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Sulforaphane inhibits blue light–induced inflammation and apoptosis by upregulating the SIRT1/PGC-1α/Nrf2 pathway and autophagy in retinal pigment epithelial cells

Po-Min Yang a,1, Kai-Chun Cheng b,c,e,1, Jing-Yao Huang d, Shih-Yun Wang d, Yung-Ni Lin d, Yen-Tzu Tseng d, Chia-Wen Hsieh d, Being-Sun Wung d,*

Abstract

The present study elucidated mechanisms through which sulforaphane (SFN) protects retinal pigment epithelial (RPE) cells from blue light–induced impairment. SFN could activate the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increase the expression of the heme oxygenease-1 (HO-1) gene and production of glutathione. SFN reduced blue light–induced oxidative stress, and effectively activated cytoprotective components including Nrf-2, HO-1, thioredoxin-1, and glutathione. The protective effect of SFN on blue light–induced injury was blocked by the Nrf2 inhibitor ML385, suggesting that the SFN-induced Nrf2 pathway is involved in the cytoprotective effect of SFN. SFN inhibited intercellular adhesion molecule-1 expression induced by TNF-α or blue light, suggesting the anti-inflammatory activity of SFN. The inhibitory effect of SFN was associated with the blocking of NF-κB p65 nuclear translocation in blue light–exposed RPE cells. SFN protected RPE cells from blue light–induced interruption of the mitochondrial membrane potential and reduction of the Bcl-2/Bax ratio and cleaved caspase-3 and PARP-1 expression, suggesting the antiapoptotic activity of SFN. SFN alone or together with blue light exposure increased the expression of the autophagy-related proteins LC3BII and p62. An autophagy inhibitor, 3-MA, inhibited the protective effect of SFN on blue light–induced cell damage. SFN increased sirtuin-1 (SIRT1) expression; however, treatment with blue light induced peroxisome proliferator- activated receptor gamma coactivator-1α (PGC-1α) expression. Our study results demonstrated that SFN exerts its protective effect under blue light exposure by maintaining the Nrf2-related redox state and upregulating SIRT1 and PGC-1α expression and autophagy.

Keywords:
Sulforaphane
Blue light
Retinal pigment epithelial cells
Nrf2
Autophagy Apoptosis

