Protective effects of the compounds isolated from the seed of Psoralea corylifolia on oxidative stress-induced retinal damage

Toxicology and Applied Pharmacology 269 (2013) 109–120

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Protective effects of the compounds isolated from the seed of Psoralea corylifolia on oxidative stress-induced retinal damage Kyung-A Kim a, 1, Sang Hee Shim b, 1, Hong Ryul Ahn a, Sang Hoon Jung a,⁎ a b

Functional Food Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangneung 210-340, Republic of Korea School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea

a r t i c l e

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Article history: Received 27 October 2012 Revised 6 March 2013 Accepted 17 March 2013 Available online 29 March 2013 Keywords: Apoptosis Bakuchiol N-methyl-D-aspartate Optic nerve crush Oxidative stress Psoralea corylifolia

a b s t r a c t The mechanism underlying glaucoma remains controversial, but apoptosis caused by increased levels of reactive oxygen species (ROS) is thought to play a role in its pathogenesis. We investigated the effects of compounds isolated from Psoralea corylifolia on oxidative stress-induced cell death in vitro and in vivo. Transformed retinal ganglion cells (RGC-5) were treated with L-buthione-(S,R)-sulfoximine (BSO) and glutamate in the presence or with pre-treatment with compound 6, bakuchiol isolated from P. corylifolia. We observed reduced cell death in cells pre-treated with bakuchiol. Moreover, bakuchiol inhibited the oxidative stress-induced decrease of mitochondrial membrane potential (MMP, ΔΨm). Furthermore, while intracellular Ca2+ was high in RGC-5 cells after exposure to oxidative stress, bakuchiol reduced these levels. In an in vivo study, in which rat retinal damage was induced by intravitreal injection of N-methyl-D-aspartate (NMDA), bakuchiol markedly reduced translocation of AIF and release of cytochrome c, and inhibited up-regulation of cleaved caspase-3, cleaved caspase-9, and cleaved PARP. The survival rate of retinal ganglion cells (RGCs) 7 days after optic nerve crush (ONC) in mice was significantly decreased; however, bakuchiol attenuated the loss of RGCs. Moreover, bakuchiol attenuated ONC-induced up-regulation of apoptotic proteins, including cleaved PARP, cleaved caspase-3, and cleaved caspase-9. Bakuchiol also significantly inhibited translocation of mitochondrial AIF into the nuclear fraction and release of mitochondrial cytochrome c into the cytosol. These results demonstrate that bakuchiol isolated from P. corylifolia has protective effects against oxidative stress-induced retinal damage, and may be considered as an agent for treating or preventing retinal degeneration. © 2013 Elsevier Inc. All rights reserved.

Introduction Glaucomatous retina is characterized pathologically by progressive degeneration of retinal ganglion cells (RGCs) as well as by damage to the visual field. Degeneration of RGCs appears to be secondary to injury to the axons of these cells, which project to the brain (Kalesnykas et al., 2012). Abbreviations: BSA, bovine serum albumin; BSO, L-buthionine-(S,R)-sulfoximine; EtBr, ethidium bromide; FG, fluorogold; Fluo-3/AM, fluo-3/acetoxymethyl ester; GCL, ganglion cell layer; GSH, glutathione; IOP, intraocular pressure; INL, inner nuclear layer; IPL, inner plexiform layer; MC, methylene chloride; MFI, mean fluorescence intensity; MMP, mitochondria membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide; NMDA, N-methyl-D-aspartate; ONC, optic nerve crush; PI, propidium iodide; RGCs, retinal ganglion cells; ROS, reactive oxygen species; RP-HPLC, reverse phase-high performance liquid chromatography; SDS, sodium dodecyl sulfate; TMRE, tetramethylrhodamine ethylester. ⁎ Corresponding author at: Functional Food Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Daejeon-dong, Gangneung 210-340, Republic of Korea. Fax: +82 33 650 3679. E-mail address: [email protected] (S.H. Jung). 1 These authors contributed equally to this work. 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.03.017

The exact mechanism underlying glaucoma remains controversial; however, elevated intraocular pressure (IOP) and ischemia are accepted important risk factors (Tataru and Bogdanici, 2009). This has led to the focusing of pharmacological and surgical treatments for glaucoma on lowering the IOP (Chang and Goldberg, 2012; Toris, 2010). However, significant numbers of patients with glaucoma continue to experience loss of visual field long after therapeutic normalization of their IOP, and ocular hypertensive patients do not all develop glaucoma (Brubaker, 1996). This indicates that glaucoma is the result of multiple, combined factors, rather than a consequence of elevated IOP per se (Varma et al., 2008). There are various hypotheses regarding the mechanism involved in the death of RGCs in glaucoma, such as ischemic insults to the ganglion cell axons (Osborne et al., 2001), neurotrophin-deprivation (Baltmr et al., 2010), dysfunction of non-neuronal components in the optic nerve head (Quigley, 1999), mechanical damage to the ganglion cell axons (Morgan, 2000) and retinal glial cell dysfunction (Zhong et al., 2007). It has also been suggested that reactive oxygen species (ROS) play a key role in the pathogenesis of glaucoma. ROS can function as

