Protective effects of protocatechuic acid on retinal ganglion cells from oxidative damage induced by H2O2 Zhuo Wang2, Xiaohong Pan1, Dan Wang3, Hongliu Sun1, Fang Han1, Changjun Lv1, Xiuli Zhang1 1

School of Pharmaceutical Sciences, Binzhou Medical University, Shandong, P.R. China, 2Department of Pharmacology, HE’s University, Liaoning, Shenyang, P.R. China, 3Department of Histology and Embryology, College of Basic Medical Science, China Medical University, Shenyang, P.R. China

Oxidative stress-induced retinal ganglion cell (RGC) death is one of the important factors in ocular disease such as glaucoma. The aim of this study is to investigate whether protocatechuic acid (PCA), a monomeric phenolic acid with strong free radical scavenging effects, could protect RGC from oxidative stress induced by H2O2 and provide a potential therapy drug. The viability of RGC was determined by the MTT reduction and lactate dehydrogenase (LDH) assay. The possible protective mechanism was investigated via detecting apoptosis by Hoechst staining, Bcl-2 and Bax protein levels by ELISA, caspase-3 activation and membrane potential by immunofluorescence, and caspase-3 mRNA expression by polymerase chain reaction (PCR) method. The results showed that PCA was effective in reducing apoptotic death induced by oxidative stress, mainly through inhibiting depolarization of membrane and activation of caspase-3, downregulating of apoptosis-related protein Bax, and up-regulating Bcl-2. Keywords: Protocatechuic acid, H2O2, Apoptosis, Caspase-3, Retinal ganglion cells

Introduction Glaucoma is a chronic neurodegenerative disease, and various mechanisms of glaucomatous optic neuropathy (GON) have been thought to cause retinal ganglion cell (RGC) death leading to visual loss.1 Retinal ganglion cell death in GON has been thought to occur by an apoptotic mechanism triggered by multiple stimuli. Recent studies demonstrated that RGC and optic nerve injury are accompanied by increased reactive oxygen species (ROS).2 Excessive amounts of ROS induced by oxidative stress can modify proteins, lipids, and DNA to alter their functions and activate signaling pathways resulting in death of retinal neurons.3 Consequently, the suggestion has been made that anti-oxidants can prevent or ameliorate the progression of neuronal death in patients with glaucoma.3 Protocatechuic acid (PCA), a phenolic compound, was first isolated from the extract of Alpinia oxyphylla fruits, and its structure was determined by spectroscopic analysis.4 Protocatechuic acid Correspondence to: Xiuli Zhang, School of Pharmaceutical Sciences, Binzhou Medical University, Shandong 264003, P.R. China. Email: [email protected] or Changjun Lv, School of Pharmaceutical Sciences, Binzhou Medical University, Shandong 264003, P.R. China. Email: [email protected]

ß W. S. Maney & Son Ltd 2015 DOI 10.1179/1743132814Y.0000000421

stimulated PC12 cellular proliferation and markedly attenuated MPPz-induced apoptotic cell death in a dose-dependent manner.4 It has a wide variety of biological activities including anti-radical, anti-oxidant, and moderate scavenger of H2O2.5–7 Recent studies have demonstrated that PCA can protect against H2O2 and MPPz-induced oxidative damage on PC12 cells, showing its significant effect on protecting PC12 cells against H2O2-induced apoptosis.4,8 In animal models, it has been found that PCA could enhance cognitive performance in aged rats and reduce oxidative stress in brain, liver, and spleen.9–11 Based on these reports, we hypothesized that PCA may play a neuroprotective role in glaucoma. So, the aim of this study is to investigate whether PCA could inhibit the oxidative damage induced by H2O2 in cultured RGC-5 cells and to explore the possible neuroprotective mechanism of PCA.

