**3. Results**

#### *3.1. E*ff*ect of H2O2 on the Viability of ARPE-19 Cells and Intracellular ROS Level*

To establish the H2O2-induced oxidative stress model in ARPE-19 cells, different concentrations of H2O2 were treated to the cells and their viability and intracellular ROS level were measured. Viability measured with MTT assay decreased as the concentration of treated H2O2 increased. When cells were treated with 0.4 mM H2O2, they showed the viability of 66% and the viability change was the greatest between 0.2 mM and 0.6 mM (Figure 1A). Crystal violet assay resulted in a similar aspect of viability change as MTT assay with 69% of viability at 0.4 mM (Figure 1B). Intracellular ROS level increased dependently to the concentration of H2O2 (Figure 1C). The mean value of the ROS level measured in 0.8 mM H2O2 increased to 176% compared to the control group. This trend of decreased cell viability after the H2O2 exposure was confirmed in bright field imaging (Figure 1D,E). As cell viability changed rapidly at 0.4 mM H2O2, 0.4 mM was set to be a lethal dose of H2O2 and 0.2 mM was set to be sublethal dose.

**Figure 1.** Change of viability and intracellular ROS level in ARPE-19 cells after exposure to H2O2. The response of ARPE-19 cells to 0–0.8 mM H2O2 exposure for MTT assay (**A**), and crystal violet assay (**B**) to determine cell viability. For intracellular ROS level, DCFH-DA was treated for 30 min after the H2O2 exposure. Exposure to H2O2 reduced the cell viability (**A**,**B**) and increased the intracellular ROS level (**C**). The cell morphology was observed with bright field microscopy (Scale bar 500 μm) (**D**) and with higher magnification (scale bar 100 μm) (**E**). Asterisks indicate a significant reduction in cell viability or increment in ROS level compared with untreated cells (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; DCFH-DA, 2-,7--dichlorodihydrofluorescein diacetate.

#### *3.2. E*ff*ect of UVB Irradiation on the Viability of ARPE-19 Cells and Intracellular ROS*

To establish the UVB-induced oxidative stress model in ARPE-19 cells, different doses of UVB were exposed to the cells and their viability and intracellular ROS level were measured. Viability measured with MTT assay decreased as the dose of UVB irradiation increased. When cells were exposed to 20 mJ/cm<sup>2</sup> UVB, they showed the viability of 80% and with 100 mJ/cm<sup>2</sup> UVB, the viability was 60% (Figure 2A). In a crystal violet assay with the same range of UVB dose, the viability dropped to 78% at 20 mJ/cm<sup>2</sup> UVB and to 72% at 100 mJ/cm<sup>2</sup> UVB (Figure 2B). Intracellular ROS level increased dependently to the UVB dose (Figure 2C). The mean value of ROS level measured at 20 mJ/cm<sup>2</sup> UVB increased to 140% and 270% at 100 mJ/cm<sup>2</sup> UVB compared to the control group. Morphological change of the cells was observed in bright field imaging. Cells became rounder and holes in the monolayer were observed as UVB dose increased (Figure 2D,E). The sublethal dose of UVB was set to be 20 mJ/cm2, where the cells show 80% of viability without significant morphological change and 100 mJ/cm<sup>2</sup> where the cells show 60% of viability with morphological change was set to be the lethal dose of UVB.

**Figure 2.** Change of viability and intracellular ROS level in ARPE-19 cells after UVB irradiation. The response of ARPE-19 cells 24 h after 0–100 mJ/cm<sup>2</sup> UVB irradiation with MTT assay (**A**), and crystal violet assay (**B**) to determine cell viability. For intracellular ROS level, DCFH-DA was treated for 30 min after the UVB irradiation. Irradiation by UVB reduced the cell viability (**A**,**B**) and increased the intracellular ROS level (**C**). The cell morphology was observed with bright field microscopy (scale bar 500 μm) (**D**) and with higher magnification (scale bar 100 μm) (**E**). Asterisks indicate a significant reduction in cell viability or increment in ROS level compared with untreated cells (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). UVB, ultraviolet B; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; DCFH-DA, 2-,7--dichlorodihydrofluorescein diacetate.

#### *3.3. Antioxidative E*ff*ect of Ascorbic Acid and Astaxanthin by Scavenging DPPH*

DPPH scavenging assay was performed with ascorbic acid and astaxanthin. Ascorbic acid, at concentrations of 0.025 mM, 0.1 mM, 0.4 mM, and 1.6 mM, dissolved in DMSO was mixed with DPPH solution and each concentration scavenged 33%, 52%, 57%, 73% of DPPH, respectively, after 30 min of reaction (Figure 3A). When 75 μM, 85 μM, 95 μM, and 105 μM astaxanthin dissolved in DMSO were reacted with DPPH solution for 30 min, 44%, 50%, 64%, and 69% of DPPH were scavenged, respectively (Figure 3B). Both ascorbic acid and astaxanthin showed antioxidative effect.

