Article pubs.acs.org/JAFC

Hydroxycinnamic Acids in Crepidiastrum denticulatum Protect Oxidative Stress-Induced Retinal Damage Hong Ryul Ahn,† Hee Ju Lee,† Kyung-A Kim,† Chul Young Kim,‡ Chu Won Nho,† Holim Jang,†,§ Cheol-Ho Pan,† Chang Yong Lee,§ and Sang Hoon Jung*,† †

Functional Food Center, Korea Institute of Science and Technology (KIST), Daejeon-dong, Gangneung 210-340, Republic of Korea College of Pharmacy, Hanyang University, Ansan 426-791, Republic of Korea § Department of Food Science, Cornell University, 411 Tower Road, Ithaca, New York 14850, United States ‡

ABSTRACT: We investigated the effects of an ethanol extract of C. denticulatum (EECD) in a mouse model of glaucoma established by optic nerve crush (ONC), and found that EECD significantly protected against retinal ganglion cell (RGC) death caused by ONC. Furthermore, EECD effectively protected against N-methyl-D-aspartate-induced damage to the rat retinas. In vitro, EECD attenuated transformed retinal ganglion cell (RGC-5) death and significantly blunted the up-regulation of apoptotic proteins and mRNA level induced by 1-buthionine-(S,R)-sulfoximine combined with glutamate, reduced reactive oxygen species production by radical species, and inhibited lipid peroxidation. The major EECD components were found to be chicoric acid and 3,5-dicaffeoylquinic acid (3,5-DCQA) that have shown beneficial effects on retinal degeneration both in vitro and in vivo studies. Thus, EECD could be used as a natural neuroprotective agent for glaucoma, and chicoric acid and 3,5-DCQA as mark compounds for the development of functional food. KEYWORDS: Crepidiastrum denticulatum, chicoric acid, glaucoma, neuroprotection, RGC-5

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INTRODUCTION The genus Crepidiastrum is distributed in east Asia, and plants of this genus are used to prepare Kimchi, a traditional Korea fermented food. Among Crepidiastrum plants, Crepidiastrum denticulatum (Asteraceae) is widely cultivated in Korea, and extensively used in herbal medicines in east Asia, especially in those used for treating conditions such as inflammation, blood circulation disorders, and eye-related diseases.1 Several compounds have been isolated from Crepidiastrum plants, including sesquiterpene glucosides of youngiaside A, youngiaside B, and youngiaside C, and hydroxycinnamic acids of chlorogenic acid, 2,3-dicaffeoyltartaric acid (chicoric acid) and 3,5-dicaffeoylquinic acid (3,5-DCQA).2−4 Hydroxycinnamic acids are the major components of C. denticulatum, of which 3,5-DCQA is the most abundant.3 These compounds have attracted attention owing to their numerous biological activities, including antioxidative effects.5 Among the group of compounds, chicoric acid has lipid peroxidation inhibitory effect by having its electron withdrawing group, and is a potential antidiabetic agent owing to its property of enhancing insulin release and glucose uptake.6 Caffeoylquinic acid derivatives are esters of caffeic and quinic acids, and exhibit antioxidative effects that contribute to neuroprotection by reducing reactive oxygen species (ROS).7 DCQAs in Brazilian green propolis have also been shown to exert neuroprotective effects via antioxidative activity when used in cases of retinal damage.8 The antioxidative properties of chicoric acid and DCQAs can contribute to retinal degeneration, because oxidative stress plays an important role in the pathogenesis of glaucomatous optic neuropathy due to apoptotic degeneration of retinal ganglion cells (RGCs).9−12 This is probably why natural © 2014 American Chemical Society

antioxidant products are putative antiglaucoma agents. Indeed, several of these natural products have been reported to protect against RGC degeneration.13−15 Glaucoma, the second leading cause of blindness, is a term used to describe a group of optic neuropathies characterized by functional and structural impairment of RGCs resulting in progressive, irreversible vision loss initially marked by loss of peripheral vision with progression to total blindness.16 Elevated intraocular pressure (IOP) is the most important risk factor for glaucoma. Ischemia also plays a major role in the development of this disease.17 The major clinical therapies for glaucoma are focused on IOP reduction by increase in uveoscleral outflow of aqueous humor and inhibition of aqueous humor production.18 However, these therapeutic approaches are ineffective in a significant number of glaucoma patients, probably because normotensive glaucoma patients have normal IOP. Moreover, not all hypertensive patients develop glaucoma.19 Thus, alternative treatment approaches are needed for glaucoma, and a treatment that protects RGCs, specifically against apoptosis, 8 without producing significant side effects might be effective.20 Various neuroprotective approaches have been evaluated for glaucoma treatment, including termination or prevention of apoptosis, inhibition of glutamate excitotoxicity, and improvement of blood flow to the optic nerve.21 In the present study, we examined the protective effect of C. denticulatum and hydroxycinnamic acids from the plant on Received: Revised: Accepted: Published: 1310

October 15, 2013 December 10, 2013 January 15, 2014 January 15, 2014 dx.doi.org/10.1021/jf4046232 | J. Agric. Food Chem. 2014, 62, 1310−1323

