Temporary Upregulation of Nrf2 by Naringenin Alleviates Oxidative Damage in the Retina and ARPE-19 Cells

Dry age-related macular degeneration (dAMD) is a chronic degenerative ophthalmopathy that leads to serious burden of visual impairment. Antioxidation in retinal pigment epithelium (RPE) cells is considered as a potential treatment for dAMD. Our previous studies have showed that naringenin (NAR) protects RPE cells from oxidative damage partly through SIRT1-mediated antioxidation. In this study, we tested the hypothesis that the Nrf2 signaling is another protective mechanism of NAR on dAMD. NaIO3-induced mouse retinopathy and ARPE-19 cell injury models were established. Immunochemical staining, immunofluorescence, and western blotting were performed to detect the protein expressions of Nrf2 and HO-1. In addition, ML385 (activity inhibitor of Nrf2) and zinc protoporphyrin (ZnPP, activity inhibitor of HO-1) were applied to explore the effect of NaIO3 or NAR. The results showed that NAR increased the protein expressions of Nrf2 and HO-1 in the retinas in mice exposed to NaIO3 at the early stage. NAR treatment also resulted in a stronger activation of Nrf2 at the early stage in NaIO3-treated ARPE-19 cells. Moreover, inhibition of HO-1 by ZnPP weakened the cytoprotective effect of NAR. The constitutive accumulation and activation of Nrf2 induced by NaIO3 led to the death of RPE cells. However, NAR decreased the protein expressions of Nrf2 and HO-1 towards normal level in the mouse retinas and ARPE-19 cells exposed to NaIO3 at the late stage. Our findings indicate that NAR protects RPE cells from oxidative damage via activating the Nrf2 signaling pathway.


Introduction
Age-related macular degeneration (AMD) is a progressive chronic ophthalmopathy with aging and eventually results in serious visual impairment. Among patients with AMD, dry AMD (dAMD) accounts for 85%-90%, and some of which may develop into geographic atrophy (GA) and/or wet AMD (wAMD). In dAMD, retinal pigment epithelium (RPE) is accepted as the primary lesion, and photoreceptors are the second one that contributes to visual impairment [1,2].
Oxidative stress is considered as a central contributor to the progress of dAMD. Prooxidative factors such as aging, smoking, and sunlight lead to the oxidative insult in RPE cells, man-ifesting as the formation of deposit and drusen between the RPE and Bruch membrane (BrM). The accumulations of deposit and drusen boost the thickness of BrM. Besides causing the death of RPE, oxidative stress compels RPE to detach from BrM and then makes RPE losing function. In dAMD patients, RPE cells detached from BrM are going forward into the nerve fiber layer or backward into the BrM [3,4]. The displacement of RPE may be the result of epithelium-to-mesenchymal transition. As a consequence, RPE cells may survive at the cost of loss of function. The death and dysfunction of RPE result in poor nutrient supply of photoreceptors, which causes the atrophy of photoreceptors gradually [2]. Therefore, antioxidation targeting to RPE cells is a potential treatment for dAMD. Naringenin (NAR), 4 ′ ,5,7-trihydroxyflavanone, is rich in fruits such as grapefruit and citrus. In our previous report, topical administration of NAR eye drops improved retinal function and structure in murine with retina degeneration induced by NaIO 3 [5,6] or N-methyl-N-nitrosourea (MNU) [7]. Antioxidant activity of NAR on age-related neurodegenerative diseases has aroused special attention [8]. We found that NAR protects RPE cells from NaIO 3 -induced oxidative damage through upregulation of SIRT1 [6]. However, inhibition of SIRT1 decreased the protective effect of NAR on cell viability by 69.2% in ARPE-19 cells incubated with NaIO 3 , which suggests that other protective pathways of NAR may exist.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential regulator of redox homeostasis. Under normal condition, Nrf2 is suppressed by Kelch-like ECH-associated protein 1 (Keap1) and anchored in the cytoplasm. While under oxidative stress, Nrf2 is dissociated from Keap1 and transits to the nucleus [9]. Nrf2 aggregates in the nucleus and then heterodimerizes with small Maf proteins and binds to the antioxidant response element (ARE), which mediates the transcription of various antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), thioredoxin reductase (TrxR), peroxiredoxin (Prx), hemeoxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1) [10]. Several evidences have demonstrated that the expression of Nrf2 and its activity decrease during aging, meaning Nrf2 is an important regulator in cell senescence [11][12][13]. Recent studies have shown that aged Nrf2 -/mice develop dAMD-like pathology presenting as vacuolation of RPE, accumulation of lipofuscin, drusen-like deposits, and sub-RPE deposits of inflammatory proteins [14,15]. Upregulation of Nrf2 rescues retinal function and protects retinal cells from oxidative damage [16][17][18]. Therefore, upregulation of Nrf2 is a potential antioxidant strategy for dAMD. Therefore, in this study, we investigated whether Nrf2/HO-1-mediated antioxidant mechanism was involved in the protective effect of NAR in NaIO 3 -treated mice in vivo and ARPE-19 cells in vitro.

