Nutraceutical effects of Emblica officinalis in age-related macular degeneration

Emblica officinalis Gaetrn (i.e., Phyllanthus emblica/ Indian gooseberry/ Amla) (EO) has been used extensively as a nutraceutical in several diseases since it is known to boost immunity and offers numerous health benefits such as antioxidant, anti-inflammatory, and anti-aging effects. The goal of our study was to test the hypothesis that EO will rescue human AMD RPE transmitochondrial cells from mitochondria-induced cellular damage. AMD RPE transmitochondrial cell lines were created by fusion of mitochondria DNA-deficient APRE-19 (Rho0) cells with platelets isolated from AMD patients, and therefore had identical nuclei but differed in mitochondrial DNA content. These AMD RPE cells were treated with EO extract followed by characterization of effects of EO using cellular and molecular assays. Herein, EO significantly improved live cell number and mitochondrial membrane potential, reduced apoptosis and oxidative stress, down-regulated VEGF, and up-regulated PGC-1α. In conclusion, EO improved cellular and mitochondrial health, thereby playing a key cytoprotective role in AMD in vitro. Further studies are required to examine the mechanisms that mediate the cytoprotective effects of EO.

Due to its high Vitamin C content which on an average is ~600 mg/100 g, EO is well-known as an immunity boosting food. In addition to vitamin C, EO is a rich source of antioxidants, including polyphenols, which confer EO its free radical scavenging potential [3]. A study by Carlson et al. revealed that EO has an antioxidant content of ~261.5 mmol/100 g which was substantially higher than numerous other plant-based foods and supplements that were tested using the FRAP assay in the same study [4]. Substantive evidence validates the antioxidant and cytoprotective properties of EO in several disease models including Alzheimer's, diabetes, cardiac diseases, inflammatory disorders, hepatic diseases, atherosclerosis, cancer, and pulmonary fibrosis [5][6][7][8][9][10][11].
The goal of the current study was to examine and characterize the nutraceutical potential of EO in a human retinal pigment epithelial (RPE) age-related macular degeneration (AMD) transmitochondrial cybrid cell model [12]. We hypothesized that EO will rescue AMD RPE transmitochondrial cells from cellular and mitochondrial damage in vitro. The results of this novel study the cytoprotective role of EO in AMD RPE cybrid cells in terms of increased viability and reduction in oxidative stress and apoptosis.

EO concentration optimization
To determine the optimum working concentration of EO for all experiments, we performed an initial concentration titration experiment wherein AMD cybrids were treated with increasing doses of EO i.e, 10, 15, 20, and 25 mg/mL of EO ( Figure 1) followed by measurement of viable cell numbers using MTT assay. Although compared to untreated AMD cybrids (Bar 1; 1 ± 0.382 (Mean ± SEM) arbitrary unit (a.u.), n=3), 10 mg/mL EOtreated AMD cybrids (Bar 3; 1.59 ± 0.191 a.u.; n=3) showed a 59 % higher viable cell number, the difference was not statistically significant (p=0.2387). However, EO-treated cybrids showed significantly improved viable cell number at 15   difference was observed between the AMD untreated (bar 1) vs. AMD solvent control (bar 2) groups. Furthermore, no statistically significant difference was observed between untreated (bar 1) and 10 mg/mL EO-treated (bar 3) AMD cybrids. Higher viable cell numbers were observed in EO-treated AMD cybrids at concentrations of 15 mg/mL (bar 4), 20 mg/mL (bar 5), and 25 mg/mL (bar 6). *** indicates p<0.001; ns indicates non-significant p-value. Data are presented as mean ± SEM and normalized to untreated AMD cybrids which were assigned a value of 1. Experiments were performed at the 24 h time-point.
These results suggest that EO can mitigate Caspase-3/7 mediated apoptosis in AMD RPE cybrids.

Effect of EO on VEGF gene expression and on cell viability and ROS levels in amyloid-β-stressed AMD cells
Since VEGF has been implicated in the etiology of AMD, we next sought to compare VEGF gene expression between untreated and EO-treated AMD cybrids. We observed significant downregulation of VEGF gene in EO-treated AMD cybrids compared to untreated cybrids (64.7 % decrease; AMD untreated: 1 ± 0.066 a.u.; AMD EO-treated: 0.353 ± 0.132 a.u.; p=0.029; n=4) ( Figure 7A).

