Antioxidative Effects of Chrysoeriol via Activation of the Nrf2 Signaling Pathway and Modulation of Mitochondrial Function

Retinal pigment epithelium (RPE) cell dysfunction caused by excessive oxidative damage is partly involved in age-related macular degeneration, which is among the leading causes of visual impairment in elderly people. Here, we investigated the protective role of chrysoeriol against hydrogen peroxide (H2O2)-induced oxidative stress in RPE cells. The cellular viability, reactive oxygen species (ROS) generation, and mitochondrial function of retinal ARPE-19 cells were monitored under oxidative stress or pre-treatment with chrysoeriol. The expression levels of mitochondrial-related genes and associated transcription factors were assessed using reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Moreover, the protein expression of antioxidant signal molecules was characterized by Western blot analysis. Chrysoeriol significantly increased cell viability, reduced ROS generation, and increased the occurrence of antioxidant molecules in H2O2-treated ARPE-19 cells. Additionally, mitochondrial dysfunction caused by H2O2-induced oxidative stress was also considerably diminished by chrysoeriol treatment, which reduced the mitochondrial membrane potential (MMP) and upregulated mitochondrial-associated genes and proteins. Chrysoeriol also markedly enhanced key transcription factors (Nrf2) and antioxidant-associated genes (particularly HO-1 and NQO-1). Therefore, our study confirms the protective effect of chrysoeriol against H2O2-induced oxidative stress in RPE cells, thus confirming that it may prevent mitochondrial dysfunction by upregulating antioxidant-related molecules.


Introduction
Age-related macular degeneration (AMD) is a serious retinal disease that causes irreversible loss of central visual function primarily in the elderly populations of developed countries. AMD is classified into two major forms-a non-neovascular dry form and a neovascular exudative wet form [1,2]. Dry AMD (atrophy-associated AMD) causes alterations in the pigment cell layer, which contains the retinal pigment epithelium (RPE) and sub-retinal sediments, due to lipid and protein accumulation between these cells and Bruch's membrane (also known as drusen). These processes eventually result in RPE cell death, photoreceptor malfunction, and central vision loss [1][2][3]. Currently, anti-vascular endothelial growth factor (anti-VEGF) therapy has been successfully used to treat wet AMD; however, effective treatments for dry AMD are not available yet [4,5] Although the etiological mechanisms of dry AMD are not known, patients with this condition exhibit elevated oxidative stress and inflammation in the RPE [5][6][7]. RPE cells exhibit high metabolic rates with enriched mitochondrial populations, and the oxidative (C) Cells were treated with H2O2 (0.125-2 mM) for 24 h, and the cell viability was measured. (D) Cells were pre-treated with CHE at the indicated concentrations or 0.1% DMSO (vehicle control) for 2 h and then incubated with or without 500 μM H2O2 for 24 h. N-acetyl-cysteine (NAC, 4 mM) was used as an antioxidant control. Cell viability was measured via the CCK-8 assay (B, C, and D). * p < 0.05, ** p < 0.01, *** p < 0.001, versus the control group; ## p < 0.01, ### p < 0.001, versus the H2O2-treated group.

Chrysoeriol Protected ARPE-19 Cells Against H2O2-induced Death
To evaluate the optimal concentration of chrysoeriol ( Figure 1A) to be used without causing cytotoxicity, ARPE-19 cells were incubated with various concentrations of chrysoeriol for 24, 48, and 72 h. As shown in Figure 1B, chrysoeriol (2.5-10 μM) did not exert any visible cytotoxic effect on ARPE-19 cells compared with the control group. In contrast, chrysoeriol concentrations between 20 and 40 μM decreased the cell viability