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signaling molecules that activate a number of stress-sensitive pathways, which cause cellular damage, and which are ultimately responsible for apoptosis of the cells (Tezel, 2006). In an epidemiological study, analysis of blood and aqueous humor demonstrated that many glaucoma patients have reduced oxidative capacity, and have markedly elevated levels of antioxidant enzymes such as superoxide dismutase and glutathione in the aqueous humor (Ferreira et al., 2004). It would, therefore, seem plausible that antioxidants, which inhibit ROS, could be therapeutic in the prevention of glaucoma. Therefore, neuroprotection, in particular, prevention of death of RGCs and their axons, including retardation of apoptosis via inhibition of ROS, has recently been considered as a new approach to glaucoma therapy (Marcic et al., 2003; Weinreb and Levin, 1999). To this end, the effectiveness of a number of substances, including natural products, to protect RGCs has been investigated (Levin and Peeples, 2008). Psoralea corylifolia L. (Fabaceae) is a plant widely used in traditional Chinese medicine, in particular, to alleviate asthma and diarrhea. Many compounds have been isolated from this plant, including bakuchiol, corylinin, neobavaisoflavone, psoralen, isopsoralen, psoralidin, and bavachalcone (Haraguchi et al., 2000; Ruan et al., 2007; Zhao et al., 2005). We have previously reported that a compound isolated from P. corylifolia was effective in inhibiting aldose reductase in vitro (Shim et al., 2009). Many pharmacological activities such as anti-inflammatory, antihyperglycemic, antidepressant, antimicrobial, antitumor, and antidermatophytic effects have been reported for this plant (Pae et al., 2001; Qiao et al., 2007). Despite this, there have been no reports investigating any potential effects of P. corylifolia on eye-related diseases such as glaucoma. In this study, we investigated whether compounds isolated from P. corylifolia could attenuate death in oxidative stress-induced transformed RGC cells (RGC-5). In addition, we investigated whether the active compound was able to inhibit ROS production in RGC-5 cells, and evaluated the effects of P. corylifolia on N-methyl-D-aspartate (NMDA)-induced and optic nerve crush (ONC)-induced retinal damage in vivo. Materials and methods Animals Male adult (8 weeks-old) Sprague–Dawley rat weighing 250–300 g and male (5 weeks old) ICR mice weighing 30–40 g (Bio-Medical Science, Seoul, Korea) were used. The animals were divided into four groups (control, vehicle, vehicle + bakuchiol 10 nmol and vehicle + bakuchiol 1 nmol) with 8 rats or mice per group and housed at 23 ± 0.5 °C and 10% humidity in a 12 h light–dark cycle. All animals were acclimated at least for one week, caged in group of five or less, and fed with a diet of animal chow and water ad lib. All animal studies were carried out in a pathogen-free barrier zone at KIST Gangneung Institute in accordance with the procedure outline in the 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. Chemicals Agarose was purchased from Lonza (Rockland, ME, USA). Hoechst 33342, propidium iodide (PI), Fluo-3/acetoxymethyl ester (Fluo-3/AM), Pluronic F-127 and FluoroGold were from Molecular Probes (Eugene, OR, USA). Anti-PARP, anti-AIF, anti-cleaved caspase-3, anti-cleaved caspase-9 and anti-cytochrome c monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin, anti-rabbit IgG and anti-mouse IgG antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA) and anti-lamin-B1, and anti-COX-IV monoclonal antibodies from Abcam

(Cambridge, UK). The VECTASHIELD Mounting Medium was purchased from Vector Laboratories (Burlingame, CA, USA). Zoletil and Rumpun were used as anesthesia and purchased from Virbac Laboratories (Fort Worth, TX, USA) and Bayer (Newbury, UK), respectively. All other chemicals and reagents were from Sigma-Aldrich (St Louis, MO, USA). Plant materials The seeds of P. corylifolia were purchased from the Daegu pharmacopoeia market in South Korea. A voucher specimen has been deposited in the Natural Products Chemistry Laboratory of the School of Biotechnology, Yeungnam University. Extraction, isolation and identification The dried psoraleae semen (1.0 kg) was extracted three times with methanol (MeOH). The extracts were suspended in distilled water and partitioned with methylene chloride (CHCl3), ethyl acetate (EtOAc), and n-butanol. The CHCl3 fraction (96.5 g) was subjected to silica gel column chromatography eluting with n-hexane and EtOAc gradient system (10:1 → 10:4) to provide 27 fractions (Fr. 1–Fr. 27). Fraction 3 gave colorless oil, compound 6. Fractions 13 and 15 were crystallized from n-hexane, EtOAc, and MeOH to yield compounds 1 and 2, respectively. Fraction 16 was subjected to repeated column chromatography with a gradient elution of n-hexane/EtOAc (10:1 → 10:1.5) resulting in 19 sub-fractions (Fr. 16-1–Fr. 16-19). Compound 7 was isolated through repeated column chromatography eluted with n-hexane and CH2Cl2 gradient solvents followed by reversed-phase HPLC (30 to 100% CH3CN in H2O over 40 min). Fraction 24 of the CHCl3 fraction was chromatographed on a silica gel column with a gradient elution of n-hexane and EtOAc (10:1 → 1:1) to afford 50 sub-fractions (Fr. 24-1–Fr. 24-50). Fr. 24-7 and Fr. 24-20 were subjected to semi-preparative HPLC (20 to 100% CH3CN in H2O over 40 min) to afford compounds 5 (8.0 mg) and 7 (2.4 mg), respectively. Compound 4 was purified from Fr. 24-26 through semipreparative HPLC (10 to 100% CH3CN in H2O over 45 min). Seven compounds were isolated and their chemical structures were elucidated by spectral analysis and direct comparison with authentic compounds (Jiangning et al., 2005; Zhao et al., 2005). Their structures were identified as psoralen (1), isopsoralen (2), isobavachromene (3), bavachinin (4), corylin (5), bakuchiol (6), and 6-prenyl naringenin (7). Culture of RGC-5 cells The transformed retinal ganglion cells (RGC-5), were kindly gifted by Alcon Research, Ltd. (Fort Worth, TX, USA) at passage 15 in March 2007. RGC-5 cells were grown in 75 cm2 culture flasks in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/ml penicillin/streptomycin (Hyclone, Logan, UT, USA). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The cells passed twice a week. The RGC-5 cells were plated at a density of 5.0 × 10 3 cells per well into 96-well. After incubating for 24 h at 5% CO2 at 37 °C, cell-culture medium was replaced with DMEM supplemented with 1% FBS, 100 U/mL penicillin/streptomycin. After 1 h pretreatment with different concentrations of bakuchiol, 0.5 mM L-buthione-(S,R)-sulfoximine (BSO) and 10 mM glutamate were added to cultures, which were then maintained for 24 h. DNA amplification and sequencing Genomic DNA in RGC-5 cells was isolated by using a QiAmp DNA Mini kit (Quagen, Valencia, CA, USA), according to the manufacturer's