Materials and Methods Cell treatment RGC-5 cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, Carlsbad, USA). Cells were

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grown in a humidified incubator of 95% air and 5% CO2 at 37uC. Cells were passaged when they were 80% confluent. The cells were cultured in the presence or absence of 0.2 mM H2O2 for 24 hours. When the effects of PCA on cells were studied, PCA (0.1, 0.2, and 0.4 mM) was added for 30 minutes prior to H2O2 treatment. After 30 minutes, 0.2 mM H2O2 was added and incubated for 24 hours in growth media. The control group was treated with PBS alone in the same manner.

Cell viability assay MTT assay was employed to examine cell viability. Briefly, MTT was added to the culture medium at the end of treatment at a final concentration of 0.5 mg/ ml. After the cells were incubated at 37uC for 4 hours, the culture medium containing MTT was removed. Dimethyl sulfoxide (DMSO) was then added into each well to dissolve the formed blue formazan. Absorbance was read at 570 nm on a microplate reader.

Assays for lactate dehydrogenase The reagents used for enzyme assays were obtained from Jiancheng Bioengineering Institute (Nanjing, China). All procedures completely complied with the manufacturer’s instructions. Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is present in all cells and rapidly released from the damaged cells cytosol into the surrounding medium following loss of membrane integrity, resulting in either apoptosis or necrosis. Lactate dehydrogenase leakage was calculated as the percentage of LDH in the medium versus total LDH activity in the cells.

Hoechst staining Apoptotic cell death was analyzed by Hoechst 33258 (Sigma, St. Louis, USA) staining. Cells were fixed with 4% paraformaldehyde and rinsed twice in PBS. Cells were then incubated with 2 mg/ml Hoechst 33258 for 30 minutes at 37uC, and visualized under UV light and photographed after a final rinse with PBS.

Measurement of glutathione (GSH) The glutathione (GSH) content was determined by using commercially available kits (Bio Vision, Mountain View, USA). All procedures completely complied with the kit instructions. RGC-5 cells were pretreated with PCA (0.2 mM) 0.5 hours, subsequently treated with H2O2 (0.2 mM) for an additional 24 hours. Then, the GSH content was determined according to the user’s manual.

Assay of intracellular ROS Reactive oxygen species were measured with the nonfluorescent probe 29,79-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA passively diffuses into cells

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and is deacetylated by esterases to form nonfluorescent 29,79-dichlorofluorescein (DCFH). In the presence of ROS, DCFH reacts with ROS to form the fluorescent product DCF, which is trapped inside the cells. Cells were pretreated with PCA (0.2 mM) 0.5 hours, then treated with H2O2 (0.2 mM) for an additional 24 hours. To obtain dissociated cells for the ROS assay, culture medium was removed and the cells were washed three times with PBS. DCFH-DA, diluted to a final concentration of 10 mM with DMEM, was added to cultures and incubated for 20 minutes at 37uC. The fluorescence was read at 485 nm for excitation and 530 nm for emission with a fluorescence plate reader (Genios, Tecan, Mannedorf, Switzerland). The increase value compared to control was viewed as the increase of intracellular ROS.

Membrane potential analysis Changes in the membrane potential were indicated using the anionic lipophilic potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] (Dojindo, Kumamoto, Japan). When cells were depolarized, dye entered the cytosol and fluorescence intensities increased. After loading with DiBAC4(3) at 37uC for 40 minutes, cells were placed in a chamber filled with PBS.

Enzyme-linked immunosorbent assay Bcl-2 and bax The level of Bcl-2 and bax was quantified using an ELISA kit (Jimro, Gunma, Japan) according to the manufacturer’s instructions. All procedures were performed at room temperature. The absorbance of the plate was measured at 490 nm using a plate reader. The same procedure was followed for the standard curve.

Immunofluorescence Briefly, RGC-5 cells growing on cover slips were fixed with 4% paraformaldehyde for 30 minutes. After being blocked with 4% bovine serum albumin (BSA) for 1 hour at room temperature, and then the cells were incubated with primary antibodies. These primary antibodies included rabbit caspase-3 (1:200). After incubating with the primary antibodies at 4uC overnight, the cells were rinsed four times in PBS. Then, they were incubated with the corresponding secondary antibodies, Cy3-conjugated anti-rabbit IgG (1:100; Sigma). DAPI (Invitrogen) was used for counterstaining. A negative control was performed by replacing the primary antibody with the corresponding animal serum.

