (b exposure.)) Relative (fluorescenceb) Relative fluorescence intensity of 8-OHdG, intensity ni oftrotyrosine, 8-OHdG, nitrotyrosine, and acrolein compared and acrolein to that compared in the control to that group. in the control group. (C: Control group. All data represent mean SD. * p < 0.05, ** p < 0.01 compared to the (C: Control group. All data represent mean ± SD. * p < 0.05,± ** p < 0.01 compared to the AST-untreated AST-untreatedblue light LED blueexposed light group; LED exposed Kruskal–Wallis group; Kruskal–Wallis test with post hoc test Dunn with posttest; hocN = Dunn3 in each test; group.) N = 3 in each group.) 2.5. Astaxanthin Suppressed Blue Light LED-Induced Mitochondrial Damage in 661W Cells 2.5. Astaxanthin Suppressed Blue Light LED-Induced Mitochondrial Damage in 661W Cells The extent of mitochondrial damage was analyzed by Mitochondrial Membrane Potential Assay The extent of mitochondrial damage was analyzed by Mitochondrial Membrane Potential Assay Kit (JC-1 method). JC-1 formed J-aggregates in healthy cells and preserved monomeric form in Kit (JC-1 method). JC-1 formed J-aggregates in healthy cells and preserved monomeric form in unhealthy or apoptotic cells. Exposure to blue light LED markedly increased JC-1 monomers in 661W unhealthy or apoptotic cells. Exposure to blue light LED markedly increased JC-1 monomers in cells. However, astaxanthin treatment decreased mitochondrial damage and a higher portion of JC-1 661W cells. However, astaxanthin treatment decreased mitochondrial damage and a higher portion of aggregation was observed after blue light LED exposure (Figure 5). Further, the extent of JC-1 JC-1 aggregation was observed after blue light LED exposure (Figure5). Further, the extent of JC-1 aggregation correlated with astaxanthin concentration. aggregation correlated with astaxanthin concentration.

Mar. DrugsMar. Drugs 20202020, 18,, x18 , 387 6 of 166 of 16

Figure 5. Astaxanthin (AST) protected 661W cells from mitochondrial damage. The 661W cells Figurewere 5. subjectedAstaxanthin to JC-1 (AST) staining, protected which 661W was analyzedcells from by mitochondrial florescence microscopy. damage. The J-aggregates 661W cells and were subjectedJC-1 monomers to JC-1 staining, were detected which using was Texas analyzed Red and by FITC,florescence respectively. microscopy. Treatment J-aggregates with astaxanthin and JC-1 monomersfacilitated were JC-1 detected aggregation using and decreasedTexas Red JC-1 and monomers FITC, inrespectively 661W cells exposed. Treatment to 2000 with lx blue astaxanthin light light-emitting diode (LED) for 24 h. (Ctrl: control group, no AST or blue light LED exposure.) facilitated JC-1 aggregation and decreased JC-1 monomers in 661W cells exposed to 2000 lx blue light 2.6.light-emitting Astaxanthin diode Induced (LED) Phase for II 24 Antioxidant h. (Ctrl: contro Enzymel group, Expression no AST and or Activated blue light PI3K LED/Akt exposure.)/Nrf2 Pathway in 661W Cells 2.6. AstaxanthinThe present Induced study Phase demonstrated II Antioxidant that Enzy astaxanthinme Expression had anti-oxidative and Activated eff PI3K/Akt/Nrf2ect against blue Pathway light in 661WLED-induced Cells 661W cell damage. In order to elucidate the underlying mechanisms of detoxification effect, we examined both the mRNA profiles and protein expression of the factors involved. Phase The present study demonstrated that astaxanthin had anti-oxidative effect against blue light II enzymes have a detoxification effect against oxidative stress. Treatment with astaxanthin induced LED-induced 661W cell damage. In order to elucidate the underlying mechanisms of detoxification an increment in mRNA and protein expression of Heme oxygenase-1 (HO-1) and NAD(P)H:quinone effect,oxidoreductase-1 we examined both (NQO1) the (Figure mRNA6), profiles which was and significant protein expression at higher concentrations of the factors (20 involved. and 50 µ PhaseM). II enzymesTreatment have witha detoxification astaxanthin effect also inducedagainst oxidative the protein stress. expression Treatment of phosphoinositide with astaxanthin 3-kinases induced an increment(PI3K), in Phospho-Akt mRNA and (p-Akt), protein and expression Nuclear factor of Heme erythroid oxygenase-1 2-related (HO-1) factor 2 and (Nrf2) NAD(P)H:quinone (Figure7) in oxidoreductase-1a dose-dependent (NQO1) manner. (Figure Consequently, 6), which we was demonstrated significant that at astaxanthin higher concentrations pretreatment activated(20 and 50 the μM). TreatmentPI3K/Akt with/Nrf2 astaxanthin pathway and also subsequently induced induced the prot theein expression expression of phase of phos II enzymesphoinositide in 661W 3-kinases cells. (PI3K), Phospho-Akt (p-Akt), and Nuclear factor erythroid 2-related factor 2 (Nrf2) (Figure 7) in a dose-dependent manner. Consequently, we demonstrated that astaxanthin pretreatment activated the PI3K/Akt/Nrf2 pathway and subsequently induced the expression of phase II enzymes in 661W cells.

