Luteolin Alleviates Epithelial-Mesenchymal Transformation Induced by Oxidative Injury in ARPE-19 Cell via Nrf2 and AKT/GSK-3β Pathway

Oxidative stress plays a critical role in age-related macular degeneration (AMD), and epithelial-mesenchymal transition (EMT) is involved in this process. The aim of this study was to investigate the protective effects of luteolin, a natural flavonoid with strong antioxidant activity, on H2O2-induced EMT in ARPE-19 cells. ARPE-19 cells were incubated with H2O2 at 200 μΜ to induce oxidative stress-associated injury. Cell viability assay showed that luteolin at 20 and 40 μM significantly promoted cell survival in H2O2-treated ARPE-19 cells. Luteolin also markedly protected ARPE-19 cells from H2O2-induced apoptosis. Cell migration assay presented that luteolin significantly reduced H2O2-induced migration in APRE-19 cells. EMT in ARPE-19 cells was detected by western blotting and immunofluorescence. The results showed that H2O2 significantly upregulated the expression of α-SMA and vimentin and downregulated the expression of ZO-1 and E-cadherin, while cells pretreated with luteolin showed a reversal. Meanwhile, the assessment of effects of luteolin on the Nrf2 pathway indicated that luteolin promoted Nrf2 nuclear translocation and upregulated the expressions of HO-1 and NQO-1. In addition, luteolin significantly increased the activities of SOD and GSH-PX and decreased intracellular levels of ROS and MDA in H2O2-treated ARPE-19 cells. Meanwhile, we observed that the expression of TGF-β2, p-AKT, and p-GSK-3β was upregulated in H2O2-treated ARPE-19 cells and downregulated in luteolin-treated cells, revealing that luteolin inhibited the activation of the AKT/GSK-3β pathway. However, these effects of luteolin were all annulled by transfecting ARPE-19 cells with Nrf2 siRNA. Our current data collectively indicated that inhibition of luteolin on EMT was induced by oxidative injury in ARPE-19 cell through the Nrf2 and AKT/GSK-3β pathway, suggesting that luteolin could be a potential drug for the treatment of dry AMD.


Introduction
Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly [1]. From current knowledge, AMD is a multifactorial degenerative disease of the retina. In addition to aging, susceptibility genes, environmental factors, chronic localized inflammation, and choroidal vascular dysfunction all play important roles in the onset and progression of AMD [2]. Retinal pigment epithelium (RPE) is a monolayer cell located between photoreceptors and Bruch's membrane, characterized by cell-cell adhesion and apical-basal polarity arrangement. RPE transports nutrients from the choroidal capillary layer to the photoreceptors and phagocyte and digests photoreceptor outer segments, which is critical for keeping the photoreceptor function [3]. Abnormal distribution and dysfunction of RPE cells lead to retinal degeneration and eventually to loss of vision.
Epithelial-mesenchymal transition (EMT) is a physiological process necessary for normal embryonic development. There are three types of EMT, of which type 2 EMT is associated with tissue regeneration and wound healing, but its abnormal activation enhances pathological tissue fibrosis [4]. Tissue damage, oxidative stress, and inflammation are important factors to activate EMT. Many literatures support the role of EMT in AMD. In dry AMD, RPE loses its characteristic shape and phenotype, showing migration in the transition zone between atrophy and normal retina [5]. Further histological examination of these "degenerating" cells showed that they may not be dying but reversibly transformed into mesenchymal cells through EMT [6]. Multiple extracellular ligands are involved in the initiation and progression of the EMT process. Transforming growth factorbeta (TGF-β) is considered to be the master regulator of the process [7]. Reactive oxygen species (ROS) can directly activate TGF-β1 and initiate the EMT process through the PI3K/AKT and MAPK pathway, which means that the control of oxidative stress has the potential to regulate EMT [8]. The inhibition of the mTORC1-NOX4 pathway by an activator of AMP-dependent protein kinase, or a NOX1/4 inhibitor, decreased ROS generation, prevented stress fiber formation, attenuated EMT, and improved the regularity of the RPE structure in vitro and in vivo [9]. The Nrf2 pathway is the most important antioxidant stress defense pathway in the human body and plays an important role in the pathogenesis of AMD [10,11]. ROS production is increased under oxidative stress, which activates the Nrf2 pathway to enhance the ability of cells to clear ROS, maintain the balance of intracellular redox state, and reduce oxidative damage. Therefore, activation of the Nrf2 pathway is considered to be a potential therapeutic strategy for AMD [12,13]. Some studies have confirmed that the Nrf2 pathway is involved in the regulation of EMT [14,15]. It is thus expected that the interaction between the Nrf2 pathway and EMT, two important events under oxidative stress, may significantly expand the role of antioxidants in the treatment of AMD.
Luteolin is a natural flavonoid found in many plants, such as carrots, broccoli, perilla leaves, and celery. Luteolin has a variety of biological effects, such as anti-inflammatory, antioxidant, and antitumor [16][17][18]. Recent studies have found that many flavonoids can inhibit EMT. Tangeretin inhibits podocyte damage and fibrosis by blocking glucoseinduced oxidative stress and hypoxia-induced podocyte EMT [19]. Galatechin-3-gallate attenuates TGF-β1-induced renal tubular cell EMT through GSK-3β/β-catenin/snail1 and Nrf2 pathways [20]. It has been confirmed that luteolin has a significant inhibition on EMT in tumor cells [21,22], but it is unclear whether it also inhibits EMT in RPE cells. In this study, we used ARPE-19 cells, a commercial human RPE cell line widely used in AMD research, to assess the effect of luteolin on H 2 O 2 -induced EMT, focusing on the involvement of the Nrf2 and AKT/GSK-3β pathway in EMT.

