Ferulic acid (FA) protects human retinal pigment epithelial cells from H2O2‐induced oxidative injuries

Abstract The aim of present study is to investigate whether Ferulic acid (FA), a natural polyphenol antioxidant, was able to protect ARPE‑19 cells from hydrogen peroxide (H2O2)‑induced damage, and elucidate the underlying mechanisms. Our results revealed that FA pre‐treatment for 24 hours can reverse cell loss of H2O2‐induced ARPE‐19 cells via the promotion of cell proliferation and prevention of apoptosis, as evidenced by 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) assay, respectively. Moreover, the addition of FA (5 mM) can decrease Bax and cleaved caspase‐3 protein expression, but increase Bcl‐2 protein expression in ARPE‐19 cells. Furthermore, H2O2‐induced oxidative stress in ARPE‐19 cells was significantly alleviated by FA, illustrated by reduced levels of ROS and MDA. In addition, the attenuated antioxidant enzymes activities of (SOD, CAT and GPX) and GSH level were reversed almost to the normal base level by the pre‐addition of FA for 24 hours. In all assays, FA itself did not exert any effect on the change of the above parameters. These novel findings indicated that FA effectively protected human ARPE‐19 cells from H2O2‐induced oxidative damage through its pro‐proliferation, anti‐apoptosis and antioxidant activity, suggesting that FA has a therapeutic potential in the prevention and treatment of AMD.

Recently, consumer preference for naturally occurring antioxidant agents (including polyphenols, flavonoids, carotenoids, vitamins and minerals), which are found to be effective in reducing the risk of developing advanced AMD, has been rising. 5,8,9 In particular, polyphenols are excellent antioxidant agent, which are abundant in plants, vegetables and fruits. Increasing evidence has suggested that some polyphenol antioxidants such as quercetin, 9 resveratrol, 10 blueberry anthocyanins 8,9 and epigallocatechin-3-gallate, 11 can afford protective effects against cell death induced by oxidative stress in RPE cells.
Ferulic acid (FA) is of particular interest as a potent natural antioxidant, which has the ability to scavenge free radicals and prevent lipid peroxidation 12 ; therefore, it has been proposed as a potential therapeutic agent for treating many radical-induced disease including cardiovascular diseases, cancer, diabetes mellitus and Alzheimer's disease (AD). 13 Recently, FA was reported to exert potent protective effect on retinal damage induced by oxidative stress in vitro and in vivo, 14,15 indicating it has potential in delaying or preventing the progression of AMD. However, the exact anti-oxidative effects of FA on the ageing RPE cells are still unclear, let alone the underlying molecular mechanism. In this regard, the purpose of this study was to investigate the effects of FA on RPE cells challenged by H 2 O 2 in detail and gain further insight into the mechanism involved in this effect.

| Cell culture and treatment
The human ARPE-19 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM/F-12 supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 4 mM L-glutamine in a humidified incubator at 37°C in the presence of 5% CO 2 . The culture medium was replaced every 2-3 days and these cells of passages 5

| MTT assay
The MTT assay was used to investigate cell viability. Briefly, after different treatment, cells were added with 200 μL medium containing 20 μL of MTT (5 mg/mL) and incubated overnight at 37°C for 4 hours. Then, the medium was aspirated and 150 μL of DMSO was added to each well to dissolve formazan crystals with gentle agitation. The absorbance was measured at a wavelength of 490 nm using a microplate reader (BioTek, Winooski, VT, USA).
Cell viability (%) was calculated as follows: [(mean absorbance of the sample-reference absorbance)/ mean absorbance of the control] ×100.

| LDH assay
ARPE-19 cells seeded in 96-well microplates at a density of 5 × 10 4 cells/well were treated FA for 24 hours, and then allowed to exposure with H 2 O 2 (300 μM) for another 4 hours. Afterwards, aliquots of the conditioned cell supernate were isolated for LDH measurement using a commercial LDH kit according referring to manufacturer's instructions. The results were expressed as the percentage of the control (100%).

| 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay
After treatment with FA as describe above in six-well tissue culture plates, cells rinsed three times with cold PBS and fixed with 4% formaldehyde in PBS for 20 minutes at room temperature. Then, the cells were washed three times with cold PBS and stained with an

| TUNEL assay
Apoptotic cell death was measured using a commercially available TUNEL assay kit following the manufacturer's protocols. Briefly, cells with different treatment on coverslips were washed with cold PBS buffer and fixed with freshly prepared 4% paraformaldehyde for 15 minutes. After twice washing with PBS, the fixed cells were permeabilized in PBS containing 0.5% Triton X-100 at 37°C for 90 minutes. Thereafter, TUNEL staining was performed for each sample in the dark at 37°C. Following 3 × PBS wash, cells were incubated with 100 μL of DAPI (1 μg/mL) to stain nuclei and photographed by using a fluorescent microscope. The apoptotic index was expressed as the percentage of apoptotic cells (TUNEL-positive) compared with total cells (DAPI-positive). Five randomly selected microscopic fields in each group were used to calculate the apoptotic index, and all the experiments were performed three times.