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recommendation and DNA quantified using a spectrophotometer (Nanodrop, Wilmington, DE, USA). Polymerase chain reaction (PCR) amplification of region of nuclear DNA achieved with primers internal to the Thy1 gene. Sequences of the Mus musculus (GI: 134152684) and Rattus norvegicus (GI: 6981653) primers were 5′-ccatccagcatgagttcagcct-3′ (forward) and 5′-accagtcacagagaaatgaagtcc-3′ (reverse). Mitochondrial DNA (mtDNA) sequences of the M. musculus (GI: 34538597) and R. norvegicus (GI: 110189714) primers were 5′-ctcaacatagccgtcaaggc-3′ (forward) and 5′-accaaacctttgtgtttatggg-3′ (reverse). The PCR was carried out with a GeneAmp PCR system 9700 (Applied Biosystems, Carlsbad, CA, USA). Thermal cycling conditions included 94 °C for 5 min, 35 cycles of 95 °C for 30 s, and 62 °C for 30 s, then 72 °C for 60 s, followed by 72 °C for 5 min. The PCR products were analyzed by electrophoresis with a 2% TAE agarose gel and staining with ethidium bromide (EtBr). PCR product bands were purified using a gel extraction kit (Bioneer, Alameda, CA, USA) and sequence similarity was analyzed by ClustalW2 program. Glutamate sensitivity assay Glutamate sensitivity assay was performed as described previously (Van Bergen et al., 2009). Briefly, plated RGC-5 cells were incubated with different concentrations of glutamate (5–50 mM) for 24 h or 48 h. Twenty four hours or 48 h later, viability of cells was evaluated by MTT assay (Mosmann, 1983). Cell viability The RGC-5 cells were plated at a density of 5.0 × 10 3 cells/mL in 96-well plates, and cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Mosmann, 1983). Briefly, the RGC-5 cells were treated with MTT solution (0.5 mg/mL final concentration) for 1 h at 37 °C. After 1 h incubation, the formazan crystals formed in intact cells were solubilized with DMSO, and absorbance at 570/590 nm was measured with a microplate reader (BioTek Instruments, Winooski, VT, USA). Results are expressed as percent MTT reduction. Microscopic analysis for cell viability by propidium iodide (PI) and Hoechst 33342 double staining Apoptotic and necrotic cell deaths were differentiated by a method using fluorescence microscopy with Hoechst 33342 and PI double staining. The RGC-5 cells were stained with 8 μM Hoechst 33342 and 1.5 μM propidium iodide (PI) for 30 min and analyzed under a fluorescence microscope (Nikon, Tokyo, Japan), which we have recently described (Jung et al., 2010). PI positive cells were counted using a cell counter under a fluorescence microscope at 100 times magnification and four representative images were used to estimate the percent of PI positive cells of total cell numbers (minimum 200 cells/ well were counted). Measurement of the mitochondria membrane potential (ΔΨm) Mitochondria membrane potential (ΔΨm) was measured by using tetramethylrhodamine ethylester (TMRE) dye, mitochondrion-specific indicator, as described previously (Panickar et al., 2007) with minor modifications. Briefly, RGC-5 cells were plated at a density of 1.0 × 105 cells per well into 24-well plate, after the treatments and the cells were incubated with 100 nM TMRE for 30 min at 37 °C in the dark. Then cells were fixed with 4% paraformaldehyde for 20 min and washed twice with D-PBS. Cells were mounted on glass slides with Fluorescent VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and examined for fluorescence using a Leica TCS SP5 confocal system (Leica, Wetzlar, Germany; Ex 543/Em