1. Introduction

Age-related macular degeneration (AMD) is the leading cause of blindness in older people. AMD results in the progressive loss of central vision because of degeneration at the ocular interface between the retina and the underlying choroid (Kokotas et al., 2011). Several risk factors for AMD progression have been identified including age, smoking, genetic factors, and sunlight exposure (Kent and Sheridan, 2003; Tak and Firestein, 2001; Nakanishi-Ueda et al., 2013). Epidemiological studies have demonstrated that sunlight exposure plays a pivotal role in the pathological process of AMD (Sui et al., 2013; Fletcher et al., 2008; Butt et al., 2011). Within the visible light spectrum, blue light is particularly implicated in causing many diseases (Vicente-Tejedor et al., 2018; Tao et al., 2019). Blue light exposure has become increasingly common in modern environments. In addition to sunlight, technological devices, such as light-emitting diodes, are the major source of light and can emit a relatively high level of blue light. Blue light has the shortest wavelength and highest energy in the visible light spectrum, and continued exposure to blue light over time can damage retinal cells (Vicente- Tejedor et al., 2018; Tao et al., 2019). Dry AMD, a nonneovascular form of AMD, is characterized by the degeneration of retinal pigment epithelial (RPE) cells and, subsequently, the overlying photoreceptors (Kokotas et al., 2011). The retinal pigment epithelium is firmly attached to the underlying choroid, and a monolayer of RPE cells is critical for maintaining the health of photoreceptors and the choriocapillaris and sustaining retinal homeostasis, eye development, and vision (Strauss, 2005). Because RPE cells are sensitive to blue light, which can induce cellular damage, we speculated that exposing RPE cells to blue light can be a suitable model for studying AMD (Algvere et al., 2006).
Patients with dry AMD exhibit a decrease in RPE cells, photoreceptors, and choroidal capillaries. By contrast, patients with wet AMD exhibit neovascularization penetrating from the choroid to retinal layers. The pathogenesis of AMD is strongly associated with inflammation, leading to choroidal neovascularization (Tan et al., 2020). Patients with neovascular AMD present a high level of tumor necrosis factor (TNF)-α, suggesting that TNF-α can serve as a biomarker for the formation of choroidal neovascularization (Cousins et al., 2004). RPE cells are a component of the outer blood–retinal barrier and may respond to the proinflammatory cytokine TNF-α secreted by macrophages. Ocular inflammation involves the migration of macrophages and lymphocytes to the posterior compartment of the eye (Tan et al., 2020). Intercellular adhesion molecule-1 (ICAM-1) is a major protein that mediates leukocyte recruitment and adhesion to the retina. A study reported a correlation between an increased soluble ICAM-1 level and choroidal neovascularization (Jonas et al., 2010). However, whether blue light can induce ICAM-1 expression as well as the underlying regulatory mechanism remain unclear.
Exposure of RPE cells to blue light at certain doses resulted in intracellular oxidative stress (Yu et al., 2017; Pavan and Dalpiaz, 2018a). Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a vital role in protecting RPE cells through Nrf2-driven enzymes (Itoh et al., 1997; Alam et al., 1999). Nrf2-driven heme oxygenase-1 (HO-1) and glutathione (GSH) have been reported to exert cytoprotective effects in vivo and in vitro (Satoh et al., 2006; Townsend et al., 2003). In addition, Nrf2 and Nrf2-related genes were found to be decreased in the RPE cells of aging mice compared with younger mice; this decrease could be attributed to an age-related increase in basal oxidative stress (Sachdeva et al., 2014). Taken together, the findings of previous studies suggest that Nrf2 regulates the cell redox state to protect RPE cells from blue light–induced damage and that oxidative stress may be a potential treatment for AMD.
In addition to the Nrf2 system, autophagy is another protective mechanism that removes aberrant molecules or dysfunctional organelles. Impaired autophagy can increase the susceptibility of RPE cells to oxidative stress and is involved in the pathogenesis of dry AMD (Mitter et al., 2014; Hyttinen et al., 2017). The protein p62 tags ubiquitinated proteins, isolates them from the cytosol in p62-LC3 (microtubule-associated protein 1A/1B light chain 3), and guides autophagosome formation (Pankiv et al., 2007). The lysosome fuses to the autophagosome, resulting in the degradation of its contents. Thus, p62 and LC3 can be used as biomarkers of autophagy activity (Pankiv et al., 2007). Moreover, p62 can interact with the Nrf2/antioxidant response element pathway by disrupting the Nrf2–Keap1 complex, leading to the nuclear translocation of Nrf2 (Liu et al., 2016). Sirtuin 1 (SIRT1) serves as a key regulator of autophagy by deacetylating diverse autophagy-related proteins including autophagy-related genes and LC3 (Lee, 2019). In addition, SIRT1 can regulate the activity of peroxisome proliferator- activated receptor gamma coactivator-1 (PGC-1) (Salminen et al., 2013). In RPE cells, PGC-1 alpha (PGC-1α) could drive mitochondrial biogenesis and activate the antioxidant defense system (Kaarniranta et al., 2018). Loss of function of PGC-1α could trigger reactive oxygen species (ROS) burst and cause significant age-dependent degeneration of the retinal pigment epithelium (Felszeghy et al., 2019). Furthermore, a recent study reported that SIRT1 exerted an antioxidative effect through the upregulation and nuclear deacetylation of Nrf2 (Ding, 2016). Therefore, we examined the regulation of SIRT1, PGC-1, Nrf2, and autophagy under blue light stress in RPE cells.
Sulforaphane (SFN), an isothiocyanate phytochemical with anticancer properties, is predominantly present in several cruciferous vegetables such as broccoli (Zhang et al., 1992). SFN could attenuate white light–induced retinal damage or protect RPE cells against ultraviolet exposure and all-trans-retinal treatment (Tanito et al., 2005; Gao and Talalay, 2004). SFN is an activator of Nrf2 and also can promote autophagy (Herman-Antosiewicz et al., 2006). The cytoprotective mechanism of SFN against blue light–induced damage in RPE cells and the molecular mechanism underlying the effect of SFN on the autophagic pathway remain unclear. Therefore, in the present study, we investigated the protective effect of SFN against blue light–induced damage and the underlying mechanism of Nrf2 and autophagy pathways.

