Cytoprotective Potential of Fucoxanthin in Oxidative Stress-Induced Age-Related Macular Degeneration and Retinal Pigment Epithelial Cell Senescence In Vivo and In Vitro

Oxidative stress is identified as a major inducer of retinal pigment epithelium (RPE) cell dysregulation and is associated with age-related macular degeneration (AMD). The protection of RPE disorders plays an essential role in the pathological progress of retinal degeneration diseases. The pharmacological functions of fucoxanthin, a characteristic carotenoid, including anti-inflammatory and antioxidant properties, may ameliorate an outstanding bioactivity against premature senescence and cellular dysfunction. This study demonstrates that fucoxanthin protects RPE cells from oxidative stress-induced premature senescence and decreased photoreceptor cell loss in a sodium iodate-induced AMD animal model. Similarly, oxidative stress induced by hydrogen peroxide, nuclear phosphorylated histone (γH2AX) deposition and premature senescence-associated β-galactosidase staining were inhibited by fucoxanthin pretreatment in a human RPE cell line, ARPE-19 cells. Results reveal that fucoxanthin treatment significantly inhibited reactive oxygen species (ROS) generation, reduced malondialdehyde (MDA) concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli was inhibited in cells treated with fucoxanthin after oxidative stress. During aging, the RPE undergoes well-characterized pathological changes, including amyloid beta (Aβ) deposition, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) expression and tight junction disruption, which were also reduced in fucoxanthin-treated groups by immunofluorescence. Altogether, pretreatment with fucoxanthin may protect against premature senescence and cellular dysfunction in retinal cells by oxidative stress in experimental AMD animal and human RPE cell models.


Introduction
While aging causes a decline in the ability to respond and adapt to the accumulative impact of different exposures, age-related disease develops when cellular dysfunction from compromised cytoprotective pathways is severe enough to cause tissue destruction [1]. Aging is also an independent risk for visual impairment. With aging, the photoprotective capability of the retinal pigment epithelium (RPE) decreases. During aging, the RPE undergoes well-characterized structural changes, including apical microvilli atrophy, accumulation of formation of drusen and progressive cell loss [2]. Many cellular stresses activate senescence, a terminal arrest of proliferation, including dysfunctional telomeres, DNA damage and oxidative stress. pain and thinning of the corneal epithelial layer [29,30]. Due to the necessary functions that keep photoreceptors healthy, the RPE is critical for maintaining eye vision; however, the effects of fucoxanthin have not been extensively examined on oxidative stress-induced AMD and premature senescence. Therefore, we conducted this in vivo and in vitro study to examine the effect of fucoxanthin on the premature senescence of RPE cells challenged by hydrogen peroxide and investigated the possible mechanisms underlying this effect.
Mar. Drugs 2021, 19, x FOR PEER REVIEW 3 of 15 ported to have antioxidant, anti-inflammatory, anticancer and antimicrobial effects in various tissues and cells, as tested by in vitro or in vivo experiments [25][26][27][28]. We demonstrated that pretreatment with fucoxanthin inhibited the ultraviolet B-induced corneal inflammatory pain and thinning of the corneal epithelial layer [29,30]. Due to the necessary functions that keep photoreceptors healthy, the RPE is critical for maintaining eye vision; however, the effects of fucoxanthin have not been extensively examined on oxidative stress-induced AMD and premature senescence. Therefore, we conducted this in vivo and in vitro study to examine the effect of fucoxanthin on the premature senescence of RPE cells challenged by hydrogen peroxide and investigated the possible mechanisms underlying this effect.

Fucoxanthin Pretrement Inhibits ROS Generation and Lipid Peroxidation in Sodium Iodate-Induced Retinal Degeneration Animal Model
The main representative of the retinal disorder induced by sodium iodate is significant and progressive damage to the RPE. Otherwise, a significant decrease in photoreceptors was also observed in the outer nuclear layer. To investigate the protective effect of fucoxanthin against sodium iodate-induced retinal degeneration, experimental animals were treated daily with fucoxanthin over a period of 1 week before sodium iodate-induced retinal degeneration. ROS generation and the MDA concentration of the retinal tissues are indicative of the oxidative damage capacity, respectively. ROS levels in retinas of the sodium iodate-treated rats were markedly increased as compared with the blank control group. However, the group pretreated with 10 mg/kg BW fucoxanthin indicated a significant decrease in ROS generation compared with the sodium iodate/vehicle group ( Figure 2A). In addition, the concentrations of MDA in retinal tissues were analyzed. The MDA levels in the ocular tissue of the sodium iodate/vehicle group were significantly increased as compared with the blank control group, but the levels were significantly decreased in groups pretreated with 1 (p < 0.05) and 10 mg/kg fucoxanthin (p < 0.01) ( Figure  2B). The results are represented as the mean ± SD (n = 5). The mean value was significantly different as compared with the sodium iodate/vehicle group (*** p < 0.01; Student's t test). The mean value was significantly different in the sodium iodate-induced group (## p < 0.05 and ### p < 0.01; one-way ANOVA followed by Bonferroni's multiple comparison test). ROS, reactive oxygen species; MDA, malondialdehyde.

