Protective Effect of Quercetin on Sodium Iodate-Induced Retinal Apoptosis through the Reactive Oxygen Species-Mediated Mitochondrion-Dependent Pathway

Age-related macular degeneration (AMD) leads to gradual central vision loss and is the third leading cause of irreversible blindness worldwide. The underlying mechanisms for this progressive neurodegenerative disease remain unclear and there is currently no preventive treatment for dry AMD. Sodium iodate (NaIO3) has been reported to induce AMD-like retinal pathology in mice. We established a mouse model for AMD to evaluate the effects of quercetin on NaIO3-induced retinal apoptosis, and to investigate the pertinent underlying mechanisms. Our in vitro results indicated that quercetin protected human retinal pigment epithelium (ARPE-19) cells from NaIO3-induced apoptosis by inhibiting reactive oxygen species production and loss of mitochondrial membrane potential as detected by Annexin V-FITC/PI flow cytometry. We also evaluated the relative expression of proteins in the apoptosis pathway. Quercetin downregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP and upregulated the expression of Bcl-2 through reduced PI3K and pAKT expressions. Furthermore, our in vivo results indicated that quercetin improved retinal deformation and increased the thickness of both the outer nuclear layer and inner nuclear layer, whereas the expression of caspase-3 was inhibited. Taken together, these results demonstrate that quercetin could protect retinal pigment epithelium and the retina from NaIO3-induced cell apoptosis via reactive oxygen species-mediated mitochondrial dysfunction, involving the PI3K/AKT signaling pathway. This suggests that quercetin has the potential to prevent and delay AMD and other retinal diseases involving NaIO3-mediated apoptosis.


Cell Viability Assay
ARPE-19 cells (1.5 × 10 5 cells/well) were seeded into 24-well plates at 1 mL volume and incubated at 37 • C for 24 h. The culture medium was subsequently replaced by a medium containing various doses of quercetin (0, 1.25, 2.5, 5, 10, and 20 µM) alone or with a co-treatment of NaIO 3 . After 24 h, 0.5 mL culture medium containing 10 µL Cell Counting Kit-8 reagent (Dojindo Molecular Technologies, Kumamoto, Japan) was added to the cells in each well and they were incubated at 37 • C for 1-4 h. The absorbance of each well was then measured at 450 nm using a ELISA reader (Multiskan Spectrum, Thermo Co., Vantaa, Finland).

Measurement of Intracellular ROS and H 2 O 2 Production
Intracellular ROS production was determined by measuring the oxidation of 2 ,7 dichlorofluorescein diacetate (DCFH-DA) to the highly fluorescent compound 2 ,7 -dichlorofluorescein (DCF). ARPE-19 cells were cultured in 12-well plates and pretreated with different concentrations (1.25, 2.5, and 5 µM) of quercetin for 1.5 h, followed by incubation with 6 mM NaIO 3 at 37 • C for 15 h. Subsequently, 10 µM DCFH-DA was added to the culture medium, and the reaction was allowed to proceed at 37 • C for 30 min. The cells were then washed with PBS and collected for flow cytometry (BD Biosciences, San Jose, CA, USA). Data analysis was performed using CellQuest.

Measurement of Mitochondrial Damage
For the analysis of mitochondrial status, the cells were incubated with JC-1 dye (2 µg/mL; Cayman Chemical, Ann Arbor, MN, USA) for 50 min, then 2 µL Hoechst33342, a DNA-specific fluorescent dye, was added and the cells were incubated in darkness at 37 • C for 10 min. The stained cells were then observed under a fluorescence microscope. JC-1 accumulates in mitochondria and appears as a red/orange fluorescence (590 nm) in healthy organelles, however, when depolarized it appears as a green fluorescence (530 nm). Fluorescence images of the cells were recorded and the relative intensities of green/red JC-1 fluorescence were quantified using Image J Software (U.S. National Institutes of Health, Bethesda, MD, USA).