Reverse-transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from RGC-5 cells following the protocol of an RNeasy mini kit. DNase I (Sigma) treatment was then used to reduce the likelihood of genomic DNA contamination. cDNA

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Protocatechuic acid protects retinal ganglion cells from oxidative injury

was synthesized by using a PrimeScript RT reagent kit (Takara, Tokyo, Japan). Polymerase chain reaction (PCR) was carried out with the following program: 94uC, 3 minutes; 94uC, 30 seconds, 52uC, 30 seconds, 72uC, 45 seconds, 35 cycles; 72uC, 5 minutes. The PCR products were electrophoresed on 1% agarose gels and detected by ultraviolet excitation.

Western blot Cell tissue was homogenized in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, complete protease inhibitor. The homogenate was incubated on ice for 30 minutes, and then the suspension was sonicated on ice using three 10-second bursts at high intensity with a 10-second cooling period between each burst. The sample were centrifuged at 13 000g for 15 minutes at 4uC. Protein concentrations in the supernatant were measured and 50 mg of each protein extract was separated on a 4–12% sodium dodecyl sulfate (SDS)polyacrylamide gel by electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20), and then probed overnight with primary antibody (cleavedcaspase-3 antibody; 1:500 in TBST/5% milk). After washing with TBST, the membrane was incubated for 1 hour in TBST containing the appropriate horseradish peroxidase-conjugated secondary antibody (1:200) and again washed. The blots were developed using ECL western blotting reagents. Densitometric analysis was performed in Scion Image software. Beta-actin normalization was performed for each sample.

Statistical analysis Data are expressed as the mean ¡ S.D. Statistical evaluation of the data was performed by one-way analysis of variance (ANOVA) followed by a Student’s t test. All estimates were conducted in triplicate. A value of P less than 0.05 was considered statistically significant.

Results and Discussion Effect of PCA on H2O2-induced RGC death

Figure 1 Protective effects of protocatechuic acid (PCA) on H2O2-induced neurotoxicity of cultured retinal ganglion cell (RGC)-5 cells. (A) H2O2-induced cytotoxicity in RGC-5 cells. (B) Effects of PCA on H2O2-induced neurotoxicity in RGC-5 cells. (C) Effects of PCA on LDH release in RGC-5 cells. Each value represents the mean ¡ S.E.M. obtained from four culture wells per experiment, determined in three independent experiments. (*) P , 0.05 in comparison with control; (**) P , 0.01 in comparison with cells exposed to H2O2 alone.

In order to select an optimal H2O2 concentration to induce an oxidative stress in RGC-5 cells, cultures were treated with different concentrations of H2O2 (0: control group, 0.1–0.8 mM) for 24 hours. Then, cell viability was measured by MTT assay. As shown in Fig. 1, cell viability was significantly reduced by H2O2 concentration-dependent manner. Consequently, 0.2 m M H2O2 (58.6 ¡ 2.7% of control cell viability) was chosen for subsequent experiments (Fig. 1A). Later, we evaluated the protective effects of PCA on MTT reduction in cultured RGC-5 cells. The

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Figure 2 Retinal ganglion cell (RGC)-5 cells were observed by optic microscope directly or fluorescence microscopy after nuclei staining with Hoechst 33258. (A) Control cells; (B) cells treated with H2O2 alone; (C) cells treated with H2O2 and PCA. The figures are representative for three different experiments.

results (Fig. 1A) showed that the survival rate of RGC-5 was about 58.41 ¡ 2.5% treated with H2O2 alone. However, pretreatment with PCA increased the viability of RGC-5 to 69.2 ¡ 2.9%, 81.5 ¡ 2.5%, and 82.9 ¡ 1.6% at final PCA concentrations of 0.1, 0.2, and 0.4 mM, respectively. Thus, we chose 0.2 mM