Fucoxanthin Pretrement Inhibits ROS Generation and Lipid Peroxidation in Sodium Iodate-Induced Retinal Degeneration Animal Model
The main representative of the retinal disorder induced by sodium iodate is significant and progressive damage to the RPE. Otherwise, a significant decrease in photoreceptors was also observed in the outer nuclear layer. To investigate the protective effect of fucoxanthin against sodium iodate-induced retinal degeneration, experimental animals were treated daily with fucoxanthin over a period of 1 week before sodium iodate-induced retinal degeneration. ROS generation and the MDA concentration of the retinal tissues are indicative of the oxidative damage capacity, respectively. ROS levels in retinas of the sodium iodate-treated rats were markedly increased as compared with the blank control group. However, the group pretreated with 10 mg/kg BW fucoxanthin indicated a significant decrease in ROS generation compared with the sodium iodate/vehicle group ( Figure 2A). In addition, the concentrations of MDA in retinal tissues were analyzed. The MDA levels in the ocular tissue of the sodium iodate/vehicle group were significantly increased as compared with the blank control group, but the levels were significantly decreased in groups pretreated with 1 (p < 0.05) and 10 mg/kg fucoxanthin (p < 0.01) ( Figure 2B). ported to have antioxidant, anti-inflammatory, anticancer and antimicrobial effects in various tissues and cells, as tested by in vitro or in vivo experiments [25][26][27][28]. We demonstrated that pretreatment with fucoxanthin inhibited the ultraviolet B-induced corneal inflammatory pain and thinning of the corneal epithelial layer [29,30]. Due to the necessary functions that keep photoreceptors healthy, the RPE is critical for maintaining eye vision however, the effects of fucoxanthin have not been extensively examined on oxidative stress-induced AMD and premature senescence. Therefore, we conducted this in vivo and in vitro study to examine the effect of fucoxanthin on the premature senescence of RPE cells challenged by hydrogen peroxide and investigated the possible mechanisms underlying this effect.

Fucoxanthin Pretrement Inhibits ROS Generation and Lipid Peroxidation in Sodium Iodate-Induced Retinal Degeneration Animal Model
The main representative of the retinal disorder induced by sodium iodate is significant and progressive damage to the RPE. Otherwise, a significant decrease in photoreceptors was also observed in the outer nuclear layer. To investigate the protective effect of fucoxanthin against sodium iodate-induced retinal degeneration, experimental animals were treated daily with fucoxanthin over a period of 1 week before sodium iodate-induced retinal degeneration. ROS generation and the MDA concentration of the retinal tissues are indicative of the oxidative damage capacity, respectively. ROS levels in retinas of the sodium iodate-treated rats were markedly increased as compared with the blank control group. However, the group pretreated with 10 mg/kg BW fucoxanthin indicated a significant decrease in ROS generation compared with the sodium iodate/vehicle group ( Figure 2A). In addition, the concentrations of MDA in retinal tissues were analyzed. The MDA levels in the ocular tissue of the sodium iodate/vehicle group were significantly increased as compared with the blank control group, but the levels were significantly decreased in groups pretreated with 1 (p < 0.05) and 10 mg/kg fucoxanthin (p < 0.01) ( Figure  2B). The results are represented as the mean ± SD (n = 5). The mean value was significantly different as compared with the sodium iodate/vehicle group (*** p < 0.01; Student's t test). The mean value was significantly different in the sodium iodate-induced group (## p < 0.05 and ### p < 0.01; one-way ANOVA followed by Bonferroni's multiple comparison test). ROS, reactive oxygen species; MDA, malondialdehyde. The results are represented as the mean ± SD (n = 5). The mean value was significantly different as compared with the sodium iodate/vehicle group (*** p < 0.01; Student's t test). The mean value was significantly different in the sodium iodate-induced group (## p < 0.05 and ### p < 0.01; one-way ANOVA followed by Bonferroni's multiple comparison test). ROS, reactive oxygen species; MDA, malondialdehyde.

Fucoxanthin Inhibits Cellular Senescence in Retinal Tissues of Sodium Iodate-Induced Retinal Degeneration In Vivo
To examine inhibitory prosenescent properties of fucoxanthin in vivo, we pretreated the animals with fucoxanthin (0.1, 1 and 10 mg/kg/day) for seven days before sodium iodate-induced retinal degeneration and examined retinal senescence and histological changes. β-Galactosidase (β-gal), a lysosomal hydrolytic enzyme with the physiological function of catalyzing the hydrolysis of glycosidic bonds which transform lactose into galactose, is known to be characteristic of senescent cells. The intense blue deposits of senescence-associated β-galactosidase (SA b-Gal) staining were observed in sodium iodateinduced experimental animals ( Figure 3C), comparable with that of the blank control group ( Figure 3A). Further, a marked decrease in photoreceptors and thinning in the outer nuclear were found in retinal degeneration animals as compared with the blank control group ( Figure 3D). These findings agree with our SA-b-Gal staining results. Although sodium iodate treatment caused severe retinal generation, sodium iodate effects were inhibited by fucoxanthin. Significant improvements in the prosenescent properties and histological changes were observed during examination of the group treated with 10 mg/kg fucoxanthin compared ( Figure 3I,J) with the retinal disorders detected in the sodium iodatetreated experimental animals ( Figure 3A,B).