Measurements of Antioxidative Capacities and H 2 O 2 Production
The activities of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) were analyzed using assay kits from Cayman according to the manufacturer's instructions (Cat. 706002, 707002 and 703002, Cayman, Ann Arbor, MI, USA).
The H 2 O 2 production in the culture medium was determined using a Biovision assay kit (Biovision Research Products, Milpitas, CA, USA) following the manufacturer's instructions. The absorbance was measured at 570 nm with an ELISA reader.

Animal Model
Forty-two-week-old BABL/c mice were purchased from Asia University Animal Care and Use Committee (IACVC No. 107-a51a-20, 01 August 2019) and housed in standard 1.
Mock group: Animals pretreated with an intraperitoneal (IP) injection of PBS and then a single intravenous (IV) injection of PBS.

2.
Vehicle-treated group: Animals pretreated with an IP injection of PBS and then a single IV injection of 40 mg/kg NaIO 3 [31].

3.
Experimental group: Animals pretreated with an IP injection of 100 mg/kg quercetin and then a single IV injection of 40 mg/kg NaIO 3 .

Histology and Immunohistochemistry
The eyeballs were fixed in Davidson's solution (containing 10% formalin, 10% glacial acetic acid, and 4% formaldehyde) for 3 days [32]. Paraffin-embedded sections (5 µm) were obtained, stained with hematoxylin and eosin (H&E), and photographed using an optical microscope (Olympus Optical, Tokyo, Japan). The thicknesses of the whole retina, outer nuclear layer (ONL), and inner nuclear layer (INL) were measured at a distance of between 600 µm and 900 µm from the optic nerve along the superior and inferior hemiretina. Data from six random sites were averaged for each eye.
Immunohistochemical staining was performed using a BondMax automated slide staining system (Vision BioSystems Ltd., Newcastle Upon Tyne, UK). The sections were subjected to antibody cleaved caspase-3 (1:100, Cell Signaling Technology, Danvers, MA, USA) and then photographed using an optical microscope (Olympus Optical). Caspase-3 staining was quantified using the Image J Immunohistochemistry Toolbox (National Institute of Health, Starkville, MD, USA).

Retinal Imaging
Optical coherence tomography (OCT) was performed using RTVue XR Avanti with AngioVue (Optovue Inc, Fremont, CA, USA). Briefly, OCT of a certain region of the retina was performed repeatedly, and the resultant scans were examined for changes. Additional information pertaining to OCT can be found in two previous studies [33,34].

Statistical Analysis
The current study was conducted using a completely randomized design. When a significant difference (p < 0.05) was detected among groups by one-way analysis of variance (ANOVA), differences among the treatments were further tested using the least significant difference (LSD) test. All statistical analyses were performed using Statistical Analysis Software (SAS Institute Inc., Cary, NC, USA, 2002).

Effect of Quercetin Pretreatment on the Viability and NaIO 3-Induced Apoptosis of ARPE-19 Cells
To determine whether quercetin is toxic to ARPE-19 cells, we exposed them to various concentrations of quercetin and then evaluated the cell viability. The cell survival rate was unaffected following treatment of cells with <5 µM quercetin ( Figure 1A). However, treatment with 10-20 µM quercetin significantly reduced the cell viability, with effects being significantly different from the untreated controls (0 µM). For this reason, 1.25, 2.5, and 5 µM quercetin were used for all subsequent experiments.
We also determined the viability of ARPE-19 cells, which were treated with various concentrations of quercetin (1.25, 2.5, and 5 µM) and 6 mM NaIO 3 ( Figure 1B). Cell viability significantly decreased from 100% in the mock group to 70% in the vehicle-treated group. All tested concentrations (1.25, 2.5, and 5 µM) of quercetin significantly increased the cell viability when ARPE-19 cells were treated with 6 mM NaIO 3 .