Chrysoeriol Protected ARPE-19 Cells against H 2 O 2 -Induced Death
To evaluate the optimal concentration of chrysoeriol ( Figure 1A) to be used without causing cytotoxicity, ARPE-19 cells were incubated with various concentrations of chrysoeriol for 24, 48, and 72 h. As shown in Figure 1B, chrysoeriol (2.5-10 µM) did not exert any visible cytotoxic effect on ARPE-19 cells compared with the control group. In contrast, chrysoeriol concentrations between 20 and 40 µM decreased the cell viability after 48 and 72 h. Therefore, chrysoeriol was administered at the concentrations of 2.5 and 5 µM in the subsequent experiments. To determine a suitable H 2 O 2 concentration for the induction of oxidative damage, the cells were exposed to various doses of H 2 O 2 for 24 h. H 2 O 2 significantly reduced the cell viability to approximately 55% when used at 500 µM, and thus this concentration was utilized in the subsequent experiments (IC 50; 528 ± 4.2 µM, Figure 1C). To test the protective effect of chrysoeriol on H 2 O 2 -induced cell death, the cells were pre-treated with chrysoeriol for 2 h before being exposed to H 2 O 2 for 24 h. As illustrated in Figure 1D, the pre-treatment with chrysoeriol significantly prevented the H 2 O 2 -induced death of ARPE-19 cells almost as much as the pre-treatment with N-acetyl-cysteine (NAC), used as an antioxidant control.

Chrysoeriol Suppressed H 2 O 2 -Induced Oxidative Stress in ARPE-19 Cells
Excessive ROS accumulation is considered to be one of the primary sources of cell damage. Using the fluorescence probes 2,7-dichlorofluorescein diacetate (H 2 DCFDA) and dihydroethidium (DHE), the intracellular ROS levels in H 2 O 2 -treated ARPE-19 cells were quantitated. As shown in Figure 2, 500 µM H 2 O 2 significantly increased the fluorescent intensities of H 2 DCFDA (A) and DHE (B) in the ARPE-19 cells compared with the control group. However, pre-treating ARPE-19 cells with chrysoeriol substantially decreased the ROS levels compared with the levels in the cells treated with H 2 O 2 alone (Figure 2A,B). The suppressive activity of chrysoeriol was also evident in the H 2 DCFDA fluorescence image ( Figure 2C). Additionally, the ROS levels in the cells treated with chrysoeriol alone were not significantly different than the levels in untreated cells. To examine the potential protective properties of chrysoeriol against oxidative damage, the expression levels of genes encoding the antioxidant proteins SOD1, SOD2, catalase (CAT), and GPx were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ( Figure 3A). According to our gene expression analyses, chrysoeriol pre-treatment significantly increased the expression of antioxidant genes (particularly SOD2 and GPx) compared with the expression levels in the cells treated with H 2 O 2 alone. Additionally, chrysoeriol pre-treatment, versus H 2 O 2 treatment alone, markedly upregulated HO-1, NQO-1, and Nrf2 expression ( Figure 3B). Moreover, as shown in Figure 3C, pre-treatment with chrysoeriol markedly enhanced Nrf2 protein expression levels, which was largely abrogated by treatment with H 2 O 2 . Although the expression of HO-1 and NQO-1 was lower than that of Nrf2, these genes exhibited the same protein expression trends as Nrf2 ( Figure 3C,D). Interestingly, Nrf2 protein level significantly increased by chrysoeriol treatment alone in normal ARPE-19 cells. These results suggest that chrysoeriol is a potent activator of Nrf2 expression in ARPE-19 cells.
pared with the expression levels in the cells treated with H2O2 alone. Additionally, chrysoeriol pre-treatment, versus H2O2 treatment alone, markedly upregulated HO-1, NQO-1, and Nrf2 expression ( Figure 3B). Moreover, as shown in Figure 3C, pre-treatment with chrysoeriol markedly enhanced Nrf2 protein expression levels, which was largely abrogated by treatment with H2O2. Although the expression of HO-1 and NQO-1 was lower than that of Nrf2, these genes exhibited the same protein expression trends as Nrf2 ( Figure 3C,D). Interestingly, Nrf2 protein level significantly increased by chrysoeriol treatment alone in normal ARPE-19 cells. These results suggest that chrysoeriol is a potent activator of Nrf2 expression in ARPE-19 cells.   Nrf2 expression level was plotted relative to the control level. * p < 0.05, ** p < 0.01, *** p < 0.001, versus the control group; # p < 0.05, ## p < 0.01, ### p < 0.01, versus the H2O2-treated group.