**Figure 3.** DPPH scavenging activity of ascorbic acid and astaxanthin. The antioxidative capacities of ascorbic acid and astaxanthin were determined by their capabilities to scavenge DPPH. Ascorbic acid (0.025–1.6 mM) was reacted with DPPH (**A**), and astaxanthin (75–105 μM) was reacted with DPPH (**B**). The compounds were diluted in DMSO. Both compounds scavenged DPPH in dose-dependent way in 30 min of reaction time. Asterisks indicate a significant increment in DPPH scavenging activity compared with controls (\*\*\* *p* < 0.001). DPPH, 2,2-diphenyl-1-picrylhydrazyl; DMSO, dimethyl sulfoxide.

#### *3.4. Antioxidative E*ff*ect of Ascorbic Acid on ARPE-19 Cells Under H2O2-Induced Oxidative Stress*

ARPE-19 cells were pretreated with various concentrations of ascorbic acid or astaxanthin for 6 h and then they were treated together with H2O2 and the same concentrations of antioxidants for another 24 h. Viability was assessed after 3 h of MTT treatment. When groups treated together with ascorbic acid and H2O2 they showed increased viability compared to controls. Cells treated only with 0.2 mM H2O2 showed the viability of 80% and groups treated together with ascorbic acid showed 81%, 107%, and 126% of viability, respectively, for 250 μM, 500 μM, and 750 μM of the drug concentration. On the other hand, astaxanthin did not show any significant effect on the viability of ARPE-19 with H2O2-induced oxidative stress (Figure 4A). For 0.4 mM H2O2 treatment, cells treated only with H2O2 showed 58% of viability, while 250 μM, 500 μM, and 750μM of ascorbic acid increased the viability to 64%, 72%, and 95%, respectively. On the other hand, astaxanthin did not show any significant effect on the viability of ARPE-19 with H2O2-induced oxidative stress (Figure 4B).

**Figure 4.** Effect of ascorbic acid and astaxanthin on H2O2-induced oxidative stress model of ARPE-19. The effect of various concentration of ascorbic acid or astaxanthin (pretreated for 6 h and co-treated with H2O2 for 24 h) on the response of ARPE-19 cells to sublethal dose of 0.2 mM (**A**) or lethal dose of 0.4 mM H2O2 (**B**). The cell viability was determined by MTT assay. Treatment of ascorbic acid (500–750 μM) significantly increased ARPE-19 cell viability following 0.2 mM H2O2 exposure. However, astaxanthin (10–40 μM) did not significantly affect the cell viability (**A**). Ascorbic acid (500–750 μM) also significantly increased the cell viability under 0.4 mM H2O2 but astaxanthin (10–40 μM) did not have significant effect on the viability (**B**). Asterisks indicate a significant increment in cell viability compared with cells treated with H2O2 only (\* *p* < 0.05, \*\* *p* < 0.01). AA, ascorbic acid; AST, astaxanthin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

#### *3.5. Antioxidative E*ff*ect of Ascorbic Acid and Astaxanthin on ARPE-19 Cells Under UVB-induced Oxidative Stress*

ARPE-19 cells were pretreated with various concentrations of ascorbic acid or astaxanthin for 6 h and then they were irradiated with UVB. Viability 24 h after the irradiation was assessed with MTT assay. When cells were pretreated with ascorbic acid and then UVB irradiated with it, the cell viability increased compared to the UVB irradiation-only group. Cells irradiated only with 20 mJ/cm<sup>2</sup> UVB showed the viability of 85% and groups treated together with ascorbic acid showed 92%, 102%, and 130% of viability, respectively, for 250 μM, 500 μM, and 750 μM of the drug concentration. Astaxanthin treated cells also showed increased viability compared to the cells irradiated only with UVB. The 10 μM, 20 μM, and 40 μM astaxanthin groups showed 95%, 101%, and 102%, respectively, after 20 mJ/cm<sup>2</sup> UVB irradiation (Figure 5A). For 100 mJ/cm<sup>2</sup> UVB irradiation, the cells irradiated only with UVB showed 66% of viability while 250 μM, 500 μM, and 750 μM of ascorbic acid increased the viability to 68%, 78%, and 109%, respectively. Astaxanthin-treated cells also showed increased viability compared to the cells irradiated only with UVB. The 10 μM, 20 μM, and 40 μM astaxanthin groups showed 67%, 74%, and 83% after 100 mJ/cm<sup>2</sup> UVB irradiation (Figure 5B).