Quercetin Reduced the Expression of Intracellular ROS and H 2 O 2 Production
ROS is closely associated with oxidative stress, which can lead to cell apoptosis [34]. Oxidative stress induced by NaIO 3 has been reported to cause apoptosis and autophagy [35]. Therefore, we assessed the effects of intracellular and mitochondrial ROS on NaIO 3-induced ARPE-19 cell death. After treatment with NaIO 3 for 15 h, the cells were labeled with DCFH-DA (an intracellular ROS probe) and analyzed through flow cytometry. As illustrated in Figure 2A,B, the mean ROS-associated fluorescence intensity in the NaIO 3 -treated group was significantly higher than in the mock group. Compared with the NaIO 3 -treated group, pretreatment with quercetin attenuated the NaIO 3 -induced intracellular ROS levels in the ARPE-19 cells ( Figure 2B). We then measured the level of hydrogen peroxide (H 2 O 2 ) again using a commercial kit, and the results again showed that the increased expression of H 2 O 2 in the NaIO 3 -induced group was significantly eliminated after treatment with quercetin (1.25-5 µM) ( Figure 2C).
with various concentrations (1.25, 2.5 and 5 µM) of quercetin for 1.5 h before treatment with NaIO3 (6 mM) for 15 h. ARPE-19 cells were then detected by flow cytometry after staining with both Annexin V-FITC and PI for 30 min. (D) The percentage of apoptotic cells in each treatment group was quantified. Values represent the mean ± SD (n = 3). Data bars without letters in common (a-d) indicate a significant difference (p < 0.05).

Quercetin Reduced the Expression of Intracellular ROS and H2O2 Production
ROS is closely associated with oxidative stress, which can lead to cell apoptosis [34]. Oxidative stress induced by NaIO3 has been reported to cause apoptosis and autophagy [35]. Therefore, we assessed the effects of intracellular and mitochondrial ROS on NaIO3induced ARPE-19 cell death. After treatment with NaIO3 for 15 h, the cells were labeled with DCFH-DA (an intracellular ROS probe) and analyzed through flow cytometry. As illustrated in Figures 2A,B, the mean ROS-associated fluorescence intensity in the NaIO3treated group was significantly higher than in the mock group. Compared with the NaIO3treated group, pretreatment with quercetin attenuated the NaIO3-induced intracellular ROS levels in the ARPE-19 cells ( Figure 2B). We then measured the level of hydrogen peroxide (H2O2) again using a commercial kit, and the results again showed that the increased expression of H2O2 in the NaIO3-induced group was significantly eliminated after treatment with quercetin (1.25-5 µM) ( Figure 2C).

Quercetin Inhibited the Reduction of Mitochondrial Membrane Potential (∆Ψm) via Modulated the Activity of Anti-Oxidants
It is well known that the activity of glutathione (GSH), superoxide (SOD), and catalase protects cells against ROS-induced oxidative damage [13]. As shown in Figure 3A-C, the expressions of anti-oxidants except SOD were dramatically decreased in the NaIO 3induced group. Quercetin (2.5 and 5 µM) treatment significantly reversed the reduced levels of catalase and GSH and the increased level of SOD, indicating that quercetin could modulate the activity of anti-oxidants. Since mitochondrial membrane integrity is sensitive to cellular ROS, we assessed disruption of mitochondrial membrane potential in ARPE-19 cells after quercetin pre-treatment. ARPE-19 cells exposed to NaIO 3 for 15 h had a decreased ∆Ψm, suggesting mitochondrial disruption, as indicated by a decrease in the red/green fluorescence intensity ratio ( Figure 3D). However, treatment with 2.5 and 5 µM quercetin significantly increased the ∆Ψm. These results indicated that quercetin could protect cells from NaIO 3 -mediated ROS injury by maintaining ROS levels and the ∆Ψm.