Materials and Methods
This study used the method of Chen et al., and the method description partly reproduced their wording [6].

NaIO 3 -Induced Retinal Degeneration and Animal
Treatment. Mice were randomly divided into three groups: normal group, model group, and 1% NAR group. Normal mice were injected with saline intraperitoneally, while others were intraperitoneally injected with a single dose of 25 mg/kg NaIO 3 . All the eyes of mice in the normal and model groups were topically administered with vehicle solution. All the eyes of mice in the 1% NAR group were topically administered with one drop of 1% NAR triple daily from 1 d before to 2 d or 10 d after injection of NaIO 3 . At 2 d or 10 d after NaIO 3 injury, all the eyes were removed for fixing with solution containing glacial acetic acid, formaldehyde, 95% ethanol, and distilled water in a ratio of 1 : 2 : 5 : 3 or collecting the retinas. Protein expressions of Nrf2 and HO-1 in the retinas were detected with immunochemical staining and western blotting.
2.4. Immunochemical Staining. Immunochemical staining procedure was carried out as our previous report [6]. Briefly, deparaffinized sections were blocked, underwent antigen retrieval, and then incubated. Sections were incubated with primary antibodies against Nrf2 (1 : 250) and HO-1 (1 : 600) at 4°C overnight. After washing, sections were then incubated with HRPconjugated anti-rabbit or anti-mouse IgG (1 : 400) for 1 h. After washing, sections were incubated with DAB and slightly stained with hematoxylin. Immunostaining images were taken by a fluorescence microscope (BX53F; OLYMPUS, Tokyo, Japan).

Normal
Model 1% NAR Figure 1: NAR eye drops improved the retinal morphology from NaIO 3 -induced injury in mice. 2.11. Measurement of Carbonyl Protein. The expression of carbonyl protein was measured using a commercial oxidized protein detection kit. The program of detecting carbonyl protein was according to our previous report [6]. Cells were seeded on glass coverslips and cultured in 24-well plates overnight. Then, cells were incubated with NaIO 3 and ML385 for 24 h. Cells were fixed with precold methanol, then incubated with 2,4-dinitrophenylhydrazine (DNP) solution, blocked with blocking buffer, and finally incubated with biotinylated anti-DNP antibody at 4°C overnight. Then, cells were incubated with streptavidin-Cy3 and then DAPI. Images were collected using a confocal fluorescence microscope.
2.12. Statistical Analysis. All data were represented as the mean ± standard error of mean (SEM) and analyzed by Statistical Package for the Social Sciences version 21.0 (SPSS 21.0). Statistical comparisons were performed using Student's t -test, one-way analysis of variance (ANOVA), or Mauchly's test of sphericity with Tukey's test. P < 0:05 was assumed to be significant.  Figure 1, when the retinas were detached from the eyes, we found that the retinas of normal mice were light yellow and manifested as a film of half cup while the retinas damaged by NaIO 3 were pale, thin, and friable. Treatment of 1% NAR eye drops for 10 d visibly improved the appearance of the retinas.  (Figures 3(a) and  3(b)). Compared to the model group, NAR treatment further increased Nrf2 and HO-1 protein levels by 122% and 145% after 2 d of NaIO 3 injury, respectively. However, NAR treatment decreased both Nrf2 and HO-1 protein expressions towards normal levels after 10 d of NaIO 3 injury.