Cell Migration Assay.
A 24-well modified Boyden chamber (Corning, NY, USA) was performed in migration assay. ARPE-19 cells (1 × 10 5 /mL) from the indicated groups were resuspended in the upper chambers with 200 μL serum-free MEM. The bottom chambers were filled with 500 μL of DMEM containing 10% FBS. The chambers were incubated at 37°C and 5% CO 2 for 24 h. Nonmigrated cells on the top surface of the filter were wiped away using cotton swabs. The cells that migrated through the filter were fixed with 4% paraformaldehyde for 10 min and stained with 1% crystal violet in methanol. After washing twice with PBS, the cell number of the bottom side was counted and plotted as the mean number of cells migrated in six nonoverlapping fields by three investigators under a microscope (Olympus IX70, Tokyo, Japan).

Determination of
2.11. Statistical Analysis. All experiments were performed at least three times. Data were presented as mean ± SD, and results were analyzed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). The significance of the difference was determined by one-way ANOVA with the post hoc Dunnett test. P < 0:05 was considered to be statistically significant.  Figure 1(b), luteolin increased cell survival rate in a dose-dependent manner. Next, apoptosis was examined using flow cytometry. As shown in Figure 1(c), ARPE-19 cells exposed to H 2 O 2 had significant apoptotic rate compared to control, but luteolin significantly reduced apoptosis. These data collectively indicated that luteolin effectively protected against cell death in ARPE-19 cells treated with H 2 O 2 .

Luteolin Promoted Nrf2 Nuclear Translocation and
Increased Antioxidant Enzyme Expressions. We assessed the effects of luteolin on the Nrf2 pathway. As shown in Figure 4(a), the level of cytoplasmic Nrf2 was decreased and the level of nuclear Nrf2 was increased in ARPE-19 cells treated with H 2 O 2 . Luteolin reduced the level of cytoplasmic Nrf2 and increased the level of nuclear Nrf2 in a dose-effect manner, indicating that luteolin promoted Nrf2 nuclear translocation. Meanwhile, we examined the expression of HO-1 and NQO-1, target proteins of the Nrf2 pathway. As shown in Figure 4(b), the expressions of HO-1 and NQO-1 were increased in ARPE-19 cells treated with H 2 O 2 and significantly upregulated when pretreated with luteolin.