PCA for the later experiments against oxidative stress induced by H2O2 in cultured RGC-5 cells (Fig. 1B). Lactate dehydrogenase is a stable cytoplasmic enzyme present in most cells, and it is rapidly released into the cell culture supernatant upon the plasma membrane damage. An increase in the number of dead or plasma membrane-damaged cells results in an increase in LDH activity in the culture supernatant. As shown in Fig. 1C, LDH leakage increased to 44.4 ¡ 4.9% compared with the control group after treatment with H2O2. Protocatechuic acid (0.2 mM) significantly attenuated H2O2-induced cell death, reducing the LDH leakage to nearly normal level.

PCA rescued H2O2-induced changes in nuclear morphology Changes in nuclear morphology after H2O2 treatment were assessed by Hoechst 33258 staining. As shown in Fig. 2, the control RGC-5 cell nuclei had a regular and oval shape. However, apoptotic nuclei, characterized by nuclear condensation and fragmentation, appeared after exposure to H2O2 for 24 h. Protocatechuic acid treatment blocked the H2O2-induced nuclear morphological change.

Measurement of GSH content To determine whether PCA could alter GSH content in the cultured cell, the cellular content of GSH was determined following incubation with H2O2 (0.2 mM) (Fig. 3A). Incubated for 24 hours with H2O2 (0.2 mM) in the absence of PCA, GSH content was decreased dramatically, only 62.1% compared with the control group. However, presence of PCA in a concentration of 0.2 mM obviously reversed the decrease of GSH induced by H2O2.

PCA inhibited H2O2-induced over-production of intracellular ROS Figure 3 Effect of protocatechuic acid (PCA) on GSH content and reactive oxygen species (ROS) formation. (A) GSH level. (B) Reactive oxygen species formation. The results are the mean ¡ SD of four culture wells per experiment, determined in three independent experiments. # P , 0.05 ##P , 0.01 in comparison with control, *P , 0.05 **P , 0.01 compared with H2O2-treated cultures alone.

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Reactive oxygen species formation was measured because it is an important biomarker of oxidative stress. The results showed that the ROS level was significantly increased by H2O2 (0.2 mM) treatment alone. However, pretreatment with PCA (0.2 mM) attenuated H2O2-induced ROS production (Fig. 3B).

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Figure 4 Effect of protocatechuic acid (PCA) on H2O2-induced cell-membrane potential alteration using DiBAC4(3) staining. (A) Retinal ganglion cell (RGC)-5 cells were untreated. (B) H2O2 injured cells. Dye entered the cytosol and fluorescence intensities increased. (C) Protocatechuic acid blocked H2O2-induced cell-membrane depolarized.

PCA blocked H2O2-induced the change in membrane potential The cytotoxic effect of H2O2 was analyzed with. The potential-sensitive fluorescent dye DiBAC4(3) was used to measure membrane potential. Treatment of RGC-5 cells with 0.2 mM H2O2 for 24 hours induced cells depolarized, dye entered the cytosol and fluorescence intensities increased. PCA prevented the H2O2-induced depolarized in RGC-5 cells (Fig. 4).

Effects of PCA on Bcl-2/Bax levels in H2O2induced RGC Extensive evidence suggests that Bcl-2 gene family shows a complex network regulating apoptosis. We, therefore, examined the effect of PCA on the cellular level of Bcl-2 and Bax proteins. After treatment of RGC-5 cells with 0.2 mM H2O2 for 24 hours, Bcl-2 level tended to decrease (Fig. 5A) and Bax level tended to increase (Fig. 5B). Simultaneous incubation with 0.2 mM PCA significantly increased the cellular level of Bcl-2 and decreased Bax level (Fig. 5A and B).