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ganglion cell markers and exhibit ganglion cell-like behavior in culture.25 In previous report, RGC-5 cells have been proven to be mice origin.26 There were 5.0 × 103 cells seeded in 96 well plates for 24 h for incubation, and they were then exposed to DMEM containing 1% FBS plus samples or plus vehicle. After 1 h pretreatment with samples, 1buthionine-(S,R)-sulfoximine (BSO) (0.5 mM) plus glutamate (10 mM) (glutamate/BSO) was added to cultures. To test cell viability, MTT was added to the cells in 96-well plates at a final concentration of 0.5 mg/mL for 1 h at 37 °C. Optical density of the solubilized formazan product in each well was measured using a spectrophotometer (BioTek Instruments, VT) with a 570 nm test wavelength and a 690 nm reference wavelength.27 Microscopic Analysis of Cell Viability by PI and Hoechst 33342 Double Staining. The cells were stained with 8 μM of Hoechst 33342 with or without 1.5 μM of PI for 30 min at 37 °C in order to determine apoptotic or necrotic cell death caused by glutamate/BSO.13 After being washed twice with serum free media, the cells were imaged using a fluorescence microscope (Olympus, Tokyo, Japan). Assessment of ROS. Intracellular ROS was quantified using the dye, dichlorodihydrofluorescein diacetate (DCFH-DA), as a radical probe.28,29 The RGC-5 cells were seeded at a density of 5.0 × 103 cells per well into 96-well plates, and 24 h later, the cell-culture medium was replaced with DMEM containing 1% FBS. The cells were loaded with the radical probe DCFH-DA (10 μM) through incubation for 20 min at 37 °C. Then, the cell-culture medium was replaced to remove the extra probe. To generate the radical species, we added H2O2 at 1 mM (H2O2 radical), H2O2 at 1 mM plus 100 μM of iron(II) perchlorate hexahydrate (•OH) or KO2 at 1 mM (O2•−), to the radical probe loading-medium. Fluorescence was then measured after ROSgenerating compounds had been present for various time-periods, using excitation/emission wavelengths of 485/535 nm (luminescence spectrometer LS50B, Perkin-Elmer Ltd., England). Superoxide radicals were assessed using the dye dihydroethidium (DHE) according to previous methods.30,31 Red fluorescence was detected using a laser scanning confocal microscope (model Leica TCS SP5; Leica, Germany). Excitation wavelength was set at 514 nm and emissions filters at 590 nm. Quantifying of images was analyzed with Leica Application Suite 2.02 software using four representative fields from each well, and images of four wells were sampled per group (n = 16 in each group). Protein Extraction and Western Blot Analysis. After the pretreatment with different concentrations of EECD for 1 h in RGC-5 cells, the cells were treated with glutamate/BSO for 24 h. For Western blot analysis using retina in rat or mice, four animals (4 retinas) were used from three independent experiments. After cells were washed with cold D-PBS, the cells were scraped using a cell scraper and centrifuged at 14 000 × g for 10 min. The cell pellets were resuspended in cell lysis buffer (1 M Tris pH 7.4, 2 M NaCl, 1 M EDTA, 10% NP40, 1 × protease inhibitors, 1 mM PMSF) and then incubated on ice for 10 min. After centrifuging at 14 000 × g for 30 min at 4 °C, the supernatant was sonicated, and the total protein concentration was determined using a Bio-Rad Protein Assay kit (BioRad Laboratories, Hercules, CA). A 10 μg sample of protein from the retina homogenates was loaded per lane on a 10% polyacrylamide gel and then transferred to a polyvinylidenedifluoride (PVDF) membrane (Hybond-P; Amersham Biosciences, GE Healthcare, U.K.). Western blot analysis was performed with the primary antibodies including anti-PARP, anticleaved caspase-3 (1:1000, Cell Signaling Technology, Beverly, MA), and secondary antibodies (1:3000, Santa Cruz Biotechnology, CA). Immunoreactive bands were detected using the enhanced chemiluminescence reagents (Amsersham Bioscience, GE Healthcare, U.K.) and were measured via densitometry using a LAS-4000 image reader and Multi Gauge 3.1 software (Fuji Photo Film, Japan) Quantification of mRNA Using Real-Time RT-PCR. Total RNA was extracted from RGC-5 cells using the RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. After quantification, total RNA (1 μg) was used to synthesize cDNA by

oxidative stress-induced cell death in vitro by using RGC-5 cells, a transformed retinal ganglion cell line. We also report neuroprotective properties in vivo against retinal damage following optic nerve crush (ONC) in mice.

