Pueraria lobata Extract Protects Hydrogen Peroxide-Induced Human Retinal Pigment Epithelial Cells Death and Membrane Permeability

Background Pueraria lobata is used in traditional Asian medicine to treat cardiovascular diseases, diarrhea, diabetes mellitus, and diabetic complications such as diabetic retinopathy. Oxidative stress in retinal pigment epithelial cells is implicated in the pathogenesis of retinopathy and age-related macular degeneration (AMD). Here, we evaluated whether the P. lobata extract can prevent cell death and decrease membrane permeability in oxidative stress-induced human retinal pigment epithelial cells. Methods The effects of P. lobata extract on hydrogen peroxide- (H2O2-) induced oxidative stress were investigated using 2′,7′–dichlorofluorescin diacetate, western blotting, and immunohistochemistry in human retinal pigment epithelial cells. The effects of puerarin, daidzein, and daidzin isolated from P. lobata extract were also studied by determining cell death, reactive oxygen species (ROS) generation, and p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation. Results Our results showed that the P. lobata extract inhibited ROS generation, suppressed the disruption of zonula occludens-1 (ZO-1), and reduced membrane permeability in H2O2-induced human retinal pigment epithelial cells. Additionally, the P. lobata extract prevented the inhibition of p38 MAPK and JNK phosphorylation. Conclusion Our findings suggest that the P. lobata extract has the potential to prevent AMD development by inhibiting the mechanism underlying oxidative stress-mediated ocular disorders.


Background
Retinal damage, also known as retinopathy, is a major cause of vision loss among middle-aged and elderly people, often resulting as a consequence of complications associated with diabetes, hypertension, atherosclerosis, blood dyscrasias, systemic infections, and exposure to radiation [1][2][3]. e retinal pigment epithelium (RPE) plays an important role in the development and maintenance of adjacent photoreceptors in the retina. RPE cells are used to investigate the pathology and physiology of diabetic retinopathy and agerelated macular degeneration (AMD). Drug candidates are tested in RPE cells to develop treatments for retinopathy and AMD [4].
Oxidative stress plays a pivotal role in the development and acceleration of retinal diseases. It increases intracellular levels of reactive oxygen species (ROS), which cause retinal damage, and is a major pathogenic component [5]. ROS increase the chronological age of cells and reduce mitochondrial function in RPE cells, causing cell damage [6]. A recent study showed that oxidative stress has a particularly significant role in the development, degeneration, dysfunction, and age-related loss of RPE [7,8]. Repeated exposure to oxidative stress from ROS, such as hydrogen peroxide (H 2 O 2 ), causes RPE damage [9]. erefore, H 2 O 2 is suitable for evaluating oxidative damage of RPE and investigating retinopathy progression. In addition, oxidative stress influences the formation of the blood-retinal barrier (BRB) by RPE via tight junctions and adherens junctions [9][10][11]. e tight junction consists of the transmembrane protein zonula occludens-1 (ZO-1) and occludins, which maintain BRB integrity [9,10]. Oxidative stress disrupts the tight junctions and increases paracellular permeability across the epithelial monolayers, thereby decreasing the localization of occludins and association of ZO-1 [10,12]. e roots of Pueraria lobata Ohwi (family: Fabaceae) are well known and used in traditional medicine. e plants are widely cultivated in East Asia and used for the treatment of diarrhea, diabetes, and cardiovascular diseases [13][14][15]. P. lobata extract and the compounds present in the extract have been shown to possess therapeutic properties, owing to their antioxidant, anti-ischemic, anticancer, antiinflammation, antifatigue, and antiretinopathic effects [14][15][16][17]. A previous study showed that the P. lobata extract prevent apoptosis of lung fibroblasts in Chinese hamsters by inhibiting hydrogen peroxide-induced oxidative stress [18]. In this study, we explored whether the P. lobata extract and their individual constituent compounds (puerarin, daidzein, and daidzin) can protect human RPE cells against oxidative stress. In addition, we evaluated the expression of tight junctions and oxidative stress-induced decrease in cell membrane permeability as well as examined the mechanisms involved in the antioxidative effects of P. lobata in RPE cells.