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590). ΔΨm was assessed by quantifying TMRE fluorescence intensity via Leica Application Suite 2.02 software (Leica, Wetzlar, Germany). Measurement of intracellular Ca 2+ level The intracellular Ca 2+ level of RGC-5 cells were measured by using Fluo-3/AM dye, Ca 2+-selective fluorescent indicator, as described previously (Ge et al., 2008) with minor modifications. Briefly following the experiment, the RGC-5 cells were cultured in 100 mm 2 dishes at a density of 6.5 × 10 4 cells/mL and incubated with 2 μM Fluo-3/AM in the presence of 0.005% Pluronic F-27 for 30 min at 37 °C. The cells were then trypsinized, washed twice with cold D-PBS, and measured by using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA; Ex 488/Em 525). The data were analyzed by using CellQuest software. The data are presented as mean fluorescence intensity (MFI) of 10,000 labeled cells after subtraction of background staining or as percent of adhesion molecule expressing cells, reflecting percent of cells with fluorescence above blank control (1%). Protein extraction and Western blot analysis Total cell or retina tissue extraction. The RGC-5 cells or retina tissues were washed with cold D-PBS. Lysis was carried out at 4 °C for 15 min in cell lysis buffer [1 M Tris pH 7.4, 2 M NaCl, 1 M EDTA, 10% NP-40, 1× protease inhibitors and 1 mM PMSF] or RIPA buffer [150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 1 × protease inhibitors and 1 mM PMSF]. After centrifugation at 14,000 g for 30 min at 4 °C and then, the supernatant was separated in a new tube. Preparation of nucleic and cytoplasmic fractions from culture cells or retina tissues. Nucleic and cytoplasmic fractions were separated from the RGC-5 cells or retina tissues using a CelLytic™ NuCLEAR Extraction kit (Sigma, St Louis, MO, USA), according to the manufacturer's recommendation. Isolation of mitochondrial fractions from culture cells or retina tissues. The isolation of mitochondria fraction in RGC-5 cells or retina tissues was using a mitochondrial rapid-isolation kit and procedures in accordance with the manufacturer's recommendation (Pierce Biotechnology, Rockford, IL, USA). Protein assay. All protein concentrations were determined with the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Berkeley, CA, USA). Bradford reagent was added and the absorbance was measured using a spectrophotometer (BioTek Instruments, Winooski, VT, USA) with a 595 nm. The protein concentration was determined based on the standard curve using a bovine serum albumin (BSA). Western blot analysis. Protein samples were electrophoresed on 10 or 12% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membrane (Hybond-P; Amersham Biosciences, GE Healthcare, Little Chalfont, UK) using a Trans-Blot® Semi-Dry Cell (Invitrogen, NY, USA). The membrane was blocked at room temperature in 5% skim milk in PBST buffer [8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L, Na2HPO4, 0.24 g/L, NaH2HPO4 and 0.1% Tween-20]. Dilutions of were anti-PARP (25.5 ng/mL) anti-AIF (31.5 ng/mL), anti-cleaved caspase-3 (28.5 ng/mL), anti-cleaved caspase-9 (33.0 ng/mL), cytochrome c (82.5 ng/mL) (Cell Signaling Technology, Danvers, MA, USA); anti-β-actin (100.0 ng/mL) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-lamin-B1 (50.0 ng/mL), anti-COX-IV (150.0 ng/mL) (Abcam, Cambridge, UK) primary antibodies were in PBST with 1% skim milk. The membranes were incubated overnight at 4 °C. Following three washes with PBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (133.0 ng/mL) or horse

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anti-mouse (133.0 ng/mL) secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBST with 1% skim milk for 1 h at room temperature. The membranes were washed again three times with PBST, and then immunoreactive bands were detected using the enhanced chemiluminescence reagents (Amersham Biosciences, GE Healthcare, Little Chalfont, UK) and measured by densitometry using a LAS-4000 image reader and Multi Gauge 3.1 software (Fuji Photo Film, Tokyo, Japan) Animal experiment. The Sprague–Dawley rats were anesthetized by intraperitoneal injection of Zoletil (1.6 μg/g, Verbac Laboratories, Fort Worth, TX, USA) and Rompun (0.05 μL/g, Bayer, Newbury, UK) mixture. Retina damage was injected intravitreally using Hamilton microsyringe with 30 gauge needles. At 10 min after anesthetization, 2 μL/eye of solution was injected into the vitreous body of the left eye. The solution was prepared in doses of 10 nmol and 1 nmol dissolved with 0.01 M phosphate-buffered saline (PBS) buffer plus 2.5 mM NMDA. The left eye has reduced risk of infection treated with 0.5% moxifloxacin ophthalmic solution (Alcon Laboratories Inc., Fort Worth, USA) after intravitreal injection. Animals were killed 1 day after the NMDA injection. The eyes were enucleated, immediately frozen in liquid nitrogen. Frozen eyes were used to Western blot analysis. Optic nerve crush (ONC) and labeling RGCs. Retrograde labeling to identify RGCs by ONC was performed according to previous report (Danias et al., 2003; Thaler et al., 2011). The optic nerve 2 mm behind the globe of the left eye was pressed for 5 s with cross-action calibrated forceps. Sample was injected to posterior to the pars plana with a 10 μL syringe (Hamilton, Reno, NV, USA) and a 30-gauge needle. After optic nerve injury, the skin over the cranium was incised, and the scalp was exposed. Holes approximately 2 mm in diameter were drilled in the skull 4 mm posterior to the bregma, 1 mm lateral to the midline, with a dentist's drill on both sides of the midline raphe. The superior colliculi were exposed by gentle aspiration of the overlying occipital cortex. A piece of Gelfoam soaked in a 3% solution of the neurotracer dye, FluoroGold was directly applied to each superior colliculus. Skull openings were then sealed with a petrolatumbased anti-biotic ointment. Seven days after the application of FluoroGold, mice were killed. The eyes were enucleated, immediately frozen in liquid nitrogen or fixed with 4% paraformaldehyde solution in PBS. The retinas were detached at the ora serrata and cut with a rephine around the optic nerve head. Four radial relaxing incisions were made and the retinas prepared as flattened whole mounts on silane-coated microscope slides. Frozen eyes were used to Western blot analysis. Statistical analysis. All data were presented as mean ± S.E.M. Statistical significance was assessed with one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple group comparison. *p b 0.05 was considered statistically significant. Results Characterization of RGC-5 cells RGC-5 cells have a number of the biochemical characteristics of RGCs; however, these cells have recently proven to originate from mice, rather than rat (Van Bergen et al., 2009). Thus, the species of origin required clarification before RGC-5 cells could be implemented. As shown in Fig. 1A, the sequence of an amplified region of Thy1 was identical between M. musculus (134152684) and RGC-5; in contrast, the similarity between this sequence in R. norvegicus (6981653) and RGC-5 was only 85.0% (Fig. 1A). Moreover, comparison of a region of mtDNA including the d-loop, showed that the homology between M. musculus mtDNA (34538597) and RGC-5 was 100.0%; in contrast, the homology