Mar. Drugs 2020, 18, 387 7 of 16 Mar. Drugs 2020, 18, x 7 of 16

Figure 6. Astaxanthin (AST) upregulated mRNA and protein expression of Phase II enzymes. FigureThe 6. 661W Astaxanthin cells, pretreated (AST) upregulated with diﬀerent mRNA AST concentrations and protein for expression 1 h, were exposedof Phase to II 2000 enzymes. lx blue The 661Wlight cells, light-emitting pretreated with diode different (LED) for AST 24 h.concentrat Relativeions expression for 1 h, of were (a) hemeexposed oxygenase-1 to 2000 lx (HO-1); blue light light-emitting(b) NAD(P)H:quinone diode (LED) oxidoreductase-1 for 24 h. Relative (NQO1). expression Evaluation of of ( thea) relativeheme oxygenase-1 mRNA expression (HO-1); by (b) quantitative reverse transcription polymerase chain reaction (qRT-PCR). (All data represent mean SD. NAD(P)H:quinone oxidoreductase-1 (NQO1). Evaluation of the relative mRNA expression± by quantitative* p < 0.05, reverse *** p < 0.001 transcription compared polymerase to the AST-untreated chain reaction blue light (qRT-PCR). LED exposed (All group data by represent ANOVA with mean ± SD. *Dunnett’s p < 0.05, *** multiple p < 0.001 comparisons compared test; to N the= 5 AST-untreated in each group.) Relativeblue light protein LED expression exposed group of (c) HO-1 by ANOVA and (d) NQO1 by Western blot analysis. β-actin was used as the internal control. (All data represent mean with Dunnett’s multiple comparisons test; N = 5 in each group.) Relative protein expression of (c) HO- SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the AST-untreated blue light LED exposed group 1 and± (d) NQO1 by Western blot analysis. β-actin was used as the internal control. (All data represent by Kruskal–Wallis test with post hoc Dunn test; N = 4 per group.) mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the AST-untreated blue light LED exposed group by Kruskal–Wallis test with post hoc Dunn test; N = 4 per group.)

Mar. Drugs 2020, 18, 387 8 of 16 Mar. Drugs 2020, 18, x 8 of 16

Figure 7. Astaxanthin (AST) activated phosphoinositide 3-kinases (PI3K)/Akt/Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. The 661W cells, pretreated with diﬀerent AST concentrations for Figure 7. Astaxanthin (AST) activated phosphoinositide 3-kinases (PI3K)/Akt/Nuclear factor 1 h, with were exposed to 2000 lx blue light light-emitting diode (LED) for 24 h. (a) Evaluation of the erythroidprotein 2-related expression factor of PI3K, 2 (Nrf2) Akt, and pathway. Nrf2 by Western The blot661W analysis. cells,β-actin pretreated was used with as the internaldifferent AST concentrationscontrol. (forb) Relative 1 h, with protein were expression exposed of to (b) 2000 PI3K, (lxc) blue Phospho-Akt light light-emitting (p-Akt)/Akt, and diode (d) Nrf2. (LED) (All data for 24 h. (a) represent mean SD. * p < 0.05, ** p < 0.01 compared to the AST-untreated blue light LED exposed Evaluation of the protein± expression of PI3K, Akt, and Nrf2 by Western blot analysis. β-actin was used as thegroup internal by Kruskal–Wallis control. (b test) Relative with post protein hoc Dunn expression test; N = 4 perof (b) group.) PI3K, (c) Phospho-Akt (p-Akt)/Akt, and2.7. (d) Astaxanthin-InducedNrf2. (All data represent Nuclear mean Translocation ± SD. * ofp < Nrf2 0.05, in ** 661W p < 0.01 Cells compared to the AST-untreated blue light LEDThe exposed results ofgroup Western by Kruskal–Wallis blot showed that test astaxanthin with post induced hoc Dunn the expressiontest; N = 4 ofper Nrf2 group.) and phase II enzymes. In order to elucidate the modulating mechanisms of Nrf2 activation, we isolated and 2.7. Astaxanthin-Inducedevaluated the expression Nuclear of Translocation nuclear and cytosolic of Nrf2 Nrf2 in 661W proteins. Cells When astaxanthin treated 661W cells were exposed to blue light LED, Nrf2 translocated from the cytoplasm to the nucleus (Figure8). TheThe results expression of Western of nuclear blot Nrf2 showed increased that with astaxa astaxanthinnthin induced concentration. the expression We demonstrated of Nrf2 that and phase II enzymes.astaxanthin In order induced to elucidate nuclear translocation the modulating of Nrf2, mechanisms which then activated of Nrf2 the activation, expression of we phase isolated II and evaluatedantioxidant the expression enzymes of in nuclear 661W cells. and cytosolic Nrf2 proteins. When astaxanthin treated 661W cells were exposed to blue light LED, Nrf2 translocated from the cytoplasm to the nucleus (Figure 8). The expression of nuclear Nrf2 increased with astaxanthin concentration. We demonstrated that astaxanthin induced nuclear translocation of Nrf2, which then activated the expression of phase II antioxidant enzymes in 661W cells.