Quercetin Inhibited the Reduction of Mitochondrial Membrane Potential (∆Ψm) via Modulated the Activity of Anti-Oxidants
It is well known that the activity of glutathione (GSH), superoxide (SOD), and catalase protects cells against ROS-induced oxidative damage [13]. As shown in Figure  3A-C, the expressions of anti-oxidants except SOD were dramatically decreased in the NaIO3-induced group. Quercetin (2.5 and 5 µM) treatment significantly reversed the reduced levels of catalase and GSH and the increased level of SOD, indicating that quercetin could modulate the activity of anti-oxidants. Since mitochondrial membrane integrity is sensitive to cellular ROS, we assessed disruption of mitochondrial membrane potential in ARPE-19 cells after quercetin pre-treatment. ARPE-19 cells exposed to NaIO3 for 15 h had a decreased ΔΨm, suggesting mitochondrial disruption, as indicated by a decrease in the red/green fluorescence intensity ratio ( Figure 3D). However, treatment with 2.5 and 5 µM quercetin significantly increased the ΔΨm. These results indicated that quercetin could protect cells from NaIO3-mediated ROS injury by maintaining ROS levels and the ΔΨm.

Effect of Quercetin on the Endogenous NaIO 3 -Induced Apoptotic Pathway
Activation of caspase-3 and a decrease in Bcl-2 are typical markers of apoptosis; Bax is also an integral part of this apoptotic pathway [36][37][38]. We found that NaIO 3 induced apoptosis. To investigate how quercetin protects ARPE-19 cells from NaIO 3 -induced apoptosis, we used Western blotting to evaluate the effect of quercetin on the expression of apoptosis-related proteins (Bcl-2, Bax, caspase-3, cleaved caspase-3, and cleaved PARP) (Figure 4). Compared with the mock group, exposure to 6 mM NaIO 3 led to a lower level of Bcl-2 and higher levels of Bax, caspase-3, cleaved caspase-3, and cleaved PARP. However, treatment with 5 µM quercetin significantly reduced the expression levels of Bax, cleaved caspase-3, and cleaved PARP, compared with the NaIO 3 -treated group. In addition, quercetin increased the protein expression of Bcl-2 compared with the mock group. These results indicated that quercetin inhibited cell apoptosis, downregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP, and upregulated the protein expression of Bcl-2 in NaIO 3 -treated ARPE-19 cells.

Effect of Quercetin on the Endogenous NaIO3-Induced Apoptotic Pathway
Activation of caspase-3 and a decrease in Bcl-2 are typical markers of apoptosis; Bax is also an integral part of this apoptotic pathway [36][37][38]. We found that NaIO3 induced apoptosis. To investigate how quercetin protects ARPE-19 cells from NaIO3-induced apoptosis, we used Western blotting to evaluate the effect of quercetin on the expression of apoptosis-related proteins (Bcl-2, Bax, caspase-3, cleaved caspase-3, and cleaved PARP) (Figure 4). Compared with the mock group, exposure to 6 mM NaIO3 led to a lower level of Bcl-2 and higher levels of Bax, caspase-3, cleaved caspase-3, and cleaved PARP. However, treatment with 5 µM quercetin significantly reduced the expression levels of Bax, cleaved caspase-3, and cleaved PARP, compared with the NaIO3-treated group. In addition, quercetin increased the protein expression of Bcl-2 compared with the mock group. These results indicated that quercetin inhibited cell apoptosis, downregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP, and upregulated the protein expression of Bcl-2 in NaIO3-treated ARPE-19 cells.

Quercetin Suppressed Apoptosis via the PI3K/AKT Signaling Pathway
The PI3K signaling pathway promotes cell survival and reportedly participates in apoptosis in the central nervous system. AKT, a serine/threonine protein kinase that is also known as protein kinase B, is the primary downstream effector of the PI3K signaling pathway [39]. In a recent study, Chan et al. (2019) reported that NaIO3 induced cytosolic ROS production by activating ERK, p38, JNK, and the AKT signaling pathway [36]. To determine whether the antiapoptotic effects of quercetin on NaIO3-treated ARPE-19 cells were mediated through the PI3K/AKT signaling pathway, we performed Western blotting to measure AKT phosphorylation.
As shown in Figure 5, the levels of PI3K and phosphorylated-AKT (p-AKT) were significantly higher in the NaIO3-treated group compared with the mock and experimental groups. This indicates that quercetin inhibited the PI3K/AKT signaling pathway and suggests that this pathway is involved in the underlying mechanism of apoptosis in NaIO3-treated ARPE-19 cells. Values represent the mean ± SD (n = 3). Data bars without letters in common (a-d) indicate a significant difference (p < 0.05).