Chrysoeriol Enhanced Mitochondrial Function by Upregulating Mitochondrion-Related Genes
Growing evidence has linked the activation of Nrf2 expression to mitochondrial function and integrity under stressful conditions [21][22][23]. Therefore, to further determine whether chrysoeriol is involved in mitochondrial function, the mitochondrial membrane potential (MMP, ∆Ψm) of ARPE-19 cells was characterized by analyzing their red/green fluorescence intensity ratio via the JC-10 assay. As shown in Figure 4A, the ARPE-19 cells exposed to 500 μM H2O2 exhibited a reduced red/green fluorescence intensity ratio, indicating ∆Ψm dissipation, as in the cells treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP; i.e., a mitochondrial oxidative phosphorylation uncoupler). However, pre-treatment with chrysoeriol at 2.5 or 5 μM for 2 h improved the H2O2-induced ∆Ψm Figure 3. Involvement of the HO-1/ Nrf2 axis in the antioxidant effects of CHE. Cells were pre-treated with 2.5 or 5 µM CHE or 0.1% DMSO (vehicle control) for 2 h and then incubated with or without 500 µM H 2 O 2 for an additional 24 h. Afterwards, the cells were harvested to extract RNA and protein. Expression analysis of antioxidant marker genes via RT-qPCR. All the gene expression data were analyzed using Student's t-test (A,B). (C) Cells were pre-treated with 5 µM CHE or 0.1% DMSO for 2 h and then incubated with or without 500 µM H 2 O 2 for an additional 24 h. HO-1, NQO-1, and Nrf2 protein levels were quantified via Western blot analysis. (D) HO-1, NQO-1, or Nrf2 expression level was plotted relative to the control level. * p < 0.05, ** p < 0.01, *** p < 0.001, versus the control group; # p < 0.05, ## p < 0.01, ### p < 0.01, versus the H 2 O 2 -treated group.

Chrysoeriol Enhanced Mitochondrial Function by Upregulating Mitochondrion-Related Genes
Growing evidence has linked the activation of Nrf2 expression to mitochondrial function and integrity under stressful conditions [21][22][23]. Therefore, to further determine whether chrysoeriol is involved in mitochondrial function, the mitochondrial membrane potential (MMP, ∆Ψm) of ARPE-19 cells was characterized by analyzing their red/green fluorescence intensity ratio via the JC-10 assay. As shown in Figure 4A, the ARPE-19 cells exposed to 500 µM H 2 O 2 exhibited a reduced red/green fluorescence intensity ratio, indicating ∆Ψm dissipation, as in the cells treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP; i.e., a mitochondrial oxidative phosphorylation uncoupler). However, pre-treatment with chrysoeriol at 2.5 or 5 µM for 2 h improved the H 2 O 2 -induced ∆Ψm reduction similarly to the antioxidant positive control (NAC) ( Figure 4A). Additionally, chrysoeriol alone did not have a significant effect on the MMP of ARPE-19 cells compared with the control cells. To understand the underlying mechanisms by which chrysoeriol protects mitochondrial function, the expression of mitochondrial respiration and mitochondrial dynamics genes was studied via RT-qPCR. As illustrated in Figure 4B, the mRNA levels of mitochondrial transcription factors (transcription factor A, mitochondrial TFAM) and DNA replication genes (polymerase (DNA-directed), gamma (POLG)) were significantly increased by chrysoeriol pre-treatment compared with H 2 O 2 -alone treatment. Moreover, the expression levels of oxidative phosphorylation (OXPHOS)-associated genes, including ATP synthase subunit O (ATP5O), COX4I1 (cytochrome c oxidase subunit 4 isoform 1), cytochrome c oxidase subunit 5B (COX5b), and NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5 (NDUFB5), were significantly increased by chrysoeriol pre-treatment compared with H 2 O 2 -alone treatment or DMSO treatment ( Figure 4C). Moreover, the expression levels of genes related to mitochondrial dynamics, such as fission 1 (FIS1) and mitofusin 1 and 2 (MFN1 and 2), were significantly upregulated in the cells pre-treated with chrysoeriol compared with the levels in H 2 O 2 -alone-treated or control cells ( Figure 4D). These results suggest that the protective effect of chrysoeriol against H 2 O 2 -induced cell damage in ARPE-19 cells is mediated through the upregulation of mitochondrial biogenesis genes.
Molecules 2021, 26, x FOR PEER REVIEW 6 of 16 subunit 4 isoform 1), cytochrome c oxidase subunit 5B (COX5b), and NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5 (NDUFB5), were significantly increased by chrysoeriol pre-treatment compared with H2O2-alone treatment or DMSO treatment ( Figure 4C). Moreover, the expression levels of genes related to mitochondrial dynamics, such as fission 1 (FIS1) and mitofusin 1 and 2 (MFN1 and 2), were significantly upregulated in the cells pre-treated with chrysoeriol compared with the levels in H2O2-alone-treated or control cells ( Figure 4D). These results suggest that the protective effect of chrysoeriol against H2O2-induced cell damage in ARPE-19 cells is mediated through the upregulation of mitochondrial biogenesis genes.