2. Materials and methods

The nuclear factor-kappa B (NF-κB)/p65 antibody was purchased from Stressgen Biotechnologies. Antibodies against poly(ADP-ribose) polymerase 1 (PARP-1), Bcl2, and Bax were purchased from Invitrogen (Thermo Fisher Scientific). The tubulin antibody was obtained from Sigma-Aldrich, Merck KGaA. Primary antibodies against ICAM-1/ CD54, LC3, p62, and SIRT1 were purchased from Cell Signaling Technology. Peroxidase-conjugated antirabbit and antimouse antibodies were obtained from Invitrogen (Thermo Fisher Scientific), and nitrocellulose was obtained from Schleicher and Schuell. All other reagents including TNF-α, ML385, and all-trans retinal (atRAL) were purchased from Sigma-Aldrich, Merck KGaA.

2.1. Cell culture and blue light exposure

The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–Ham’s F12 (1,1; Invitrogen) containing 10% fetal bovine serum (Invitrogen) and antibiotics (100mg/mL of streptomycin and 100U/mL of penicillin). The cells were grown until they reached 90%–100% confluence. Subsequently, the culture medium was replaced with serum-free DMEM–Ham’s F12, and the cells were incubated for an additional 12 h prior to experimental treatment. The ARPE- 19 cells were cultured in the dark or irradiated with blue light (400 nm) at an intensity of 2000 ± 500 lx for 24 h to establish the model of light- induced injury.

2.2. Cytotoxicity assay

Cytotoxicity was examined by performing the Alamar Blue assay. Briefly, the ARPE-19 cells were cultured in 96-well plates with or without different treatments. Prior to experimental treatment, 25 μL of Alamar Blue dye (Serotec, Oxford, UK) was added to each well, and the mixture was incubated for 2 h at 37 ◦C. Subsequently, 100 μL of the solution from each well was transferred to a 96-well culture plate. Cytotoxicity was examined following manufacturer’s instructions. The excitation and emission wavelength settings were adjusted to 530 and 590 nm, respectively.

2.3. Morphological analysis after DAPI staining

The cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min. After washing with phosphate-buffered saline (PBS), the cells were analyzed using a fluorescence microscope (Axiovert S100, Zeiss, Germany). The nucleus of a normal cell is round and exhibits clear edges and uniform staining, whereas that of an apoptotic cell exhibits nuclear pyknosis, irregular edges, and heavy staining.

2.4. Analysis of intracellular ROS

To examine intracellular ROS production, the RPE cells were seeded in DMEM–Ham’s F12 into 96-well plates (2 × 104 cells/well) and incubated for 24 h; the cells were then cultured in serum-free DMEM–Ham’s F12 for 12 h prior to experimental treatment. Subsequently, the cells were incubated with 20 μM 5-(and-6)-carboxy-2,7, dichlorodihydrofluorescein diacetate (a cell-permeant fluorogenic probe; carboxy-H2DCFDA; Molecular Probes, Eugene, OR, USA) for 30 min. After washing with PBS twice (5–10 min per wash), the cells were solubilized with 1% sodium dodecyl sulfate (SDS) and 5 mM Tris-HCl (pH 7.4). H2DCFDA fluorescence was measured at an excitation and emission wavelength of 450 and 520 nm, respectively, by using a spectrofluorophotometer (Shimadzu, Rf-5301PC). Images were observed and photographed using a fluorescence microscope (Axiovert S100, Zeiss, Germany).