DISCUSSION
In the present study, we report the protective role of Emblica officinalis in rescuing human AMD RPE cybrid cells from damage. Herein, we analyzed the effects of exogenously added EO on the viable cell numbers, ROS levels, mitochondrial membrane potential, gene expression of Caspase-3, SOD2, PGC-1α, MT-RNR2, and VEGF, and against amyloid-β-induced toxicity in AMD RPE transmitochondrial cybrid cell lines in vitro.
We began our EO study by testing a varying range of concentrations of EO i.e., 10, 15, 20, and 25 mg/mL in AMD RPE cybrids in vitro and chose EO concentration of 25 mg/mL as the final optimal working concentration for all experiments. To our knowledge, this is the first study investigating the effects of EO on RPE cybrids containing damaged AMD mitochondria [13]. According to previous literature, varying doses of EO fruit extract are administered depending on the species, the model system, and nature of the study. For instance, Rao et al. found clinically relevant concentrations at 1-100 µg/ml of EO fruit extract to be effective in human umbilical vein endothelial cells (HUVEC) in vitro [15]. Yamamoto et al. demonstrated that treatment with 100-200 µg/ mL EO was effective in C2C12 myoblasts, a skeletal muscle cell line, in vitro [16]. However, in vivo and human studies required higher concentrations of EO fruit extract. For example, Lim et al. showed administration of 300 mg/kg EO to Sprague-Dawley (SD) rats was effective [17]. In diabetic studies, human subjects were given oral concentrations of EO at 1 g/mL, 2 g/mL, and 3 g/mL in water [6].
We next characterized the cytoprotective role of EO in AMD cybrids. Comparison of cell viability between untreated and EO-treated AMD cybrids demonstrated consistently higher viable cell numbers in EO-treated AMD cybrids at 24 h, 48 h and 72 h post EO-treatment.
Our observations were consistent with previous studies that have highlighted the cytoprotective role of EO. For example, EO at a concentration of 500 mg/kg significantly increased cell viability, thereby rescuing splenocytes from arsenic-induced cell damage in mice [18]. Another recent finding revealed attenuation of t-BHP-induced cytotoxicity by pretreatment with EO for 48 h in a murine skeletal muscle cell line [16]. EO was shown to inhibit chromium-induced toxicity by enhancing percent cell survival and cell proliferation in an in vitro murine macrophage model [19]. In addition, EO prevented apoptotic cell death and enhanced cell proliferation in lymphocytes isolated from Sprague-Dawley rats [20]. Another study demonstrated the role of EO as a cytoprotectant in vivo [21].
It has been established previously that AMD RPE transmitochondrial cybrid cells are damaged due to diseased AMD mitochondria and undergo apoptotic cell death [13][14]. Therefore, to examine the effects of EO on apoptosis markers, we next examined the effects of EO administration on Caspase-3/7 activity using IncuCyte® live-cell imaging system and reagents. The NucLight reagent (Red) stains nuclei in live cells and the Caspase-3/7 reagent (Green) enables real-time quantification of cells undergoing caspase-3/7 mediated apoptosis. Our data revealed that EO-treated AMD cybrids had reduced Caspase-3/7 activity compared to their untreated counterparts. Moreover, AMD cybrids treated with EO had reduced expression levels of Caspase-3 gene compared to untreated AMD cybrids. To our knowledge, this is the first study to report such EO effects in human AMD cybrids. However, addition of EO has been reported to decrease caspase-3 activity and to protect against arsenic-induced toxicity in thymocytes of mice [22]. Another study reported that co-treatment with EO reduced caspase-3 activity in splenocytes in vitro [18]. The same study used Annexin V/PI binding experiment to demonstrate that cotreatment with EO reduced the number of apoptotic and necrotic cells [18].
Enhanced production of reactive oxygen species (ROS) is associated with damaged mitochondria and deterioration of mitochondrial health and function [23]. AMD RPE transmitochondrial cybrid cells used in this study have higher levels of mitochondrial ROS compared to normal RPE transmitochondrial cells [13]. In the current study, we used H2DCFDA, a chemically reduced form of fluorescein, as an indicator of ROS in AMD cybrids and observed that EO induces a consistent ROSreducing effect in AMD cybrids at 24 h, 48 h, and 72 h post treatment. This finding is critical since elevated ROS levels have been implicated in the pathogenesis of numerous aging-related diseases [24][25][26] including retinal diseases such as AMD, diabetic retinopathy, glaucoma, etc. [27]. Quantitative RT-PCR analyses revealed that treatment with EO increased the transcript levels of