Chrysoeriol Regulated Mitochondrial Process Proteins
Oxidative stress is one of the major contributors to mitochondrial function, due to its modulatory effects on mitochondrial fusion and fission dynamics [24][25][26]. To further characterize the protective role of chrysoeriol in mitochondrial processes triggered by H2O2, Western blot analyses were conducted to quantify the levels of crucial mitochondrial pro-

Chrysoeriol Regulated Mitochondrial Process Proteins
Oxidative stress is one of the major contributors to mitochondrial function, due to its modulatory effects on mitochondrial fusion and fission dynamics [24][25][26]. To further characterize the protective role of chrysoeriol in mitochondrial processes triggered by H 2 O 2, Western blot analyses were conducted to quantify the levels of crucial mitochondrial proteins. As shown in Figure 5, when ARPE-19 cells were exposed to chrysoeriol, TOM-20 (i.e., an outer mitochondrial membrane (OMM) marker) and MFN2 were significantly upregulated compared with the levels in cells treated with H 2 O 2 alone or in the standard control. Interestingly, 5 µM chrysoeriol treatment alone upregulated TOM20 but not MFN2. Compared with the levels in the control cells, 500 µM H 2 O 2 upregulated optic atrophy protein 1 (OPA1) and dynamin-related protein 1 (DRP1). However, chrysoeriol pre-treatment increased OPA1 and decreased DRP1 levels compared with the levels in the H 2 O 2 -alonetreated group. Additionally, DRP1 Ser 616 phosphorylation was substantially reduced upon chrysoeriol pre-treatment compared with H 2 O 2 -alone treatment ( Figure 5B). All expression levels were normalized to those of GAPDH. Together, our findings demonstrate that chrysoeriol may balance mitochondrial dynamics in ARPE-19 cells. MFN2. Compared with the levels in the control cells, 500 μM H2O2 upregulated optic atrophy protein 1 (OPA1) and dynamin-related protein 1 (DRP1). However, chrysoeriol pretreatment increased OPA1 and decreased DRP1 levels compared with the levels in the H2O2-alone-treated group. Additionally, DRP1 Ser 616 phosphorylation was substantially reduced upon chrysoeriol pre-treatment compared with H2O2-alone treatment ( Figure  5B). All expression levels were normalized to those of GAPDH. Together, our findings demonstrate that chrysoeriol may balance mitochondrial dynamics in ARPE-19 cells.