2.5. GSH assay

The intracellular level of reduced GSH was determined as described previously (Kamencic et al., 2000). Briefly, the cells were incubated with 40 μM monochlorobimane, a GSH fluorescent probe, for 20 min in the dark. After washing twice with PBS, the cells were solubilized with 5 mM Tris-HCl (pH 7.4) and 1% SDS. Fluorescence was measured using a spectrofluorophotometer (Shimadzu, Rf-5301PC, Kyoto, Japan) at an excitation and emission wavelength of 390 and 520 nm, respectively. The GSH level is expressed as the percentage relative to the GSH level in control cells (control = 100%).

2.6. Monocyte adhesion assay

The cells grown to 90%–100% confluence in a 96-well plate were pretreated with SFN and then treated with TNF-α or blue light for 24 h. The cells were cocultured with 5 × 105 calcein-labeled THP-1 cells for 30 min. After washing twice with RPMI-1640 medium, the adherent cells were examined using an enzyme-linked immunosorbent assay plate reader (FL × 800, Bio-Tek Instruments) at an excitation and emission wavelength of 485 and 538 nm, respectively.

2.7. Western blot

Whole-tissue protein was extracted from the ARPE-19 cells by using RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor mixture). The protein was then separated through 10% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA) in Tris-glycine buffer at 10 V for 1.5 h. Subsequently, the membranes were blocked using PBS containing 5% nonfat milk and incubated with antibodies for 2 h at 4 ◦C with gentle shaking. Immunoreactive proteins were visualized through electrochemiluminescence. Images were quantified using ImageJ, version 1.8.0 (National Institutes of Health).

2.8. Mitochondrial membrane potential assay

The ARPE-19 cells (1.5 × 104 cells/well) were cultured and exposed to blue light for 24 h. The mitochondrial membrane potential was estimated using JC-1 dye (mitochondrial membrane potential probe; Invitrogen). The cells were washed and incubated with 10 μg/mL JC-1 dye at 37 ◦C for 15 min in the dark. Images were obtained using a fluorescence microscope (Axiovert S100, Zeiss, Germany), which showed healthy cells with JC-1 J-aggregates (excitation and emission wavelengths =540 and 605 nm, respectively) and unhealthy cells with mostly JC-1 monomers (excitation and emission wavelengths = 480 and 510 nm, respectively). Images were quantified using ImageJ, version 1.8.0 (National Institutes of Health).

2.9. Preparation of cytosolic and nuclear lysates

To separate the nuclear fraction from the cytosol, the cell pellet was first collected through scraping in cold PBS and then lysed in a lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, and 0.3% nonidet P- 40). The cell lysate was centrifuged at 3000 rpm for 5 min at 4 ◦C. The collected supernatant represented the cytoplasmic fraction. Nuclear proteins were extracted using a buffer (25% glycerol, 20 mM HEPES, 0.6 M KCl, 1.5 mM MgCl2, and 0.2 mM ethylenediaminetetraacetic acid).

2.10. RNA isolation and reverse transcription-polymerase chain reaction

Total cellular RNA was isolated using Trizol reagent (Invitrogen), and 5 μg of RNA from the cells was reverse transcribed using 50 U of Superscript II (Invitrogen) for 50 min at 42 ◦C. After reverse transcription, cDNA was amplified in a polymerase chain reaction buffer (10 mM Tris- HCl, 50 mM KCl, 5 mM MgCl2, and 0.1% Triton X-100; pH 9.0) containing 0.6 U of Taq DNA polymerase (Promega) and 30 pmol primers. Specific primers used for the amplification of Nrf2, HO-1, TRX-1, GCLC, GCLM, HO-1, ICAM-1, and GAPDH were as follows: Nrf2, forward 5′- CGG TAT GCA ACA GGA CAT TG-3′ and reverse 5′-ACT GGT TGG GGT CTT CTG TG-3′; HO-1, forward 5′-GGT AAG GAA GCC AGC CAA GAG-3′ and reverse 5’-GCC AGC AAC AAA GTG CAA GA T-3′; TRX-1, forward 5′- ACG TGA TAT TCC TTG AAG TAG-3′ and reverse 5′-GGC ATG CAT TTG ACT TCA; GCLC, forward 5′-GTT CTT GAA ACT CTG CAA GAG AAG-3′ and reverse 5′-ATG GAG ATG GTG TAT TCT TGT CC-3′; GCLM, forward 5′-CAG CGA GGA GCT TCA TGA TTG-3′ and reverse 5′-TGA TCA CAG AAT CCA GCT GTG C-3′; ICAM-1, forward 5′-AGC AAT GTG CAA GAA GAT AGC CAA-3′ and reverse 5′-GGT CCC CTG CGT GTT CCA CC-3′; and GAPDH, forward 5′-TAT CGT GGA AGG ACTC ATG ACC-3′ and reverse 5′-TAC ATG GCA ACT GTG AGG GG-3′. Reaction products were electrophoretically separated on 2.5% agarose gel and stained with ethidium bromide.