NAR Regulates Nrf2
Expression at Early and Late Stages in NaIO 3 -Stimulated ARPE-19 Cells. We next investigated the Nrf2 signaling pathway regulated by NAR in NaIO 3stimulated ARPE-19 cells. We first assayed Nrf2 protein expression in ARPE-19 cells incubated with 10 mM NaIO 3 and NAR for 6 h and 24 h. NaIO 3 exposure for 6 h significantly increased the expression of Nrf2 about 5.5-fold higher than that in normal ARPE-19 cells (Figure 4). The protein level of Nrf2 in cells treated with 3 and 10 μM NAR for 6 h was increased by about 9.5-fold as compared with that of normal cells. Interestingly, 10 μM NAR decreased Nrf2 towards normal level after 24 h treatment.   Oxidative Medicine and Cellular Longevity

NAR Activates Nrf2 Much Earlier and Stronger in NaIO 3 -Treated ARPE-19 Cells.
We also analyzed the dynamic activation of Nrf2 to clarify whether NAR stimulated Nrf2 activation. Firstly, we analyzed the protein expression of Nrf2 in the nuclei by western blotting and immunostaining. As shown in Figure 5(a), nuclear Nrf2 level continuously increased during incubation with NaIO 3 from 3 h to 24 h. Compared with the NaIO 3 group, NAR increased nuclear Nrf2 protein content to almost twofold in the early stage of NaIO 3 -induced damage (1-3 h) and then significantly decreased nuclear Nrf2 protein in the late stage (6-24 h). Immunostaining result showed that Nrf2 protein expression in the nuclei of cells was decreased after 24 h treatment of NAR ( Figure 5(b)). Then, HO-1 protein expression was determined after 6 h and 24 h incubation with 10 mM NaIO 3 . Compared with the NaIO 3 group, HO-1 protein expression in the NAR group was increased after 6 h treatment and then decreased after 24 h (Figure 5(c)). In addition, ZnPP, an HO-1 inhibitor, significantly suppressed the protective effect of NAR by 36.9% ( Figure 5(d)).
3.6. The Abnormal Nrf2 Signaling Pathway Induced by NaIO 3 Promotes the Death of ARPE-19 Cells. In animal and cell models, we found that high protein expressions of Nrf2 and HO-1 were induced by NaIO 3 . To evaluate the effect of increased Nrf2, ML385, an inhibitor of Nrf2 activation, was used. NaIO 3 increased protein expressions of Nrf2 and HO-1 in a concentration-dependent manner (Figure 6(a)). Interestingly, 24 h treatment with ML385 reduced the levels of ROS and carbonyl protein (Figure 6(b)). Moreover, 24 h treatment with ML385 improved viability by 45.1% in 10 mM NaIO 3stimulated cells (Figure 6(c)), but 48 h treatment significantly accelerated the death of cells treated with 8 mM (Figure 6(d)) or 10 mM NaIO 3 (Figure 6(c)). In addition, ML385 in combination of different concentrations of NAR for 24 h showed the same protective effects on cells injured by NaIO 3 (Figure 6(e)).

Discussion
Our previous study showed that NAR significantly improved retinal function and morphology in NaIO 3 -treated mice and protected ARPE-19 cells partly via SIRT1-mediated antioxi-dation [6]. Here, we revealed that NAR activates the Nrf2 signaling pathway to protect RPE cells and the retinas from NaIO 3 -induced oxidative damage in the early stage.
Nrf2 is an essential regulator of redox homeostasis. However, Nrf2 expression and activity decrease with aging, which means the burden of oxidative stress gradually increases during lifetime [11][12][13]. Recent researches have shown that Nrf2 deficiency results in RPE cell damage and ocular pathology similar to human dAMD [14,15], meaning that Nrf2 is a target for the treatment of dAMD. In response to weak oxidative stress, Nrf2 increases and induces antioxidant genes such as HO-1 and NQO1 to protect cells [19,20]. We found that Nrf2 and HO-1 protein expressions were increased and mainly localized in POS and RPE in NaIO 3 -stimulated mice (Figures 2 and 3), which indicates that RPE is the major location to suffer oxidative insult from NaIO 3 . The upregulation of Nrf2 protein expression and activity was also observed in ARPE-19 cells stimulated by NaIO 3 in vitro (Figures 4-6). These results indicate that NaIO 3 induces oxidative stress and the adaptive protective response of Nrf2 is triggered in the early stage of oxidative damage in RPE cells [21].
However, if the stress is too severe, adaptive stress response may reach their boundary of activation and then lead to prohibit full recovery and provoke cell death [21]. It is reported that Nrf2 knockdown prevents cells from severe DNA damage [21], meaning high protein expression of Nrf2 is a risk factor when cells face to severe damage. Zucker et al. have demonstrated that according to different ROS levels, Nrf2 induces accumulation or removal of ROS via two separate regulatory circuits that are Klf9-dependent and HO-1/NOQ1-relative, respectively [19]. Upon severe oxidative stress, Klf9-dependent accumulation of ROS holds dominant position via repressing antioxidant enzymes such as TrxR2 and Prx6 to compel cell death [19,20]. Our results showed that NaIO 3 promoted the protein expression of Nrf2 in ARPE-19 cells in a concentration-dependent manner (Figure 6(a)), while inhibition of Nrf2 activation by ML385 promoted cell survival and reduced the levels of ROS and carbonyl protein (Figures 6(b) and 6(c)). These findings suggest that NaIO 3 induces serious oxidative stress and Nrf2 amplifies oxidative stress.