between R. norvegicus mtDNA (110189714) and RGC-5 (Fig. 1B) was 79.1%. These results are similar to those from a previous report; thus, the RGC-5 cells used in this study were proven to have a murine origin by direct DNA sequencing by PCR (Van Bergen et al., 2009). Next, in order to establish whether the RGC-5 cells used in this study are glutamate sensitive, we measured cell viability in the presence of various concentrations of glutamate with 24- and 48 hincubations as shown in Fig. 1C. Similar to the previous report, we saw that sensitivity to glutamate was reduced, as compared to earlier stock of the RGC-5 cell line, and found 92% and 86% cell survival after incubation with 5 mM glutamate for 24 and 48 h, respectively (Fig. 1C) (Van Bergen et al., 2009). Thus, our data indicated that RGC-5cells are mouse neuronal precursor cells. Effects of P. corylifolia on BSO and glutamate-induced cell death To determine the effects of P. corylifolia, cell viability assays were performed (Fig. 2). As shown in Fig. 2A, cell viability was significantly decreased in RGC-5 cells treated with 0.5 mM BSO and 10 mM glutamate for 24 h, compared to the control cells. The methylene chloride fraction of P. corylifolia extracts partially, attenuated the negative effect of BSO plus glutamate. The methylene chloride fraction was subsequently subjected to RP-HPLC, yielding 27 fractions (Fr. 1–Fr. 27). Fractions 13 and 15 were further analyzed after crystallization from n-hexane, EtOAc, and MeOH, yielding 2 identifiable compounds. Moreover, after RP-HPLC and Sephadex LH-20 chromatography, fraction 24 generated 3 identifiable compounds. Two further identifiable compounds were obtained from fractions 3 and 16. Thus, in total, 7 identifiable compounds were obtained from the methylene chloride fraction. Following spectral analysis, these 7 compounds were identified as psoralen (compound 1), isopsoralen (compound 2), isobavachromene (compound 3), bavachinin (compound 4), corylin (compound 5), bakuchiol (compound 6), and 6-prenyl naringenin (compound 7), as shown in Fig. 3. All these purified substances were solubilized in 0.5% dimethyl sulfoxide (DMSO), and tested for their capacity to counteract the toxic effects of BSO and glutamate on RGC-5 cells in culture. First, the viability of cells pre-treated with each of these 7 compounds prior to exposure to BSO and glutamate was assessed (Fig. 2B). As shown in Fig. 2B, only bakuchiol (compound 6) showed protective effects, and these were in a concentration-dependent manner. In addition, to further evaluate whether bakuchiol protects cells against BSO and glutamate-induced cell death, cells were double-stained with Hoechst 33342 and PI to detect both apoptosis (bright blue staining) and necrosis (red staining). A markedly higher number of PI-positive cells (approximately 60%) were detected in the cultures treated with BSO and glutamate than in the control cells (Fig. 2D); however, the percentage of PI-positive cells was significantly reduced when the cells were pretreated with bakuchiol. To evaluate the effects of bakuchiol on the cell viability of differentiated RGC-5 cells, we tested various concentrations of bakuchiol for protective effects against BSO and glutamate-induced, staurosporine-mediated differentiation of RGC-5 cells. We found that 0.01 μM of bakuchiol protected against the effect of BSO and glutamate on staurosporinemediated differentiation of RGC-5 cells (data not shown). Bakuchiol attenuates BSO and glutamate-induced depolarization of the mitochondrial membrane potential (MMP, ΔΨm) To examine whether excitotoxicity was associated with the change in cell viability, we measured the mitochondrial membrane potential (MMP, ΔΨm) of RGC-5 cells, by using the mitochondrion-specific dye, TMRE. Fig. 4A a shows representative confocal microscopy image:

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Fig. 1. Re-identification of cultured transformed retinal ganglion cells (RGC-5 cells). (A) Thy1 and (B) mitochondrial DNA (mtDNA) d-loop DNA sequences of RGC-5 cells as compared to published genes: M. musculus (GI: 134152684), R. norvegicus (GI: 6981653), M. musculus mtDNA (GI: 34538597), and R. norvegicus mtDNA (GI: 110189714). (C) Glutamate sensitivity of RGC-5 cells. Viability of RGC-5 cells after treatment with 5–50 mM of glutamate for 24 h or 48 h, as measured by MTT assay. Experimental values are expressed as a percentage of the viable cells with error bars indicating ± S.E.M., from 3 independent experiments.