Fucoxanthin Inhibits Cellular Senescence in Retinal Tissues of Sodium Iodate-Induced Retinal Degeneration in Vivo
To examine inhibitory prosenescent properties of fucoxanthin in vivo, we pretreated the animals with fucoxanthin (0.1, 1 and 10 mg/kg/day) for seven days before sodium iodate-induced retinal degeneration and examined retinal senescence and histological changes. β-Galactosidase (β-gal), a lysosomal hydrolytic enzyme with the physiological function of catalyzing the hydrolysis of glycosidic bonds which transform lactose into galactose, is known to be characteristic of senescent cells. The intense blue deposits of senescence-associated β-galactosidase (SA b-Gal) staining were observed in sodium iodate-induced experimental animals ( Figure 3C), comparable with that of the blank control group ( Figure 3A). Further, a marked decrease in photoreceptors and thinning in the outer nuclear were found in retinal degeneration animals as compared with the blank control group ( Figure 3D). These findings agree with our SA-b-Gal staining results. Although sodium iodate treatment caused severe retinal generation, sodium iodate effects were inhibited by fucoxanthin. Significant improvements in the prosenescent properties and histological changes were observed during examination of the group treated with 10 mg/kg fucoxanthin compared ( Figure 3I,J) with the retinal disorders detected in the sodium iodate-treated experimental animals ( Figure 3A,B).

Fucoxanthin Affects Oxidative Stress-Induced ROS Generation and Mitochondria Respiration
To investigate the cytoprotective effect of fucoxanthin and hydrogen peroxide in human ARPE-19 cells, the cells were pretreated with 1, 5 or 10 μM fucoxanthin for 48 h and with 500 μM hydrogen peroxide for an additional 48 h. Hydrogen peroxide increased the ROS level in the ARPE-19 cells and fucoxanthin exhibited an inhibitory effect on oxidative

Fucoxanthin Affects Oxidative Stress-Induced ROS Generation and Mitochondria Respiration
To investigate the cytoprotective effect of fucoxanthin and hydrogen peroxide in human ARPE-19 cells, the cells were pretreated with 1, 5 or 10 µM fucoxanthin for 48 h and with 500 µM hydrogen peroxide for an additional 48 h. Hydrogen peroxide increased the ROS level in the ARPE-19 cells and fucoxanthin exhibited an inhibitory effect on oxidative stress-induced ROS production. Compared with the ROS generation detected in the hydrogen peroxide-exposed groups, drastically decreased ROS generation was observed Mar. Drugs 2021, 19, 114 5 of 15 in the group treated with 5 and 10 µM fucoxanthin ( Figure 4A). Moreover, mitochondria respiration was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compared with hydrogen peroxide-exposed groups, mitochondria respiration was increased in fucoxanthin pretreatment groups and a marked difference was observed in 5 and 10 µM fucoxanthin groups ( Figure 4B). Thus, fucoxanthin is shown to suppress ROS generation and promote mitochondria respiration. stress-induced ROS production. Compared with the ROS generation detected in the hydrogen peroxide-exposed groups, drastically decreased ROS generation was observed in the group treated with 5 and 10 μM fucoxanthin ( Figure 4A). Moreover, mitochondria respiration was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compared with hydrogen peroxide-exposed groups, mitochondria respiration was increased in fucoxanthin pretreatment groups and a marked difference was observed in 5 and 10 μM fucoxanthin groups ( Figure 4B). Thus, fucoxanthin is shown to suppress ROS generation and promote mitochondria respiration. The value of the control group was considered 100%. Values presented are mean ± S.D (n = 5). The mean value was significantly different as compared with the hydrogen peroxide/vehicle group (*** p < 0.01; Student's t test). The mean value was significantly different in the hydrogen peroxide exposure group (## p < 0.05 and ### p <0.01; one-way ANOVA followed by Bonferroni's multiple comparison test). ROS, reactive oxygen species; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fucoxanthin Protects ARPE-19 Cells from Hydrogen Peroxide-Induced Cellular Senescence and DNA Damage Response
Hydrogen peroxide is a potent senescence inducer and plays an important role in the induction of senescence. It is well established that hydrogen peroxide triggers potent senescence and DNA damage by various signaling cascades through oxidative stress. To investigate whether fucoxanthin protects against oxidative stress-induced cellular senescence and DNA damage, ARPE-19 cells were pretreated with fucoxanthin for 24 h and then exposed to 500 μM hydrogen peroxide for another 24 h. The cellular senescence was determined with galactosidase activity. The proportion of intense blue deposits after SAb-Gal staining cells exhibited a statistically significant increase in the ARPE-19 cultures treated with 500 μM hydrogen peroxide for 24 h alone ( Figure 5B), compared with the untreated control cultures ( Figure 5A). However, the ARPE-19 cells pretreated with 10 μM fucoxanthin for 24 h before hydrogen peroxide exposure exhibited a reduced number of hydrogen peroxide-induced SA-b-Gal-stained cells compared with the hydrogen peroxide group ( Figure 5C,D). This result suggests that fucoxanthin could inhibit hydrogen peroxide-induced cellular senescence in ARPE-19 cells. Cell viability was analyzed by ROS generation (A) and MTT (B) assay. The value of the control group was considered 100%. Values presented are mean ± SD (n = 5). The mean value was significantly different as compared with the hydrogen peroxide/vehicle group (*** p < 0.01; Student's t test). The mean value was significantly different in the hydrogen peroxide exposure group (## p < 0.05 and ### p < 0.01; one-way ANOVA followed by Bonferroni's multiple comparison test). ROS, reactive oxygen species; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fucoxanthin Protects ARPE-19 Cells from Hydrogen Peroxide-Induced Cellular Senescence and DNA Damage Response
Hydrogen peroxide is a potent senescence inducer and plays an important role in the induction of senescence. It is well established that hydrogen peroxide triggers potent senescence and DNA damage by various signaling cascades through oxidative stress. To investigate whether fucoxanthin protects against oxidative stress-induced cellular senescence and DNA damage, ARPE-19 cells were pretreated with fucoxanthin for 24 h and then exposed to 500 µM hydrogen peroxide for another 24 h. The cellular senescence was determined with galactosidase activity. The proportion of intense blue deposits after SA-b-Gal staining cells exhibited a statistically significant increase in the ARPE-19 cultures treated with 500 µM hydrogen peroxide for 24 h alone ( Figure 5B), compared with the untreated control cultures ( Figure 5A). However, the ARPE-19 cells pretreated with 10 µM fucoxanthin for 24 h before hydrogen peroxide exposure exhibited a reduced number of hydrogen peroxide-induced SA-b-Gal-stained cells compared with the hydrogen peroxide group ( Figure 5C,D). This result suggests that fucoxanthin could inhibit hydrogen peroxide-induced cellular senescence in ARPE-19 cells.
Moreover, there are well-known molecular triggers for the senescence response, including the DNA damage response, so we also examined the protective effect of fucoxanthin from DNA damage in ARPE-19 cells after oxidative stress. Immunofluorescence staining for γH2AX, the substitute marker of the DNA damage reaction, revealed that oxidative stress commanded to nuclear cH2AX deposition, compared with the significant increase in nuclear γH2AX observed in the hydrogen peroxide-treated groups ( Figure 6D-F), which was reduced with fucoxanthin treatment (Figure 6G-I). These results demonstrate that fucoxanthin suppresses ARPE-19 cells from oxidative stress-induced DNA damage ( Figure 6J).  Moreover, there are well-known molecular triggers for the senescence response, including the DNA damage response, so we also examined the protective effect of fucoxanthin from DNA damage in ARPE-19 cells after oxidative stress. Immunofluorescence staining for γH2AX, the substitute marker of the DNA damage reaction, revealed that oxidative stress commanded to nuclear cH2AX deposition, compared with the significant increase in nuclear γH2AX observed in the hydrogen peroxide-treated groups ( Figure  6D-F), which was reduced with fucoxanthin treatment ( Figure 6G-I). These results demonstrate that fucoxanthin suppresses ARPE-19 cells from oxidative stress-induced DNA damage ( Figure 6J).

