Current Eye Research, Early Online, 1–9, 2014 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.903497
ORIGINAL ARTICLE
Antiapoptotic Effects of Anthocyanin from the Seed Coat of Black Soybean Against Oxidative Damage of Human Lens Epithelial Cell Induced by H2O2 Jee Won Mok1, Dong-Jin Chang2,3 and Choun-Ki Joo1,2,3
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Catholic Institute for Visual Science, The Catholic University of Korea, Seoul, Korea, Department of Ophthalmology and Visual Science, The Catholic University of Korea, Seoul, Korea, and 3Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
ABSTRACT Purpose: To describe the protective effect of anthocyanin from black soybean in human lens epithelial cell line (HLE-B3) under H2O2-induced oxidative stress. Methods: Cytotoxicity of anthocyanin and H2O2 were determined by Cell Counting Kit-8 test. Viability of HLE-B3 cells under various H2O2 concentration (0, 50 and 100 mM) with or without pretreatment of anthocyanin (0, 50, 100 and 200 mg/ml) was measured. After quantifying the percentage of the apoptosis by Annexin V assay and APO-BrdU TUNEL assay, we conducted western blot and immunostaining of apoptosis-related molecules; Bcl2, BAD, BAX, p53 and caspase-3. To confirm the effect of anthocyanin on an ex vivo model, its effect on cultures of the lenses of porcine were examined. Results: Anthocyanin reduced cell death of HLE-B3 under H2O2-induced oxidative stress in a dose-dependent manner. In Annexin V analysis, anthocyanin protected HLE-B3 cells from apoptosis. H2O2 increased the expression of BAX, BAD, p53 and caspase-3 in a time-dependent manner, those of which anthocyanin significantly decreased. On the other hand, Bcl2 was increased from anthocyanin-treated lens cells. And in anthocyanin-treated lens organ culture, transparency was maintained. Conclusions: This study showed that anthocyanin protects HLE-B3 cells under oxidative stress from apoptosis, and the mechanism of the effect is related to the intrinsic pathway of apoptosis. Anthocyanin has a potential in prevention of cataract. Keywords: Anthocyanin, apoptosis, cataract, human lens epithelial cells, oxidative stress
INTRODUCTION
Anthocyanin contents in various food source were investigated, and plants rich in anthocyanins are Vaccinium species, such as blueberry, cranberry and bilberry, Rubus berries including black raspberry, red raspberry and blackberry, blackcurrant, cherry, eggplant peel, black rice, Concord grape and muscadine grape, red cabbage and violet petals.3 The highest recorded amount is specifically in the seed coat of black soybean (Glycine max L. Merr.) containing over 2000 mg per 100 g.4
Anthocyanins (Greek a´0& (anthos), flower and Greek 0& (kyanos) blue), used originally to describe the blue pigment of the cornflower Centaurea cyanus, is one of the subclasses of flavonoids which occur widely in the fruits and vegetables.1,2 By the year 2000, Over 500 of anthocyanin structures were reported and they occur in 27 families of plants.1
Received 30 October 2013; revised 2 March 2014; accepted 4 March 2014; published online 18 April 2014 Correspondence: Choun-Ki Joo, MD, PhD, Department of Ophthalmology, Catholic Institute for Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, #505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea. Tel: 82-2-2258-7621. Fax: 82-2-5333801. E-mail: [email protected]
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Number of studies have investigated healthpromoting effects of the anthocyanin including antioxidant, anti-allergic, anti-inflammatory, anti-viral, anti-proliferative, anti-mutagenic, anti-microbial and anti-carcinogenic protection from cardiovascular damage and allergy, microcirculation improvement, peripheral capillary fragility prevention, diabetes prevention and vision improvement.2 Beneficial effect of the anthocyanins on the eye also has been studied. Orally administered black-currant (BC) extract significantly inhibited enlargement of vitreous-chamber depth, axial and ocular lengths in a dose-dependent manner, and intravenously administered BC anthocyanins inhibited elongation of vitreous-chamber depth and axial length in chick myopia model.5 It was also effective in the prevention of macular degeneration and cataract in senescenceaccelerated OXYS rats.6 The lens of the eye is a unique organ in that it is nonvascularized and noninnervated and contains a single layer of epithelial cells on its anterior surface. The epithelial cells remain quiescent in the central section, divide toward the equatorial area and terminally differentiate into fiber cells in the equatorial region.7,8 This single layer of lens epithelial cells is essential for maintaining the metabolic homeostasis and transparency of the entire lens. When the ocular lens faces environmental stress such as oxidative stress, UV, etc., it can detoxify such stress if a strong defense system exists and the level of insult is not overwhelming. However, with the defense system being weakened, moderate environmental stress can trigger lens epithelial cell apoptosis that probably initiates human cataract.9 For this reason, researchers try to find molecules that could block the apoptosis in lens epithelial cells to make them as a preventive agent of cataract.10 In this study, we investigated the effect of anthocyanin on apoptosis of human lens epithelial (HLE-B3) cells induced by H2O2 and the possible molecular mechanisms involved.