Mar. Drugs 2020, 18, 387 9 of 16 Mar. Drugs 2020, 18, x 9 of 16

FigureFigure 8. 8.Astaxanthin Astaxanthin (AST) (AST) induced induced the the nuclear nuclear translocation translocation of of nuclear nuclear factor factor erythroid erythroid 2-related 2-related factorfactor 2 2 (Nrf2) (Nrf2) in in 661W 661W cells. cells. The The 661W 661W cells cells were were exposed exposed to to 2000 2000 lx lx blue blue light light light-emitting light-emitting diode diode (LED)(LED) for for 24 24 h after h after treatment treatment with with different different concentrations concentrations of AST of for AST 1 h. for (a) 1 Evaluation h. (a) Evaluation of the protein of the expressionprotein expression of Nrf2 in of cytosol Nrf2 andin cytosol nucleus and by nucl Westerneus by blot Western analysis. blot Glyceraldehyde-3-phosphate analysis. Glyceraldehyde-3- dehydrogenasephosphate dehydrogenase (GAPDH) and (GAPDH) histone and H3 werehistone used H3 aswere the used internal as the controls internal in controls cytosol andin cytosol nucleus, and respectively.nucleus, respectively. (b) Relative (b protein) Relative expression protein expression of Nrf2 in (b) of cytosolNrf2 in and(b) cytosol (c) nucleus. and ( (Allc) nucleus. data represent (All data mean SD. C: control group, no AST or blue light LED exposure. * p < 0.05 compared to the represent± mean ± SD. C: control group, no AST or blue light LED exposure. * p < 0.05 compared to the AST-untreated blue light LED exposed group by Kruskal–Wallis test with post hoc Dunn test; N = 3 AST-untreated blue light LED exposed group by Kruskal–Wallis test with post hoc Dunn test; N = 3 per group.) per group.) 3. Discussion 3. Discussion We demonstrated that astaxanthin had a protective effect on 661W photoreceptor cells against damageWe mediated demonstrated by blue that light astaxanthin LED exposure. had a protective Astaxanthin effect reduced on 661W the photoreceptor production of cells ROS against and otherdamage oxidative mediated stress by blue biomarkers light LED as exposure. well as stabilized Astaxanthin the reduced mitochondrial the production membrane of ROS potential and other of cells.oxidative Furthermore, stress biomarkers it up-regulated as well Nrf2-regulated as stabilized phase the mitochondrial II enzymes (HO-1 membrane and NQO1) potential through of cells. the activationFurthermore, of PI3K it /up-regulatedAkt pathway. Nrf2-regulated Subsequently, itphase increased II enzymes the expression (HO-1 ofand anti-apoptotic NQO1) through protein the Bcl-2,activation thereby of inhibitingPI3K/Akt cellpathway. apoptosis Subsequently, and promoting it increased cell survival. the expression A previous of anti-apoptotic study demonstrated protein thatBcl-2, astaxanthin thereby inhibiting analogs reducecell apoptosis white fluorescentand promot light-induceding cell survival. photoreceptor A previous study degeneration demonstrated [20]. However,that astaxanthin blue light analogs LEDshave reduce higher white energy fluorescent emission light-induced and therefore photoreceptor can induce more degeneration damage to the[20]. retina.However, To the blue best light of our LEDs knowledge have higher the present energy study emission is the and first therefore to investigate can induce the protective more damage effects of to astaxanthinthe retina. onTo bluethe best light of hazards. our knowledge the present study is the first to investigate the protective effectsOxidative of astaxanthin stress has on been blue implicatedlight hazards. in the pathogenesis of diabetic, hypertensive and ischemic proliferativeOxidative retinopathy stress has [ 21been]. Previous implicated studies in the have path shownogenesis the of potential diabetic, benefitshypertensive of astaxanthin and ischemic in retinopathyproliferative associated retinopathy with [21]. oxidative Previous stress studies [22–26 have]. A strong shown correlation the potential between benefits oxidative of astaxanthin stress and in light-inducedretinopathy retinalassociated damage with has oxidative been previously stress [22–26 reported]. A strong [6,27]. Thecorrelation production between of ROS oxidative causes tissue stress injuryand light-induced via the peroxidation retinal damage of DNA, has lipids, been proteins, previously and reported carbohydrates [6,27]. The [28]. production Therefore, of we ROS detected causes antissue increase injury in thevia expressionthe peroxidation of oxidative of DNA, biomarkers, lipids, proteins, such as and 8-OHdG, carbohydrates nitrotyrosine, [28]. andTherefore, acrolein, we indetected 661W cells an increase after blue in lightthe expression exposure. of Oxidative oxidative stressbiomarkers, also induces such as mitochondrial 8-OHdG, nitrotyrosine, dysfunction, and acrolein, in 661W cells after blue light exposure. Oxidative stress also induces mitochondrial dysfunction, and eventually decreases cell viability [29]. Astaxanthin is a strong free radical