Figure 6.
Effects of fucoxanthin on DNA damage after hydrogen peroxide exposure. Immunofluorescence staining of nuclear γH2AX was compared between the following groups: blank control (A-C), hydrogen peroxide (D-F) and hydrogen peroxide/fucoxanthin (G-I). DAPI counterstaining shows the nuclear localization of γH2AX. Positively stained cells were quantified and are shown as percentages of the total number of cells and the results are presented as means ± SD (J). (*** p < 0.01 as compared with blank control group; ### p < 0.01 as compared with hydrogen peroxide exposure group; Student's t test). γH2AX, nuclear phosphorylated histone. DAPI, 40,6-diamidino-2phenylindole.

Fucoxanthin Promotes Cell Junction and Morphogenesis of Apical Microvilli
To test the cell structure protective effects of fucoxanthin, scanning electron microscopy (SEM) was used to investigate the ultrastructure morphological changes. The intact cell junction of cultured cells was observed by electron microscopy. The RPE operates specialized metabolic and transport functions critical for homeostasis, thereby forming a  Moreover, there are well-known molecular triggers for the senescence res cluding the DNA damage response, so we also examined the protective effect o thin from DNA damage in ARPE-19 cells after oxidative stress. Immunoflu staining for γH2AX, the substitute marker of the DNA damage reaction, rev oxidative stress commanded to nuclear cH2AX deposition, compared with the increase in nuclear γH2AX observed in the hydrogen peroxide-treated grou 6D-F), which was reduced with fucoxanthin treatment (Figure 6G-I). The demonstrate that fucoxanthin suppresses ARPE-19 cells from oxidative stres DNA damage ( Figure 6J). Figure 6. Effects of fucoxanthin on DNA damage after hydrogen peroxide exposure. Im rescence staining of nuclear γH2AX was compared between the following groups: blan (A-C), hydrogen peroxide (D-F) and hydrogen peroxide/fucoxanthin (G-I). DAPI coun shows the nuclear localization of γH2AX. Positively stained cells were quantified and a as percentages of the total number of cells and the results are presented as means ± SD ( 0.01 as compared with blank control group; ### p < 0.01 as compared with hydrogen pe posure group; Student's t test). γH2AX, nuclear phosphorylated histone. DAPI, 40,6-dia phenylindole.

Fucoxanthin Promotes Cell Junction and Morphogenesis of Apical Microvilli
To test the cell structure protective effects of fucoxanthin, scanning electro copy (SEM) was used to investigate the ultrastructure morphological changes. cell junction of cultured cells was observed by electron microscopy. The RPE specialized metabolic and transport functions critical for homeostasis, thereby Figure 6. Effects of fucoxanthin on DNA damage after hydrogen peroxide exposure. Immunofluorescence staining of nuclear γH2AX was compared between the following groups: blank control (A-C), hydrogen peroxide (D-F) and hydrogen peroxide/fucoxanthin (G-I). DAPI counterstaining shows the nuclear localization of γH2AX. Positively stained cells were quantified and are shown as percentages of the total number of cells and the results are presented as means ± SD (J). (*** p < 0.01 as compared with blank control group; ### p < 0.01 as compared with hydrogen peroxide exposure group; Student's t test). γH2AX, nuclear phosphorylated histone. DAPI, 40,6-diamidino-2-phenylindole.