Effects of PCA on caspase-3 protein in H2O2induced RGC

Figure 5 ELISA analysis of the change of Bcl-2 and Bax protein levels in retinal ganglion cell (RGC)-5 cells. (A) Bcl-2 level. (B) Bax level. RGC-5 cells were treated with 0.2 mM H2O2 in the presence of PCA for 24 hours. Each value represents the mean ¡ S.E.M. obtained from four culture wells per experiment, determined in three independent experiments. (*) P , 0.05 in comparison with control; (**) P , 0.01 in comparison with cells exposed to H2O2 alone.

The cellular pathway of H2O2-induced cell death was examined by assessing cleaved-caspase-3 activation and expression, which plays a critical role in apoptosis. RGC-5 cells were treated with 0.2 mM H2O2 for 24 hours and caspase-3 activation and expression were analyzed by immunofluorescence and western blot (Fig. 5A and B). After 24 hours H2O2 treatment, the mean percentage of caspase-3 activated cells and caspase-3 expression were increased more than normal control cells. However, the activation and expression of caspase-3 were significantly decreased in PCA-treated RGC-5 cells (Fig. 6A and B).

Effects of PCA on caspase-3 mRNA in RGC-5 cells induced by H2O2 We investigated whether PCA could modulate RGC5 cells survival after H2O2 treated by analyzing the transcription changes of related genes of caspase-3.

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Figure 6 Effects of PCA on capase-3 activation and expression. (A) The capase-3 activation was examined by immunocytochemistry in RGC-5 cells treated with H2O2 and PCA. (B) The capase-3 expression was examined by western blot. (C) Effects of PCA on H2O2-induced mRNA expression of caspase-3 in RGC-5. DNA strips show expression levels of caspase-3 on agarose gel electrophoresis.

Representative reverse-transcription-polymerase chain reaction (RT-PCR) results for caspase-3 mRNA expression are shown in Fig. 6. Compared with control group, caspase-3 mRNA expression was significantly up-regulated when treated with H2O2. In the PCA group, caspase-3 mRNA expression was significantly lower than that in the H2O2 group (Fig. 6C).

Discussion Oxidative stress is thought to play a major role in the cause of visual loss,3,12–14 and oxidative stressinduced RGC death is one of important factors in ocular disease in glaucoma.15 In this study, RGC-5

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induced damage by H2O2 was used as an in vitro model of glaucoma because it has many of the characteristics of primary RGCs.16 It is known that H2O2 is highly membrane permeable and readily induces cytotoxicity17 by increasing intracellular ROS accumulation,15,18 which has been widely used as an inducer of oxidative stress in a wide range of cell types including neurites and spiral ganglion cells.19 Reactive oxygen species are reactive molecules that include superoxide anion, H2O2, hydroxyl radical, nitric oxide, peroxyl radical, and singlet oxygen, which can ultimately lead to apoptotic or necrotic cell death.20 Therefore, removal of excess ROS or