Quercetin Suppressed Apoptosis via the PI3K/AKT Signaling Pathway
The PI3K signaling pathway promotes cell survival and reportedly participates in apoptosis in the central nervous system. AKT, a serine/threonine protein kinase that is also known as protein kinase B, is the primary downstream effector of the PI3K signaling pathway [39]. In a recent study, Chan et al. (2019) reported that NaIO 3 induced cytosolic ROS production by activating ERK, p38, JNK, and the AKT signaling pathway [36]. To determine whether the antiapoptotic effects of quercetin on NaIO 3 -treated ARPE-19 cells were mediated through the PI3K/AKT signaling pathway, we performed Western blotting to measure AKT phosphorylation.
As shown in Figure 5, the levels of PI3K and phosphorylated-AKT (p-AKT) were significantly higher in the NaIO 3 -treated group compared with the mock and experimental groups. This indicates that quercetin inhibited the PI3K/AKT signaling pathway and suggests that this pathway is involved in the underlying mechanism of apoptosis in NaIO 3treated ARPE-19 cells.

Protective Effects of Quercetin on Retinal Degeneration
To confirm the association between in vitro and in vivo experimen damage caused by NaIO3, we established a retinal degeneration mouse m days of NaIO3 treatment, OCT was used to observe the thickness of the who and ONL, and the results were validated by histological analyses (H&E stai OCT is a noninvasive technique based on optical reflectivity and it is a retinal thickness and structure to produce three-dimensional, cross-sec images [34]. It has been clinically adopted as the standard method to obse changes associated with retinopathy. In the current study, the OCT images o treated mice showed that their total retinal thickness was significantly lower in the mock group ( Figure 6). However, quercetin treatment was shown restore the retinal thickness.

Protective Effects of Quercetin on Retinal Degeneration
To confirm the association between in vitro and in vivo experiments on retinal damage caused by NaIO 3 , we established a retinal degeneration mouse model. After 7 days of NaIO 3 treatment, OCT was used to observe the thickness of the whole retina, INL and ONL, and the results were validated by histological analyses (H&E staining).
OCT is a noninvasive technique based on optical reflectivity and it is able to analyze retinal thickness and structure to produce three-dimensional, cross-sectional, retinal images [34]. It has been clinically adopted as the standard method to observe structural changes associated with retinopathy. In the current study, the OCT images of day 7 NaIO 3treated mice showed that their total retinal thickness was significantly lower than the mice in the mock group ( Figure 6). However, quercetin treatment was shown to effectively restore the retinal thickness.
Representative images are shown for the retinal sections from the three study groups ( Figure 7A). The thicknesses of the total retina, INL and ONL were measured. In the NaIO 3treated mice, the thicknesses of the total retina, INL and ONL were significantly lower than in the mice from the mock group (p < 0.05). In addition, disruption between the inner segment and the outer segment of the photoreceptor (IS/OS) was found in NaIO 3 -treated mice ( Figure 7A). Representative images are shown for the retinal sections from the three study groups ( Figure 7A). The thicknesses of the total retina, INL and ONL were measured. In the NaIO3-treated mice, the thicknesses of the total retina, INL and ONL were significantly lower than in the mice from the mock group (p < 0.05). In addition, disruption between the inner segment and the outer segment of the photoreceptor (IS/OS) was found in NaIO3-treated mice ( Figure 7A).
Quercetin treatment protected the retinal layers against loss and caused partial recovery of the IS/OS disruption ( Figure 7A). INL and ONL thicknesses were also lower in the NaIO3-treated mice; however, the degeneration was almost alleviated via quercetin treatment ( Figure 7B,C). The degree of retinal degeneration was quantified by measuring the ONL thickness. The number of ONL nuclei was counted to demonstrate the degeneration of the retinal layers. Treatment of NaIO3-treated mice with quercetin prevented the decrease in ONL thickness and nuclei counts. Overall, quercetin administration effectively prevented retinal degeneration caused by NaIO3.