Fucoxanthin Promotes Cell Junction and Morphogenesis of Apical Microvilli
To test the cell structure protective effects of fucoxanthin, scanning electron microscopy (SEM) was used to investigate the ultrastructure morphological changes. The intact cell junction of cultured cells was observed by electron microscopy. The RPE operates specialized metabolic and transport functions critical for homeostasis, thereby forming a part of the blood-retina barrier. The apical surface of RPE cells radiates long and thin microvilli that establish a complex of close structures ( Figure 7A). The cellular morphological changes reflected that RPE senescence occurred. The effects of losing RPE-RPE adhesion and the broad space between adjoining cells after hydrogen peroxide exposure were also investigated. Some cells shrank and became round. The elaboration of apical microvilli presented a decrease in hydrogen peroxide-treated RPE cells as compared with blank con-trol cells. Stubby apical microvilli ( Figure 7B) were revealed. Compared with the hydrogen peroxide-exposed group, mildly disordered cell junctions and remarkably long microvilli were demonstrated in the fucoxanthin-pretreated group ( Figure 7C). These results suggest that fucoxanthin may protect the RPE cells from essentially changed microvilli and cell junctions induced by oxidative stress.
part of the blood-retina barrier. The apical surface of RPE cells radiates long an microvilli that establish a complex of close structures ( Figure 7A). The cellular m logical changes reflected that RPE senescence occurred. The effects of losing RP adhesion and the broad space between adjoining cells after hydrogen peroxide ex were also investigated. Some cells shrank and became round. The elaboration of microvilli presented a decrease in hydrogen peroxide-treated RPE cells as compare blank control cells. Stubby apical microvilli ( Figure 7B) were revealed. Compared w hydrogen peroxide-exposed group, mildly disordered cell junctions and remarkab microvilli were demonstrated in the fucoxanthin-pretreated group ( Figure 7C). Th sults suggest that fucoxanthin may protect the RPE cells from essentially changed villi and cell junctions induced by oxidative stress. 19 cells displayed an apical surface densely covered by microvilli (A). After hydrogen pero exposure, the number of apical microvilli was decreased as compared with the blank contro group. In contrast, not only the number, but also the morphology of microvilli was affected treatment of the cultured cells with fucoxanthin. Scale bars: 5 μm.

Fucoxanthin Protects Hydrogen Peroxide-Induced Degradation of Cytoskeleton Actin Disrupyion of Cell Junction
As the abovementioned experiment observed the protective effect of fucoxan microvilli formation following exposure to hydrogen peroxide, further study w formed to examine the morphogenesis of the cytoskeleton and cell junction. The e fucoxanthin on the fluorescence staining of tight junction protein zonular occluden 1) and F-actin in ARPE-19 exposure to hydrogen peroxide was investigated by im fluorescence microscopy. The results show that the expression continuous around t 1 cells and the filamentous structure of actin were observed in the ARPE-19 mon culture ( Figure 8A-D). With hydrogen peroxide exposure, cell junction networks amentous actin collapsed rapidly in the ARPE-19 cells. The abnormal distribution 1 typically manifested as fragmental staining and actin was charlatanically exhib diffused staining ( Figure 8E-H). An organized cell junction and filamentous cytosk were detected in fucoxanthin-pretreated ARPE-19 cells (Figure 8I-L). From this ex tion, we found that significantly protective effects on the cell junction and actin cy eton structure can be observed from cells pretreated with fucoxanthin. Figure 7. Morphological changes of apical microvilli by scanning electron microscopy. Ultrastructure of microvilli in a monolayer of cultured ARPE-19 cells was compared between the following groups: blank control (A), hydrogen peroxide (B) and hydrogen peroxide/fucoxanthin (C). ARPE-19 cells displayed an apical surface densely covered by microvilli (A). After hydrogen peroxide exposure, the number of apical microvilli was decreased as compared with the blank control group. In contrast, not only the number, but also the morphology of microvilli was affected by the treatment of the cultured cells with fucoxanthin. Scale bars: 5 µm.

Fucoxanthin Protects Hydrogen Peroxide-Induced Degradation of Cytoskeleton Actin C and Disrupyion of Cell Junction
As the abovementioned experiment observed the protective effect of fucoxanthin on microvilli formation following exposure to hydrogen peroxide, further study was performed to examine the morphogenesis of the cytoskeleton and cell junction. The effect of fucoxanthin on the fluorescence staining of tight junction protein zonular occludens (ZO-1) and F-actin in ARPE-19 exposure to hydrogen peroxide was investigated by immunofluorescence microscopy. The results show that the expression continuous around the ZO-1 cells and the filamentous structure of actin were observed in the ARPE-19 monolayer culture ( Figure 8A-D). With hydrogen peroxide exposure, cell junction networks and filamentous actin collapsed rapidly in the ARPE-19 cells. The abnormal distribution of ZO-1 typically manifested as fragmental staining and actin was charlatanically exhibited as diffused staining ( Figure 8E-H). An organized cell junction and filamentous cytoskeleton were detected in fucoxanthin-pretreated ARPE-19 cells (Figure 8I-L). From this examination, we found that significantly protective effects on the cell junction and actin cytoskeleton structure can be observed from cells pretreated with fucoxanthin.

