**1. Introduction**

Diabetes is a major metabolic disease that can affect different organs through microvascular and macrovascular damages [**?** ]. Diabetic retinopathy (DR), a leading microvascular complication of diabetes, is characterized by a progressive increase in vascular permeability, retinal ischemia and edema, and neovascularization, which can result in visual impairment and even legal blindness [**?** ]. Despite the improved understanding of its pathogenesis and the advances in available treatments, the long-term outcome of DR remains poor owing to its complex pathogenesis [**? ?** ]. Therefore, the continuous search for new modalities to prevent and treat this debilitating complication is essential.

Hyperglycemia induces oxidative stress and generates reactive oxygen species (ROS) within the retina [**? ?** ]; however, the activity of cellular antioxidant enzymes responding to ROS is insufficient to prevent the consequent damages. Yeh et al. reported a positive correlation between ROS levels in vitreous fluid and the severity of DR [**?** ]. In addition, several studies have indicated chronic oxidative stress as one of the primary causes of DR [**?????** ]. Normally, the retina already presents

substantial lipid oxidation and ROS production due to the high content of unsaturated fatty acids and high oxygen uptake, which render it more vulnerable to oxidative stress damage than other tissues [**? ?** ]. Furthermore, ROS accumulation alters the homeostasis of retinal cells, triggers cellular apoptosis, and leads to increased vascular permeability and basement membrane leakage in the retina. These pathological changes may lead to a breakdown of the blood–retinal barrier and the development of DR [**? ?** ]. Thus, reducing oxidative stress could inhibit apoptosis in retinal cells and reduce the risk of DR progression.

Astaxanthin (AST) is a member of the xanthophyll family of oxygenated carotenoid derivatives. AST has been recently recognized as a strong free radical scavenger and an excellent anti-inflammatory agen<sup>t</sup> that suppresses the expression of proinflammatory chemokines and cytokines [**? ?** ]. The unique molecular structure of AST, which contains hydroxyl and keto moieties on each ionone ring, explains its high antioxidant activity [**?** ]. Mechanistically, AST terminates the free radical chain reaction by donating electrons and reacting with free radicals to convert them to more stable products [**? ?** ]. AST can, therefore, act as a powerful antioxidant in numerous organisms. In fact, AST has been attributed as having the potential to defend organisms against a broad range of diseases, including cardiovascular disease [**?** ], ischemic brain damage [**?** ], cataracts [**?** ], diabetes [**?** ], and diabetic nephropathy [**? ?** ]. AST has also shown protective e ffects in ocular diseases, including neuroprotection in retinal ganglion cells [**?** ], suppression of choroid neovascularization development [**?** ], and reduction of endotoxin-induced uveitis [**?** ].

Recent studies have shown that AST can activate the Nrf2–antioxidant responsive element (ARE) pathway in di fferent cell types and organs [**?????** ]. The Nrf2–ARE pathway is an important endogenous mechanism that can attenuate oxidative stress within the cell [**? ?** ]. Nrf2 induces the expression of Phase II enzymes, including NAD(P)H dehydrogenase (NQO1) and heme oxygenase-1(HO-1), thereby limiting the subsequent generation of ROS and promoting the formation of antioxidant bilirubin [**????** ]. An earlier study indicated that retinal pigment epithelial (RPE) cells could be protected against oxidative damage by activating the Nrf2–ARE pathway and increasing expression of Phase II enzymes [**?** ]. A later study further showed the crucial role of the PI3K/Akt pathway in modulating Nrf2–ARE-dependent protection against oxidative stress in RPE cells [**?** ].

Therefore, in this study, we hypothesized that AST could counteract high glucose-induced oxidative stress and attenuate oxidative stress-induced apoptosis by modulating Nrf2 expression in retinal photoreceptor cells. To test our hypotheses, we first investigated the role of high glucose-induced oxidative stress in initiating retinal photoreceptor cell (661W) apoptosis and evaluated the antioxidative and antiapoptotic e ffects of AST. Moreover, we analyzed the modulatory e ffect of AST on Nrf2 expression and analyzed the related signaling pathway. Our results showed that AST reduced high glucose-induced oxidative stress and attenuated apoptosis of photoreceptor cells by induction of antioxidant enzymes via the PI3K/Akt/Nrf2 pathway.