Chrysoeriol-Mediated Activation of p38 and Mitochondrial-Related Genes
The p38 mitogen-activated protein kinase (MAPK) activation is related to redox signaling transduction via stimulation of stress responses, including mitochondrial dysfunction [27][28][29][30][31]. Therefore, the protective antioxidant effect of chrysoeriol on the p38 MAPK pathway was determined in ARPE-19 cells. Western blot analysis results reveal that the total intracellular p38 level was largely unaffected; however, the activated form of p38 (pp38) was significantly upregulated in the H2O2-treated group. In contrast, when ARPE-19 cells were co-exposed to chrysoeriol and H2O2, the pp38 protein level was substantially decreased compared with that of the H2O2 control ( Figures 6A,B). Next, we sought to determine whether MAPK inhibitors mediate the phosphorylation of p38 and the protective effects of chrysoeriol. Cell viability was increased in the cells pre-treated with chrysoeriol compared with H2O2-alone-treated cells. The antioxidant effects of chrysoeriol were not affected by the other inhibitors (PD 98059; MEK/ERK pathway inhibitor, SP600125; c-Jun N-terminal kinase (JNK) inhibitor, and LY 294002; phosphatidylinositol 3-kinase (PI3K) inhibitor). However, treatment with SB203580 (p38 inhibitor) significantly decreased cell

Chrysoeriol-Mediated Activation of p38 and Mitochondrial-Related Genes
The p38 mitogen-activated protein kinase (MAPK) activation is related to redox signaling transduction via stimulation of stress responses, including mitochondrial dysfunction [27][28][29][30][31]. Therefore, the protective antioxidant effect of chrysoeriol on the p38 MAPK pathway was determined in ARPE-19 cells. Western blot analysis results reveal that the total intracellular p38 level was largely unaffected; however, the activated form of p38 (pp38) was significantly upregulated in the H 2 O 2 -treated group. In contrast, when ARPE-19 cells were co-exposed to chrysoeriol and H 2 O 2 , the pp38 protein level was substantially decreased compared with that of the H 2 O 2 control ( Figure 6A,B). Next, we sought to determine whether MAPK inhibitors mediate the phosphorylation of p38 and the protective effects of chrysoeriol. Cell viability was increased in the cells pre-treated with chrysoeriol compared with H 2 O 2 -alone-treated cells. The antioxidant effects of chrysoeriol were not affected by the other inhibitors (PD 98059; MEK/ERK pathway inhibitor, SP600125; c-Jun N-terminal kinase (JNK) inhibitor, and LY 294002; phosphatidylinositol 3-kinase (PI3K) inhibitor). However, treatment with SB203580 (p38 inhibitor) significantly decreased cell viability in the chrysoeriol pre-treatment group ( Figure 6C). To characterize the role of p38 activation in Nrf2 signaling regulation, ARPE-19 cells treated with a p38 inhibitor were also assessed by Western blot analysis. The chrysoeriol and H 2 O 2 co-treated group exhibited an upregulation in Nrf2 and HO-1 expression, whereas the SB203580-treated group exhibited a decrease in Nrf2 and HO-1 expression ( Figure 6D). The protein expression levels in Figure 6D were normalized to those of GAPDH ( Figure 6E). SB203580 significantly blocked chrysoeriol-induced antioxidant molecules such as Nrf2 and HO-1 compared to the chrysoeriol and H 2 O 2 co-treatment group. These findings indicate that the activation of the p38 pathway mediates the antioxidant effects of chrysoeriol and the activation of Nrf2 signaling in H 2 O 2 -induced damaged ARPE-19 cells. group exhibited a decrease in Nrf2 and HO-1 expression ( Figure 6D). The protein expression levels in Figure 6D were normalized to those of GAPDH ( Figure 6E). SB203580 significantly blocked chrysoeriol-induced antioxidant molecules such as Nrf2 and HO-1 compared to the chrysoeriol and H2O2 co-treatment group. These findings indicate that the activation of the p38 pathway mediates the antioxidant effects of chrysoeriol and the activation of Nrf2 signaling in H2O2-induced damaged ARPE-19 cells. The quantitative analysis of the p38, pp38, HO-1, NQO-1, and Nrf2 levels was performed via densitometric measurements relative to the control. ** p < 0.01, *** p < 0.001, versus control group; ## p < 0.01, ### p < 0.001, versus the H2O2-treated group; ++ p < 0.01, +++ p < 0.001, versus the H2O2 and chrysoeriol-treated group.

