**3. Results**

#### *3.1. Astaxanthin Protects 661W Cells from Apoptosis under High Glucose Conditions*

To evaluate the potential role of AST as a therapeutic agent, we first tested the safety of different concentrations (1, 5, 10, 20, and 50 μM) of AST on 661W cells. The cell viability was not affected by treatment with up to 50 μM AST, as shown by MTT assay (Figure **??**A). Therefore, 10, 20, and 50 μM AST were used for the following experiments. To evaluate the protective effect of AST in 661W cells under high glucose conditions, we used the TUNEL assay to detect apoptosis. The results demonstrated a dose-dependent protective effect of AST against 661W cell apoptosis in a high glucose environment (Figure **??**B), with a significantly decreased number of apoptotic cells upon treatment with 20 and 50 μM AST. The antiapoptotic effect of AST was further confirmed by decreased caspase-3 cleavage and decreased PARP in AST-treated 661W cells (Figure **??**C).

**Figure 1.** Effects of AST on cell viability and high glucose-induced apoptosis in 661W cells. (**A**) Cell viability was evaluated using MTT assay upon treatment with different concentrations of AST for 24 h. AST-treated and control cells showed similar cell viability. (**B**) Increased cell apoptosis was noted under high glucose environment, demonstrated by TUNEL assay and protein expression (western blots, **C**). AST reduced apoptosis at 20 and 50 μM concentration. (**C**) Decreased cleaved caspase-3 and PARP protein expression seen in western blots indicated that apoptosis was attenuated by AST. AST = astaxanthin; HG = high glucose; LG = low glucose; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; PARP = poly(ADP-ribose) polymerase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase. \* Significantly different from control group (*p* < 0.05); # significantly different from high glucose-treated group without AST (*p* < 0.05). The Mann–Whitney *U* test was used to calculate the differences between the means of different experimental groups (three repeats per experiment, three wells per repeat, five images per well analyzed).

#### *3.2. Astaxanthin Reduces ROS and ROS-Related Mitochondrial Damage*

To understand if the observed attenuation in apoptosis resulted from the antioxidative effect of AST, we measured the intracellular ROS levels in cells treated with different concentrations of this compound. AST reduced the levels of ROS increased by high glucose treatment in 661W cells at 20 μM and 50 μM concentration (Figure **??**A). The ROS-related mitochondrial damages, detected by JC-1 staining, were also reduced by AST treatment (Figure **??**B).

**Figure 2.** AST reduced ROS and ROS-related mitochondrial damage. (**A**) 661W cells pretreated with different concentrations of AST for 2 h were cultured under high glucose environment. Intracellular ROS levels were detected by measuring the green fluorescence of 2-,7--dichlorodihydrofluorescein diacetate oxidation. ROS levels were significantly reduced in cells pretreated with 20 and 50 μM of AST. (**B**) Mitochondrial membrane potential was evaluated by JC-1 staining. The results showed decreased mitochondrial damage after AST treatment. AST = astaxanthin; HG = high glucose; LG = low glucose; ROS = reactive oxygen species. \* Significantly different from control group (*p* < 0.05); # significantly different from high glucose-treated group without AST (*p* < 0.05). The Mann–Whitney *U* test was used to calculate the differences between the means of different experimental groups (three repeats per experiment, three wells per repeat, five images per well analyzed).

#### *3.3. Astaxanthin Reduces ROS-Related Lipid, Protein, and DNA Damage*

Increased intracellular ROS levels can cause further damage to lipids, proteins, and DNA, which may eventually lead to apoptosis. We therefore evaluated the expression of acrolein, nitrotyrosine, and 8-OHdG using different concentrations of AST. The levels of all three surrogate markers decreased with increasing concentrations of AST, indicating the antioxidative capacity of AST treatment (Figure **??**). Significantly reduced damage to lipids and DNA (indicated by acrolein and 8-OHdG, respectively) and to proteins (indicated by nitrotyrosine) was observed with at least 10 or 20 μM AST, respectively.

**Figure 3.** AST reduces ROS-related lipid, protein, and DNA damage in high glucose-treated 661W cells. To confirm that high glucose-induced ROS-related damage was also attenuated by AST treatment, we measured the expression of acrolein (**A**), nitrotyrosine (**B**), and 8-OHdG (**C**) by immunocytochemistry to indicate the oxidative damages to lipids, proteins, and DNA, respectively. Significantly decreased oxidative damage was noted under AST treatment. AST = astaxanthin; HG = high glucose; LG = low glucose; ROS = reactive oxygen species. \* Significantly different from control group (*p* < 0.05); # significantly different from high glucose-treated group without AST (*p* < 0.05). The Mann–Whitney *U* test was used to calculate the differences between the means of different experimental groups (three repeats per experiment, three wells per repeat, five images per well analyzed).