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MATERIALS AND METHODS

Chemicals. First grade solvents were used for extraction fractionation, and column chromatography. Kiesel gel 60 (70−230 mesh, Art, 7734 Merck) was used as the column packing material. Kiesel 60 F254 (precoated plate, Art. 5559, Merck) was used for thin layer chromatography (TLC). Iodine vapor and 10% H2SO4 were used for TLC detection. Hoechst 33342 and propidium iodide (PI) were from Molecular Probes (Eugene, OR). Antipoly (ADP-ribose) polymerase (PARP) and anticleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). All other chemicals and reagents were purchased from Sigma-Aldrich (St Louis, MO). Animals. All animal studies were carried out in a pathogen-free barrier zone at the KIST Gangneung Institute and were done in accordance with the procedure outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Procedures used in this study were approved by the Animal Care and Use Committee of KIST. In the present study, male adult Sprague−Dawley rats weighing between 250 and 300 g (8 weeks of age) and male ICR mice weighing between 30 and 35 g (8 weeks of age) were used for the protective effects on NMDA and ONC induced retinal damage, and were acclimated for one week, caged in groups of five for mice and two for rat, and were fed with a diet of animal chow and water ad lib. They were housed at 23 ± 0.5 °C and 10% humidity with a 12 h light−dark cycle. Plant Material and Isolation of Compounds. The C. denticulatum samples were collected in the vicinity of Gangneung, Korea, and the voucher specimen (Voucher No. D-043) was deposited at the Herbarium of Functional Food Center, KIST Gangneung Institute, Korea. The dried aerial section of deposited sample (10.0 kg) was extracted three times with hot 94% EtOH (57.0 L) for 4 h. This residue was evaporated in vacuo to yield the total extract (1.4 kg). Dried plants (1.0 kg) were extracted three times by sonication with 94% ethanol for 4 h at room temperature. The residue was evaporated to yield ethanol extract of C. denticulatum (EECD, 70 g) under reduced pressure. High-performance liquid chromatography (HPLC)−ultraviolet chromatograms of EECD were obtained as follows. The HPLC system was equipped with an Agilent (Palo Alto, CA) series 1200 liquid chromatograph and a Shiseido MG II C18 column packed with 5 μm particles (4.6 mm × 250 mm, film thickness 4 μm) (Shiseido Inc., Japan). The temperature of the column oven was 35 °C, and the injection volume was 10 μL. Separations and simultaneous determination were performed using a mixture of acetonitrile and 0.3% formic acid in water (18:82) as a mobile phase. The flow rate was 1.0 mL/min, and UV detection was performed at 330 nm. EECD (2.0 g) was chromatographed on a Sephadex LH-20 column (2.5 cm × 65 cm, 100% methanol) to yield fractions 1−9. Chicoric acid was isolated by semipreparative HPLC using the YMC Hydrosphere C18 column (20 mm × 250 mm, film thickness 5 μm) with acetonitrile containing 0.1% trifluoroacetic acid in an aqueous gradient (20−30% acetonitrile for 40 min, 10 mL/min) from fraction 4. 3,5-Dicaffeoylquinic acid (3,5-DCQA) was purified using semipreparative HPLC (acetonitrile containing 0.1% trifluoroacetic acid in aqueous isocratic 25% acetonitrile for 40 min at a flow rate of 2 mL/ min, using the YMC Hydrosphere C18 column [10 mm × 250 mm, film thickness 5 μm]) from fraction 5. Chemical structures were confirmed by 1H and 13C nuclear magnetic resonance spectroscopy and compared with the literature.22−24 The contents of chicoric acid and 3,5-DCQA in 1 g of EECD were found to be 12.44 mg/g ± 0.25 and 3.80 mg/g ± 0.10, respectively. Culture of RGC-5 Cells and Cell Viability. RGC-5 cells were used in this study, which have been previously shown to express 1311

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Figure 1. (A) Effect of EECD on the viability of RGC-5 cells exposed to 10 mM glutamate plus 0.5 mM BSO for 24 h as measured by the MTT assay. 1 mM N-acetyl-L-cysteine (NAC) was used as a positive control. The results are mean values with error bars indicating ± SEM where n = 6 independent experiment (** p < 0.01, *** p < 0.001). (B) Representative fluorescence microscopy of PI (red) and Hoechst 33342 (blue) staining. (a) Control RGC-5 cells in culture. (b) 10 mM glutamate plus 0.5 mM BSO treated RGC-5 cells in culture. (c−e) Pretreatment with EECD (0.1−10 μg/mL concentration) followed by 10 mM glutamate plus 0.5 mM BSO. (f) Pretreatment with NAC (1 mM concentration) followed by 10 mM glutamate plus 0.5 mM BSO. Scale bar = 50 μm. (C) Representative fluorescence microscopy of Hoechst 33342 stained RGC-5 cells. (a) Control RGC-5 cells in culture. (b) 10 mM glutamate plus 0.5 mM BSO treated RGC-5 cells in culture. (c−e) Pretreatment with EECD (0.1−10 μg/mL concentration) followed by 10 mM glutamate plus 0.5 mM BSO. (f) Pretreatment with NAC (1 mM concentration) followed by 10 mM glutamate plus 0.5 mM BSO. Scale bar = 50 μm. reverse transcription with the Omniscript reverse transcription kit and the T7-(dT)24 primer (Qiagen). Quantitative real-time RT-PCR was performed to evaluate mRNA expression for PARP and caspase-3 with the Light-Cycler Real-Time PCR System (Roche, Manheim, Germany). The primers for PARP were 5′-AGGCCGCCTACTCTATCCTC-3′ (sense) and 5′-GATTCACTGCTGCCTTGAGA-3′ (antisense). The primers for caspase-3 were 5′-GAGGCTGACTTCCTGTATGCTT-3′ (sense) and 5′AACCACGACCCGTCCTTT-3′ (antisense). The primers for GAPDH as control were 5′-AGGCAAAAGACACCGTCAAG-3′ (sense) and 5′-CACAAGAAGATGCGGCTGT-3′ (antisense). Analysis was based on the results of three independent experiments. Lipid Peroxidation Assay. Lipid peroxidation was tested for the amount of formation for thiobarbituric acid reactive species (TBARS) as described in previous studies.13,32 The rat brain was homogenized in ice-cold 0.9% saline, centrifuged at 1000 × g for 10 min at 4 °C, and the supernatant was used for the lipid peroxidation assays. The lipid