Fucoxanthin Inhibits Hydrogen Peroxide Exposure up-Regulated Cellular Expressions of Aβ1-42 and Beta-Site Amyloid Precursor Protein-Cleaving Enzyme 1 (BACE1)
BACE1 is a transmembrane protease responsible for the β-site cleavage of the amyloid precursor protein to produce Aβ, and Aβ is an important component of plaques in neurological disease and drusen deposits in AMD. To test the protective effect of fucoxanthin on the AMD cellular model, fucoxanthin was added to ARPE-19 cells exposed to hydrogen peroxide. To further confirm the involvement of AMD, the expression of its various drusen-related proteins including Aβ1-42 and BACE1 was examined by immunofluorescence assays. Hydrogen peroxide exposure up-regulated cellular expressions of Aβ1-42 and BACE1 ( Figure 9E-H) compared with the control group ( Figure 9A-D). These results reveal that hydrogen peroxide activated the expression of drusen-related proteins in ARPE-19 cells. However, the increased expressions of Aβ1-42 and BACE1 were consequently down-regulated with fucoxanthin pretreatment (Figure 9I-L), suggesting a protective role of fucoxanthin on the ARPE-19 cells in reducing formation of Aβ deposition.

Fucoxanthin Inhibits Hydrogen Peroxide Exposure Up-Regulated Cellular Expressions of Aβ1-42 and Beta-Site Amyloid Precursor Protein-Cleaving Enzyme 1 (BACE1)
BACE1 is a transmembrane protease responsible for the β-site cleavage of the amyloid precursor protein to produce Aβ, and Aβ is an important component of plaques in neurological disease and drusen deposits in AMD. To test the protective effect of fucoxanthin on the AMD cellular model, fucoxanthin was added to ARPE-19 cells exposed to hydrogen peroxide. To further confirm the involvement of AMD, the expression of its various drusenrelated proteins including Aβ1-42 and BACE1 was examined by immunofluorescence assays. Hydrogen peroxide exposure up-regulated cellular expressions of Aβ1-42 and BACE1 ( Figure 9E-H) compared with the control group ( Figure 9A-D). These results reveal that hydrogen peroxide activated the expression of drusen-related proteins in ARPE-19 cells. However, the increased expressions of Aβ1-42 and BACE1 were consequently downregulated with fucoxanthin pretreatment (Figure 9I-L), suggesting a protective role of fucoxanthin on the ARPE-19 cells in reducing formation of Aβ deposition.

Discussion
This study demonstrated that fucoxanthin has a cytoprotective effect on retinal cell degeneration in a dose-response fashion in experimental animal and cultured cell models. Treatment with fucoxanthin substantially inhibited the DNA strand damage marker, and nuclear γH2AX deposition and premature SA β-galactosidase staining were observed following fucoxanthin pretreatment. An administration of fucoxanthin significantly inhibited ROS generation, reduced MDA concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli, Aβ deposition, Beta-secretase 1 (BACE1) expression and tight junction disruption were also reduced and inhibited in cells treated with fucoxanthin after oxidative stress.
AMD is a complex eye disease and is classified into wet or dry forms. Wet AMD is characterized by the sprouting of new vessels from choriocapillaris through Bruch's membrane. Drusen and RPE alterations are the hallmark of dry AMD [31]. Oxidative stress plays an important role in the development of AMD. As revealed, RPE degeneration, accumulation of lipofuscin, formation of drusen and microvilli atrophy have been associated with the pathogenesis of dry AMD in relation to increased oxidative stress [32]. Oxidative stress is also a potent inducer of inflammatory cytokines promoting inflammation and macrophage infiltration in retinal tissues [33]. Studies revealed significant changes in fucoxanthin-regulated oxidative stress and inflammatory responses in various tissues [29,34]. To elucidate the potential protective effects against oxidative stress in retinal cells, ROS generation and the peroxidation index were measured. In this work, fucoxanthin provided protection against hydrogen peroxide-induced ROS in human RPE cells. So-