Chrysoeriol Protected ARPE-19 Cells from Sodium Iodate-Induced Oxidative Damage
Sodium iodate (NaIO3) is an oxidizing agent that promotes ROS generation and is specifically toxic to RPE cells by inducing mitochondrial dysfunction [32][33][34][35]. To investigate this effect further, cells were exposed to NaIO3. A NaIO3 concentration of 20 mM sig-  [32][33][34][35]. To investigate this effect further, cells were exposed to NaIO 3 . A NaIO 3 concentration of 20 mM significantly reduced cell viability by approximately 50%, whereas chrysoeriol pre-treatment significantly suppressed the NaIO 3 -induced reduction in ARPE-19 cell viability ( Figure 7A). As shown in Figure 7B, compared with the control group, 20 mM NaIO 3 caused a significant increase in the fluorescence intensities of H 2 DCFDA (B) and DHE (C) in the ARPE-19 cells. However, pre-treatment with chrysoeriol in the ARPE-19 cells substantially suppressed these increases compared with the levels in NaIO 3 treatment alone ( Figure 7B,C). When exposed to 20 mM NaIO 3 , the cells exhibited a reduction in MMP, whereas pre-treatment with chrysoeriol for 2 h suppressed this effect compared with the cells exposed to NaIO 3 alone ( Figure 7D). Together, our findings suggest that chrysoeriol could effectively protect RPE cells against pathological oxidative damage.

Sodium iodate (NaIO 3 ) is an oxidizing agent that promotes ROS generation and is specifically toxic to RPE cells by inducing mitochondrial dysfunction
Molecules 2021, 26, x FOR PEER REVIEW 9 of 16 ARPE-19 cells. However, pre-treatment with chrysoeriol in the ARPE-19 cells substantially suppressed these increases compared with the levels in NaIO3 treatment alone (Figure 7B,C). When exposed to 20 mM NaIO3, the cells exhibited a reduction in MMP, whereas pre-treatment with chrysoeriol for 2 h suppressed this effect compared with the cells exposed to NaIO3 alone ( Figure 7D). Together, our findings suggest that chrysoeriol could effectively protect RPE cells against pathological oxidative damage.

Discussion
Age is a key risk factor for the development of AMD, which is largely caused by oxidative stress resulting from elevated ROS levels. AMD is characterized by abnormal RPE cell layers, whereby superimposing foveal photoreceptor dysfunction ensues. Additionally, substantial oxidative stress build-up may fully disrupt the antioxidant systems and result in irreversible retinal damage [2,36]. Here, we investigated the protective effects of chrysoeriol (a flavonoid compound) against oxidative damage in human RPE cells. At the mitochondrial level, chrysoeriol exerts its protective effects via MMP mediation and