Quercetin Reduced NaIO3-Induced Retinal Apoptosis in Mice
We further explored the protective effects of quercetin on NaIO3-induced retinal cell injury. After 7 days of NaIO3 treatment, the mock, vehicle-treated, and experimental groups were subjected to immunohistochemical staining for cleaved caspase-3. Compared with the other two groups, the expression of cleaved caspase-3 was significantly higher in the vehicle-treated group (Figure 8), indicating that quercetin significantly reduced the production of NaIO3-induced cleaved caspase-3. Quercetin treatment protected the retinal layers against loss and caused partial recovery of the IS/OS disruption ( Figure 7A). INL and ONL thicknesses were also lower in the NaIO 3-treated mice; however, the degeneration was almost alleviated via quercetin treatment ( Figure 7B,C). The degree of retinal degeneration was quantified by measuring the ONL thickness. The number of ONL nuclei was counted to demonstrate the degeneration of the retinal layers. Treatment of NaIO 3 -treated mice with quercetin prevented the decrease in ONL thickness and nuclei counts. Overall, quercetin administration effectively prevented retinal degeneration caused by NaIO 3 .

Quercetin Reduced NaIO 3 -Induced Retinal Apoptosis in Mice
We further explored the protective effects of quercetin on NaIO 3 -induced retinal cell injury. After 7 days of NaIO 3 treatment, the mock, vehicle-treated, and experimental groups were subjected to immunohistochemical staining for cleaved caspase-3. Compared with the other two groups, the expression of cleaved caspase-3 was significantly higher in the vehicle-treated group (Figure 8), indicating that quercetin significantly reduced the production of NaIO 3 -induced cleaved caspase-3.

Quercetin Reduced NaIO3-Induced Retinal Apoptosis in Mice
We further explored the protective effects of quercetin on NaIO3-induced retinal cel injury. After 7 days of NaIO3 treatment, the mock, vehicle-treated, and experimenta groups were subjected to immunohistochemical staining for cleaved caspase-3. Compared with the other two groups, the expression of cleaved caspase-3 was significantly higher in the vehicle-treated group (Figure 8), indicating that quercetin significantly reduced th production of NaIO3-induced cleaved caspase-3.