Discussion
This study demonstrated that fucoxanthin has a cytoprotective effect on retinal cell degeneration in a dose-response fashion in experimental animal and cultured cell models. Treatment with fucoxanthin substantially inhibited the DNA strand damage marker, and nuclear γH2AX deposition and premature SA β-galactosidase staining were observed following fucoxanthin pretreatment. An administration of fucoxanthin significantly inhibited ROS generation, reduced MDA concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli, Aβ deposition, Beta-secretase 1 (BACE1) expression and tight junction disruption were also reduced and inhibited in cells treated with fucoxanthin after oxidative stress.
AMD is a complex eye disease and is classified into wet or dry forms. Wet AMD is characterized by the sprouting of new vessels from choriocapillaris through Bruch's membrane. Drusen and RPE alterations are the hallmark of dry AMD [31]. Oxidative stress plays an important role in the development of AMD. As revealed, RPE degeneration, accumulation of lipofuscin, formation of drusen and microvilli atrophy have been associated with the pathogenesis of dry AMD in relation to increased oxidative stress [32]. Oxidative stress is also a potent inducer of inflammatory cytokines promoting inflammation and macrophage infiltration in retinal tissues [33]. Studies revealed significant changes in fucoxanthin-regulated oxidative stress and inflammatory responses in various tissues [29,34]. To elucidate the potential protective effects against oxidative stress in retinal cells, ROS generation and the peroxidation index were measured. In this work, fucoxanthin provided protection against hydrogen peroxide-induced ROS in human RPE cells. Sodium iodate-and hydrogen peroxide-induced ROS are well known for studying RPE cell senescence models [15,35,36]. Here, our results demonstrate that hydrogen peroxide can lead to cell senescence and increased ROS generation, MDA production and DNA damage in human RPE cells, but this process was substantially inhibited with fucoxanthin treatment. These findings suggest that fucoxanthin reduces hydrogen peroxide-triggered cell senescence in human RPE cells, which may be strongly correlated with the antioxidative effects of fucoxanthin.
Mitochondrial dysfunction and oxidative damage are appreciably improved with senescence and aging-related diseases. During aging, increased ROS disrupt mitochondrial DND, lipids and structure and limit energy production in AMD [37][38][39]. ROS and imbalanced mitochondrial calcium cause the mitochondrial permeability transition pore to open and lead to cell death [40]. Studies demonstrated that mitochondrial function is significantly affected by AMD in RPE cells and mitochondrial disorders are involved in AMD pathology [41,42]. Oxidative stress-induced mitochondrial damage of the RPE has a secondary effect on photoreceptors in the AMD progression [43]. Here, mitochondria respiration was determined by the MTT assay. Compared with hydrogen peroxide-exposed groups, mitochondria respiration was increased in human RPE cells of the fucoxanthin pretreatment groups Thus, fucoxanthin is shown to inhibit ROS generation and promote mitochondria function.
The actin cytoskeleton is a highly dynamic structure that participates in the morphogenesis of apical microvilli. Due to phototoxicity, the daily renewal of the outer segment of the photoreceptor is ensheathed by microvilli arising from the surface of the pigment epithelial cells and is intensely important to the survival of photoreceptors [7,44]. Disrupted apical microvilli of the RPE are accompanied by photoreceptor cell death [3]. Here, we demonstrated that oxidative stress expression leads to structural changes and cellular senescence in the RPE and a decreased number of photoreceptors. However, significant improvement in the histological changes and prosenescent properties was observed with fucoxanthin treatment. The detailed mechanisms of which signaling pathways are involved in these regulations are still undergoing further studies. Besides microvilli, the actin cytoskeleton is also found at lateral contacts between epithelial cells and co-localizes with tight junctions [45]. To seal the adhesion between cells and to ensure integrity of the bloodretina barrier of tight junctions are essential to maintain visual physiology [46]. In AMD, breakdown of the blood-retina barrier, resulting in disruption of ZO-1 organization in tight junctions, increased the monolayer permeability. It is the integrity of the blood-retina barrier that keeps the choroidal vascular response from invading the retina and causing AMD [47]. We confirmed that fucoxanthin effectively stabilized the morphology of tight junctions and elevated the filamentous cytoskeleton protein F-actin in cultured RPE cells with the immunocytochemical staining assay.
The Aβs, from the amyloid β precursor protein cleaved by BACE1, are associated with the pathogenesis of Alzheimer's disease [48]. Aβ is associated with the progression of physical disorders, and the results of several studies showed that Aβ is involved in the pathogenesis of AMD [49,50]. Studies also observed that Aβ production is involved in the development of dry AMD with the expression of some cytokines [12]. While RPE dysfunction caused by Aβ and drusen is essential for AMD pathogenesis, we determined whether fucoxanthin was involved in the oxidative stress-stimulated Aβ production. Our results show that the expression of Aβ and BACE1 decreased upon exposure to hydrogen peroxide. Since Aβ-containing elements are associated with drusen and are considered to be the initial characteristic in retinal tissues with AMD pathogenesis, decreased expression of Aβ suggests it is related to the inhibition of drusen formation. However, cross-talk between Aβ and cellular senescence was not investigated in this study. In addition, we did not examine the functional assessments of the RPE cells, including the RPE barrier or their phagocytic function. Further experiments are needed for understanding the functional assessments of RPE and photoreceptor pathogenesis.

Sodium Iodate-Induced Retinal Degeneration in Rat Model
Healthy 4-5-week-old male Sprague-Dawley rats (BioLASCO Taiwan, Taipei City, Taiwan) weighing 200-300 g were used. All care and treatments of experimental animal studies were approved and monitored by the Mackay Medical College Institutional Animal Care Committee (IACUC-A1070033) in accordance with institutional animal ethical guidelines. Standard diet and tap water were provided ad libitum.
The sodium iodate-induced AMD animal model has been widely used for studying retinal degeneration diseases and drug treatment effects. To induce retinal generation, experimental methods followed a previously described protocol [4] with slight modifications. Here, 50 rats were randomly split into five groups. Group I: blank control (injected phosphate-buffered saline (PBS) via the sublingual vein). Group II (injected sodium iodate (Sigma-Aldrich, St. Louis, MO, USA) via the sublingual vein at a dose of 40 mg/kg). Group III: 0.1 mg/kg fucoxanthin + sodium iodate (oral administration of 0.1 mg/kg fucoxanthin (Sigma-Aldrich) in 0.1% dimethyl sulfoxide solution mixed with 0.1 mL PBS daily for 2 weeks prior to sublingual vein injection with sodium iodate). Group IV: 1 mg/kg fucoxanthin+ sodium iodate (oral administration of 1 mg/kg fucoxanthin daily for 2 weeks prior to injection with sodium iodate). Group V: 10 mg/kg fucoxanthin+ sodium iodate (oral administration of 10 mg/kg fucoxanthin daily for 2 weeks prior to injection with sodium iodate). After 7 days of sodium iodate injection, the animals were sacrificed for follow-up experiments.