peroxidation in rat homogenate was evaluated using the TBARS method as described previously.33 Absorbance of the samples was measured at 532 nm, and the amount of TBARS was determined using a standard curve of the malondialdehyde (MDA) derivative 1,1,3,3tetraethoxypropane (0.63−100 μM). Protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA) using bovine serum albumin as the standard. NMDA-Induced Retinal Damage and Histological Analysis. Retinal damage caused by NMDA was determined according to previous methods.14 Rats (n = 7) were put under anesthesia with a Zoletil (1.6 μg/g, Verbac Laboratories 06515, France) and Rompun (0.05 μL/g, Bayer) mixture, and retinal damage was induced using an injection (2 μL/eye) of NMDA (2.5 mM dissolved in 0.01 M PBS) to the left eye using a 30 gauge Hamilton syringe (final concentration 5 nmol/eye).34 Seven days after the injection, the eyeballs were enucleated for histological analysis. Previous study has shown that 18 h after an NMDA injection into a rabbit retina, typical apoptotic 1312

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Figure 2. (A) Expression of apoptotic protein levels (PARP, cleaved caspase-3) in RGC-5 cells subjected to 10 mM glutamate plus 0.5 mM BSO with or without EECD treatment. Protein level values are expressed as mean ± SEM from three independent experiments. (B) Expression of mRNA level (PARP and caspase-3) in RGC-5 cells subjected to 10 mM glutamate plus 0.5 mM BSO with or without EECD treatment. Values are expressed as mean ± SEM from three independent experiments. features in degenerative cells in the retinal ganglion cell layer (GCL) and the inner nuclear layer (INL) were noted by electron microscopy. TUNEL positive nuclei were detected in these layers as early as 4 h showing maximal numbers at 18 h.35,36 For this reason, the animals were killed 18 h after the NMDA injection and fixed for the TUNEL assay, and 7 days after the NMDA injection they were fixed for histological analysis. The enucleated eyes were fixed in 10% formalin for 24 h, embedded in paraffin, and sectioned through an equatorial plane at 4 μm thickness using a HM340E microtome (Walldof, Germany). Four sections (n = 4) were used for analysis. Hematoxylin solution was added to the retina section (0.1% hematoxylin, 10% ammonium) for 8 min. The sections were then washed three times with distilled water. Bluing reagent (0.2% lithium carbonate solution) was added to the section for 1 min. The sections were quickly rinsed in 95% alcohol, and 1% Eosin Y solution was added to the sections for 1 min. Eosin Y was washed off with 95%

alcohol three times, and the sections were coverslipped with a mounting medium and observed under a light-microscope (Olympus, Tokyo, Japan). TUNEL Staining. To analyze TUNEL-positive cells, the sections were submitted to enzymatic digested with 20 μg/mL of proteinase K for 15 min, then washed with PBS and incubated with 3% hydrogen peroxide in PBS for 5 min at room temperature, and twice rinsed in PBS. They were then immersed, incubated with a stock solution of terminal deoxynucleotidyl transferase (TdT) reaction enzyme in a humidified chamber at 37 °C for 1 h, and then washed three times in PBS for 1 min. The sections were subsequently incubated with antidigoxygenin peroxidase conjugate and peroxidase substrate to detect signs of apoptosis, which appear brown. The histological aspect of apoptosis was observed using a light microscope (Olympus, Tokyo, Japan). Optic Nerve Crush (ONC) and Quantification of Retinal Ganglion Cells. Mice (n = 8) were put under anesthesia by 1313

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intraperitoneal injection of Zoletil (1.6 μg/g, Verbac Laboratories 06515, France) and Rompun (0.05 μL/g, Bayer) mixture, and retinal damage was induced using optic nerve crush.37 The optic nerve of the left eye was exposed by opening the meninges of the optic nerve with the sharp tips of a forceps, followed by blunt dissection. ONC was crushed 2 mm behind the globe for 7 s with a cross-action calibrated forceps. The samples were injected intravitreally 1 h before ONC, and the animals were killed 7 days after the ONC and the eyeballs enucleated immediately after sacrifice. The eyeballs were fixed for retrograde label of RGCs. RGC Labeling and Retinal Flat Mount Preparation. Mice were anesthetized by intraperitoneal administration of a mixture of Zoletil (1.6 μg/g, Verbac Laboratories 06515, France) and Rompun (0.05 μL/g, Bayer), and to the superior colliculi in mice was applied 5% solution of the neurotracer dye Fluorogold (Invitrogen, NY) by a piece of soaked gelfoam. Skull openings were then sealed with a petrolatum based antibiotic ointment. The overlying skin was sutured and antibiotic ointment applied externally. Seven days after the application of Fluorogold, eyes were enucleated, and the retinas were detached at the ora serrata and cut with a trephine around the optic nerve head. Four radial relaxing incisions were made and the retinas prepared as flattened whole mounts on silane-coated microscope slides.38 Statistical Analysis. The data are expressed as a mean percentage of the control value plus standard error of the mean (SEM). Statistical comparisons were made using a one-way ANOVA followed by a Dunnett’s test. Statistical analyses were conducted using GraphPad Prism (version 4.0) (GraphPad, San Diego, CA). p < 0.05 was considered significant.