TMRE-fluorescence intensity was decreased in RGC-5 cells that had been treated with BSO and glutamate alone, compared to the control. However, pre-treatment with bakuchiol in a dose-dependent manner attenuated the negative effects of BSO and glutamate in RGC-5 cells. Quantification data (Fig. 4B) demonstrated that, after treatment with BSO plus glutamate alone, only 40% of MMP was retained. In contrast, approximately 80% of MMP could be preserved by pretreatment with 10 μM of bakuchiol, as indicated by a high level of TMRE fluorescence intensity. Effects of bakuchiol isolated from P. corylifolia on intracellular [Ca 2+] level To further examine the protective effects of bakuchiol isolated from P. corylifolia, we investigated its effects on changes in the concentration of cytosolic Ca2+ in RGC-5 cells. The intracellular Ca 2+-concentration was measured using the membrane-permeable Ca2+-selective fluorescent indicator, Fluo-3/AM, by flow cytometry. Control cells showed very little fluorescence, and cells treated with bakuchiol showed a similarly low pattern of intracellular Ca2+-concentration (Figs. 4C and D). Treatment with BSO and glutamate for 24 h increased the fluorescence

intensity of Fluo-3/AM; thus, the increase in intracellular Ca2+-levels was effectively blocked by bakuchiol in a concentration-dependent manner.

Bakuchiol blocks oxidative stress-induced apoptosis pathways in RGC-5 cells Bakuchiol-treated cells were evaluated by Western blot analysis to determine whether this treatment affects the cleavage or activation of apoptosis-associated proteins, such as PARP, caspase-3, -9, AIF, and cytochrome c (Fig. 5). The protein levels of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 were up-regulated in cells treated with BSO and glutamate, while pre-treatment with bakuchiol suppressed this up-regulation in the total cell lysate (Fig. 5A). Moreover, Western blot analysis of the nucleic, cytoplasmic, and mitochondrial fractions was used to trace the intracellular migration of AIF and cytochrome c in RGC-5 cells (Figs. 5B–D). AIF was translocated from the mitochondrial fraction to the nucleic fraction, and cytochrome c was released from the mitochondrial fraction to the cytoplasmic fraction in RGC-5 cells, after 24 h of exposure to BSO and glutamate.

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However, pre-treatment with bakuchiol significantly reduced the translocation of AIF and the release of cytochrome c (Figs. 5B–D). Effects of bakuchiol in the NMDA-induced retinal damage model To examine the effect of bakuchiol on retinal damage, we used a rat model of NMDA-induced retinal damage. Western blot analysis was conducted to investigate the in vivo effect of bakuchiol on PARP, AIF, cleaved caspase-3, cleaved caspase-9, and cytochrome c expression, after NMDA-induced retinal damage in the rat retina. The expression of cleaved PARP, cleaved caspase-3, and cleaved caspase-9 appeared to be higher in retinal tissue 24 h after intravitreal injection of NMDA, compared to control retinal tissue (Fig. 6). In contrast, the levels of these cleaved proteins decreased in the bakuchiol-injected retinal tissue (Fig. 6A). Injection of NMDA into the retina resulted in the translocation of AIF and the release of cytochrome c; in contrast, the translocation of mitochondrial AIF into the nuclear fraction and the release of mitochondrial cytochrome c into the cytosol were significantly inhibited by bakuchiol (Figs. 6B–D). Effects of bakuchiol on ONC-induced loss of RGCs To examine the protective effect of bakuchiol on loss of RGCs, ONC-induced retinal damage in mice was investigated. The number of surviving RGCs was determined by counting retrograde-labeled cells in flat-mount retinal preparations (Fig. 7A). Seven days after ONC, the survival rate of RGCs was reduced by 21.2% ± 7.06% compared with control, however, mice treated with 1 or 10 nmol bakuchiol after ONC showed significantly less loss of RGCs compared with mice that had received ONC only (Fig. 7B). Fig. 8 shows the results of Western blot analysis for PARP, AIF, cleaved caspase-3, cleaved caspase-9, and cytochrome c proteins in the control and ONC mice in the presence and absence of 10 or 1 nmol bakuchiol treatment. The expression of cleaved PARP, cleaved caspase-3, and cleaved caspase-9 appeared to be higher in retinal tissue 7 days after ONC, compared to the levels in control retinal tissue (Fig. 8). In contrast, the levels of these cleaved proteins decreased in the bakuchiol-injected retinal tissue (Fig. 8A). In mice, ONC resulted in the translocation of AIF and the release of cytochrome c; in contrast, the translocation of mitochondrial AIF into the nuclear fraction and the release of mitochondrial cytochrome c into the cytosol were significantly inhibited by bakuchiol (Figs. 8B–D). Discussion In this study, we set out to identify active compounds from P. corylifolia that have protective effects on retinal cells; we used a combination of BSO and glutamate to induce cell injury and screened these compounds for protective activity in RGC-5 cells, even though these cells have limited RGC properties. To identify the active compounds from P. corylifolia, we fractionated the extract to yield 3 fractions. These 3 fractions were then tested for their effect on the oxidative stress response in RGC-5 cells. The methylene chloride fraction attenuated BSO and glutamate-induced RGC-5 cell death (Fig. 2A). We further isolated 7 compounds from the methylene chloride fraction (Fig. 3) including bakuchiol, which showed the most effective protection against BSO and glutamate-induced cell death in a concentration-dependent manner (Figs. 2B–D). After identifying an

Fig. 3. Chemical structures of the 7 compounds isolated from P. corylifolia.