Staining for SA b-Gal
β-Galactosidase activity is known to be characteristic of senescent cells and is used as a biomarker for senescent and aging cells. To evaluate the protective effect of fucoxanthin on cellular senescence, galactosidase activity was analyzed. For senescence assay, experimental samples were performed according to the protocol of the SA b-Gal Staining Kit (Cell Signaling Technology, Danvers, MA, USA). Tissue and cultured cell samples were incubated overnight at 37 • C with 5% CO 2 . The next day, β-galactosidase staining was identified by development of blue color using microscopy.

ROS Generation
Tissues or cells were harvested and incubated in PBS containing 10 mM general oxidative stress Indicator CM-H2DCFDA (the chloromethyl derivative of 20,70-dichlorodihydrofluorescein diacetate; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min to 1 h at 37 • C in the dark to allow loading of dye into the cells. This test compound is nonfluorescent when chemically reduced, but after intracellular esterases and oxidation occur within the cell, it becomes fluorescent. The intracellular production of ROS was monitored by a microplate reader with excitation at 490 nm to obtain the absorbance value. The results were expressed as percentage of change and the blank control group was taken as 100%.

MDA Assay for Lipid Peroxidation
The thiobarbituric acid (TBA) reactive substances of product MDA assay (Sigma-Aldrich) was conducted for an index of lipid peroxidation and oxidative stress of retinal tissues. The method involved heating up the assay mixture comprising tissue homogenates. MDA-TBA adduct was prepared by adding TBA solution into each vial containing the standard and the sample was incubated at 95 • C for 60 min. After cooling to room temperature in an ice bath for 20 min, the reaction mixture was centrifuged for 10 min at 16,000× g at 4 • C. Later, the absorbance of the supernatant obtained was measured spectrophotometrically at 532 nm according to the manufacturer, and the analyzed data were expressed as malondialdehyde equivalents (nmol/mg tissue protein). These analyzed results were expressed as percentage of change and the blank control group was taken as 100%.

Cells and Treatments
Human RPE cell line ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) were grown in DMEM/F12 media with 10% fetal bovine serum (FBS) and standard antibiotics (100 IU/mL penicillin, and 100 µg/mL streptomycin, Sigma-Aldrich) at 5% CO 2 and 37 • C in a humidified incubator. When cells reached confluency, cells were pretreated with various concentrations of fucoxanthin for 3 days and replaced every 24 h for the duration of the experiment. After a brief wash with medium, cells were incubated with 500 µM hydrogen peroxide in culture media for oxidative stress. All experiments were performed in triplicate.

MTT Assay for Mitochondrial Metabolic Rate
MTT assay (Thermo Fisher Scientific) was used to evaluate cellular metabolic activity as an indicator of cell viability and cytotoxicity. After the experimental incubation period, cells were washed once and then incubated with 0.5 mg/mL MTT labeling reagent at 37 • C for 4 h. Solubilization solution was added to solubilize the produced purple formazan crystals (MTT metabolic product). The formazan was then solubilized, and its concentration was determined by optical density at absorbance of 570 nm using a microplate reader.

DNA Strand Damage Marker γ-H2AX
For DNA strand damage assay, cells were fixed with 4% paraformaldehyde in PBS for 30 min at 25-27 • C and then anti-γ-H2AX (Cell Signaling Technology) at 4 • C overnight. After washing, the cells were incubated with a fluorochrome-conjugated secondary antibody at 27 • C for 1 h. After additional rinsing three times in PBS (10 min each), the cells were stained with DAPI (40,6-diamidino-2-phenylindole) nuclear probe (Roche, Basel, Switzerland) at RT for 2 min. After drying and fixation, the samples were visualized with a fluorescence microscope.

Scanning Electron Microscopy
Briefly, experimental cells were fixed in 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.125 M cacodylate buffer (pH 7.4) with 2 mM CaCl2. Upon postfixing with 2% osmium tetroxide in 0.1 M cacodylate buffer, experimental cells were dehydrated through a graded series of ethanol-water mixtures and then dried by the critical point method. After drying, the sample was sputter coated with gold, and cells were examined on a JEOL 100CX transmission electron microscope.

Immunocytochemical Staining Assay
Human RPE cell line ARPE-19 cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeated in 0.05% Triton-X 100 for 15 min and blocked with 4% FBS in PBS for 30 min. Anti-ZO-1 antibody (Abcam, Cambridge, UK) was used to determine the expression of junctional proteins. Anti-Amyloid β 42 (Aβ42) (Abcam) and anti-BACE1 against β-secretase (BACE1, Abcam) antibodies were used to determine the expression of amyloid β peptides. Hoechst 33,342 or DAPI (40,6-diamidino-2-phenylindole, Thermo Fisher Scientific) was used to stain nucleic acids for the nuclear staining. Images on slides were taken using a fluorescence microscope system.

Statistical Analysis
Statistical data were analyzed using the SPSS program for Windows software (SPSS, Inc., Chicago, IL, USA). Means and standard deviations (SD) were presented for all experimental values in this study. The Kolmogorov-Smirnov normality test was performed to verify the normal distribution of the data. The Mann-Whitney test was used to analyze the non-parametric values. Student's t-test was evaluated to compare between any two groups. One-way analysis of variance (ANOVA) followed by Dunnett's or Bonferroni's multiple comparison test was evaluated to analyze the parametric value groups. Statistically significant differences between groups were established when p-values were less than 0.05.

Data Availability Statement:
The data presented in this study are available in the main text.