#### **2. Materials and Methods**

#### *2.1. Cell Culture and Experimental Design*

In this study, we used the 661W cell line, obtained from Dr. M. Al-Ubaidi (University of Houston), to evaluate the response of photoreceptor cells to AST treatment. The 661W cells, exhibiting biochemical features characteristic of cone photoreceptor cells, constitute an immortalized mouse photoreceptor cell line (by the expression of simian virus 40 T antigen) [**?** ]. We cultured the 661W cells in Dulbecco's modified Eagle's media (DMEM) containing 10% phosphate bu ffer solution (PBS) and 1% penicillin–streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 ◦C and 5% CO2. Cells were passaged by trypsinization every 3–4 days and used for experiments at the second to fifth passage. For the experiment, the cells were exposed to either normal (5 mM/mL) or high glucose (35 mM/mL) concentration. In addition, the cells were pretreated with 10, 20, or 50 μM AST (Sigma, St. Louis, MO, USA) for 2 h prior to high glucose treatment. After 24 h treatment, the cells were collected

and further analyzed. Two di fferent inhibitors, PI3K inhibitor (LY294002, 20 μM, (Sigma, St. Louis, MO, USA)) and Nrf2 inhibitor (ML385, 10 μM, (Selleck Chemicals, Houston, TX, USA)) were used to determine the causal relationship between AST and the changes in other antioxidative molecules. The cultured cells were pretreated with either of the inhibitors and 50 μM AST simultaneously for 2 h, followed by high glucose treatment for 24 h.

#### *2.2. Cell Viability Assay*

Cell viability assay was performed with CyQUANT MTT Cell Viability Assay Kit (Invitrogen-Life Technologies Inc., Gaithersburg, MD, USA). The cells were seeded in 96-well plates (1 × 10<sup>4</sup> cells per well) and incubated at 37 ◦C. Di fferent concentrations of AST were added to the cells exposed to high glucose. After 24 h incubation, 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated at 37 ◦C for 4 h. The culture medium supernatant was removed, and the formazan was dissolved with dimethyl sulfoxide (DMSO) for 30 min at 25 ◦C. The absorbance was measured at 570 nm with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

#### *2.3. Analysis of Apoptosis by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL)*

We used TUNEL to detect 661W cell apoptosis. TUNEL was performed using a commercial kit (Millipore Corp., Billerica, MA, USA) according to the manufacturer's instructions. Positive controls were cultured cells incubated with DNase I prior to the labeling procedure, and sham controls were cells stained with label solution containing no terminal transferase.

#### *2.4. Detection of Intracellular ROS*

We used Image-IT Green Reactive Oxygen Species Detection Kit (Invitrogen-Life Technologies Inc., Gaithersburg, MD, USA) to determine the level of ROS under di fferent conditions. Intracellular ROS levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (2',7'-DCFDA) oxidation. For the detection of ROS, 661W cells were exposed to 10 μM 2',7'-DCFDA; in the presence of ROS, bright green fluorescence could be detected by a fluorescence microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA).

#### *2.5. Quantitative Detection of Nrf2 and ROS-Induced Cellular Oxidation Using Immunocytochemistry (ICC)*

DNA oxidation, lipid peroxidation, and protein oxidation levels were determined by detecting the expression of 8-hydroxydeoxyguanosine (8-OHdG), acrolein, and nitrotyrosine, respectively, using ICC according to a previously published protocol [**?** ]. In brief, the cultured cells were simultaneously blocked and permeabilized with 0.2% Triton in PBS containing 5% goa<sup>t</sup> serum for 1 h at room temperature. Then, cells were incubated with primary antibodies diluted in blocking solution overnight at 4 ◦C, followed by incubation with the appropriate fluorescent secondary antibodies (all diluted 1:200) in blocking solution for 3 h at room temperature. Primary antibodies included anti-Nrf2, antiacrolein, antinitrotyrosine, and anti-8-OHdG (all from Abcam, Cambridge, MA, USA). Nuclei were counterstained with DAPI.

The quantitative protein expression measurements were done by densitometric methods as previously described [**?** ]. The relative density of immunostaining was analyzed by an immunostaining index comparing 661W cells treated with di fferent AST concentrations and high glucose condition versus low glucose condition (as normal reference).

#### *2.6. Determination of Changes in Mitochondrial Membrane Potential*

The mitochondrial membrane potential was measured using a fluorescence reader with JC-1 stain (Sigma, St. Louis, MO, USA) and a lipophilic cationic probe to detect the extent of mitochondrial dysfunction. The 661W cells were seeded in 96-well plates (1 × 10<sup>4</sup> cells per well) and incubated

at 37 ◦C. Then, different concentrations of AST were added to the cells exposed to high glucose. After 24 h incubation, 5 μL of JC-1 staining solution was added to each well, and the plate was incubated at 37 ◦C for 15 min. The fluorescence intensity 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 measured with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For microscopy, the cells were incubated in 24-well plates. Then, 50 μL of JC-1 staining buffer was added to 1 mL of culture medium. After incubation at 37 ◦C for 30 min, the cells were observed using fluorescence microscopy. J-aggregates and JC-1 monomers were detected with settings designed to detect Texas Red and FITC, respectively.