MATERIALS AND METHODS
FIGURE 1. The chromatogram of the crude anthocyanin obtained by HPLC. Anthocyanin was composed of the following three components: (1) delphinidin-3-O-glucoside (Dp3glc), (2) cyanidin-3-O-glucoside (Cy3glc) and (3) petunidine-3-Oglucoside (Pt3glc). The relative amount of the contents were 25.2%, 68.3% and 6.5%, respectively.
Crude anthocyanin from the coats of black soybean was composed of delphinidin-3-O-glucoside, Cy3glc and petunidine-3-O-glucoside. The relative amount of the contents were 25.2%, 68.3% and 6.5%, respectively (Figure 1).11,12
Cell Culture Conditions HLE-B3 cells were cultured in minimum essential medium (MEM; GIBCO, CA) with 20% fetal bovine serum (FBS; GIBCO, CA) and 50 mg/ml gentamicin (GIBCO, CA) at 37 C with 5% CO2. For experiments, the cells were grown to 70% confluence and then were treated with various concentrations of H2O2 (Sigma-Aldrich, St. Louis, MO) and anthocyanin in serum free media for the indicated times.13
Preparation of Anthocyanin Assay for Cell Viability Anthocyanin, purified cyanidin-3-O-glucoside (Cy3glc), was offered from Rural Developmental Administration, Republic of Korea. Brief explanation of the extraction procedure is as follows: Hand-peeled seed coats (500 g) from Cheongja 3 black soybeans were soaked in 80% ethanol (0.1% acetic acid), and the solution was filtered through a 0.45 mm filter. After the concentration in a rotary evaporator at 35 C, the freeze-drying technique was used to obtain crude anthocyanins. HPLC of the crude anthocyanins was performed to extract purified components.