| Flow cytometry
The assessment of apoptosis was performed by employing the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's protocol as described previously. 15 Briefly, after treatment, the cells were harvested, washed twice with ice-cold PBS,

| Western blot assay
After treatment, the harvested cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer. The lysates were centrifuged at 13 000 rpm for 15 minutes at 4°C, and the resulting supernatants were collected to determine the protein content with a BCA protein assay kit according to the manufacturer's instructions. Each sample with 20 μg of total protein was decentralized by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes. 5% non-fat milk was added to block the membrane for 1 hour at room temperature and then incubated overnight with the primary antibody (Bax, Bcl-2 and cleaved caspase-3) at a dilution of 1:1,000 at 4°C. After being rinsed with PBS for three times, the samples were incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:5000) at room temperature for 1 hour. The protein bands image was developed using an enhanced chemiluminescence (ECL) reagents, and band intensities were quantified with ImageQuant LAS 4000 (Pittsburgh, PA, USA). β-actin was used as an internal control. All experiments were repeated three times.

| Measurement of intracellular ROS
The level of intracellular ROS formation was determined using 2,7-dichlorofluorescein diacetate (DCFH-DA) as previously described. 7 Briefly, treated cells were stained with 10μM DCFH-DA at 37°C in the dark for 30 minutes followed by twice PBS wishing, and immediately imaged on a fluorescence microscope before being subjected

| Antioxidant assay
After treating cells in different conditions, total cell extracts were prepared and protein concentration was determined by Bradford

| Statistical analysis
All data were expressed as means ± standard deviation (SD). All statistical analyses were conducted using GraphPad Prism software version 5.0. The statistical differences were carried out using oneway ANOVA followed by Tukey's multiple comparison. A value of P < .05 were regarded statistically significant. All experiments were performed at least in triplicates.

| FA prevented H 2 O 2 -induced cell damage and death in ARPE-19 cells
The cytotoxicity of FA on human ARPE-19 cells was first examined after 24 hours treatment. As shown in Figure 1A

| FA attenuated H 2 O 2 -induced apoptosis in ARPE-19 cells
Next, in order to evaluate if this protective effect of FA on H 2 O 2induced cell death was related to apoptosis, ARPE-19 cells pre-treated with or without FA (5 mM) for 24 hours were further exposed to 300 μM H 2 O 2 for 4 hours, and then subject to TUNEL staining assay.
As shown in Figure 3A

| FA ameliorated H 2 O 2 -induced oxidative stress and lipid peroxidation in ARPE-19 cells
The ROS secretion in different group was measured by DCFH-DA staining ( Figure 5A). Compared with the control cells, FA alone treatment did not cause any ROS change. Exposure of ARPE-19 cells to H 2 O 2 lead to a 2.7-fold increase of ROS generation compared with control, but pre-treatment of cells with FA lowered down this increase to 1.5-fold of control cells. As for MDA, FA (5 mM) showed the same tendency as observed for ROS in ARPE-19 cells ( Figure 5B).

Increased MDA level by H 2 O 2 was significantly mitigated in ARPE-19
cells pre-treated with FA for 24h. Similarly, no change of MDA was observed in FA alone treated group compared with control (P > .05).

| FA improved antioxidant enzymes activities and glutathione content in ARPE-19 cells
As shown in Figure 6, exposure of ARPE-19 cells to 300μM H 2 O 2 resulted in a significant decline of SOD, CAT and GSH-PX activities, as well as GSH in ARPE-19 cells when compared with untreated control (P < .01). When ARPE-19 cells were pre-cultured with FA (5 mM) for 24 hours and then co-incubated with H 2 O 2 (300 μM) for another 4 hours, SOD, CAT, and GSH-PX and GSH levels were increased in different scale to approach the normal level, which were all statistically different from H 2 O 2 group (P < .05 or P < .01). In addition, no change of these oxidative stress biomarkers was observed in cells treated with FA alone.

| D ISCUSS I ON AND CON CLUS I ON S
The RPE is a single layer of pigmented cells locating adjacent to the outer retina layer, where it forms partial blood-retina barrier and performs functions that are essential for maintaining the structural integrity of the retina. 17,18 This process involves transportation of oxygen and nutrients, uptake of circulating vitamin A, elimination of wastes of photoreceptors metabolism and secretion of essential factors for facilitating the regeneration and repair of retina. 19 It is found that the retina contains highest oxygen consumption than other tissues, which suggests that RPE cells are susceptible to oxidative stress, particularly when exposed to high levels of reactive oxygen species (ROS). 20 Furthermore, RPE is lack of ability to renew itself following differentiation 7  Recent studies have suggested that H 2 O 2 -induced apoptosis was believed to be related to increased levels of ROS in the mitochondria of RPE cells and this over-production of ROS can be reversed by enhancing antioxidant enzymes, thus in turn alleviating apoptotic status of aged RPE cells. 28,29 As such, DCFH-DA staining was first used to determine the ROS production in ARPE-19 cells In summary, the present findings demonstrated for the first time that FA was able to effectively protect H 2 O 2 -induced cell damage via suppression of apoptosis, promotion of cell proliferation and activation of anti-oxidative activity, which shed light on the potential use of FA for the prevention or treatment of AMD.

CO N FLI C T O F I NTE R E S T
The authors have no commercial or other associations that might pose a conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.