Discussion
Age is a key risk factor for the development of AMD, which is largely caused by oxidative stress resulting from elevated ROS levels. AMD is characterized by abnormal RPE cell layers, whereby superimposing foveal photoreceptor dysfunction ensues. Additionally, substantial oxidative stress build-up may fully disrupt the antioxidant systems and result in irreversible retinal damage [2,36]. Here, we investigated the protective effects of chrysoeriol (a flavonoid compound) against oxidative damage in human RPE cells. At the mitochondrial level, chrysoeriol exerts its protective effects via MMP mediation and its related effector genes, including OXPHOS genes, mitochondrial process genes, and mitochondrial DNA replication and transcription genes. Notably, chrysoeriol may prevent oxidative damage by increasing the expression of antioxidant enzymes, mainly HO-1 and SOD2, via the upregulation of Nrf2 and mitochondrial molecules in ARPE-19 cells. Furthermore, we confirmed that the p38 signaling pathway was involved in chrysoeriol-mediated RPE cell death. Therefore, our findings highlight the potential therapeutic applicability of chrysoeriol to prevent or treat AMD, a disease initiated by cell death caused by oxidative stress and RPE dysfunction.
These findings indicate that chrysoeriol may possess the ability to scavenge oxygen free radicals, thereby indirectly combating oxidative stress. Numerous studies have reported that Nrf2 activation is a major regulator of several antioxidant and detoxification genes in RPE cells, including downstream targets of Nrf2 [39][40][41][42][43][44]. Our study suggests that chrysoeriol activates Nrf2 and increases HO-1 and NQO-1 activity in H 2 O 2 -exposed cells, as demonstrated by RT-qPCR and Western blot analyses.
Mitochondrial dysfunction is a key factor that leads to AMD pathological changes, including MMP (∆Ψm) reduction and mitochondrial DNA damage [45,46]. Additionally, previous studies have reported an overall reduction in mitochondria numbers in the RPE of elderly individuals, which was even more severe in AMD patients [47][48][49]. In this study, we found that pre-treatment with chrysoeriol significantly increased the MMP compared to H 2 O 2 treatment alone. Next, we analyzed mitochondrial-related gene expression, and our results demonstrate that chrysoeriol significantly upregulated TFAM, POLG, ATP5O, COX4I1, COX5B, NDUFB5 MFN1, and MFN2. Mitochondria continuously undergo fission and fusion processes to maintain mitochondrial function. Mitochondrial fission and fusion enable the recycling of damaged elements via the segregation of injured organelles and the exchange of materials with healthy mitochondria [50][51][52]. DRP1 is a key mediator of mitochondrial fission, whereas OPA1 is pivotal for mitochondrial membrane fusion and maintaining proper mitochondrial cristae architecture [50,53,54]. Therefore, we investigated the expression of crucial mitochondrial process genes including TOM20, MFN2, OPA1, and DRP1. Our results indicated that chrysoeriol significantly increased TOM20, MFN2, and OPA1 and decreased DRP1, whereas H 2 O 2 induced DRP1 activation. Thus, chrysoeriol may improve mitochondrial function and biogenesis, thereby alleviating H 2 O 2 -induced oxidative stress.
MAPKs, ERK1/2, p38, and JNKs, all of which are well-characterized mitogen-activated protein kinases, are known to be activated by oxidative stress and are involved in cell growth and death [55,56]. Increasing evidence suggests that regulating the MAPK signaling pathway, particularly p38 activation, is critical to protect cells from ROS injury and cellular death [57]. Here, H 2 O 2 -induced oxidative stress significantly promoted p38 phospho-rylation [58]. Compared with the H 2 O 2 group, p38 phosphorylation was lower in the chrysoeriol group, suggesting that chrysoeriol exerts its antioxidant effect through the modulation of the ROS-mediated p38 MAPK signaling pathway. Next, we confirmed that chrysoeriol treatment decreased p38 phosphorylation, whereas pre-treatment with a p38 inhibitor and chrysoeriol attenuated the cytoprotective effect of chrysoeriol against H 2 O 2 in ARPE-19 cells. The p38 pathway negatively regulated the chrysoeriol-induced Nrf2/HO-1 expression, and treatment with a p38 inhibitor reduced the chrysoeriol-induced antioxidant protein expression. Recent studies have reported that p38 induces mitochondrial fragmentation by mediating DRP1. It has also been reported that PKCδ-activated p38 MAPK directly phosphorylates DRP1 to induce its mitochondrial translocation and subsequent mitochondrial fission. Based on our results, inhibition of p38 MAPK prevents mitochondrial dysfunction via inhibition of DRP1 phosphorylation and activation of Nrf2/HO-1; thus, our study demonstrates that chrysoeriol could lead to improved mitochondrial function and biogenesis against H 2 O 2 -induced oxidative stress in ARPE-19 cells. Furthermore, we demonstrate that chrysoeriol not only exerts antioxidant effects in H 2 O 2 -exposed cells but also in cells exposed to NaIO 3 , an RPE-specific oxidizing agent that induces mitochondrial dysfunction.
In conclusion, our study demonstrates the protective effect of chrysoeriol against oxidative damage to ARPE-19 cells in experimental conditions that were meant to mimic AMD pathological development. Chrysoeriol exhibited potent antioxidant effects, as observed by increased cell survival, increased antioxidant enzyme expression, decreased ROS accumulation, increased OXPHOS, and increased mitochondrial-related gene expression, leading to increased MMP and mitochondrial function via the modulation of mitochondrial gene expression. The mechanisms by which chrysoeriol exerted these effects included the regulation of mitochondrial quality control molecules (e.g., DRP1 and OPA1) via the modulation of p38/ Nrf2/HO-1 signaling, as well as an increase in antioxidant molecules. Our study is the first to demonstrate the potential therapeutic applicability of chrysoeriol for the prevention of dry AMD. However, further studies are needed to determine the physiological function and biological efficacy of chrysoeriol in both primary human RPE cells (or at least fully differentiated ARPE-19 cell models) and in vivo models.