Cytotoxicity assays were performed using the Cell Counting Kit-8 (CCK-8) as described by the manufacturer (Dojindo Laboratory, Kumamoto, Japan). Cells were seeded onto 96-well plates and grown in a final volume of 100 ml media per well. After treating as indicated in the text, 10 ml of kit reagent was added and incubated for an additional 4 h. Absorbance was measured at a wavelength of 450 nm using a microplate reader (Synergy HT, BioTek, Winooski, VT). Current Eye Research
Effect of Anthocyanin on HLE
Migration Assay The HLE-B3 cells were seeded at a density of 1 105 cells in six-well plate chambers with MEM including 20% FBS. Confluent cells in six-well plates were wounded by manual scraping four times with a blue pipette tip. After the cells were carefully washed, they were exposed to 2 ml of 100, 200 and 400 mg/ml anthocyanin and serum-containing culture reagent (positive control). After 24 h, cells stained in 0.5% crystal violet and observed by microscope with digital image system. (Carl Zeiss AG, Oberkochen, Germany).14
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Annexin V Assay and TUNEL Assay The HLE-B3 cells were seeded at a density of 1 106 cells in MEM including 20% FBS. After reaching confluence, cells were treated with H2O2 (0, 50, 100 and 200 mM), anthocyanin (0, 100, 200 and 400 mg/ml) and both. Quantification of oxidative stress-induced apoptotic and/or necrotic cells was determined by FITCconjugated annexin V/PI (Annexin V-FITC Apoptosis Kit; Invitrogen, CA) and Alexa FluorÕ 488 dye-labeled anti-BrdU antibody (APO-BrdU TUNEL assay kit; Invitrogen, CA). For annexin V apoptosis assay, after treatment, cells were washed in phosphate-buffered saline (PBS) and centrifuged at 1000 g for 5 min at 4 C. Cellular pellets were resuspended in annexin V labeling solution and incubated for 15 min in the dark according to manufacturer’s instructions. Stained cells were dispersed with a pipette and apoptosis was carried out using a FACS LSR Fortessa (BD Biosciences Immunocytometry Systems, San Jose, CA). For APO-BrdU TUNEL assay, after treatment, cells were washed in PBS and centrifuged at 1000g for 5 min at 4 C. Cellular pellets were resuspended in DNA labeling solution included TdT enzyme and BrdUTP and incubated Alexa FluorÕ 488 dye-labeled anti-BrdU antibody in the dark according to manufacturer’s instructions. Stained cells were dispersed with a pipette, and apoptosis was carried out using a FACS LSR Fortessa (BD Biosciences Immunocytometry Systems).
Protein Extraction and BCA Assay Cells were washed with ice-cooled PBS. After incubation with RIPA buffer (Pierce, Rockford, IL), the supernatant was centrifuged at 12,000 rpm, 4 C for 10 min. The pellet was collected and vigorously vortexed in a 2 ml tube for 30 min in a 4 C-cold room. Centrifugation was done at 14,000 rpm, 4 C for 30 min, and the supernatant was saved for further procedures. Bovine serum albumin (BSA) was serially diluted form the concentration of 2 mg/ml to !
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0.03125 mg/ml to be used as standards. Standard curve, based on the protein standards, was plotted to determine the obtained protein samples according to the BCA Protein Assay Kit instructions (Pierce).
Western Blot Analysis and Immunostaining Antibodies used in this study were obtained as follows: Bcl2 (Santa Cruz, TX), BAX (Santa Cruz, TX), BAD (Santa Cruz, TX), p53 (Santa Cruz, TX), and caspase-3 (Santa Cruz, TX). For western blotting, treated HLE cells were washed in ice-cold PBS (pH 7.4) and collected in cell lysis buffer. Crude proteins were extracted, quantified and separated by electrophoresis (100 V, 2.5 h). After appropriate primary and secondary antibody incubations, protein signals were developed using chemiluminescence detection reagents (Bio-Rad, Hercules, CA). Membranes were exposed to Kodak X-OMAT AR film (Rochester, NY) for an appropriate length of time and developed according to manufacturer’s recommendations. Prestained molecular weight standards were purchased from Elpis-Biotech (Daejeon, Korea). Cells were cultured in four-well multi chamber slides (Invitrogen, CA) and then treated with anthocyanin and H2O2. Treated cells were rinsed in PBS, fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton-X 100 in PBS. After blocking, the cells were incubated with Bcl2, BAX and caspase-3. After blocking, the cells were incubated with Bcl2, Bax and Caspase-3. Primary antibodies detection were with the appropriate Alexa 488 or Alexa 594- conjugated secondary antibodies (Abcam, USA) and counterstaining with Hoechst33342 and images were acquired on confocal microscopy (Leica TSC 4D, Nussulock, Germany).