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RESULTS Effect of EECD on the Apoptotic Cell Death Caused by an Insult of Glutamate/BSO. Figure 1 shows the protective effects of EECD against oxidative stress-induced RGC-5 cell death (Figure 1). When glutamate/BSO was added to RGC-5 cells for 24 h, cell viability was significantly decreased by 36.7%. However, this decrease in cell viability was significantly attenuated by EECD in a concentration-dependent manner (Figure 1A). The protective effect of EECD caused by oxidative stress was confirmed by microscopic analysis using a Hoechst 33342/PI double staining method for DNA (Figure 1B). Hoechst 33342 is a blue fluorescent dye that is permeable to live or fixed cells, while PI is a red membrane-impermeant dye and is, therefore, commonly used to identify dead cells.13 As shown in Figure 1B, there were few red-stained nuclei in cells under basal conditions. However, cells treated with glutamate/BSO showed numerous red-stained nuclei. Pretreatment with EECD attenuated red-stained nuclei and chromatin condensation in a concentration-dependent manner (Figure 1B,C). To determine whether EECD’s protective effects on oxidative stress-induced RGC-5 cell death were due to antiapoptotic activities, Western blot analysis was performed to evaluate the change in apoptotic protein levels for cleaved PARP and cleaved caspase-3 (Figure 2). Exposure to glutamate/BSO resulted in the up-regulation of protein levels for cleaved PARP and cleaved caspase-3; however, EECD significantly reduced this up-regulation in a concentrationdependent manner (Figure 2A). Moreover, EECD significantly reduced the mRNA expression of PARP and caspase-3 caused by glutamate/BSO in a concentration-dependent manner (Figure 2B). Effect of EECD on the Intracellular Levels of ROS. Inhibitory effects of EECD on ROS production were determined by DCFH-DA as a ROS fluorescence probe. ROS

Figure 3. Radical scavenging capacities of EECD against the production of three radical species (H2O2, O2•−, and •OH) in RGC5. ROS production was stimulated with H2O2 at 1 mM, or with KO2 at 1 mM, or with H2O2 at 1 mM plus ferrous perchlorate(II) at 100 μM. (A) H2O2-induced oxidation of DCFH in RGC-5, (B) O2•−-induced oxidation of DCFH in RGC-5, and (C) •OH-induced oxidation of DCFH in RGC-5. Results are mean values with error bars indicating ± SEM where n = 3 (* p < 0.05, *** p < 0.001).

was induced by different oxidative insults including H2O2, O2•−, and •OH. 1314

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Figure 4. Effect of EECD on ROS levels in RGC-5 cells subjected to oxidative stress induced by 10 mM glutamate plus 0.5 mM BSO for 24 h. DHE assay was used to demonstrate the intensity of intracellular ROS generated by oxidative stress. Images were obtained by confocal microscopy (400×). DAPI was used to demonstrate live cells. (a) Control RGC-5 cells in culture. (b) 10 mM glutamate plus 0.5 mM BSO treated RGC-5 cells in culture. (c−e) Pretreatment of EECD (0.1−10 μg/mL concentration) with 10 mM glutamate plus 0.5 mM BSO. (f) Pretreatment of NAC (1 mM concentration) with 10 mM glutamate plus 0.5 mM BSO. (g) Quantification of DHE fluorescence was analyzed with Leica Application Suite 2.02 software (Leica, Germany) using four representative fields from each well, and images of four wells were sampled per group (n = 16 in each group). Scale bar = 50 μm. The results are mean values with error bars indicating ± SEM (*** p < 0.001).

able to penetrate live cell membranes and to bind doublestranded DNA in the nucleus, and to dye live cells (Figure 4). When the cells were exposed to glutamate/BSO, the number of live cells decreased while red fluorescence increased by approximately 9-fold (relative DHE fluorescence 97.3 ± 8.2) compared to control. However, enhancement of this red fluorescence was clearly attenuated in a concentration-dependent manner by pretreatment of EECD, with 10 μg/mL showing the highest potency (DHE fluorescence 10.8 ± 5.2 relative to control) (Figure 4). EECD Inhibition of Sodium Nitropruside (SNP)Induced Lipid Peroxidation in Rat Forebrain Homogenates. Malondialdehyde (MDA) is the final product of lipid peroxidation and a marker for oxidative stress-induced injury.

ROS activity was increased more than 13 times when the RGC-5 cells were exposed to H2O2 at 1 mM, from the basal level of 100.63 ± 27.36 to 1343.13 ± 103.27 (Figure 3A). However, pretreatment with EECD markedly inhibited ROS production in a concentration-dependent manner (Figure 3A). Moreover, EECD also scavenged O2•− and •OH radicals in a similar manner (Figure 3B,C). To test the ability of EECD to inhibit the superoxide production caused by glutamate/BSO, DHE staining was used, and the results are shown in Figure 4. DHE fluorescence is blue in the cell cytoplasm, but is converted to red fluorescence by ROS production upon DNA intercalation. DAPI staining was used to verify the DHE staining of live cells because DAPI is 1315