SOD2, the mitochondrial superoxide dismutase which plays an antioxidant role in preserving cellular health in AMD. [28] This is a key finding since SOD2 deficiency contributes to oxidative damage in RPE and development of AMD pathogenesis [29]. Our current results are consistent with previous studies that have highlighted the crucial role of EO as a potential antioxidant [4,[30][31] in combating oxidative stress in aging-related diseases/disorders such as diabetes, renal dysfunction, hyperlipidaemia, etc. [32][33][34]. Elevated ROS levels can cause VEGF (Vascular Endothelial Growth Factor) activation thereby triggering angiogenesis and subsequent choroidal neovascularization in wet AMD [35][36]. In our previous studies, we found significant up-regulation of VEGF gene in AMD RPE transmitochondrial cybrid cells compared to agematched normal RPE transmitochondrial cybrid cells (Data not shown). Interestingly, in the present study, EO decreased the VEGF gene levels in AMD cybrids, suggesting that EO might help in reducing VEGFinduced neovascularization in AMD. This data is consistent with reports by Lu et al. wherein an EO preparation inhibited VEGF-induced angiogenesis via suppression of VEGF receptor activity [37].
Using the JC-1 dye, we compared the mitochondrial membrane potential (ΔΨm) between untreated and EOtreated AMD cybrids, and found significantly higher ΔΨm in EO-treated AMD cybrids. Therefore, EO can enhance the oxidative redox state of AMD cybrids, contributing to preservation of mitochondrial integrity and function in AMD cybrids. Our data were in agreement with recent studies showing that EO positively contributes to mitochondrial health by enhancing the spare respiratory capacity [16]. QRT-PCR analyses demonstrated that EO-treated AMD cybrids had significant upregulation of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) gene compared to untreated AMD cybrids. PGC-1α is an important regulator of mitochondrial biogenesis and its downregulation contributes to AMD pathology [38]. Moreover, PGC-1α drives human RPE mitochondrial function and induces antioxidant capacity [39].
Drusen deposit formation under the retina is a characteristic feature in AMD [40]. Amyloid-β is a common protein found in retinal drusen deposits [41][42] and in the brains of patients with Alzheimer's disease [43][44]. Therefore, we tested the effects of EO on amyloid-β-induced toxicity. The AMD cybrids were pretreated with EO followed by exposure to amyloid-β peptides. The EO pretreatment rescued AMD cybrids from amyloid-β-induced cellular damage as demonstrated by higher cell viability and lower ROS levels. Similar results were observed in an in vivo model of Alzheimer's disease wherein oral administration of EO attenuated amyloid-induced toxicity [5].
To speculate a mechanism by which EO mediates its protective action in AMD cybrids, we measured the expression of MT-RNR2 gene using TaqMan probes. Significant up-regulation of the MDP-coding MT-RNR2 gene was observed in EO-treated AMD cybrids compared to the untreated cells. Since MDPs have been assigned a cytoprotective role in AMD [13][14] and other age-related diseases [45], EO-mediated cytoprotection in the AMD cybrids may be partly attributed to higher expression of the MT-RNR2 gene. Another plausible mechanism by which EO confers its protective effects in AMD cybrids could be via aldose reductase inhibition. It is known that tannins, which are one of the components of EO, possess aldose reductase inhibitor activity [46]. Aldose reductase, an enzyme involved in glucose metabolism, has been associated with the pathogenesis of retinal diseases including diabetic retinopathy and cataract [47][48][49][50]. Chang et al. demonstrated that overexpression of aldose reductase is associated with activation of retinal microglia in mice. Since retinal microglia are immune cells that mediate inflammatory responses in the eye, their activation causes secretion of pro-inflammatory cytokines thereby contributing to the pathogenesis of eye diseases. However, suppression of aldose reductase prevents retinal microglia activation and migration, subsequently preventing ocular inflammation and disease development [51].
In conclusion, treatment with purified EO extract preserves mitochondrial and cellular health and function in human AMD RPE cybrids, implying that EO mitigates aging-related damage in AMD. Since EO extract is an over-the-counter nutraceutical and is available in both liquid and capsule forms for easy consumption, it might serve as an effective, inexpensive, and non-invasive therapeutic option for treatment of AMD. Further studies are required to fully understand the precise mechanisms that orchestrate the protective events post EO treatment in AMD cells.