Porcine Lens Organ Culture Porcine eyes were obtained from a local abattoir and were stored at 4 C until used. Whole lenses from porcine were incubated in Medium 199 (SigmaAldrich, St. Louis, MO) containing 0.1% BSA (GIBCO, CA) and 50 mg/ml gentamicin at 37 C with 5% CO2. The next day, the medium was changed and 100 mM of H2O2, 200 mg/ml anthocyanin and both was added for one day and were observed daily under a stereomicroscope and photographed.13
Statistical Analysis The data were analyzed with the paired t-test and a one-way analysis of variance followed by Tukey’s multiple comparison test. Statistical analysis was performed by SigamPlotÕ 11.0 (Systat Software, Inc., Chicago, IL).
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FIGURE 2. Cell viability by CCK-8 test. Cell viability was significantly decreased from the concentration of 75 mM of H2O2 (A) and 500 mg/ml of anthocyanin (B). *p50.05.
FIGURE 3. Migration assay at different concentrations anthocyanin treated HLE-B3 cells. Wounds in the serum containing positive control (A); 100, 200 and 400 mg/ml anthocyanin (B–D) healed after 24 h.
Current Eye Research
Effect of Anthocyanin on HLE
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cell was significantly higher than that in the 100 mg/ ml or 400 mg/ml anthocyanin-treated HLE-B3 cell.
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Cell Culture and Migration Assay Figure 2 shows the absorbance of culture media in CCK-8 assay, which is believed to be proportional to the number of living cells. H2O2 treatment elicited dose-dependent cytotoxicity on HLE-B3 cells. H2O2 mildly reduced cell survival at a concentration of 100 mM. Marked reduction of cell survival occurred at the concentration of 125 mM. At a concentration of 500 mg/ml, anthocyanin showed significant toxicity than those at lower concentrations. Treatment concentrations of H2O2 were determined as 0, 50 and 100 mM, and anthocyanin as 0, 100, 200 and 400 mg/ml. Figure 3 compares the migration assay rates in various concentration of anthocyanin. The migration rate in the 200 mg/ml anthocyanin-treated HLE-B3
Prevention of Apoptosis in HLE-B3 Cell Under H2O2-Induced Oxidative Stress Examination of cell viability, apoptosis and morphologic change of HLE-B3 cells treated with H2O2 in the presence or absence of anthocyanin or only with anthocyanin were performed. As shown in Figure 4, the pretreatment of anthocyanin inhibited cell death and morphologic change. Morphologic observation of HLE cells treated with H2O2 in the presence or absence of anthocyanin or only with anthocyanin was performed. After H2O2 treatment, a high fraction of cells exhibited apoptosis-like morphologic changes, such as detachment, and cytoplasmic-condensation
FIGURE 4. Morphologic change of HLE-B3 cells under H2O2-induced oxidative stress with or without anthocyanin pretreatment. Anthocyanin inhibited morphological changes of HLE cells induced by H2O2. When treated by H2O2 (50 mM and 100 mM) for 24 h, a large fraction of cells demonstrated apoptosis signs such as detachment and cytoplasmic condensation leading to rounding. However, the proportion of cells with abnormal morphology decreased in parallel with the concentrations of anthocyanin. !
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FIGURE 5. Anti-apoptosis effects of anthocyanin on H2O2 treated HLE-B3 cells by (A) Annexin V assay and (B) APO-BrdU TUNEL assay. Anthocyanin inhibited apoptosis of HLE-B3 cells induced by H2O2. When treated by H2O2 (100 mM) for 24 h, 52.4% of cells went apoptosis, respectively. However, the proportion of the cells decreased in parallel with the concentrations of anthocyanin.