#### *3.4. Astaxanthin Reduces ROS through Upregulation of Phase II Enzymes*

The Phase II enzymes HO-1 and NQO1, which exhibit antioxidative properties and are known to reduce intracellular ROS, have been reported to be upregulated by AST [**???** ]. To confirm if the reduction in ROS induced by AST in our study was associated with a change in Phase II enzyme expression, we evaluated both the mRNA and protein levels of HO-1 and NQO1. We found that Phase II enzymes were upregulated both at the mRNA and protein levels after AST treatment in a dose-dependent fashion (Figure **??**).

**Figure 4.** AST promotes the expression of Phase II enzymes HO-1 and NQO1 in 661W cells. The expression of HO-1 and NQO1 was determined in AST-treated 661W cells by measuring the mRNA levels with qPCR (**A**) and the corresponding protein levels by western blot (**B**). The measurements were normalized to GAPDH. AST = astaxanthin; HO-1 = Heme oxygenase-1; HG = high glucose; LG = low glucose; NQO1 = NAD(P)H dehydrogenase. \* Significantly different from control group (*p* < 0.05); # significantly different from high glucose-treated group without AST (*p* < 0.05). The Mann–Whitney *U* test was used to calculate the differences between the means of different experimental groups.

#### *3.5. Astaxanthin Activates PI3K*/*Akt Pathway and Upregulates the Expression of Nrf2*

We further evaluated the activation of the PI3K/Akt pathway and the expression of Nrf2 under AST treatment to better understand how AST protects 661W cells from high glucose-induced damage. Treatment of 661W cells cultured in high glucose with 20 and 50 μM AST increased PI3K protein levels, which further increased the downstream p-Akt/Akt ratio (Figure **??**A,B). In the same culture conditions, we found increased nuclear expression of Nrf2 upon treatment with AST (Figure **??**C). Moreover, activation of Nrf2 was observed 30 min after treatment with 50 μM AST (Figure **??**D). These results indicate that AST activates the PI3K/Akt/Nrf2 pathway, which further contributes to decreasing the ROS generated in a high glucose environment.

**Figure 5.** AST upregulates the expression of the PI3K/Akt/Nrf2 pathway in 661W cells. (**A**,**B**) The expression of PI3K and phosphorylated Akt proteins were detected by western blot. 661W cells were pretreated with different concentrations of AST for 2 h and grown in high glucose for 24 h. (**C**) Nrf2 levels in nuclear protein extracts were determined by electrophoretic mobility shift assay. (**D**) Immunocytochemistry confirmed the increased expression of Nrf2 after AST treatment. AST = astaxanthin; HG = high glucose; LG = low glucose. \* Significantly different from control group (*p* < 0.05); # significantly different from high glucose-treated group without AST (*p* < 0.05). The Mann–Whitney *U* test was used to calculate the differences between the means of different experimental groups.

#### *3.6. Inhibition of Both PI3K and Nrf2 Attenuate the Antioxidative E*ff*ect of AST*

To confirm whether the protective effects of AST act through the PI3K/Akt/Nrf2 pathway, we added the PI3K inhibitor LY294002 (20 μM) and Nrf2 inhibitor ML385 (10 μM) and observed the corresponding changes in Phase II enzyme production and ROS levels. In the presence of PI3K inhibitor, downstream p-Akt decreased accordingly. Moreover, the levels of both HO-1 and NQO1 were lower upon treatment with the PI3K inhibitor than with AST alone (Figure **??**A). The Nrf2 inhibitor also downregulated the AST-enhanced expression of HO-1 and NQO1 while not affecting p-Akt. Both PI3K and Nrf2 inhibitors attenuated the reduction in ROS induced by AST (Figure **??**B). These results confirmed the sequential changes and causal relationship between PI3K/Akt pathway, Nrf2 and Phase II enzyme expression, and apoptosis with the protective effects of AST in photoreceptor cells.

**Figure 6.** Blocking the PI3K/Akt/Nrf2 pathway abolishes the antioxidative effect of AST in 661W cells. Cells were administered PI3K inhibitor (LY294002, 20 μM) and Nrf2 inhibitor (ML385, 10 μM) along with AST. (**A**) The protein expression of Akt, p-Akt, HO-1, and NQO1 was detected by western blot. The PI3K inhibitor resulted in lower p-Akt/Akt ratio, HO-1, and NQO1 expression compared with AST treatment alone. Meanwhile, Nrf2 inhibition resulted in decreased expression of HO-1 and NQO1 but not the p-Akt/Akt ratio. (**B**) Both inhibitors counteracted the protective effect of AST and significantly increased the ROS levels, AST = astaxanthin; ROS = reactive oxygen species. \* Significantly different from control group (*p* < 0.05); # significantly different from high glucose-treated group without AST (*p* < 0.05); @ significantly different from high glucose-treated group with 50 μM AST. The Mann–Whitney *U* test was used to calculate the differences between the means of different experimental groups (three repeats per experiment, three wells per repeat, five images per well analyzed).