Mar. Drugs 2020, 18, 387 10 of 16 and eventually decreases cell viability [29]. Astaxanthin is a strong free radical scavenger and its antioxidant properties are attributed to the physical and chemical interactions with cell membranes. The conjugated double bonds in the polyene chain of astaxanthin trap and scavenge radicals in the cell membrane, quench singlet oxygen, and terminate free radical chain reactions [30]. Taken together, our results demonstrated that astaxanthin attenuated ROS production, decreased mitochondrial damage, and reduced the generation of oxidation products, including 8-OHdG, nitrotyrosine, and acrolein, subsequently enhancing 661W cell survival. In addition to validating the antioxidant properties of astaxanthin, our study demonstrated that astaxanthin upregulated the Nrf2-antioxidant response element (ARE) pathway and facilitated the expression of phase II antioxidant enzymes, HO-1 and NQO-1. The Nrf2–ARE pathway is an important mechanism to suppress oxidative stress [31]. This mechanism also reportedly protected RPE cells against oxidative damage [32,33]. In the absence of oxidative stress, Nrf2 is bound to the chaperone Kelch-like ECH-associated protein 1 (KEAP1) and is localized in the cytosol. However, in an oxidant environment, the structure of KEAP1 changes and Nrf2 is released, which then translocates to the nucleus and binds to the ARE promoter. Phase II antioxidant enzymes, such as HO-1, and NQO1, are subsequently activated to neutralize oxidative stress [34]. The activation of Nrf2–ARE pathway by astaxanthin was reported in previous studies [35–37]. Using RT-PCR and Western blot analysis, our study showed that astaxanthin induced the expression of Nrf2, HO-1, and NQO1 in a dose-dependent manner. ERK, and PI3K/Akt are the two possible mechanisms involved upstream of the Nrf2–ARE pathway [38]. Moreover, both can be activated by astaxanthin [39,40]. However, in previous studies, the PI3K/Akt pathway was found to play important roles in modulating Nrf2–ARE–phase II antioxidant enzymes in response to oxidative stress in RPE cells [33,41]. Although we used 661W cells instead of ARPE-19 cells as our model of blue light LED-induced retinal injury, we observed that astaxanthin induced the expression of PI3K and the phosphorylation of Akt in a dose-dependent manner. In summary, our results indicated that astaxanthin activated the PI3K/Akt pathway, induced the nuclear translocation of Nrf2, increased the expression of phase II antioxidant enzymes, and eventually protected 661W cells against blue light LED-induced oxidative stress. Astaxanthin has two hydroxyl groups that can span the bi-lipid layer and link the inside and outside of the cell membrane [30]. Moreover, as it has both lipophilic and hydrophilic properties and is fat-soluble, it could be carried by fat molecules to multiple organs and tissues, such as the brain, muscle, and retina [42]. It also has better biological activity than other antioxidants [43] and no toxic effects have been reported. In fact, the United States Food and Drug Administration has approved the use of astaxanthin as a dietary supplement since 1999 [44]. As a potent antioxidant and anti-inflammatory agent, astaxanthin has anti-cancer, anti-diabetic, and immunomodulatory activity [30] as wells as provides protective effects in cardiovascular and neurodegenerative diseases [38,45]. Although astaxanthin is not a component of human retina, it could cross the blood–retinal barrier and exerts anti-oxidative effects on retinal ganglion cells [46]. There were a few limitations in our study. First, our study was conducted in vitro. The protective effects of astaxanthin against blue light damage need to be demonstrated in animal models. Second, the intensity of light exposure in our study was much higher than the typical human daily exposure. Blue light-induced retinal damage is a gradual process and the protective effects of nutrient supplements is difficult to determine. Last but not the least, the blue light-absorbing properties of astaxanthin may also play some roles in its protective effects. However, the results of this study provided strong evidence that astaxanthin may be useful for the prevention of blue light LED-induced retinal injury. Moreover, we proved that the protective effects were mainly attributed to the free radical scavenging activity of astaxanthin and the activation of the PI3K/Akt/Nrf2 pathway. Mar. Drugs 2020, 18, 387 11 of 16

4. Materials and Methods

4.1. 661W Cell Culture The 661W cell line was obtained from Dr. M. Al-Ubaidi (University of Houston). It is a mouse photoreceptor cell line immortalized by the expression of simian virus 40 T antigen, which displays biochemical features of cone cells [47]. The 661W cells were maintained in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% phosphate buffer solution (PBS), 100 units/mL penicillin, and 100 µg/mL streptomycin in a 5% CO2 atmosphere at 37 ◦C. Cells were passaged by trypsinization every 3–4 days. Cells from the second to fifth passages were used in the subsequent experiments.

4.2. Blue Light LED and Astaxanthin Treatment of 661W Cells A blue light LED tube (wavelength 450 20 nm, 20 W) was placed at the bottom of a special ± framework, and we used an illuminometer to measure and conform the light intensity at the cell surface. The distance between the light tube and cell surface was adjusted according to light intensity. The 661W cells seeded on a 96-well plate received LED light exposure directly to minimize the screening eﬀect of astaxanthin in the medium. To ensure a stable environment for 661W cells, the device was placed in the CO2 incubator. Temperature was maintained at 36.5–37.5 ◦C. Astaxanthin (average molecular weight 596.84) was purchased from Sigma-Aldrich (CAS Number 472-61-7, St. Louis, MO, USA). Cells were treated with astaxanthin at various concentrations (0, 5, 10, 20, 50, 100 µM) for 1 h and then exposed to blue light LED with an intensity of 2000 lx for 24 h. Cells were cultured for 24–48 h after exposure. Astaxanthin remained in the medium during the LED exposure.

4.3. CCK-8 Assay for Cell Viability The viability of astaxanthin-treated and blue light LED exposed cells was determined using the CCK-8 assay (TEN-CCK8, Biotools, New Taipei City, Taiwan), according to manufacturer’s instructions. The 661W cells were cultured in 96-well plates and then incubated for 24 h in a 5% CO2 atmosphere at 37 ◦C. The absorbance was measured at 450 nm with a microplate reader (Bio-Rad Laboratories, CA, USA).

4.4. TUNEL Assay TUNEL assay was performed by a FragELTM DNA fragmentation detection kit (Calbiochem, Darmstadt, Germany). The TUNEL enzyme and peroxidase converter were added to the cells after incubation for 5 min in a permeabilizing solution containing 0.1% Triton-X and 0.1% sodium citrate. After adding FITC–Avidin, image analysis was used to detect the ﬂuorescent signals of the TUNEL–positive cells in four random slides per sample.

4.5. Detection of Intracellular ROS

Intracellular ROS levels were quantified using 20,70-dichlorodihydrofluorescein diacetate (20,70-DCFDA; Sigma-Aldrich, MO, USA) oxidation. To astaxanthin pretreated and blue light LED exposed cells, 10 mM 5-(and-6)-chloromethyl-20,70-DCFDA, acetyl ester (CM-H2DCFDA), a free radical probe, was added and incubated for 1 h at 37 ◦C. The radical probe was converted to 20,70-dichlorodihydrofluorescein (DCFH) by intracellular esterase. Intracellular DCFH (non-fluorescent) was oxidized to 20,70-dichlorfluorescein (DCF, fluorescent) by intracellular ROS. Fluorescence was measured by a microplate reader (Bio-Rad Laboratories, CA, USA) at 485 nm (excitation) and 535 nm (emission).