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stimulation of TBARS induced by SNP, with an IC50 value of 0.12 μg/mL (Table 1). Effect of EECD on NMDA-Induced Retinal Damage. To determine whether EECD had neuroprotective effects in vivo, the changes in ganglion cell numbers and IPL thickness caused by the intravitreal injection of NMDA into rat retinas were determined. After intravitreal injection of NMDA, significant thinning of the IPL was observed (compare Figure 5b to Figure 5a). However, this IPL thinning was attenuated by treatment with EECD (2 μg/mL, intravitreal injection) (Figure 5c,e). The antiapoptotic properties of EECD against NMDAinduced damage to ganglion cells were tested using TUNEL staining (Figure 6). There were few TUNEL-positive cells in the normal control (Figure 6a,e). At 24 h after the intravitreal injection of NMDA, prominent TUNEL-positive cells were found in the GCL as shown in Figure 6b,e. After pretreatment with 2 μg/mL of EECD, the number of TUNEL-positive cells was decreased as shown in Figure 6c−e. Effect of EECD on Optic Nerve Crush (ONC)-Induced Retinal Damage. The neuroprotective effect of EECD was tested following ONC in mice by injecting the fluorescent retrograde tracer Fluorogold into the superior colliculus. RGC death was observed in mice after ONC by the loss of fluorescent Fluorogold-labeled RGCs (Figure 7b,e,h), as compared with the normal control (Figure 7a,d,g). However, the degeneration of RGCs caused by ONC was significantly attenuated by pretreatment with EECD (Figure 7c,f,i). Major Compounds in EECD and Their Protective Effects on Oxidative Stress-Induced RGC-5 Cells and

Table 1. Effect of EECD on Inhibition of Lipid Peroxidation Induced by Sodium Nitropruside (SNP) in Rat Forebrain Homogenatesa lipid peroxidation sample

conc (μg mL)

EECD EECD EECD EECD EGCG EGCG EGCG EGCG

2.5 1.25 0.625 0.3125 0.23 0.115 0.0575 0.028 75

inhibition (%)

IC50 (μg mL)

± ± ± ± ± ± ± ±

1.72 1.72 1.72 1.72 0.12 0.12 0.12 0.12

57.78 42.93 33.19 27.22 63.06 48.72 38.16 27.53

16.41 13.84 10.59 11.20 7.48 7.66 12.40 12.67

SNP at a concentration of 20 μM caused a submaximum stimulation of TBARS in a previous study; therefore, this SNP concentration was used to determine the inhibitory effects in the samples. Different concentrations of EECD were preincubated with brain homogenates at 37 °C for 5 min. SNP was then added at the concentrations indicated, and the homogenates were incubated for a further 10 min. Results are means values ± SEM of three independent experiments. a

The TBARS method was used to assess the production of MDA in rat brains. As shown in Table 1, MDA levels were increased following SNP (20 μM) stimulation in rat forebrain homogenates as compared to the control group. However, pretreatment with EECD inhibited the production of MDA in a concentration-dependent manner, with an IC50 value of 1.72 μg/mL (Table 1). EGCG as a positive control inhibited the

Figure 5. Effect of EECD on retinal damage induced by intravitreous injection of N-methyl-D-aspartate (NMDA) in rats. Representative photomicrographs showing the histologic appearance of retinal cross sections with hematoxylin and eosin staining at 7 days after treatment with or without NMDA. (a) Nontreated, (b) NMDA (5 nmol), (c) NMDA (5 nmol) plus EECD (2 μg/mL), (d) NMDA (5 nmol) plus EECD (0.2 μg/ mL). Quantification of the thickness of the IPL is shown in part e. The scale bar represents 50 μm. Results are the mean values with error bars indicating ± SEM from six independent experiments (*** p < 0.001). 1316

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Figure 6. Antiapoptotic effect of EECD demonstrated by a TUNEL assay. Representative photomicrographs showing the histologic appearance of retinal cross sections with TUNEL stain after 18 h with or without NMDA. Retinal damage was induced by an intravitreous injection of NMDA. (a) Nontreated, (b) NMDA (5 nmol), (c) NMDA (5 nmol) plus EECD (2 μg/mL), (d) NMDA (5 nmol) plus EECD (0.2 μg/mL). The arrows indicate TUNEL-positive cells (brown stain). Quantification of the TUNEL positive cells is shown in part e. The scale bar represents 50 μm.

Optic Nerve Crush (ONC)-Induced Retinal Damage. As shown in Figure 8, the HPLC profile of EECD was recorded at 330 nm. Matching the compounds isolated from EECD with HPLC data indicated that the major constituents of EECD were chicoric acid and 3,5-DCQA. It has also reported from our laboratory that 3,5-DCQA isolated from Gymnaster koraiensis can protect RGCs in vitro and in vivo;14 thus, we have focused on the effects of chicoric acid in this study. Chicoric acid showed protective effects against glutamate/ BSO-induced RGC-5 cell death in a concentration-dependent manner (Figure 9A). This was confirmed by the Western blot analysis, and chicoric acid significantly reduced this upregulation of protein levels for cleaved PARP and cleaved caspase-3 in a concentration-dependent manner (Figure 9B). Moreover, chicoric acid significantly reduced the mRNA expression of PARP and caspase-3 caused by glutamate/BSO (Figure 9C). From the ONC model, RGCs death caused by ONC was also significantly attenuated by pretreatment with 5 nmol/eye (Figure 9Dc,g,E) and 25 nmol/eye chicoric acid (Figure 9Cd,h), as compared with vehicle group (Figure 9Db,f,E), although the attenuation by 5 nmol/eye chicoric acid-treated group was not statistically significant (Figure 9E).