Discussion
AMD is the primary cause of blindness in the elderly population of developed countries. Millions of patients with AMD experience gradual vision loss year by year and no preventive treatment yet exists for decreasing dry AMD, which can progress to wet AMD [40][41][42]. Therefore, the identification of potential anti-AMD agents is extremely important. AMD is associated with several risk factors, and many of them are highly linked to increased ROS. The current literature also indicates that excessive ROS triggers AMD development, leading to apoptosis of RPE cells [13]. Previous in vivo and in vitro studies have reported that inhibitors of caspases and necroptotic signaling pathways inhibit NaIO 3 -induced RPE and photoreceptor cell death [23,35,43,44]. Chan et al. reported that NaIO 3 -induced AKT activation was partially dependent on ROS production, eventually leading to cell death [36]. In addition, blue light can induce apoptosis by increasing the cleaved form of caspase-3 and the Bax/Bcl-2 ratio in RPE cells [45,46]. PARP-1 plays a crucial role in DNA repair, replication, and cell death. PARP-1 cleavage is observed during apoptosis, which is induced by caspase-3 activation [36].
In order to investigate the effects of quercetin on the retina, an NaIO 3 -induced experimental model of AMD was established in vitro and in vivo. In this study, we found that NaIO 3 -induced ROS production caused apoptosis by upregulating the expressions of Bax, cleaved caspase-3, and cleaved PARP, and downregulating Bcl-2 expression through the PI3K/AKT signaling pathway in ARPE-19 cells (Figures 4 and 5). In addition, we found that quercetin inhibited NaIO 3 -induced cell apoptosis by mediating PI3K/AKT inactivation, thereby downregulating the expressions of Bax, cleaved caspase-3, and cleaved PARP and upregulating Bcl-2 expression (Figure 4). Consistent with these findings, we found that quercetin attenuated NaIO 3 -induced increases in cleaved caspase-3 levels. Taken together, our findings suggest that the antiapoptotic effects of quercetin are mediated by the inhibition of apoptotic effectors. The results of this study showed that quercetin significantly protected ARPE-19 cells against NaIO 3 -induced toxicity, high ROS levels and apoptosis in vitro. NaIO 3 -induced retinochoroidal degeneration is used as an animal model for AMD, because IS/OS disruption of retinal tissue has been observed in mice treated with NaIO 3 ( Figure 7A) [19,47]. Previous studies have reported endogenous retinal repair following rapid RPE damage induced by relatively high doses of NaIO 3 (50-100 mg/kg). The administration of 40mg/kg NaIO 3 has been reported to be sufficient to generate disturbances in retinal function by retinal degeneration in mice and rats [48,49]. In the current study, we observed that the intravenous administration of NaIO 3 (40 mg/kg) induced RPE degeneration in 7 days ( Figure 6). We also found that quercetin treatment ameliorated NaIO 3 -induced retinochoroidal degeneration in vivo. NaIO 3 -induced RPE degeneration has been shown to lead to the production of pigment-rich substances, and reductions in the thickness of both the outer and inner segments of the photoreceptor and the ONL [18,49,50]. In addition, exposure of RPE cells to NaIO 3 has been shown to induce a loss of their integrity and cause enlargement [51]. In this study, we found that the thicknesses of the total retina, INL, and ONL of NaIO 3treated mice were significantly lower compared with animals in the mock group at day 7, however, quercetin treatment could prevent the retinal degeneration caused by NaIO 3 (Figures 6 and 7).
Manganese superoxide dismutase (SOD2) is a primary antioxidant located on the matrix side of inner mitochondrial membranes, where it dismutates the superoxide byproducts of oxidative phosphorylation (OXPHOS) to H 2 O 2 , and the latter is further reduced by glutathione peroxidases and catalase [52]. In this study, we demonstrated that NaIO 3 could induce H 2 O 2 accumulation due to cell death caused by ROS through enhanced SOD activity and the inhibition of GSH and CAT activity ( Figure 3A,B). In addition, we showed that quercetin attenuated the NaIO 3 -induced intracellular cell death caused by ROS via increases in catalase and GSH activity and reduced SOD activity in the ARPE-19 cells. There were a number of limitations to the present study. First, fundoscopy and electroretinogram (ERG) studies were not performed, and these examinations should be conducted in future studies to evaluate retinal function.
To the best of our knowledge, this is the first study to demonstrate the protective effects of quercetin on NaIO 3 -induced retinal degeneration in a mouse model. To conclude, we found that quercetin reduced NaIO 3 -induced cell apoptosis by downregulating the expressions of Bax, cleaved caspase-3, and cleaved PARP, and upregulating the expression of Bcl-2 via the PI3K/AKT signaling pathway in ARPE-19 cells (Figure 9). Moreover, in vivo experiments demonstrated that quercetin could protect against NaIO 3-induced retinal damage. We believe that our results will encourage further studies on the potential use of quercetin as a treatment for AMD. effects of quercetin on NaIO3-induced retinal degeneration in a mouse mod conclude, we found that quercetin reduced NaIO3-induced cell apoptos downregulating the expressions of Bax, cleaved caspase-3, and cleaved PARP upregulating the expression of Bcl-2 via the PI3K/AKT signaling pathway in AR cells ( Figure 9). Moreover, in vivo experiments demonstrated that quercetin could p against NaIO3-induced retinal damage. We believe that our results will encourage fu studies on the potential use of quercetin as a treatment for AMD. Figure 9. Illustration of the mechanism of NaIO3-induced retinal injury and the effects of quercetin. NaIO3 induce mitochondrial dysfunction through the PI3K/AKT signaling pathway and then Bax activates Cytochrome C, which cause apoptosis. Finally, IS-OS disruption occurs, which mimics AMD changes within the retinal tissue. Quercetin can activat Bcl-2 and inhibit apoptosis, thereby reducing the accumulation of oxidative damage.