Extraction of P. lobata and Isolation of Single Compounds.
e roots of P. lobata Ohwi (P. lobata) were collected from Kyonggi-do, Gachun University, Korea, and were identified by Professor J.-H. Kim. A voucher specimen (no. KIOM-P041) has been deposited at the Herbarium of Korea Institute of Oriental Medicine. e air-dried plant material (4.9 kg) was extracted with 20 L of EtOH three times by maceration. e extract was combined and concentrated in vacuo at 40°C to yield an EtOH extract (665 g). is extract was subjected to a series of chromatographic procedures, using open silica gel and RP-18 column and HPLC, leading to the isolation of three main compounds. By comparing their physicochemical and spectral data with those in the literature [19], these compounds were identified as puerarin, daidzein, and daidzin ( Figure 1). incubator, and the culture medium was replaced every 48 h. After the seeded cells reached confluency, they were rinsed with phosphate-buffered saline (PBS) and then incubated with 0.5% trypsin-EDTA (Gibco) for 5 min at 37°C.

Cell Viability Assay.
Cell viability was determined using the cell counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Japan). RPE cells (0.5 × 10 4 cells/well) were seeded into each well of a 96-well plate containing F12/ DMEM with 10% FBS and then were incubated for 24 h. After cell attachment, the cells were pretreated with the P. lobata extract and its individual compounds (puerarin, daidzein, and daidzin) in serum-free medium for 1 h and with H 2 O 2 for 24 h. After incubation, 10 μL of CCK-8 was added to each well of the 96-well plate and incubated at 37°C under 5% CO 2 in a humidified incubator for 2 h. e absorbance was measured at 450 nm using a Multidetection Microplate Reader (BioTek, Synergy HT, Winooski, VT).

Measurement of ROS Production
. ROS production was measured by using the 2′,7′-dihydrodichlorofluorescein diacetate (DCF-DA, Invitrogen, USA) staining assay. After pretreatment with the P. lobata extract, puerarin, daidzein, and daidzin for 1 h, the cells were cotreated with H 2 O 2 (200 μM) and 25 μM DCF-DA in a humidified 5% CO 2 incubator at 37°C for 30 min. e cells were then washed with PBS and treated with 0.5% trypsin-EDTA. After the cells were detached, they were harvested in PBS. ROS production was immediately analyzed by using BD FACSCalibur ™ (San Jose, CA, USA).

Permeability Assay.
To evaluate the inhibitory effect of the P. lobata extract on oxidative stress-induced alteration in cell membrane permeability, the cells were seeded on a Transwell upper chamber (24 wells, PE, 0.4 μm pore diameter, Corning Inc., Tewksbury, MA, USA) at a density of 0.3 × 10 4 cells/well. After the cells formed a monolayer, the medium was replaced with a serum-free medium. e P. lobata extract, puerarin, daidzein, and daidzin as well as FITC-conjugated dextran (50 kDa; Sigma-Aldrich, St. Louis, MO, USA) were added into the Transwell and incubated for 1 h. Next, H 2 O 2 (200 μM) was added and the plate was incubated for 24 h at 37°C under 5% CO 2 in a humidified incubator. FITC was measured at an excitation wavelength of 485 nm and an emission wavelength of 525 nm using Synergy ™ HT Multidetection Microplate Reader (BioTek).
2.6. Immunohistochemistry. After treatment, the cells were washed with PBS and fixed with 2% paraformaldehyde for 20 min at 4°C. Next, the cells were washed twice with PBS and permeabilized using 0.2% Triton X-100 in PBS for 15 min at room temperature. en, they were again washed twice and blocked using blocking buffer (PBS containing 0.3% Triton X-100 and 5% normal serum) at room temperature.
e cells were subsequently incubated with a primary antibody against ZO-1 in blocking buffer (1 : 1000) overnight at 4°C, washed three times with PBS, and incubated with a secondary antibody in blocking buffer (1 : 1000) for 2 h at room temperature. e cells were then treated with DAPI for visualization of the nuclei. Immunofluorescent images of the cells were captured using a fluorescence microscope (BX51, Olympus microscope, Japan).