Using quantitative analysis of cell death, our in vitro studies showed a concentration-dependent effect of EECD to protect against the retinal degeneration caused by glutamate/BSOinduced oxidative stress (Figure 1). It has been shown that glutamate/BSO can induce apoptosis in RGC-5 cells and up-regulate cleaved caspase-3 and cleaved PARP proteins.39 We confirmed these observations in our study and also found that pretreatment with EECD inhibited in a concentration-dependent manner this up-regulation of apoptotic proteins and mRNA level (Figure 2). This suggests that EECD protects against the retinal degeneration from antiapoptotic effects by inhibiting the activation of apoptotic proteins. Retinal degeneration may occur by various mechanisms including neurotrophic factor deprivation, ischemia, glial cell activation, glutamate excitotoxicity, and abnormal immune response.40 These mechanisms can induce oxidative stress caused by imbalance the production of ROS and have been recognized as critical mediators in RGC death.41 Increased levels of ROS cause significant molecular damage within cells, which can lead to neuronal death by several mechanisms including protein modification and DNA damage.42 In addition, accumulating evidence from experimental models indicates that oxidative stress, including oxidative damage in the trabecular meshwork and retina, is implicated in glaucoma.43,44 It is therefore proposed that antioxidants that regulate oxidative imbalance may be therapeutic strategies for retinal degeneration. Furthermore, natural antioxidants may be

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DISCUSSION In this study, we presented in vitro and in vivo evidence that EECD and its isolated compounds protects against retinal degeneration. 1317

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Figure 7. Fluorogold labeled RGCs in the mouse 1 week after optic nerve crush (ONC). Retrogradely labeled RGCs of mice with uninjured and injured optic nerves. RGCs were labeled by injecting 3% Fluorogold into the superior colliculi of the brain. The figure shows representative micrographs of normal retina (a) and damaged retina 1 week after ONC with (c) or without EECD (b). Scale bars in parts a−c represent 500 μm. Low-magnification (×100) images of labeled RGCs in a−c are shown in d−f. Scale bars in d−f represent 100 μm. High magnification (×200) images of a−c are shown in g−i. Scale bars in g−i represent 50 μm. The results showed a significant increase in RGC survival after EECD treatment compared with vehicle treatment (j). Experimental values are expressed as percentage of surviving RGCs with error bars indicating ± SEM from three independent experiments (** p < 0.01).

peroxidation is implicated in glaucoma.47 In a study using UVspectrophotometry and fluorescent analysis of lipid extracts from lenses, aqueous humor, and blocks containing trabecular and Schlemms canal tissues in primary open-angle glaucoma (POAG), significant changes of lipid peroxidation in comparison with the control were observed.48 MDA is final product of lipid peroxidation, and MDA levels were estimated in rat brain homogenates after induction by SNP using a TBARS assay.13 In our hands, treatment with EECD clearly inhibited lipid peroxidation in a concentration-

putative agents for treating or preventing retinal degeneration such as in glaucoma.45 In view of the roles of oxidative stress in the retinal degeneration, we have shown that in RGC-5 cells EECD ameliorates the effects of the ROS production induced by H2O2, O2•−, or •OH by scavenging radicals (Figure 3). Moreover, the inhibitory effects of EECD on ROS were clearly confirmed by using the DHE assay, which directly measures intracellular O2•− (Figure 4).46 Lipid peroxidation can be referred to as oxidative degeneration of lipids, and it has been shown that lipid 1318

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Figure 8. Chromatogram of the HPLC profile for EECD at 330 nm. Peaks: (1) chicoric acid, (2) 3,5-dicaffeoylquinic acid.

blockade of the downstream signaling pathways involved in NMDA-induced excitotoxicity. We also determined whether EECD was effective in optic nerves to protect RGC following optic nerve crush (ONC) (Figure 7). ONC is a common experimental model of chronic glaucoma in which the optic nerve is surgically exposed and clamped for several seconds.55 There are several possible mechanisms for the degeneration of RGCs by ONC, including blockade of axonal transport resulting in inadequate supply of neurotrophic factors, disturbances of intracellular calcium homeostasis, activation of cell death genes, and local excitotoxicity due to uncontrolled activation of NMDA receptors.56,57 Another mechanism of degeneration may be through ROS production, which causes the slow, chronic, and synchronous death of RGCs.58 In our study the density of RGCs was decreased by ONC; however, pretreatment with EECD significantly reduced the loss of RGCs (Figure 7). Our HPLC results identified the major components in EECD as chicoric acid and 3,5-DCQA (Figure 8). Previous studies have shown that 3,5-DCQA from propolis extracts have neuroprotective effects on oxidative stress induced retinal damage.8 It was also reported that 3,5-DCQA isolated

dependent manner (Table 1). Thus, taken together, our results suggest that the neuroprotective effects of EECD in vitro occur through antiapoptotic and antioxidative mechanisms. In order to confirm the neuroprotective effects of EECD, we examined the number of TUNEL-positive cells in the GCL and the thickness of IPL following a single intravitreal injection of NMDA because excessive activation of the excitatory amino acid glutamate has been implicated in retinal neuronal cell death and in the pathogenesis of glaucoma.49 NMDA receptors have been identified on RGC and amacrine cells, and NMDAreceptor activation causes apoptosis via ROS production through intracellular Ca2+ influx.50 It was previously reported that NMDA administered in rat eye caused cell death in the GCL and reduction of IPL thickness, and that the blockade of excessive glutamate could be a possible strategy for development of neuroprotective agents.51−53 Moreover, IPL thicknesses are well correlated with retinal nerve fiber layer thickness and visual field sensitivity, and the IPL thickness can provide the index of INL damage after retinal injury.54 The present study showed that EECD attenuated the detrimental effects caused by NMDA (Figures 5 and 6). These results indicate that, in addition to its direct inhibition of oxidative stress, EECD protection of RGC may be due to 1319