2.11. Statistical analysis

Data are expressed as the mean ± standard error (SE) of at least three experiments. Statistical significance was assessed using one-way analysis of variance, followed by Tukey’s test. Statistical analyses were performed using SigmaPlot, version 12 (Systat Software, San Jose, CA, USA). Confidence limits with P < 0.05 were considered statistically significant. 3. Results 3.1. Cytoprotective effect of SFN on RPE cells First, we assessed the cytotoxic effect of SFN by incubating the ARPE- 19 cells with 5–25 μM SFN (Fig. 1A). As illustrated in Fig. 1A, the cells incubated with 5–25 μM SFN for 24 h exhibited no cell toxicity. Subsequently, we determined whether continued blue light exposure can induce cytotoxicity. As shown in Fig. 1B, the RPE cells exposed to blue light for 24 h showed 20% cytotoxicity. Accordingly, a blue light exposure duration of 24 h was used in subsequent experiments. We examined the protective effects of SFN against blue light–induced cytotoxicity in the RPE cells. Pretreatment of cells with 5–25 μM SFN for 3 h significantly prevented cytotoxicity (Fig. 1C). This finding suggests that 5 μM SFN is sufficient to protect cells against blue light exposure. The compound atRAL is a major intermediate of the visual cycle. A previous study reported that atRAL exerted a toxic effect by causing mitochondrial damage, leading to apoptosis (Maeda et al., 2009). We examined the protective effects of SFN by treating the cells with 5 μM atRAL for 6 h. The results revealed that SFN could not fully protect the cells from atRAL; however, it still partially protected the cells pretreated with 10 and 15 μM SFN (Fig. 1D). 3.2. SFN induces Nrf2-related gene expression SFN can activate the Nrf2 pathway in various systems. We confirmed this finding in this study (Fig. 2A) and observed SFN-induced Nrf2 accumulation in the nuclear fraction. Furthermore, we examined whether the Nrf2-related gene HO-1 is activated by SFN. We observed that the HO-1 protein level increased in cells treated with 5 μM SFN (Fig. 2B) for 6 h (Fig. 2C). GSH is the most abundant antioxidative enzyme and plays a crucial role in maintaining the cellular redox state (Tan et al., 2020). The enzyme in the de novo synthesis of GSH is GCL, which is regulated by Nrf2-mediated expression of GCLC and GCLM. As shown in Fig. 2D, the GSH level increased after 12 h of SFN treatment. These findings suggest that SFN induced Nrf2-related gene expression in the RPE cells. 3.3. Nrf2 protects RPE cells from blue light–induced cytotoxicity Blue light exposure can increase oxidative stress (Tao et al., 2019). We performed a assay to observe that blue light exposure induced ROS generation in the RPE cells (Fig. 3A). Moreover, pretreatment with SFN reduced the intracellular ROS level (Fig. 3B). Although blue light exposure for 24 h increased oxidative stress, treatment with SFN increased the expression of Nrf2 and Nrf2-related genes, namely HO-1, Trx-1, GCLC, and GCLM (Fig. 3C). Similarly, the GSH level was higher in SFN-treated cells exposed to blue light than in SFN-treated cells not exposed to blue light (Fig. 3D). The cytoprotective effect of SFN was significantly decreased after treatment with ML385, an Nrf-2 inhibitor (Fig. 3E). These results suggest that the expression of Nrf2 and Nrf2- related genes plays a key role in the protective effect of SFN under blue light exposure. 3.4. SFN exerts anti-inflammatory effects on RPE cells treated with TNF-α or blue light The upregulation of ICAM-1 expression contributes to leukocyte adhesion at inflammation sites (Cousins et al., 2004). Therefore, we examined the effects of SFN on blue light– and TNF-α-induced monocyte adhesion in the ARPE-19 cells. We found that pretreatment with SFN inhibited blue light– and TNF-α-induced monocyte adhesion (Fig. 4A). Furthermore, we observed that the pretreatment of cells with SFN for 6 h significantly inhibited TNF-α-induced ICAM-1 expression at the mRNA (Fig. 4B) and protein levels (Fig. 4C). By contrast, pretreatment of the ARPE-19 cells with SFN inhibited blue light–induced ICAM-1 expression (Fig. 4D). In addition, we examined whether SFN regulates blue light–induced NF-κB activation. The results of Western blotting revealed that SFN inhibited NF-κB nuclear translocation (Fig. 4E). Collectively, our results demonstrated that SFN inhibited monocyte adhesion through activation of NF-κB and inducement of ICAM-1 expression under blue light exposure. 