**Figure 5.** Effect of ascorbic acid and astaxanthin on UVB-induced oxidative stress model of ARPE-19. The effect of various concentration of ascorbic acid and astaxanthin (pretreated for 6 h and additional 24 h after UVB irradiation) on the response of ARPE-19 cells to sublethal dose of 20 mJ/cm<sup>2</sup> (**A**) or lethal dose of 100 mJ/cm<sup>2</sup> UVB (**B**). The cell viability was determined by MTT assay 24 h after the irradiation. Treatment of ascorbic acid (500–750 μM) and astaxanthin (20–40 μM) significantly increased ARPE-19 cell viability following 20 mJ/cm<sup>2</sup> UVB irradiation (**A**). Ascorbic acid (500–750 μM) and astaxanthin (20–40 μM) also significantly increased the cell viability after 100 mJ/cm<sup>2</sup> UVB irradiation (**B**). Asterisks indicate a significant increment in cell viability compared with cells treated with UVB only (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). UVB, ultraviolet B; AA, ascorbic acid; AST, astaxanthin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

#### *3.6. E*ff*ect of Ascorbic Acid on the Intracellular ROS Level of ARPE-19*

The effect of ascorbic acid on the intracellular ROS level of ARPE-19 cells was studied with DCFH-DA assay. The intracellular ROS level was measured after cells were treated with UVB with or without 500 μM ascorbic acid. UVB of 20 mJ/cm<sup>2</sup> and 100 mJ/cm<sup>2</sup> increased the intracellular ROS level to 123% and 234%, respectively, and 500 μM ascorbic acid treatment reduced the ROS level to 105% and 115% (Figure 6A). This trend between groups were confirmed with fluorescence microscopy (Figure 6B).

**Figure 6.** Intracellular ROS level of ARPE-19 after UVB treatment with ascorbic acid. The effects of ascorbic acid on the intracellular ROS level of ARPE-19 under UVB-induced oxidative stress were examined by DCFH-DA assay. Ascorbic acid at 500 μM significantly reduced the ROS level after UVB irradiation (20–100 mJ/cm2) compared to groups with UVB irradiation only (**A**). The green fluorescence of the reacted DCFH-DA which indicates the ROS level, was observed with fluorescence microscopy (scale bar 250 μm) (**B**). Asterisks indicate a significant reduction in ROS level compared with control cells only with UVB exposure without ascorbic acid treatment (\* *p* < 0.05, \*\* *p* < 0.01). ROS, reactive oxygen species; UVB, ultraviolet B; DCFH-DA, 2-,7--dichlorodihydrofluorescein diacetate; AA, ascorbic acid.

#### *3.7. Antioxidative E*ff*ect of Astaxanthin and Ascorbic Acid by Reducing Intracellular ROS in ARPE-19 Cells*

H2O2 of 0.2 mM and 0.4 mM increased the intracellular ROS level to 123% and 135% compared to the nontreated group, while ascorbic-acid-treated group showed reduced ROS level of 33% and 34%, respectively (Figure 7A).

ARPE-19 cells were pretreated with either 20 μM astaxanthin, 90 μM ascorbic acid, or a mixture of 20 μM astaxanthin and 90 μM ascorbic acid. When cells were exposed to 0.2 mM H2O2 for 24 h, the viability decreased to 75%. The 20-μM-astaxanthin- and 90-μM-ascorbic-acid-treatment could increase the viability to 97% and 93%, respectively. The mixture of 20 μM astaxanthin and 90 μM ascorbic acid increased the viability to 129% (Figure 7B). Each drug could also decrease the intracellular ROS level. When cells were treated with 0.2 mM H2O2 for 24 h, the intracellular ROS level increased to 200%. The 20-μM-astaxanthin- and 90-μM-ascorbic-acid-treatment reduced the ROS level to 169%, and 135%, respectively. The mixture of 20 μM astaxanthin and 90 μM ascorbic acid decreased the ROS level to 104% (Figure 7C).

**Figure 7.** Intracellular ROS level and cell viability of ARPE-19 after H2O2 exposure with ascorbic acid and the mixture of ascorbic acid and astaxanthin. Ascorbic acid at 500 μM significantly reduced the intracellular ROS level under sublethal and lethal dose of H2O2 (0.2–0.4 mM) compared to the control group without ascorbic acid treatment ( **A**). The e ffect of 20 μM astaxanthin, 90 μM ascorbic acid, and the mixture of the two compounds on the cell viability of ARPE-19 under H2O2-induced oxidative stress was examined by MTT assay. Cell viability was significantly increased when the cells were pretreated with 20 μM astaxanthin, 90 μM ascorbic acid, and the mixture of the two compounds for 6 h and with 0.2 mM H2O2 for 24 h, compared to H2O2 only (**B**). ROS level was significantly decreased when the cells were pretreated with 20 μM astaxanthin, 90 μM ascorbic acid, and the mixture of the two compounds for 6 h and with 0.2 mM H2O2 for 24 h, compared to H2O2 only. Asterisks indicate a significant di fference between increment in cell viability and reduction in intracellular ROS level compared to control cells only with H2O2 exposure without antioxidant treatment ( **C**). (\*\* *p* < 0.01, \*\*\* *p* < 0.001). AST, astaxanthin; AA, ascorbic acid; ROS, reactive oxygen species; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