#### *2.7. Preparation of RNA and cDNA*

Total RNA was extracted from the retinas using TRIzol reagen<sup>t</sup> (Invitrogen-Life Technologies Inc., Gaithersburg, MD, USA). For each sample, 1 μg of total RNA was incubated with 300 ng of Oligo dT for 5 min at 65 ◦C and reverse-transcribed into cDNA using 80 U of Moloney murine leukemia virus reverse transcriptase per 50 μg reaction sample for 1 h at 37 ◦C. The reaction was stopped by heating the samples for 5 min at 90 ◦C.

#### *2.8. Quantitative Analysis of mRNA Levels*

We performed polymerase chain reaction (PCR) on the resultant cDNA from each sample using specific primers. The amplification was performed using a thermocycler (Applied Biosystems, Waltham, MA, USA). The 25 μL reaction mixture contained 5 μL of cDNA, 1 μL of sense and antisense primers, 200 μM of each deoxynucleotide (DTT), 5 μL of 10× Taq polymerase buffer, and 1.25 U of GoTaq polymerase (Applied Biosystems, Waltham, MA, USA). The PCR reactions were performed using an annealing temperature of 56 ◦C with GoTaq polymerase, cDNA, and the following primers: sense 5--ATGACACCAAGGACCAGAGC and antisense 5--GTAAGGACCCATCGGAGAAGC for HO-1; sense 5--TATCCTGCCGAGTCTGTTCTG and antisense 5'-AACTGGAATATCACAAGGTCTGC for NQO1; and sense 5--CGACTTCAACAGCAACTCCCACTC and antisense 5'-TGGGTGGTCCAGGGTTTCTTACTC for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The DNA fragments were amplified for 25–30 cycles (30 s at 94 ◦C; 1 min at 50–52 ◦C; 1 min at 72 ◦C), followed by a final 7 min extension step at 72 ◦C. The amplified products were further analyzed. Levels of each mRNA were normalized to those of GAPDH mRNA.

#### *2.9. Protein Extractions and Western Blot Analysis*

We extracted total proteins from the 661W cells using radioimmunoprecipitation assay (RIPA) lysis buffer (0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, and 10% protease inhibitors). Before analysis, the protein samples were separated on a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membranes were probed using the following antibodies: anti-HO-1; anti-NQO1 (from Abcam, Cambridge, MA, USA); anti-PI3K; anti-Akt; anti-phospho-Akt(p-Akt); anti-caspase-3; anti-poly(ADP-ribose) polymerase (PARP) (from Cell Signaling Technology, Danvers, MA, USA); and anti-GAPDH (Millipore Corp., Billerica, MA, USA). Immunodetections were done using enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instructions. Protein levels were determined using densitometry analysis of the protein bands.

#### *2.10. Nuclear Protein Extraction and Electrophoretic Mobility Shift Assay (EMSA)*

The 661W cells treated with low glucose, high glucose, and different concentrations of AST were harvested separately. Nuclear proteins were prepared as described before [**?** ]. In brief, the cells were trypsinized, resuspended, and homogenized in ice-cold buffer A (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.9), 1.5 mM KCl, 10 mM MgCl2, 1.0 mM dithiothreitol (DTT), and 1.0 mM phenylmethylsulfonyl fluoride (PMSF)). The cell suspensions

were homogenized and centrifuged for 10 min at 5000× *g* at 4 ◦C. The sediment was resuspended in 200 μL of buffer B (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, and 4 μM leupeptin). Then, the samples were incubated for 30 min on ice and centrifuged for another 30 min at 12,000× *g* at 4 ◦C. The supernatants containing the nuclear proteins were collected for further analysis. A bicinchoninic acid assay kit, with bovine serum albumin as the standard, was used to determine the protein concentration. EMSA was performed with a Nrf2 DNA-binding protein detection system (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Ten micrograms of nuclear protein was incubated with a biotin-labeled Nrf2 consensus oligonucleotide probe (5--GCACAAAGCGCTGAGTCACGGGGAGG-3-) for 30 min in binding buffer. The specificity of the DNA/protein binding was then determined by adding a 100-fold molar excess of unlabeled Nrf2 oligonucleotide for competitive binding, 10 min before adding the biotin-labeled probe.