The Source of Chrysoeriol
We cultivated perilla (cultivar name Anyu, Perilla frutescens) seeds in an experimental field at the National Institute of Crop Science (NICS) in the Rural Development Administration (RDA), Jeonbuk, Korea, 2019. After harvesting, perilla seeds were immediately freeze-dried and stored at −40 • C.

Cell Culture and Cell Viability Assay
ARPE-19 cells were routinely maintained in DMEM/F12 media supplemented with 10% FBS and 1% penicillin/streptomycin at 37 • C in a 5% CO 2 atmosphere. Cell viability was assessed using the CCK-8 assay according to the manufacturer's instructions. Each experiment was repeated three times with triplicate samples.

ROS Measurement
Intracellular ROS levels were examined using H 2 DCF-DA or DHE according to the manufacturer's instructions. The fluorescence intensity was measured using a fluorescence plate reader (Bio-Tek) at Ex/Em = 495/527 nm for H 2 DCF-DA and Ex/Em = 535/610 nm for DHE. Afterward, H 2 DCFDA-stained cell images were obtained using an IX51 fluorescent microscope coupled with a DP microscope camera controller (Olympus Optical, Japan).

Mitochondrial Membrane Potential (MMP) Assay
The MMP assay was conducted using the JC-10 MMP assay kit (Merck, St. Louis, MO, USA) according to the manufacturer's instructions. In brief, ARPE-19 cells (5 × 10 3 cells/well) seeded in a 96-well transparent-bottom black plate (Eppendorf Ltd., Germany) were treated as described above. After 24 h, the JC-10 dye solution (JC-10 and assay buffer A 1:100 v/v) was added (50 µL/well) to the control and treated cells. Following the treatment, the plate was incubated in dark conditions for 30 min. Afterward, assay buffer B (50 µL/well) was added, and the fluorescence intensity was measured at 490/525 nm (red) and 540/590 nm (green) using a multimode plate reader (Bio-Tek). The red/green fluorescence intensity ratio was used to determine the MMP. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and N-acetyl-cysteine (NAC) were used as negative and antioxidant controls, respectively.

Western Blotting
Cells were lysed in RIPA buffer with protease inhibitors. An equal amount of protein was separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk in TBST, incubated with a primary antibody overnight at 4 • C, and then allowed to react with the secondary antibodies for 2 h at room temperature. Protein expression levels were evaluated with an enhanced chemiluminescence kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.