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Figure 9. (A) Effect of chicoric acid on the viability of RGC-5 cells exposed to 10 mM glutamate plus 0.5 mM BSO for 24 h as measured by the MTT assay. 1 mM N-acetyl-L-cysteine (NAC) was used as a positive control. The results are mean values with error bars indicating ± SEM where n 1320

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Figure 9. continued = 5 independent experiment (** p < 0.01, *** p < 0.001). (B) Expression of apoptotic protein levels (PARP, cleaved caspase-3) in RGC-5 cells subjected to 10 mM glutamate plus 0.5 mM BSO with or without chicoric acid treatment. Protein level values are expressed as mean ± SEM from three independent experiments. (C) Expression of mRNA level (PARP and caspase-3) in RGC-5 cells subjected to 10 mM glutamate plus 0.5 mM BSO with or without chicoric acid treatment. Values are expressed as mean ± SEM from three independent experiments. (D) Fluorogold labeled RGCs in the mouse 1 week after optic nerve crush (ONC). Retrogradely labeled RGCs of mice with uninjured and injured optic nerves. RGCs were labeled by injecting 3% Fluorogold into the superior colliculi of the brain. The figure shows representative micrographs of normal retina (a) and damaged retina 1 week after ONC with 5 nmol (c) and 25 nmol chicoric acid (d), or without chicoric acid (b). Scale bars in parts a−d represent 500 μm. High magnification (×100) images of parts a−d are shown in parts e−h. Scale bars in parts e−h represent 100 μm. (E) The results showed a significant increase in RGC survival after chicoric acid treatment compared with vehicle treatment. Experimental values are expressed as percentage of surviving RGCs with error bars indicating ± SEM from three independent experiments (** p < 0.01).

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ABBREVIATIONS USED EECD, ethanol extract of Crepidiastrum denticulatum; ONC, optic nerve crush; RGC, retinal ganglion cell; IOP, intraocular pressure; NMDA, N-methyl-D-aspartate; TLC, thin layer chromatography; PI, propidium iodide; ARVO, Association for Research in Vision and Ophthalmology; HPLC, highperformance liquid chromatography; 3,5-DCQA, 3,5-dicaffeoylquinic acid; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; BSO, 1-buthionine-(S,R)-sulfoximine plus glutamate; NAC, N-acetyl-L-cysteine; MTT, (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species; DCFH-DA, dichlorodihydrofluorescein diacetate; DHE, dihydroethidium; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; TBARS, thiobarbituric acid reactive species; MDA, malondialdehyde; PBS, phosphate-buffered saline; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; SEM, standard error of the mean; SNP, sodium nitroprusside; GCL, ganglion cell layer; POAG, primary open-angle glaucoma; RT-PCR, reverse transcription-polymerase chain reaction

from Gymnaster koraiensis can protect RGCs from oxidative stress in vitro and from NMDA-induced excitotoxicity in vivo.14 We have proven that 3,5-DCQA has protective effects on retinal degeneration in previous research, thus focused on the effects of chicoric acid in this study. Our present study supported that chicoric acid, the major phenolic compound of EECD, has protective effects both in vitro and in vivo (Figure 9), which could be postulated that chicoric acid and 3,5-DCQA are active compounds for the RGCs protective effects of EECD. This is the first report of the presence of chicoric acid in C. denticulatum and the potential value in retinal degeneration. Variations in food matrices and/or phenolic composition can contribute to having a major influence on intestinal bioavailability, since there are controversial results by observing the oral bioavailability of hydroxycinnamic acids intake.59 Thus, further study will be needed for the bioavailability of chicoric acid and 3,5-DCQA with the C. denticulatum as food matrices. This is supported by another report that chicoric acid strengthens the anti-inflammatory activity of luteolin through NF-κB attenuation.60 In conclusion, the present study demonstrated that EECD and its isolated compound of chicoric acid can ameliorate RGC degeneration caused by oxidative stress both in vitro and in vivo, and that this protective effect may be mediated by its antioxidative and antiapoptotic effects. Taken together, these findings support EECD as a potential bioactive wild vegetable that could be used as functional food for the prevention of neurodegenerative diseases such as glaucoma and age-related macular degeneration. Moreover, chicoric acid and 3,5-DCQA isolated from C. denticulatum are suggested to be offered as mark compounds for the standardization in the development of functional food.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +82-33-650-3653. Fax: +82-33-650-3679. Notes

The authors declare no financial conflict of interest. The authors declare no competing financial interest.

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REFERENCES

ACKNOWLEDGMENTS

The RGC-5 cells were a generous gift from Alcon Research, Ltd. This work was financially supported by an intramural grant (2Z03850) from the Korea Institute of Science and Technology (KIST), Republic of Korea. 1321

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