active compound from P. corylifolia, we investigated the effects of this compound on RGCs in vivo. We showed that P. corylifolia significantly attenuates oxidative stress-induced retinal damage both in vitro and in vivo. We verified that the RGC-5 cells used in this study are a lineage of mouse neuronal precursor cells, as shown in Fig. 1. Moreover, these cells have reduced glutamate sensitivity, as compared to the initial RGC-5 cells (Van Bergen et al., 2009). It was previously demonstrated that RGC-5 cells are not affected by glutamate alone; hence, the reduced cell viability caused by the combination of BSO and glutamate may be due to oxidative stress (Harper et al., 2009; Osborne et al., 2012). In this model, ROS is elevated by a breakdown of DNA, expression of phosphatidylserine, and the activation of p38 MAPK (Osborne et al., 2012), and upon induction of oxidative stress, GSH is depleted, inhibiting cysteine uptake, which relies on ROS produced endogenously by the mitochondria (Maher and Hanneken, 2005; Nakajima et al., 2007). The in vitro model used in our study induces oxidative stress through activation of glutamate receptors; we then measured the MMP and the intracellular Ca2+ concentration after exposure of cells to these compounds. Treatment with BSO and glutamate induced an elevation of cytoplasmic Ca2+, while pre-treatment with bakuchiol protected the cells by preventing Ca 2+-influx (Fig. 4). Ca2+-overload can enhance intracellular ROS production and depolarization of the MMP (Kaushik et al., 2003). However, we found that cells that were exposed to BSO and glutamate for 24 h, but which have been pre-treated with bakuchiol, demonstrated significantly increased MMP compared to the non-pre-treated cells (Fig. 4). Thus, pre-treatment of cells with bakuchiol before exposure to BSO and glutamate led to recovery of the MMP decline, and inhibition of the increase in cytoplasmic Ca2+ concentration. Apoptosis is closely associated with cell survival and cell death. Many studies have reported that oxidative stress induces changes in mitochondrial function as well as over-expression of the apoptosis-associated proteins in neuronal cells (Mattson, 2000; Schrier and Falk, 2011). Our study

Fig. 2. Effects of P. corylifolia on RGC-5 cells subjected to 0.5 mM L-buthione-(S,R)-sulfoximine (BSO) and 10 mM glutamate for 24 h. (A) Treatment with BSO and glutamate reduced the viability of RGC-5 cells to approximately 60% compared that of to the control. The methylene chloride (MC) fraction of P. corylifolia attenuated the negative effect of BSO and glutamate. (B) Effects of compounds 1–7 isolated from P. corylifolia on the viability of RGC-5 cells after treatment with BSO and glutamate for 24 h, as measured by MTT assay. (C) Bakuchiol significantly attenuated the negative effects of BSO and glutamate, in a dose-dependent manner. (D) Fluorescence microscopy analysis of RGC-5 cells by double staining with PI and Hoechst 33342. Cells were exposed to normal medium (control), BSO and glutamate for 24 h, or were pre-treated with 10 μM bakuchiol, 1 μM bakuchiol, or 0.1 μM bakuchiol. Scale bars = 50 μm. Experimental values are expressed as a percentage of the viable cells with error bars indicating ± S.E.M., from 4 independent experiments. *p b 0.05, **p b 0.01, *p b 0.001, indicating statistically significant differences from the cells exposed to BSO and glutamate alone.

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Fig. 5. Protein expression levels in RGC-5 cell lysates. Western blot analysis showing (A) total lysates, (B) nucleic fraction, (C) cytoplasmic fraction, and (D) mitochondrial fraction of RGC-5 cells. The bands at 116 and 89 kDa correspond to PARP and cleaved PARP, respectively; those at 67 kDa, AIF; those at 37 kDa, cleaved caspase-9; those at 19 and 17 kDa, cleaved caspase-3; and those at 15 kDa, cytochrome c. Expression of β-actin (43 kDa; a cytosolic marker), lamin-B1 (68 kDa; a nuclear marker), and COX-IV (15 kDa; a mitochondrial marker) was used as controls for gel loading. Results shown are representative of 3 independent experiments.

Fig. 6. Expression of protein in the whole retina after retinal damage was induced by an intravitreal injection of NMDA. Western blot analysis showing (A) total lysate, (B) nucleic fraction, (C) cytoplasmic fraction, and (D) mitochondrial fraction from retinal tissue. In all blots, the bands at 116 and 89 kDa, as assessed by molecular weight markers, correspond to PARP and cleaved PARP, respectively; those at 67 kDa, AIF; those at 37 kDa, cleaved caspase-9; those at 19 and 17 kDa, cleaved caspase-3; and those at 15 kDa, cytochrome c. Expression of β-actin (seen at 43 kDa; a cytosolic marker), lamin-B1 (68 kDa; a nuclear marker), and COX-IV (15 kDa; a mitochondrial marker) was used as control for gel loading. Results shown are representative of 3 independent experiments (n = 8 rats per group).

confirmed that oxidative stress-related apoptosis pathways induce retinal cell death. Our results indicated that bakuchiol has protective effects against oxidative stress-induced apoptosis pathways. In particular, exposure to BSO and glutamate for 24 h caused an increase in the levels of cleaved PARP, cleaved caspase-3, and cleaved caspase-9 in total protein lysates, which also occurs in response to oxidative stress (Cheng et al., 2008). Pre-treatment with bakuchiol inhibited this up-regulation in a dose-dependent manner (Fig. 5). These data suggest that bakuchiol may protect RGC-5 cells from apoptotic death by inhibiting the activation of these apoptotic proteins. A previous study had shown that translocation of AIF and release of cytochrome c, following oxidative stress-induced apoptosis in neuronal

cells, can lead to the induction of cell death (Camins et al., 2008). Here, we demonstrated that bakuchiol inhibited translocation of AIF, and release of cytochrome c in its protective effect against oxidative stress-induced apoptosis (Figs. 5B–D). These results suggest that the neuroprotective properties of bakuchiol could be due to its anti-apoptotic effects. We used intravitreal NMDA injection of the rat retina as an in vivo model of oxidative stress to evaluate the effects of bakuchiol on NMDA-induced retinal damage. NMDA has been reported to induce thinning of the IPL as well as apoptotic cell death in the GCL and the inner part of INL (Lam et al., 1999). We then examined the expression levels of apoptosis-associated proteins in rat retina. Similar to our cell culture results, we found that the levels of cleaved PARP, cleaved