3.5. SFN reduces blue light–induced apoptosis We performed DAPI staining to determine whether SFN attenuates blue light–induced cell apoptosis. First, the condensed nuclei of the ARPE-19 cells exposed to blue light for 24 h were evaluated through DAPI staining. We observed that the number of pyknotic nuclei increased in the cells exposed to blue light but decreased in the cells pretreated with SFN (Fig. 5A). A study demonstrated that blue light–induced ROS accumulation in the mitochondria led to cell death (Tao et al., 2019). Subsequently, we used JC-1 fluorescence to detect mitochondrial activity by examining the mitochondrial membrane potential in the RPE cells exposed to blue light. We compared the effects of SFN with those of blue light on mitochondrial activity by using JC-1 fluorescence. Red fluorescence indicates healthy mitochondria, whereas green fluorescence indicates unhealthy mitochondria. The results revealed that the red fluorescence intensity was increased in the cells pretreated with SFN compared with the cells exposed to blue light only. Furthermore, we observed a decrease in the Bax protein level and an increase in the Bcl-2 protein level in cells treated with SFN compared with cells exposed to blue light; however, the ratio of Bcl-2/Bax recovered after SFN pretreatment (Fig. 5C). The increase in mitochondrial membrane potential and Bax/Bcl-2 expression by SFN prompted us to investigate the expression of caspase-3, a terminal protein in the apoptosis pathway, and PARP-1, a downstream effector. As shown in Fig. 5D and E, the blue light–induced expression of cleaved caspase-3 and PARP-1 was markedly suppressed after pretreatment with SFN. These results suggest that SFN effectively inhibited blue light–induced apoptosis by maintaining mitochondrial function. 3.6. SFN increases autophagy and protects cells against blue light exposure SFN may protect RPE cells against blue light exposure through the involvement of autophagy, a major mechanism responsible for the renewal of all the cytoplasmic components of postmitotic cells (Kaarniranta et al., 2018). Thus, we examined the effects of SFN on the autophagy of RPE cells. LC3B is a classic autophagic marker, and the ratio of conversion from LC3BI to LC3BII is closely correlated with the extent of autophagosome formation. We evaluated the expression of LC3BII autophagic markers. As shown in Fig. 6A, treatment with SFN for 6 h increased the expression of LC3-II and p62. Furthermore, the expression of LC3-II and p62 was enhanced in the cells treated with both SFN and blue light compared with those treated with SFN alone (Fig. 6B). This finding indicates that SFN enhances the autophagic process in cells treated with blue light. To examine whether autophagy is involved in the protective effects of SFN on cells exposed to blue light, we pretreated cells with the autophagy inhibitor 3-MA. The results revealed that incubation with 3-MA significantly reduced the viability of the SFN-treated cells exposed to blue light (Fig. 6C). Because SFN did not exert the antioxidative effect on the cells exposed to blue light, we examined whether antioxidant activity is involved in the enhancement of blue light–induced autophagy. However, enhanced autophagy was not observed in cells pretreated with catalase or acetylcysteine, unlike in cells pretreated with SFN. These results suggest that SFN-induced autophagy activation promoted the cell viability of RPE cells; however, the antioxidative effect of SFN did not play a role in this induction. 