Human Subjects
The Institutional review board of the University of California Irvine approved research with human subjects (Approval #2003-3131). All participating subjects provided informed consent and clinical investigations were performed according to the tenets of Declaration of Helsinki.

Cell culture
Human AMD RPE transmitochondrial cells were created by fusing mitochondria DNA-deficient APRE-19 (Rho0) cells with platelets isolated from AMD patient's blood as described previously. [13] Passage 5 cells were used for all experiments (n=3-6).

Treatment with Emblica Officinalis (EO)
Purified EO extract was obtained and used at a concentration of 25 mg/mL for all experiments. DMSO was used as an initial solvent. EO was subsequently dissolved in culture media for cell treatment.

Cell viability assay (MTT assay)
The numbers of viable cells were measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Cat.# 30006, Biotium, Fremont, CA). Cells were plated in 96-well tissue culture plates and treated with 25 mg/mL EO for 24 h and 48 h. Cells were incubated with MTT reagent at 37 °C for 1 h, followed by addition of DMSO. Signal absorbance was measured at 570 nm and background absorbance was measured at 630 nm. Normalized absorbance values were obtained by subtracting background absorbance from signal absorbance. The colorimetric signal obtained was proportional to the cell number.

IncuCyte Live-cell imaging
The IncuCyte NucLight Rapid Red Reagent (Cat.# 4717, IncuCyte, Ann Arbor, MI) is a cell permeable DNA stain that specifically stains nuclei in live cells and enables real-time quantification of cell proliferation.
The IncuCyte Caspase-3/7 Green Apoptosis Reagent (Cat.# 4440, IncuCyte) couples the activated Caspase-3/7 recognition motif (DEVD) to a DNA intercalating dye and enables real-time quantification of cells undergoing caspase-3/7 mediated apoptosis. This reagent is an inert, non-fluorescent substrate which when added to culture medium, crosses the cell membrane where it is cleaved by activated caspase-3/7 resulting in the release of the DNA dye and fluorescent staining of the nuclear DNA.
Cells were seeded in 96-well plates at a density of 5000 -10,000 cells/well, treated with EO, followed by staining with IncuCyte® NucLight Rapid Red (1:500) and Caspase-3/7 Green (1:1000) labeling reagents. Stained cell plates were placed into the IncuCyte® livecell analysis system and allowed to warm to 37 °C for 30 min prior to scanning. Phase Contrast, Green, and Red channels were selected, 5 images were taken per well with an average scan interval of ~2 h until the experiment was complete. Fluorescent objects were quantified using the IncuCyte® integrated analysis software that minimizes background fluorescence.

Reactive Oxygen Species (ROS) assay
To quantitate ROS levels, the cell-permeant H2DCFDA (2', 7'-dichlorodihydrofluorescein diacetate) was used as an indicator for ROS in cells. Stock solution of 5mM H2DCFDA was prepared in DMSO. Stock solution was then diluted in DPBS to obtain a working concentration of 10 µM. Cells were plated in 96-well tissue culture plates followed by treatment with 25 mg/mL EO. 10 µM H2DCFDA solution was added to cells and incubated for 30 min at 37 °C. H2DCFDA was then replaced with DPBS. Fluorescence which was measured at excitation 4924 nm and emission 520 nm was proportional to ROS levels in cells.

Quantitative Real-Time PCR
RNA extraction, cDNA synthesis, and qRT-PCR analysis from EO-treated AMD cybrids were performed as described previously. [14] QuantiTect Primer Assays were used to study the expression of Caspase-3 gene (Cat. # QT00023947, Qiagen, Germantown, MD), and SOD2 gene (Cat. # QT01008693, Qiagen). KiCqStart® SYBR® green primers were used to examine the expression of PGC-1α and VEGF genes (Cat. # kspq12012, Sigma, St. Louis, MO). Specific housekeeper genes used were HPRT1 (Cat. # QT00059066, Qiagen) and HMBS (Cat. # QT00014462). TaqMan gene expression master mix (Cat. # 4369016, Life Technologies) and TaqMan gene expression assays were used to examine the expression of the MT-RNR2 gene (Assay ID: Hs02596860_s1, Life Technologies), for which GAPDH (Assay ID: Hs02786624_g1, Life Technologies) was used as a housekeeper gene. Data analysis was performed using ∆∆Ct method which was calculated by subtracting ∆Ct of the AMD group from ∆Ct of the normal group. ∆Ct was the difference between the Cts (threshold cycles) of the target gene and Cts of the housekeeper gene (reference gene). Fold change was calculated using the following formula: Fold change = 2 ΔΔCt .

Statistical analysis
Non-parametric Mann-Whitney test (for 2 groups) or one-way ANOVA (for 3 or more groups) followed by AGING post-hoc Tukey-Kramer test (GraphPad Prism 5.0; GraphPad Software, CA, USA) were performed to analyze data between groups. P values < 0.05 were considered statistically significant.