Fig. 4. Tetramethylrhodamine ethyl ester (TMRE)-staining for analysis of mitochondrial membrane potential (ΔΨm) and intracellular Ca2+ levels in RGC-5 cells following treatment with bakuchiol. (A) BSO and glutamate-treated cells were stained with 100 nM TMRE (nuclei were stained with DAPI) and analyzed by confocal microscopy (original magnification, 400×). (B) Quantification of fluorescent levels; fluorescent levels are expressed as relative value. RGC-5 cells were loaded with Fluo-3/AM, and the intracellular Ca2+-levels were determined by FACSCalibur flow cytometry, with or without pre-treatment with bakuchiol (0.1–10 μM) for 1 h prior to exposure to BSO plus glutamate. (C, D) Distribution of the fluorescence intensity of Fluo-3/AM in RGC-5 cultures; relative amount of total Fluo-3/AM fluorescence intensity in RGC-5 cells. Experimental values are expressed as mean ± S.E.M. from 3 independent experiments (*p b 0.05, **p b 0.01, ***p b 0.001).

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Fig. 7. Effect of bakuchiol on RGCs with optic nerve crush (ONC). Retrogradely labeled RGCs from mice with uninjured and injured optic nerves. RGCs were labeled by injecting 3% FluoroGold (FG) into the superior colliculi of the brain. (A) The figure shows representative micrographs (40×) of (a) control and damaged retinas 7 days after ONC, with (c, d) or without bakuchiol (b). Scale Bars: 500 μm. (e), (f), (g) and (h): high-magnification (100×). Scale bars: 100 μm. (i), (j), (k) and (l): high magnification (200×). Scale bars: 50 μm. (B) Quantitative analysis of the survival (%) rate of RGCs 7 days after ONC. The results showed a significant increase in survival rate of RGCs after bakuchiol treatment compared to vehicle treatment. Experimental values are expressed as mean ± S.E.M. *p b 0.05, ***p b 0.001 from 4 independent experiments. (n = 8 mice per group).

caspase-9, and cleaved caspase-3 were increased in the rat retina after intravitreal injection of NMDA, while bakuchiol significantly prevented this increase in cleaved proteins (Fig. 6A). Injection of NMDA also caused translocation of AIF from the mitochondria to the nucleus, release of cytochrome c from the mitochondria. However, bakuchiol inhibited these responses to NMDA in rat retina (Figs. 6B– D). Accordingly, these findings suggest that bakuchiol can protect against NMDA-induced retina damage. To confirm the protective effect of bakuchiol on retinal damage, we performed ONC in mice using retrograde-labeling with Fluoro-Gold in the superior colliculus (Fig. 7). RGC degeneration occurs after ONC, and mimics chronic glaucoma. Survival rate of RGCs after ONC has been reported to depend on the severity and time of injury (Johnson and Tomarev, 2010). Laboratory studies have previously shown that the survival rate after ONC is about 40% at 4 weeks; however, other studies have shown an almost 90% decrease of survival rate in RGCs at 5 days after ONC (Levkovitch-Verbin et al., 2000; Maeda et al., 2004; Yoles and Schwartz, 1998).

In this study, the density of RGCs was significantly decreased after ONC, with a survival rate in RGCs of 21.2% ± 7.06%; however, bakuchiol significantly decreased the loss of RGCs (Fig. 7). Bakuchiol also significantly prevented the up-regulation of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 after ONC (Fig. 8A). Moreover, ONC in mice caused translocation of AIF from the mitochondria to the nucleus, release of cytochrome c from the mitochondria; however, again, bakuchiol inhibited these responses (Figs. 8B–D). Thus, it was clear that bakuchiol was very effective in attenuating ONC-induced RGC loss in mice (Fig. 8). In conclusion, our findings suggest that bakuchiol isolated from P. corylifolia protects against retinal degeneration both in vitro and in vivo. Bakuchiol, therefore, has protective effects on retinal damage, which may prove to be useful against retinal diseases such as glaucoma. Conflict of interest The authors declare no financial conflict of interest.

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References

Fig. 8. Expression of protein in the whole retina after retinal damage was induced by ONC. Western blot analysis showing (A) total lysates, (B) nucleic fraction, (C) cytoplasmic fraction, and (D) mitochondrial fraction from retinal tissue. In all blots, the bands at 116 and 89 kDa, as assessed by molecular weight markers, correspond to PARP and cleaved PARP, respectively; those at 67 kDa, AIF; those at 37 kDa, cleaved caspase-9; those at 19 and 17 kDa, cleaved caspase-3; and those at 15 kDa, cytochrome c. Expression of β-actin (seen at 43 kDa; a cytosolic marker), lamin-B1 (68 kDa; a nuclear marker), and COX-IV (15 kDa; a mitochondrial marker) was used as control for gel loading. Results shown are representative of 4 independent experiments (n = 8 mice per group).

Acknowledgments The RGC-5 cells were kindly gifted by Alcon Research, Ltd. This work was financially supported by the Ministry of Education, Science Technology (MEST), Gangwon Province, Gangneung City, Gangneung Science Industry Foundation (GSIF) as the R&D Project for Gangneung Science Park Promoting Program, and by an intramural grant (2Z03850) from the Korea Institute of Science and Technology (KIST), Republic of Korea.

Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.taap.2013.03.017.

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