3.7. SFN induces SIRT1 expression and increases PCG-1α in blue light–exposed RPE cells Loss of the SIRT1 or PGC-1α gene can induce the aging process (Fletcher et al., 2008; Kaarniranta et al., 2018; Sridevi et al., 2020). To explore the protective mechanisms of SFN, the expression of SIRT1 and PGC-1α was examined through Western blot. As shown in Fig. 7A and B, the protein expression of SIRT1 was considerably increased in the SFN- treated cells exposed to blue light compared with the cells treated with SFN only. In addition, our results revealed that the expression of the PGC-1α gene was induced in not only the SFN-treated cells exposed to blue light but also in the cells treated with SFN only (Fig. 7C). Therefore, SFN-enhanced SIRT1 and PGC-1α expression may contribute to the protective mechanism against blue light–induced cell injury. 4. Discussion SFN is a naturally occurring chemopreventive agent that is predominantly present in cruciferous vegetables, especially conventional Chinese cabbage and broccoli (Zhang et al., 1994). In the present study, we observed that the cytoprotective effects of the noncytotoxic drug concentrations of SFN against blue light–induced injury are mediated by the inhibition of oxidative stress, NF-κB activation, and apoptosis. However, these cytoprotective effects may depend upon the expression of Nrf2- related genes and PGC-1α in SFN-treated RPE cells. Autophagy is crucial for maintaining the homeostasis of RPE cells because it removes dysfunctional organelles and proteins (Kaarniranta et al., 2018). One finding of this study is that SFN or blue light increased the expression of LC3-II and p62 (Fig. 6A); in addition, SFN enhanced LC3-II and p62 expression in cells exposed to blue light (Fig. 6B). An autophagy inhibitor, 3-MA, significantly reduced the viability of the cells exposed to blue light only and the cells treated with both blue light and SFN (Fig. 6B). These results suggest that SFN reduced the damage caused by blue light in RPE cells partially through autophagy. The highly specialized barrier characteristics of RPE and retinal vascular endothelial cells should be maintained to sustain normal retinal function. Because RPE and retinal vascular cells experience high metabolic demand, they are highly vulnerable to disease. Therefore, their dysfunction can lead to severe consequences. RPE cells form the outer blood–retinal barrier by a tight junction and are located between the choroidal capillary bed and photoreceptors in the retina. They are vital for protecting the retina from excessive light exposure, oxidative stress, and immune responses (Sparrow et al., 2010; Pavan and Dalpiaz, 2018b). In many retinal diseases, including AMD, the dysfunction of RPE cells is a crucial event in the disease process (Kokotas et al., 2011). However, the mechanism through which SFN crosses the blood–retinal barrier is not completely understood. SFN is a hydrophobic, lipid-soluble compound that is known to cross biological membranes at a low working concentration of 5 μM. This implies that SFN can trigger protective mechanisms in the retina. In the present study, we investigated mechanisms through which SFN protect cells against oxidative stress, inflammation, and cell apoptosis induced by blue light exposure. RPE cells are sensitive to blue light. A previous study demonstrated that blue light–induced ROS accumulation in the mitochondria led to cell death (King et al., 2004). Consistently, we found that blue light exposure induced cell injury and caused cell apoptosis (Fig. 1B). Our results revealed an increase in the ROS level in blue light–exposed RPE cells and indicated that SFN could reduce ROS production (Fig. 3B). Because blue light–induced cell damage results from oxidative stress, SFN, an Nrf2 activator, could reduce blue light–induced cytotoxicity (Fig. 1C). By examining the apoptotic markers, pyknosis cells, mitochondrial membrane potential, ratio of Bcl-2/Bax or cleaved caspase-3 and PARP-1, we found that SFN effectively inhibited these apoptotic markers in the RPE cells exposed to blue light (Fig. 5A-E). These findings imply that mitochondrion-dependent apoptosis is the potential protective mechanism of SFN. A previous study indicated that PGC-1α regulated respiration and mitochondrial biogenesis, upregulated antioxidant genes in the retinal pigment epithelium, and protected RPE cells from oxidative stress (Kaarniranta et al., 2018). SFN was observed to induce PGC-1α expression and improve lipid metabolism by altering mitochondrial biogenesis (Lei et al., 2019). SIRT1 is an upstream regulator of PGC-1α (Salminen et al., 2013). In the present study, we found that SFN increased SIRT1 expression (Fig. 7B and B). Furthermore, we demonstrated that SFN treatment only or SFN treatment with blue light exposure increased the expression of PGC-1α in RPE cells. The role of PGC-1α in the protective mechanism warrants further detailed investigation. Oxidative stress plays a crucial role in mechanisms underlying AMD progression under sunlight exposure, which increases oxidative damage in the retina (Beatty et al., 2000). Nrf2-related enzymes protect against oxidative stress in AMD by promoting cell viability (Sachdeva et al., 2014). Previous studies have indicated that many phytochemicals, such as EGCG, lycopene, and cinnamaldehyde, could protect cells against oxidative stress by activating the Nrf2 pathway (Wu et al., 2006; Liao et al., 2008; Yang et al., 2016). An electrophilic phytochemical, curcumin, was observed to exert a protective effect on retinal cells against light-induced cell death by upregulating Nrf2-related antioxidant enzymes (Mandal et al., 2009). We previously showed that lycopene, a natural carotenoid, could increase GSH to protect RPE cells from oxidative stress–induced cytotoxicity (Yang et al., 2016). GSH protects cells from oxidative stress and maintains the cellular thiol redox state. In the present study, we determined that SFN increased the intracellular GSH level (Fig. 2D). Additional experiments demonstrated that the SFN- treated cells exposed to blue light still showed increased GCLM gene expression and GSH levels (Fig. 3C and D). The Nrf2 inhibitor ML385 blocked the action of SFN in the blue light–exposed RPE cells (Fig. 3E). Thus, the increased activity of the Nrf2-related pathway plays a major role in the cytoprotective effects of SFN against blue light–induced cytotoxicity in RPE cells. In addition, autophagy can activate Nrf-2 by disrupting the Nrf-2–Keap1 complex (Liu et al., 2016), and SIRT1 exerts an antioxidative effect through the nuclear deacetylation of Nrf2 (Ding, 2016). Our data showed that SFN enhanced autophagy (Fig. 6A) and SIRT1 expression (Fig. 7A), suggesting that SFN-induced Nrf2-related antioxidant activity may involve the upstream regulation of autophagy and SIRT1 expression. Chronic inflammation is a common and crucial risk factor for age- related diseases such as AMD (Algvere et al., 2006; Tan et al., 2020). Chronic inflammation is a prolonged process that coexists with tissue damage and repair, contributing to tissue remodeling and dysfunction. Our study results demonstrated that SFN treatment exerted an anti- inflammatory effect on both cytokine- and blue light–treated RPE cells and that SFN ameliorated the suppression of ICAM-1 expression. The inhibition of blue light–induced ICAM-1 expression was mediated through NF-κB translocation. Our previous studies have demonstrated that the Nrf2-dependent redox state is a major modulator of the anti- inflammatory effects of CO on endothelial cells (Yang et al., 2014; Yeh et al., 2014). A notable finding is that the levels of the Nrf2-related cytoprotective enzyme HO-1, an enzyme that generates CO, increased during blue light exposure in the SFN-treated RPE cells. In summary, our study demonstrated that blue light–induced ROS production activates inflammation, autophagy, and apoptosis in ARPE- 19 cells. However, we revealed the protective effects of SFN on blue light–induced oxidative injury, inflammation, and apoptosis. 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