4.6. Immunoﬂuorescence (IF) Detection of Oxidative Stress Biomarkers IF was performed by blocking and permeabilizing the cells with 0.2% Triton in PBS containing 5% goat serum for 1 h at 25 ◦C, then incubating them with primary antibodies diluted in blocking solution Mar. Drugs 2020, 18, 387 12 of 16

overnight at 4 ◦C, and ﬁnally incubating with the appropriate ﬂuorescent-labelled secondary antibodies (diluted 1:1000 in blocking solution; Santa Cruz, TX, USA) for 3 h at 25 ◦C. Nuclei were counterstained with 40,6-diamidino-2-phenylindole (DAPI). Primary antibodies included mouse anti-rat 8-OHdG (JaICA, Shizuoka, Japan), mouse anti-rat nitrotyrosine (Abcam, Hong Kong, China), and mouse anti-rat acrolein (Abcam, Hong Kong, China). Fluorescence was measured by a microplate reader (Bio-Rad Laboratories, CA, USA).

4.7. Determination of Mitochondrial Dysfunction The mitochondrial membrane potential of cells was analyzed by the JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical Company, MI, USA). After 24 h of 2000 lx blue LED light exposure, 50 µL of JC-1 staining buffer was added to 1 mL of culture medium. The fluorescence signals for both J-aggregates with Texas Red (healthy cells, excitation/emission = 560/595 nm) and JC-1 monomers with FITC (apoptotic or unhealthy cells, excitation/emission = 485/535 nm) were captured by a fluorescence microscope. The number of cells (red or green stained cells) were counted in a blinded manner by Image-J.

4.8. RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction RNA was extracted from 661W cells by TRIzol reagent (Invitrogen-Life Technologies, MD, USA). For each sample, 1 µg of total RNA was incubated with 300 ng oligo(dT) (Promega, WI, USA) for 5 min at 65 ◦C and reverse transcribed into cDNA using 80 U of Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Thermo Fisher Scientific, MA, USA) per 50 µg of reaction sample for 1 h at 37 ◦C. The reaction was terminated by heating the samples at 90 ◦C for 5 min. The resultant cDNA product was subjected to PCR with specific primers. The primer sequences were as follows: HO-1: 50-AAGATTGCCCAGAAAGCCCTGGAC-30 (forward), 50-AACTGTCGCCACCAGAAAGCTGAG-30 (reverse); NQO1: 50-AAGGATGGAAGAAACGCCTGGAGA-30 (forward), 50-GGCCCACAGAAAGGCCAAATTTCT-30 (reverse); Bcl-2: 50-CTGGTGGACAACATCGCTCTG-30 (forward), 50-GGTCTGCTGACCTCACTTGTG-30 (reverse); Bax: 50-TGGTTGCCCTTTTCTACTTTG-30 (forward), 50-GAAGTAGGAAAGGAGGCCATC-30 (reverse); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 50-ACCACAGTCCATGCCATCAC-30 (forward), 50-TCCACCACCCTGTTGCTGTA-30 (reverse). The 50 µL reaction mixture contained 5 µL cDNA, 1 µL sense and antisense primers each, 200 µM of each deoxynucleotide, 5 µL of 10 Taq × polymerase buffer, and 1.25 U Taq polymerase (Promega, WI, USA). A thermocycler (MJ Research, MA, USA) was used for amplification with the following conditions: Initial 1 min denaturation at 94 ◦C and 3 min extension at 72 ◦C. The annealing temperature between 42 ◦C and 62 ◦C was decreased by 1 ◦C decrements, which was followed by 21 cycles at 55 ◦C. Finally, the temperature was elevated to 72 ◦C for 10 min and then reduced to 4 ◦C. The products were subjected to electrophoresis on a 2% agarose gel and analyzed by a gel analyzer system. Each mRNA level was standardized based on the intensity of GAPDH.

4.9. Western Blot Analysis 661W cells were pretreated with different concentrations of astaxanthin for 1 h, followed by exposure to 2000 lx blue light LED for 24 h. Then, the cells were washed with PBS, lysed in RIPA buffer (Sigma-Aldrich, MO, USA) containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, MO, USA), and harvested. Lysates were centrifuged at 12,000 g for 15 min at 4 ◦C. Protein concentrations were quantified by a BCA Protein Assay Kit (Bio-Rad Laboratories, CA, USA) with bovine serum albumin as the standard. The protein samples were separated by 10% SDS-PAGE gradient electrophoresis and then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, MA, USA). The primary antibodies utilized in the experiment were as follows: rabbit anti- HO-1 (1:2500, Abcam, Hong Kong, China), mouse anti- NQO1 (1:2000, Abcam, Hong Kong, China), rabbit anti- PI3K (1:1000, Cell Signaling Technology, Mar. Drugs 2020, 18, 387 13 of 16

MA, USA), rabbit anti-Akt (1:1000, Cell Signaling Technology, MA, USA), rabbit anti- p-Akt (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-Bax (1:1000, Cell Signaling Technology, MA, USA), rabbit anti- Bcl-2 (1:500, Proteintech, IL, USA), rabbit anti-Nrf2 (1:500, Abcam, Hong Kong, China), and mouse anti-β-actin (1:5000, Sigma-Aldrich, MO, USA). A horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody and an HRP-conjugated goat anti-mouse antibody were used as secondary antibodies. Immunodetection was performed by enhanced chemiluminescence (Pierce Biotechnology, MA, USA). Protein levels were measured using densitometry analysis of the bands. β-actin was used as an internal control.