Luteolin Exhibits Potent
Antioxidative Activities in ARPE-19 Cells Treated with H 2 O 2 . We next examined the effects of luteolin on enzyme activities of SOD and GSH-PX. The results demonstrated that the activities of SOD and GSH-PX were significantly reduced in H 2 O 2 -treated ARPE-19 cells, but luteolin significantly restored the activities of these enzymes (Figures 5(a) and 5(b)). The exposure of the retina to ROS is thought to be a crucial factor in the development of AMD. ROS causes lipid peroxidation of the biomembrane, which leads to the production of a large amount of MDA [25]. Therefore, we measured the levels of        Oxidative Medicine and Cellular Longevity AKT, p-AKT, GSK-3β, and p-GSK-3β were evaluated. We found that p-AKT and p-GSK-3β significantly increased in H 2 O 2 -treated cells and decreased in luteolin-treated cells ( Figure 6). Taken together, these results indicated that luteolin inhibited the activation of the AKT/GSK-3β pathway, thereby alleviating EMT mediated by TGF-β2.  (Figure 7(a)). Western blot results showed that the expression of α-SMA and vimentin was significantly upregulated, and ZO-1 and E-cadherin were downregulated in siNrf2transfected cells compared with the H 2 O 2 control cells.
Compared to the H 2 O 2 control cells, the expression of α-SMA and vimentin was significantly downregulated, and ZO-1 and E-cadherin were upregulated in luteolinpretreated cells without siNrf2 transfection, but not in siNrf2-transfected cells (Figures 7(b) and 7(d)-7(g)).
To explore the possible association between the AKT/GSK-3β and Nrf2 pathway in H 2 O 2 -induced EMT, we examined the expressions of AKT, p-AKT, GSK-3β, and p-GSK-3β. The results showed that the expressions of p-AKT and p-GSK-3β were significantly increased in siNrf2-transfected cells compared with the H 2 O 2 control cells. Compared to the H 2 O 2 control cells, the expressions of p-AKT and p-GSK-3β were decreased in luteolin-pretreated cells without siNrf2-transfection, but not in siNrf2-transfected cells (Figures 7(c), 7(h), and 7(i)). Our results strongly suggested that inhibition of luteolin on H 2 O 2 -induced EMT in ARPE-19 cell through the AKT/GSK-3β pathway is dependent on the activation of the Nrf2 pathway.