Western Blot Analysis.
After treatment, the cells were washed with PBS and harvested using Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Total protein concentrations were determined using a BCA Protein Assay Kit (Pierce Chemical, Grand Island, NY, USA). To prepare the protein samples for analysis, they were boiled. e sample was separated into equal amounts by 10% SDS-PAGE gel electrophoresis and transferred to a nitrocellulose blotting membrane (GE Healthcare Life Science, Germany). e membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and then incubated with a diluted primary antibody in TBST overnight at 4°C. e membranes were then washed three times with TBST and incubated with a diluted secondary antibody in TBST for 2 h at room temperature. e membranes were further washed with TBST and detected using EzWestLumi One (ATTO Corp., Japan) with LAS 3000 (Fujifilm, Tokyo, Japan).

Statistical Analysis.
e data are expressed as the mean ± SEM of multiple experiments. Paired Student's ttests were used to compare two groups, and ANOVA with Tukey's test was used for multiple comparison tests using Prism 5.0 software (GraphPad 5.0, San Diego, CA, USA). Values of p < 0.05 indicated statistical significance.

HPLC Analysis of Puerarin, Daidzein, and Daidzin in P. lobata Extract.
e HPLC method was applied to the quantitative analysis of puerarin, daidzein, and daidzin in the P. lobata extract. e identification and quantitative determination of puerarin, daidzein, and daidzin in the extract were accomplished by a comparison of the retention time and area with those of standard three compounds (Figure 1(b)). e linearity of the HPLC method was checked by injecting five concentrations of standard solutions (Figure 1(a)). e calibration curves of puerarin, daidzein, and daidzin showed good linearity (r 2 > 0.9999) within given concentration ranges (Table 1). e contents of puerarin, daidzein, and daidzin in the P. lobata extract were 188.90, 2.73, and 32.54 mg/g, respectively (   induced RPE cell death, the CCK-8 assay was performed, which showed that H 2 O 2 reduced RPE cell viability in a concentration-dependent manner (Figure 2(a)). H 2 O 2 at 300-500 μM reduced the cell viability index (%) at 24 h in a dose-dependent manner, and the difference was significant compared with the value of the control group ( * * * p < 0.001, * * * * p < 0.0001 vs. control). Next, to test the effect of the P. lobata extract on cell viability, the cells were treated with various concentrations (0.5-20 μg/mL) of the extract for 24 h. A high dose of the extract and single compounds did not alter cell viability (Figure 2(b)). To examine whether the P. lobata extract can protect against H 2 O 2 (300 μM) induced cell death, the cells were treated with the P. lobata extract and H 2 O 2 . As shown in Figure 2(c), the P. lobata extract (0.5 and 1 μg/mL) significantly inhibited H 2 O 2 -induced cell death ( * * * p < 0.001 vs. control; # p < 0.05 vs. H 2 O 2 -treated cells).

Effect of P. lobata Extract on H 2 O 2 -Induced Intracellular ROS Generation.
To determine the concentration of the P. lobata extract and its single compounds that do not affect cell viability, the cells were treated with the P. lobata extract and its single compounds and with 200 μM H 2 O 2 for 24 h. Before evaluating H 2 O 2 -induced intracellular ROS generation, the time required for intracellular ROS generation detected as H 2 DCF-DA was determined by FACS analysis. As expected, intracellular ROS generation was markedly increased for a 30 min treatment with H 2 O 2 treatment (data not shown). H 2 O 2 at a concentration of 200 μM showed no effect on cell viability; however, it markedly increased ROS generation (Figures 2(a) and 3(b)). Next, we examined the effect of the P. lobata extract and its individual components (puerarin, daidzein, and daidzin) on ROS generation. e P. lobata extract (10 μg/mL) inhibited H 2 O 2 -induced intracellular ROS generation, with the levels reaching those of the normal controls (Figures 3(c) and  3(g)). Puerarin, daidzein, and daidzin (1 μM) did not show any effect on H 2 O 2 -induced intracellular ROS generation (Figures 3(d)∼3(f )). Taken together, these data suggest that the P. lobata extract could inhibit oxidative damage in RPE cells.