4.10. Nuclear and Cytosolic Protein Extraction and Analysis of Nrf2 Protein Expression Nuclear and cytosolic proteins were extracted separately using NE-PER (Nuclear and Cytoplasmic Extraction Reagent; Pierce Biotechnology, MA, USA) according to the manufacturer’s instructions. Proteins were quantiﬁed by a BCA Protein Assay Kit (Bio-Rad Laboratories, CA, USA) and Western blot was performed with anti-Nrf2 antibody (1:500, Abcam, Hong Kong, China). GAPDH (1:5000, Abcam, Hong Kong, China) was used as the internal control in cytosol and histone H3 (1:1000, Cell Signaling Technology, MA, USA) was used as the internal control in nucleus.

4.11. Statistical Analyses Data are presented as mean SD. To compare numerical data among the groups, one-way ANOVA ± or Kruskal–Wallis test, followed by post hoc test was used. p < 0.05 was considered statistically signiﬁcant. Statistical analysis was performed by SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA).

5. Conclusions Our study was the first effort to investigate and demonstrate the protective effects of astaxanthin on retinal photoreceptor cells against damage mediated by blue light LED exposure. Astaxanthin upregulated the PI3K/Akt/Nrf2-ARE signaling pathway and subsequently activated the expression of phase II antioxidant enzymes (HO-1 and NQO1). As a strong free radical scavenger, it also reduced the production of ROS, up-regulated the anti-apoptotic protein Bcl-2, and stabilized the mitochondrial membrane. Thus, this study supported the idea that astaxanthin could be potentially used to prevent blue light-induced retinal damage.

Author Contributions: Conceptualization, C.-W.L., C.-M.Y., and C.-H.Y.; Data curation, C.-W.L. and C.-H.Y.; Formal analysis, C.-W.L. and C.-H.Y.; Funding acquisition, C.-W.L., C.-M.Y., and C.-H.Y.; Investigation, C.-W.L. and C.-H.Y.; Methodology, C.-W.L., C.-M.Y., and C.-H.Y.; Project administration, C.-M.Y. and C.-H.Y.; Resources, C.-M.Y. and C.-H.Y.; Software, C.-M.Y. and C.-H.Y.; Supervision, C.-M.Y. and C.-H.Y.; Validation, C.-W.L.; Visualization, C.-W.L.; Writing—original draft, C.-W.L.; Writing—review and editing, C.-M.Y. and C.-H.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by National Taiwan University Hospital (Grant 109-N4473). Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Chamorro, E.; Bonnin-Arias, C.; Pérez-Carrasco, M.J.; Luna, J.M.; Vázquez, D.; Sánchez-Ramose, C. Eﬀects of light-emitting diode radiations on human retinal pigment epithelial cells in vitro. Photochem. Photobiol. 2013, 89, 468–473. [CrossRef][PubMed] 2. Parver, L.M.; Auker, C.R.; Fine, B.S. Observations on monkey eyes exposed to light from an operating microscope. Ophthalmology 1983, 90, 964–972. [CrossRef] 3. Jaadane, I.; Boulenguez, P.; Chahory, S.; Carre, S.; Savoldelli, M.; Jonet, L.; Behar-Cohen, F.; Martinsons, C.; Torriglia, A. Retinal damage induced by commercial light emitting diodes (LEDs). Free Radic. Biol. Med. 2015, 84, 373–384. [CrossRef][PubMed] Mar. Drugs 2020, 18, 387 14 of 16