leading to rounding. However, the proportion of cells with abnormal morphology suggestive of apoptosis decreased with the increasing anthocyanin doses, indicating a dose-dependent prevention effect by anthocyanin. The viability of cells incubated with H2O2 alone was 41.61% and 7.95% at the concentration of 100 mM and 200 mM, compared to that of controls, whereas anthocyanin significantly suppressed H2O2-induced cell death. The percentage of apoptotic cells was detected by Annexin V-FITC/PI double staining and APO-BrdU TUNEL Assay to obtain a definitive quantification of the effect of different concentrations of anthocyanin (Figure 5). A significant increase of apoptosis was observed in HLE-B3 cells treated with 50 and 100 mM H2O2 compared with the control (control versus 50 mM versus 100 mM = 13.2% versus 38.5% and 67.4%, respectively). The percentages of apoptotic cells treated together with H2O2 and anthocyanin of 100 and 200 mg/ml were 56.2% and 33.3%, respectively. Anthocyanin significantly reduced the percentage of apoptotic cells compared with the H2O2-treated cells in a dose-dependent manner (p50.01 at all three concentrations).
Anthocyanin Reduced Pro-Apoptotic Protein Expression Induced by H2O2 After confirming that anthocyanin can protect HLE-B3 cells from H2O2-induced apoptosis, an effort to
investigate the potential molecular mechanisms involved was made. The expression of Bcl2, BAX, BAD, P53 and caspase-3 at the protein levels were measured. Western blot analysis revealed in increase in BAX, BAD, p53 and caspase-3 after treatment with 100 mM H2O2. The data in Figure 5 demonstrate increase in Bcl2 at 200 mm anthocyanin, whereas significantly inhibited the protein expression of BAX, BAD, p53 and caspase-3. And also anthocyanin at concentrations of 200 mM significantly inhibited the protein expression of BAX, BAD, p53 and caspase-3 and promoted that of Bcl2 in HLE-B3 cells after 24 h treatment of H2O2, as shown in Figure 6. In addition, immunofluorescence staining of H2O2 or anthocyanin-treated HLE-B3 cells showed that the levels of Bcl2 increased, where BAX and caspase-3 declined in the HLE-B3 cells treated with 200 mg/ml anthocyanin (Figure 7). To confirm these conclusions ex vivo, we used the intact porcine lens system; when porcine lenses were incubated in the presence of 100 mM H2O2, opacification was observed. But in exposure to 200 mg/ml anthocyanin, transparency of porcine lens was maintained (Figure 8).
DISCUSSION Apoptosis of lens epithelial cells is generally accepted as a causal factor of the development of cataracts, and oxidative stress is regarded as a major cause of Current Eye Research
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FIGURE 6. Western blot analysis of the expression of Bcl2, BAX, BAD, p53 and caspase-3. H2O2 increased the expression of BAX, BAD, p53 and caspase-3. Anthocyanin significantly decreased the expression of BAX, BAD, p53 and caspase-3 in H2O2-treated HLE-B3 cells.
FIGURE 7. Immunostaining of Bcl2, BAX and caspase-3 in cultured HLE-B3 cells. Cells were cocultured anthocyanin and H2O2 for 24 h. Treated HLE cells were stained with Hoechst (blue), Bcl2 (red), BAX (green) and caspase-3 (green). !
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FIGURE 8. Effects of anthocyanin on lens transparency. Whole porcine lenses were cultured for seven days with H2O2 and anthocyanin. With anthocyanin, lenses remained clear.