Discussion
Clinically, AMD is divided into two types: dry and wet. The treatment of wet AMD, which accounts for about 15% of AMD, has yielded encouraging outcome with antivascular endothelial growth factor (VEGF) drugs. However, there are no available options for the treatment or prevention of dry AMD, which is the more common type, comprising 85% of all AMD cases [27]. Thus, intense efforts have been made to identify possible therapeutic targets to reduce disease progression. Luteolin is the main active ingredient of Fructus Broussonetiae, an herb commonly used for clinical treatment of agerelated eye disease, including AMD. Therefore, luteolin was   Oxidative Medicine and Cellular Longevity selected for the preliminary study on the prevention and treatment of AMD. Oxidative damage from various sources, such as light exposure, inflammation, and oxidative stress, to the retina has been strongly linked with AMD [28,29]. Therefore, an antioxidant could have therapeutic potential in the protection of RPE cells from oxidative damages and thus, it could be beneficial against AMD, at least against the dry form of the disease. The AREDS study suggested that antioxidant supplementation could have protective effects against AMD [30]. Many natural flavonoids show strong antioxidant activity and have the potential to treat dry AMD. Morin protected RPE cells from cigarette smoke extract through reduced oxidative stress, ER stress, and lipid accumulation via activated AMPK-Nrf2-HO-1 signaling pathway [31]. Our previous studies confirmed that quercetin and apigenin reduce oxidative damage in ARPE-19 cells and retina in model mice of dry AMD by activating the Nrf2 pathway [13,24]. It also was reported that luteolin activates the Nrf2 pathway. For example, luteolin enhanced autophagy and antioxidative processes in both in vivo and in vitro models of intracerebral hemorrhage [32], protected heart tissues in STZ-induced diabetic mice through modulating Nrf2-mediated oxidative stress and NF-κB-mediated inflam-matory responses, and alleviates aflatoxin B 1 -induced apoptosis and oxidative stress in the liver of mice through activation of the Nrf2 pathway [33]. The current results showed that luteolin promoted nuclear translocation of Nrf2, upregulated the expressions of HO-1 and NQO-1, increased the activities of SOD and GSH-PX, and decreased intracellular levels of ROS and MDA, suggesting that luteolin activated the Nrf2 pathway, thereby improving the survival rate of oxidative damaged ARPE-19 cells. It is well documented that oxidative stress would also induce EMT [34][35][36]. However, available reports on EMT-related ophthalmic diseases have focused more on proliferative vitreoretinopathy than AMD [37,38]. In the present study, we found that H 2 O 2 significantly increased the number of cell migration, upregulated the expression of α-SMA and vimentin, and downregulated the expression of ZO-1 and E-cadherin, which was consistent with the EMT events in APRE-19 cells. Simultaneously, treatment of ARPE-19 cells with luteolin inhibited H 2 O 2 -induced cell migration and reversed the expression of epithelial markers ZO-1 and Ecadherin and mesenchymal markers α-SMA and vimentin. These results strongly suggested that the protective effect of luteolin on H 2 O 2 -induced EMT may be closely related to the activation of the Nrf2 pathway. 9 Oxidative Medicine and Cellular Longevity RPE is the main source of TGF-β in the outer retina. TGF-β can be activated in various ways such as heat, ultraviolet radiation, acidic pH, and ROS [39]. Once activated, TGF-β binds to specific transmembrane serine/threonine kinase receptors to transduce its intracellular signal by phosphorylating the canonical Smad pathway or noncanonical pathways, including the MAPK, PI3K/AKT, and mTOR that intricately modulate distinct downstream TGF-β responses [40]. It was reported that activation of the PI3K/AKT pathway leads to the phosphorylation and inactivation of GSK-3β, which triggers EMT [41]. In this study, we observed that the expression of TGF-β2 was upregulated in H 2 O 2 -treated ARPE-19 cells and downregulated in luteolin-treated cells. Correspondingly, the expressions of p-AKT and p-GSK-3β were increased in H 2 O 2 -treated ARPE-19 cells and decreased in luteolin-treated cells. These data indicated that luteolin inhibited the activation of the AKT/GSK-3β pathway.
Studies have supported that there is a strong relationship between the Nrf2 pathway and AKT/GSK-3β pathway in the process of EMT. Melatonin inhibits LPS-induced EMT in human alveolar epithelial cells depending on upregulation of the Nrf2 pathway mediated by the PI3K/GSK-3β axis [42]. In renal interstitial fibrosis of unilateral ureteral obstruction mice, the antifibrotic effect of Nrf2 is imparted by the inactivation of PI3K/Akt signaling [15]. In this study, we found that cell migration number was increased and the expression of α-SMA and vimentin was upregulated, and ZO-1 and E-cadherin were downregulated in siNrf2transfected cells than in siNrf2-untransfected cells. Luteolin could inhibit cell migration and reversed the expressions the EMT-related proteins in siNrf2-untransfected cells but had no effects in siNrf2-transfected cells. Further, we also found that the expressions of p-AKT and p-GSK-3β were increased in siNrf2-transfected cells than in siNrf2untransfected cells and decreased in luteolin-pretreated siNrf2-untransfected cells but did not in siNrf2-transfected cells. These results further illustrate that the inhibition of luteolin on EMT in H 2 O 2 -treated ARPE-19 cells through the AKT/GSK-3β pathway is dependent on the activation of the Nrf2 pathway.
In conclusion, in this study, we simultaneously studied the effects of oxidative stress on the Nrf2 pathway and EMT and discussed the relationship between two signaling pathways and the influence of luteolin intervention. The results showed that the inhibition of luteolin on EMT induced by oxidative injury in ARPE-19 cell through the AKT/GSK-3β pathway is dependent on the activation of the Nrf2 pathway. Our current study indicated that luteolin could be a potential drug for the treatment of dry AMD. However, luteolin has the disadvantages of poor water and fat solubility and low bioavailability. Further research is needed to change its dosage forms, such as preparation of phospholipid complex and solid dispersion; to improve its bioavailability; and then to verify its effect on dry AMD through in vivo and in vitro experiments. It may be expected that luteolin will have a good application prospect in the treatment of dry AMD after solving the problem of low biological activity.