4. Shang, Y.M.; Wang, G.S.; Sliney, D.; Yang, C.H.; Lee, L.L. White light-emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model. Environ. Health Perspect. 2014, 122, 269–276. [CrossRef] [PubMed] 5. Wu, J.; Seregard, S.; Algvere, P.V. Photochemical damage of the retina. Surv. Ophthalmol. 2006, 51, 461–481. [CrossRef] 6. Organisciak, D.T.; Vaughan, D.K. Retinal light damage: Mechanisms and protection. Prog. Retin. Eye Res. 2010, 29, 113–134. [CrossRef] 7. Hollyfield, J.G.; Bonilha, V.L.; Rayborn, M.E.; Yang, X.; Shadrach, K.G.; Lu, L.; Ufret, R.L.; Salomon, R.G.; Perez, V.L. Oxidative damage-induced inflammation initiates age-related macular degeneration. Nat. Med. 2008, 14, 194–198. [CrossRef] 8. Nakamura, M.; Kuse, Y.; Tsuruma, K.; Shimazawa, M.; Hara, H. The involvement of the oxidative stress in murine blue LED light-induced retinal damage model. Biol. Pharm. Bull. 2017, 40, 1219–1225. [CrossRef] 9. Friedman, D.S.; O’Colmain, B.J.; Muñoz, B.; Tomany, S.C.; McCarty, C.; de Jong, P.T.; Nemesure, B.; Mitchell, P.; Kempen, J. Prevalence of age-related macular degeneration in the United States. Arch. Ophthalmol. 2004, 122, 564–572. [CrossRef] 10. Bloch, S.B.; Larsen, M.; Munch, I.C. Incidence of legal blindness from age-related macular degeneration in Denmark: Year 2000 to 2010. Am. J. Ophthalmol. 2012, 153, 209–213.e2. [CrossRef] 11. Gao, X.; Talalay, P. Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage. Proc. Natl. Acad. Sci. USA 2004, 102, 10446–10451. [CrossRef][PubMed] 12. Lin, C.W.; Huang, H.H.; Yang, C.M.; Yang, C.H. Protective effect of chitosan oligosaccharides on blue light light-emitting diode induced retinal pigment epithelial cell damage. J. Funct. Foods 2018, 49, 12–19. [CrossRef] 13. Ogawa, K.; Kuse, Y.; Tsuruma, K.; Kobayashi, S.; Shimazawa, M.; Hara, H. Protective effects of bilberry and lingonberry extracts against blue light-emitting diode light-induced retinal photoreceptor cell damage in vitro. BMC Complement. Altern. Med. 2014, 2, 120. [CrossRef][PubMed] 14. Choi, S.; Koo, S. Efficient Syntheses of the Keto-carotenoids Canthaxanthin, Astaxanthin, and Astacene. J. Org. Chem. 2005, 70, 3328–3331. [CrossRef] 15. Jackson, H.; Braun, C.L.; Ernst, H. The chemistry of novel xanthophyll carotenoids. Am. J. Cardiol. 2008, 101, 50D–57D. [CrossRef] 16. Kurashige, M.; Okimasu, E.; Inoue, M.; Utsumi, K. Inhibition of oxidative injury of biological membranes by astaxanthin. Physiol. Chem. Phys. Med. NMR 1990, 22, 27–38. 17. Ohgami, K.; Shiratori, K.; Kotake, S.; Nishida, T.; Mizuki, N.; Yazawa, K.; Ohno, S. Effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo. Investig. Ophthalmol. Vis. Sci. 2003, 44, 2694–2701. [CrossRef] 18. Spiller, G.A.; Dewell, A. Safety of an astaxanthin-rich Haematococcus pluvialis algal extract: A randomized clinical trial. J. Med. Food. 2003, 6, 51–56. [CrossRef] 19. Stewart, J.S.; Lignell, A.; Pettersson, A.; Elfving, E.; Soni, M.G. Safety assessment of astaxanthin-rich microalgae biomass: Acute and subchronic toxicity studies in rats. Food Chem. Toxicol. 2008, 46, 3030–3036. [CrossRef] 20. Inoue, Y.; Shimazawa, M.; Nagano, R.; Kuse, Y.; Takahashi, K.; Tsuruma, K.; Hayashi, M.; Ishibashi, T.; Maoka, T.; Hara, H. Astaxanthin analogs, adonixanthin and lycopene, activate Nrf2 to prevent light-induced photoreceptor degeneration. J. Pharmacol. Sci. 2017, 134, 147–157. [CrossRef] 21. Opatrilova, R.; Kubatka, P.; Caprnda, M.; Büsselberg, D.; Krasnik, V.; Vesely, P.; Saxena, S.; Ruia, S.; Mozos, I.; Rodrigo, L.; et al. Nitric oxide in the pathophysiology of retinopathy: Evidences from preclinical and clinical researches. Acta Ophthalmol. 2018, 96, 222–231. [CrossRef][PubMed] 22. Otsuka, T.; Shimazawa, M.; Inoue, Y.; Nakano, Y.; Ojino, K.; Izawa, H.; Tsuruma, K.; Ishibashi, T.; Hara, H. Astaxanthin protects against retinal damage: Evidence from in vivo and in vitro retinal ischemia and reperfusion models. Curr. Eye Res. 2016, 41, 1465–1472. [CrossRef][PubMed] 23. Baccouche, B.; Benlarbi, M.; Barber, A.J.; Ben Chaouacha-Chekir, R. Short-Term Administration of astaxanthin attenuates retinal changes in diet-induced diabetic Psammomys obesus. Curr. Eye Res. 2018, 43, 1177–1189. [CrossRef] Mar. Drugs 2020, 18, 387 15 of 16