lens opacity.9,15,16 This study examined the effect of anthocyanin against H2O2-induced apoptosis of HLE-B3 cells and the possible molecular mechanisms involved. This study demonstrates that anthocyanin could suppress H2O2-induced HLE cells apoptosis in a dose-dependent manner in vitro. Under the oxidative stress which the treatment of H2O2 produced, a high fraction of the HLE-B3 cells showed apoptotic signs, which were significantly decreased in the presence of anthocyanin.2 With the increasing appreciation of the potential benefits on human health, a number of studies have researched on anthocyanin recently. Effects such as antioxidant, anti-allergic, anti-inflammatory, anti-viral, anti-proliferative, anti-mutagenic, antimicrobial, anti-carcinogenic, protection from cardiovascular damage and allergy, microcirculation improvement, peripheral capillary fragility prevention, diabetes prevention and vision improvement have been presented.17–26 In the point of view toward antioxidant effect, Zhang et al. suggested that anthocyanin in Chinese bayberry extract protect pancreatic b cells from H2O2-induced cell injury via ERK1/2 - and PI3K/Akt-mediated HO-1 upregulation.27 Speciale et al. demonstrate that Cy3glc, which comprised 68.3% of anthocyanins of our study, was able to
protect human endothelial cells against alterations induced by tumor necrosis factor alpha (TNF-a), including the activation of NF-kB, increased gene expression of adhesion molecules, leukocyte adhesion to endothelium and intracellular accumulation of H2O2 and lipid peroxidation byproducts.28 Guo et al. described that Cy3glc exerts a protective role against H2O2- or TNF-a-induced insulin resistance in 3T3-L1 adipocytes by inhibiting the JNK signal pathway.29 But there was no report about the antioxidative effect of anthocyanin in HLE-B3. In this in vitro work, anthocyanin increased the expression of Bcl2, an antiapoptotic factor. On the other hand, it reduced the expression of BAD and BAX, which are pro-apoptotic members of the Bcl-2 protein family, and also reduced that of p53 tumor suppressor, a sequence-specific transcription factor which promotes apoptotic pathway, and caspase-3, a protease which is associated with the activation of cell death. The change of expression of apoptosis-associated proteins implies that the anthocyanin suppress the extrinsic apoptotic pathways in HLE cells under H2O2-induced oxidative stress. This study showed the flavonoids from the coat of black soybean, anthocyanin, can protect HLE cells under H2O2-induced oxidative stress from apoptosis by promoting the expression of Bcl2 and inhibiting Current Eye Research
Effect of Anthocyanin on HLE the activation of BAD, BAX and caspase-3. Our study suggests that anthocyanin have potential in the prevention of cataract.
DECLARATION OF INTEREST Author has no conflict of interests to be disclosed. This research was supported by the Biogreen21 Program (Code#PJ007186), Rural Development Administration, Korea.
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REFERENCES 1. Pietta PG. Flavonoids as antioxidants. J Nat Prod 2000;63: 1035–1042. 2. Ghosh D, Konishi T. Anthocyanins and anthocyanin-rich extracts: role in diabetes and eye function. Asia Pac J Clin Nutr 2007;16:200–208. 3. Wu X, Gu L, Prior RL, McKay S. Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity. J Agric Food Chem 2004;52:7846–7856. 4. Choung MG, Baek IY, Kang ST, Han WY, Shin DC, Moon HP, et al. Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.). J Agric Food Chem 2001;49:5848–5851. 5. Iida H, Nakamura Y, Matsumoto H, Takeuchi Y, Harano S, Ishihara M, et al. Effect of black-currant extract on negative lens-induced ocular growth in chicks. Ophthalmic Res 2010;44:242–250. 6. Fursova A, Gesarevich OG, Gonchar AM, Trofimova NA, Kolosova NG. [Dietary supplementation with bilberry extract prevents macular degeneration and cataracts in senesce-accelerated OXYS rats]. Adv Gerontol 2005;16: 76–79. 7. Papaconstantinou J. Molecular aspects of lens cell differentiation. Science 1967;156:338–346. 8. Piatigorsky J. Lens differentiation in vertebrates. A review of cellular and molecular features. Differentiation 1981;19: 134–153. 9. Li WC, Kuszak JR, Dunn K, Wang RR, Ma W, Wang GM, et al. Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals. J Cell Biol 1995;130:169–181. 10. Yao H, Tang X, Shao X, Feng L, Wu N, Yao K. Parthenolide protects human lens epithelial cells from oxidative stressinduced apoptosis via inhibition of activation of caspase-3 and caspase-9. Cell Res 2007;17:565–571. 11. Jang H, Ha US, Kim SJ, Yoon BI, Han DS, Yuk SM, et al. Anthocyanin extracted from black soybean reduces prostate weight and promotes apoptosis in the prostatic hyperplasia-induced rat model. J Agric Food Chem 2010; 58:12686–12691. 12. Ha YA, Lee MH, Park CH, Pae SB, Shim KB, Ko JM, et al. Identification and characterization of anthocyanins in yard-long beans (Vigna unguiculata ssp. sesquipedalis L.) by high-performance liquid chromatography with diode array detection and electrospray ionization/mass spectrometry (HPLC-DAD-ESI/MS) analysis. J Agric Food Chem 2010;58:2571–2576.