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suppression of their generation by anti-oxidants may be effective in preventing oxidative cell death. Protocatechuic acid occurs naturally in many Chinese herbal medicines such as Salvia miltiorrhiza (Danshen)21 and Hibiscus sabdariffa L.10 Recently, it has been demonstrated to attenuate apoptosis induced by H2O28 and reduce oxidative stress in vivo.9 After the 24 hours expose to the H2O2, the cell viability decreased to58.6 ¡ 2.7% of control cell viability. However, pretreatment with PCA increased the viability of 69.2 ¡ 2.9%, 81.5 ¡ 2.5%, and 82.9 ¡ 1.6%.8 In this experiment, we have demonstrated that PCA can prevent RGC-5 against oxidative stress induced by H2O2 in vitro. The data presented in this report showed that PCA can protect RGCs from H2O2-induced cell death by increasing cell viability (Fig. 1). To investigate the possible mechanism of PCA protecting RGCs from H2O2-induced cell death by H2O2, cell-membrane damage, Bcl-2 family, and caspase-3 activity were also investigated. Treatment with 0.2 mM H2O2 for 24 hours induced cells membrane to become depolarized; dye entered the cytosol and fluorescence intensities increased. These findings suggest that hydrogen peroxide treatment of RGC-5 leads to cell death and that the mechanism of this cell death involves apoptotic processes. In contrast, treatment with PCA significantly reduced the membrane depolarized of apoptotic cells. The protective effect of PCA against H2O2-induced apoptotic cell death was associated with Bcl-2 family and caspase-3 expression. Members of the Bcl-2 family are pivotal regulators of the apoptotic process,22 and they play a major role in the apoptosis process of RGCs in glaucoma. Bax and Bcl-2, which are putative members of the Bcl-2 family, primarily regulate cell apoptosis.23 It is known that oxidative stress causes Bcl-2 is downregulated in apoptosis.24 Up-regulation of Bax expression with a concomitant decrease in the expression of Bcl-2 changes the permeability of the mitochondrial membrane. Our present study showed that H2O2 had profound effect on the Bcl-2 family proteins in RGC-5 cells. H2O2 had up-regulated Bax expression and down-regulated level of Bcl-2 expression (Fig. 5A). Our results showed that PCA treatment promoted the expression of anti-apoptotic protein Bcl-2 and suppressed the expression of the pro-apoptotic protein Bax (Fig. 5B). Hence, restoration of mitochondrial membrane potential, modulation of Bax, and Bcl-2 expression might be some of the major mechanisms whereby PCA protects against neuronal cell apoptosis induced by H2O2. Caspases are known to be markers of apoptosis.25,26 Caspases play an important role in the apoptotic

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process in two ways, the death receptor pathway and the mitochondrial apoptotic pathway.27 In the receptor-mediated pathway, caspase-8 is the initiator protein that can directly activate the final executioner caspase-3.28 Capase-3 activates DNA fragmentation factor, which in turn activates endonucleases to cleave nuclear DNA and ultimately leads to cell death. RGC5 treated with H2O2 (0.2 mM) for 24 hours were stained using the caspase-3 antibody to demonstrate caspase-3 activation. We also detected cleaved-caspase3 expression by western blot. Figure 6A ad B showed that the activation and expression of caspase-3 induced by H2O2 were inhibited by pretreatment of PCA. Caspase-3 mRNA expression dramatically increased following 24 hours treatment with H2O2. Addition of PCA attenuated H2O2-induced caspase-3 mRNA expression (Fig. 6C).

Conclusion In summary, our data indicated that PCA exerted an inhibitory effect on cell apoptosis death induced by H2O2 via normalizing morphological changes in nuclei, decreasing LDH release, caspase-3 activation and Bax expression, and increasing Bcl-2 expression and mitochondrial membrane potential. Thus, PCA may be used for the treatment of progressive neurodegenerative disorders such as glaucoma. However, further studies are necessary to be done for further neuroprotective mechanisms of PCA before definite conclusions can be drawn.

Disclaimer Statements Contributors Changjun Lv and XiuLi Zhang designed the experiments. Zhuo Wang wrote this article and cultured cells. Xiaohong Pan, Dan Wang and Hongliu Sun did some experiments, Fang Han revised the paper. Funding This work was supported by projects of education of Liaoning Province Science and Technology (L2013515) and the National Natural Science Foundation of China (Grant Nos. 81102828, 81273037, and 81202516) and the Natural Science Foundation of Shandong Province of China (No. ZR2011HM011). The authors also wanted to show appreciation to Project of Shandong Province Higher Educational Science and Technology Program (J11LF86 and J10LC66) and Development Programs in Medicine & Health Science and Technology of Shandong Province (HW004) and the project of Taishan Scholars Construction Engineering to HANFANG. Conflicts of interest None of the authors have conflict of interest with the submission. Ethics approval There is no ethical approval problem in this study.

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Protective effects of protocatechuic acid on retinal ganglion cells from oxidative damage induced by H2O2.

Oxidative stress-induced retinal ganglion cell (RGC) death is one of the important factors in ocular disease such as glaucoma. The aim of this study i...
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