24. Benlarbi-Ben Khedher, M.; Hajri, K.; Dellaa, A.; Baccouche, B.; Hammoum, I.; Boudhrioua-Mihoubi, N.; Dhifi, W.; Ben Chaouacha-Chekir, R. Astaxanthin inhibits aldose reductase activity in Psammomys obesus, a model of type 2 diabetes and diabetic retinopathy. Food Sci. Nutr. 2019, 7, 3979–3985. [CrossRef][PubMed] 25. Giannaccare, G.; Pellegrini, M.; Senni, C.; Bernabei, F.; Scorcia, V.; Cicero, A.F.G. Clinical applications of astaxanthin in the treatment of ocular diseases: Emerging insights. Mar. Drugs 2020, 18, 239. [CrossRef] 26. Landon, R.; Gueguen, V.; Petite, H.; Letourneur, D.; Pavon-Djavid, G.; Anagnostou, F. Impact of astaxanthin on diabetes pathogenesis and chronic complications. Mar. Drugs 2020, 18, 357. [CrossRef] 27. Shang, Y.M.; Wang, G.S.; Sliney, D.; Yang, C.H.; Lee, L.L. Light-emitting-diode induced retinal damage and its wavelength dependency in vivo. Int. J. Ophthalmol. 2017, 10, 191–202. [CrossRef] 28. Cutler, R.G. Oxidative stress profiling: Part I. Its potential importance in the optimization of human health. Ann. N. Y. Acad. Sci. 2005, 1055, 93–135. [CrossRef] 29. Godley, B.F.; Shamsi, F.A.; Liang, F.Q.; Jarrett, S.G.; Davies, S.; Boulton, M. Blue light induces mitochondrial DNA damage and free radical production in epithelial cells. J. Biol. Chem. 2005, 280, 21061–21066. [CrossRef] 30. Ambati, R.R.; Phang, S.M.; Ravi, S.; Aswathanarayana, R.G. Astaxanthin: Sources, extraction, stability, biological activities and its commercial applications—A review. Mar. Drugs 2014, 12, 128–152. [CrossRef] 31. Park, S.H.; Jang, J.H.; Chen, C.Y.; Na, H.K.; Surh, Y.J. A formulated red ginseng extract rescues PC12 cells from PCB-induced oxidative cell death through Nrf2-mediated upregulation of heme oxygenase-1 and glutamate cysteine ligase. Toxicology 2010, 278, 131–139. [CrossRef][PubMed] 32. Ha, K.N.; Chen, Y.; Cai, J.; Sternberg, P., Jr. Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE: Implication for protection against oxidative stress. Investig. Ophthalmol. Vis. Sci. 2006, 47, 2709–2715. [CrossRef][PubMed] 33. Li, Z.; Dong, X.; Liu, H.; Chen, X.; Shi, H.; Fan, Y.; Hou, D.; Zhang, X. Astaxanthin protects ARPE-19 cells from oxidative stress via upregulation of Nrf2-regulated phase II enzymes through activation of PI3K/Akt. Mol. Vis. 2013, 19, 1656–1666. 34. Zhao, C.R.; Gao, Z.H.; Qu, X.J. NRF2-ARE signaling pathway and natural products for cancer chemoprevention. Cancer Epidemiol. 2010, 34, 523–533. [CrossRef][PubMed] 35. Tripathi, D.N.; Jena, G.B. Astaxanthin intervention ameliorates cyclophosphamide-induced oxidative stress, DNA damage and early hepatocarcinogenesis in rat: Role of Nrf2, p53, p38 and phase-II enzymes. Mutat. Res. 2010, 696, 69–80. [CrossRef] 36. Wu, Q.; Zhang, X.S.; Wang, H.D.; Zhang, X.; Yu, Q.; Li, W.; Zhou, M.L.; Wang, X.L. Astaxanthin activates nuclear factor erythroid-related factor 2 and the antioxidant responsive element (Nrf2-ARE) pathway in the brain after subarachnoid hemorrhage in rats and attenuates early brain injury. Mar. Drugs 2014, 12, 6125–6141. [CrossRef] 37. Wang, H.Q.; Sun, X.B.; Xu, Y.X.; Zhao, H.; Zhu, Q.Y.; Zhu, C.Q. Astaxanthin upregulates heme oxygenase-1 expression through ERK1/2 pathway and its protective effect against beta-amyloid-induced cytotoxicity in SH-SY5Y cells. Brain Res. 2010, 1360, 159–167. [CrossRef] 38. Wu, H.; Niu, H.; Shao, A.; Wu, C.; Dixon, B.J.; Zhang, J.; Yang, S.; Wang, Y. Astaxanthin as a Potential Neuroprotective Agent for Neurological Diseases. Mar. Drugs 2015, 13, 5750–5766. [CrossRef] 39. Kim, J.H.; Nam, S.W.; Kim, B.W.; Choi, W.; Lee, J.H.; Kim, W.J.; Choi, Y.H. Astaxanthin Improves Stem Cell Potency via an Increase in the Proliferation of Neural Progenitor Cells. Int. J. Mol. Sci. 2010, 11, 5109–5119. [CrossRef] 40. Kim, Y.H.; Koh, H.K.; Kim, D.S. Down-regulation of IL-6 production by astaxanthin via ERK-, MSK-, and NF-κB-mediated signals in activated microglia. Int. Immunopharmacol. 2010, 10, 1560–1572. [CrossRef] [PubMed] 41. Wang, L.; Chen, Y.; Sternberg, P.; Cai, J. Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE. Investig. Ophthalmol. Vis. Sci. 2008, 49, 1671–1678. [CrossRef][PubMed] 42. Higuera-Ciapara, I.; Felix-Valenzuela, L.; Goycoolea, F.M. Astaxanthin: A review of its chemistry and applications. Crit. Rev. Food Sci. Nutr. 2006, 46, 185–196. [CrossRef][PubMed] 43. Yuan, J.P.; Peng, J.; Yin, K.; Wang, J.H. Potential health promoting effects of astaxanthin: A high-value carotenoid mostly from microalgae. Mol. Nutr. Food Res. 2011, 55, 150–165. [CrossRef][PubMed] 44. Guerin, M.; Huntley, M.E.; Olaizola, M. Haematococcus astaxanthin: Applications for human health and nutrition. Trends Biotechnol. 2003, 21, 210–216. [CrossRef] Mar. Drugs 2020, 18, 387 16 of 16

45. Fassett, R.G.; Coombes, J.S. Astaxanthin: A potential therapeutic agent in cardiovascular disease. Mar. Drugs 2011, 9, 447–465. [CrossRef][PubMed] 46. Nakajima, Y.; Inokuchi, Y.; Shimazawa, M.; Otsubo, K.; Ishibashi, T.; Hara, H. Astaxanthin, a dietary carotenoid, protects retinal cells against oxidative stress in-vitro and in mice in-vivo. J. Pharm. Pharmacol. 2008, 60, 1365–1374. [CrossRef] 47. Tan, E.; Ding, X.Q.; Saadi, A.; Agarwal, N.; Naash, M.I.; Al-Ubaidi, M.R. Expression of cone-photoreceptor-speciﬁc antigens in a cell line derived from retinal tumors in transgenic mice. Investig. Ophthalmol. Vis. Sci. 2004, 45, 764–768. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).