Effect of P. lobata Extract on Paracellular Permeability.
e RPE cells form the outer layer of the BRB, and the tight junctions expressed in the outer BRB regulate entry of fluids and solutes into the retina that are essential for retinal homeostasis. We explored the effect of the P. lobata extract on the function of the RPE barrier by measuring paracellular permeability of 50 kDa dextran in RPE cells. As shown in Figure 4(a), cell systems treated with FITCdextran have been used for in vitro permeability assays [2,20]. As shown in Figure 4(b), oxidative damage increased the diffusion of FITC-dextran and the P. lobata extract inhibited this increase by almost 33.3%. However, the individual compounds (at 1 μM concentration) could not inhibit this oxidative stress-induced diffusion. ese results suggest that the P. lobata extract could decrease the membrane permeability in oxidative stress-induced RPE cells and protect the BRB.
Paracellular permeability is related to the altered expression of tight junction proteins such as ZO-1. We evaluated the alterations in ZO-1 expression in response to oxidative damage for 24 h using immunoblotting. P. lobata extract and single compounds were examined for their inhibitory effects. e expression of ZO-1 decreased in RPE cells exposed to oxidative stress, and P. lobata extract attenuated the decrease in the expression of ZO-1 as compared to the level of normal control ( Figure 5(a)). In addition, ZO-1 expression on cell membranes was evaluated using immunohistochemistry in RPE cells; ZO-1 expression was altered in response to oxidative damage. Fluorescence intensity and areas of discontinuity of ZO-1 were reduced in H 2 O 2 -treated RPE cells compared to those of the normal control ( Figure 5(b)-b, white arrows). When treated with P. lobata extract, H 2 O 2 -treated RPE cells showed normalization of ZO-1 immunostaining ( Figure 5(b)-c). ese data demonstrate that the P. lobata extract inhibited the disruption of the tight junction protein in oxidative stressinduced RPE cells.

Inhibitory Effects of P. lobata Extract on H 2 O 2 -Induced Phosphorylation of p38 MAPK and JNK in RPE Cells.
To identify the signaling pathway through which the P. lobata extract exhibits their effects in H 2 O 2 -treated RPE cells, we examined the effects of the P. lobata extract on the phosphorylation of p38 MAPK and JNK. Figure 6 shows the representative immunoblots of phosphorylated p38 MAPK and JNK in H 2 O 2 -treated RPE cells. Phosphorylation of p38 MAPK and JNK was increased by 3-fold and 2.5-fold, respectively, after H 2 O 2 treatment in RPE cells. e P. lobata extract significantly inhibited phosphorylation of p38 MAPK (Figure 6(a)) and JNK (Figure 6(b)) in RPE cells.