Oxidative Medicine and Cellular Longevity
The constitutive accumulation or activation of Nrf2 is also not beneficial for individual. The abnormal Nrf2 signaling pathway results in drug resistance in cancer cells [22,23]. Several researches have shown sustained activation of the Nrf2/Keap1 redox sensing pathway, which ultimately led to age-dependent cardiac dysfunction and hypertrophic cardiomyopathy [24,25]. In this study, with the relative persistence treatment by NAR, exorbitant protein expressions of Nrf2 and HO-1 were inhibited to normal level (Figures 2 and 3), which indicates that NAR may alleviate oxidative stress of the retina via inhibiting Nrf2/HO-1 in NaIO 3 -treated mice.
NAR was reported to ameliorate age-related neurodegenerative diseases through activating Nrf2 [26,27]. Therefore, we next wanted to confirm whether NAR directly inhibits the Nrf2 signaling pathway to protect RPE or not. In the present study, NAR further increased protein expression of total Nrf2 at the early stage (within 6 h of NaIO 3 damage) (Figures 3 and 5). Moreover, NAR treatment resulted in an earlier and stronger activation of Nrf2 in ARPE-19 cells than that in the NaIO 3 alone group (Figures 5(a)-5(c)). These results indicate that NAR enhances the Nrf2 signaling pathway at the early stage of oxidative damage. Our finding is strengthened by a previous study which reveals that RS9, an Nrf2-specific activator, protects ARPE-19 cells from NaIO 3induced damage [28]. Activation of Nrf2 by RS9 accelerates the autophagic degradation of abnormal cytotoxic proteins in ARPE-19 cells after stimulating by NaIO 3 within 6 h. In our study, the Nrf2 signaling pathway was activated strongly only within 1 h treatment with NAR, which indicates that NAR may protect ARPE-19 cells from oxidative stress through temporary upregulation of Nrf2.
Besides, we also observed that ZnPP, the special inhibitor of HO-1, suppressed the protective effect of NAR ( Figure 5(d)), meaning that NAR protects RPE cells via the Nrf2/HO-1 pathway. Our result was similar to previous researches that 10 μM ZnPP reversed the protective effect of NAR and pinocembrin against H 2 O 2 -induced cell death in SH-SY5Y cells [29,30]. However, the effect of HO-1 inhibition was not consistent with the cytoprotective effect of Nrf2 inhibition. The contradictory results indicate that NAR protects RPE cells through enhancing the Nrf2/HO-1 pathway at the early stage and then decreases ROS, which may inhibit the Nrf2/Klf9 pathway and avoid cell death. Similar protective effects in cell viability were observed in ML385 and ML385 in combination with NAR in NaIO 3treated cells (Figure 6(e)). The reason may be that inhibition of Nrf2 activity inhibits not only the Nrf2/Klf9 and Nrf2/HO-1 pathways regulated but exorbitant ROS (NaIO 3 ) but also the Nrf2/HO-1 pathway regulated by NAR. How NAR regulates the Nrf2 signaling needs to be further studied.

Conclusion
Taken together, NAR temporarily upregulated Nrf2 and HO-1 protein expressions and activated Nrf2 in NaIO 3 -treated ARPE-19 cells, suggesting that NAR prevents dAMD from oxidative stress through activating the Nrf2 signaling pathway.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Materials
The protein expressions of Nrf2 and HO-1 in the retinas of each NaIO 3 -induced injury mice were showed in Figures S1(a) and S1(b). The total protein expressions of Nrf2 and HO-1 in ARPE-19 cells stimulated by NaIO 3 were showed in Figures S1(c) and S1(e). The nuclear protein expressions of Nrf2 in ARPE-19 cells stimulated by NaIO 3 were showed in Figure S1(d). Figure