Discussion
In this study, the P. lobata extract were tested for their potential inhibitory effect against H 2 O 2 -induced RPE cell death and membrane permeability. e P. lobata extract significantly inhibited cell death and ROS generation. Membrane permeability was prevented, and the expression of the tight junction protein ZO-1 was increased in H 2 O 2treated RPE cells following treatment with the P. lobata extract. Additionally, H 2 O 2 -induced p38 MAPK and JNK phosphorylation was reduced after treatment of RPE cells with the P. lobata extract.
AMD is the leading cause of blindness in the elderly. e pathogenesis of AMD is related to oxidative stress, formation of drusen, accumulation of lipofuscin, and inflammation in the retina. e retina consumes oxygen causing it to be particularly susceptible to oxidative stress.
Oxidative stress causes RPE cell death in AMD and then shows a lack of chromatin condensation and DNA fragmentation [21]. One of the targets of many drugs aiming to treat or prevent AMD is the inhibition of oxidative stress or its downstream pathways: inflammation, pathological neovascularization, and miRNA [22][23][24].
Recently, small-molecule drugs from single compounds were shown to have therapeutic effects in AMD by inhibiting oxidative stress [23]. Curcumin exhibits a strong antioxidant activity, upregulates heme oxygenase-1 (HO-1) (the oxidative stress defense enzyme), and may Evidence-Based Complementary and Alternative Medicine protect RPE cells against oxidative stress by reducing ROS levels [25]. Paeoniflorin also protects RPE cells against oxidant stress, and canolol prevents oxidative stress-induced cell damage [23,26].
A previous study showed that the antioxidative activity of P. lobata was attributable to higher contents of the isoflavonoids puerarin, daidzein, and daidzin. P. lobata water extract and puerarin (IC 50 value of 756.2 μM) have antioxidant effects against free-radical-mediated damage of red blood cells [27]. Daidzein and daidzin do not possess strong antioxidative effects, with IC 50 values over 1000 μM [27]. Our results also showed that the P. lobata extract had an antioxidant effect in H 2 O 2 -treated RPE cells (Figure 3(g), 10 μg/mL) and prevented cell death (Figure 2(c), 0.5 and 1 μg/mL). However, the individual components (1 μM) from the P. lobata extract could not significantly inhibit H 2 O 2induced oxidation in RPE cells. ese results indicate that the P. lobata extract, comprising the three individual components, has synergistic effects on antioxidation in RPE cells. Pretreatment with the P. lobata extract showed a preventive effect against H 2 O 2 -induced oxidation and cell death.
Tight junctions primarily establish a permeable retinal barrier across epithelial sheets. Occludin is the first transmembrane protein of tight junctions to be identified, and ZO-1 is the first identified tight junction component [28,29]. e effects of the P. lobata extract on the expression of tight junction proteins and membrane permeability have not been evaluated thus far. In the present study, we reported for the first time that the expression of tight junction proteins decreased in H 2 O 2 -treated RPE cells and that the P. lobata extract inhibited the disruption of ZO-1 expression, as observed by western blotting and immunohistochemistry (Figures 4 and 5). Tight junction disruption is related to the p38 MAPK signaling pathway [30,31]. Inhibition or knockdown of JNK attenuates tight junction disruption [32]. e P. lobata extract inhibited p38 MAPK and JNK phosphorylation related to H 2 O 2induced tight junction disruption and barrier dysfunction. Pretreatment with P. lobabta extract showed preventive effects against oxidation, cell death, and tight junction disruption in H 2 O 2 -treated RPE cells. However, pretreatment with the individual compounds from the P. lobata extract did not show high efficiency compared to that obtained with P. lobata extract pretreatment. erefore, these findings indicate that the P. lobata extract displays a synergistic effect of the three compounds. P. lobata extract is a crude herbal extract and is difficult to apply direct to the eyes now. However, following preclinical studies in the animal model for retinopathy and AMD and    Evidence-Based Complementary and Alternative Medicine clinical studies, we can consider its clinical use via oral administration or eye drops. A variety of herb eye drops for allergies or eye health are currently on the market [33]. Future preclinical and clinical studies are required to further establish the effects of P. lobata extract.

Conclusions
e present study showed that the preventive effect of the P. lobata extract involved the inhibition of ROS generation and cell death in RPE cells. Furthermore, the P. lobata extract prevented tight junction disruption via the p38 MAPK and JNK signaling pathways. Together, these results strongly suggest that the P. lobata extract could be a potential alternative for preventing the development of oxidative stressrelated ocular disorders such as AMD.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
NRK and YSK designed the experiments. NRK, BJP, DHJ, ISL, and CSK performed the cell culture experiments and contributed to the isolation of the extract and single compounds. NRK and YSK wrote the manuscript. JSK supervised the work and reviewed the manuscript. All authors provided comments on initial and final drafts of the manuscript.