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13. Seomun Y, Kim JT, Kim HS, Park JY, Joo CK. Induction of p21Cip1-mediated G2/M arrest in H2O2-treated lens epithelial cells. Mol Vis 2005;11:764–774. 14. Choi JA, Oh TH, Choi JS, Chang DJ, Joo CK. Impact of b-1,3-glucan Isolated from Euglena gracilis on corneal epithelial cell migration and on wound healing in a rat alkali burn model. Curr Eye Res 2013;38:1207–1213. 15. Li WC, Spector A. Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract. Free Radic Biol Med 1996;20:301–311. 16. Li WC, Kuszak JR, Wang GM, Wu ZQ, Spector A. Calcimycin-induced lens epithelial cell apoptosis contributes to cataract formation. Exp Eye Res 1995;61:91–98. 17. Al-Awwadi NA, Araiz C, Bornet A, Delbosc S, Cristol JP, Linck N, et al. Extracts enriched in different polyphenolic families normalize increased cardiac NADPH oxidase expression while having differential effects on insulin resistance, hypertension, and cardiac hypertrophy in high-fructose-fed rats. J Agric Food Chem 2005;53: 151–157. 18. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 1993;90:7915–7922. 19. Cohen-Boulakia F, Valensi PE, Boulahdour H, Lestrade R, Dufour-Lamartinie JF, Hort-Legrand C, et al. In vivo sequential study of skeletal muscle capillary permeability in diabetic rats: effect of anthocyanosides. Metabolism 2000;49:880–885. 20. Duthie GG, Duthie SJ, Kyle JA. Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr Res Rev 2000;13:79–106. 21. Galvano F, La Fauci L, Lazzarino G, Fogliano V, Ritieni A, Ciappellano S, et al. Cyanidins: metabolism and biological properties. J Nutr Biochem 2004;15:2–11. 22. Hollman PC, Katan MB. Dietary flavonoids: intake, health effects and bioavailability. Food Chem Toxicol 1999;37: 937–942. 23. Jayaprakasam B, Vareed SK, Olson LK, Nair MG. Insulin secretion by bioactive anthocyanins and anthocyanidins present in fruits. J Agric Food Chem 2005;53:28–31. 24. Lee J, Lee HK, Kim CY, Hong YJ, Choe CM, You TW, et al. Purified high-dose anthocyanoside oligomer administration improves nocturnal vision and clinical symptoms in myopia subjects. Br J Nutr 2005;93:895–899. 25. Levy Y, Glovinsky Y. The effect of anthocyanosides on night vision. Eye (Lond) 1998;12:967–969. 26. Morimitsu Y, Sugihara N, Furuno K. Inhibitory effect of flavonoids on sulfo- and glucurono-conjugation of acetaminophen in rat cultured hepatocytes and liver subcellular preparations. Biol Pharm Bull 2004;27:714–717. 27. Zhang B, Kang M, Xie Q, Xu B, Sun C, Chen K, et al. Anthocyanins from Chinese bayberry extract protect beta cells from oxidative stress-mediated injury via HO-1 upregulation. J Agric Food Chem 2011;59:537–545. 28. Speciale A, Canali R, Chirafisi J, Saija A, Virgili F, Cimino F. Cyanidin-3-O-glucoside protection against TNF-alphainduced endothelial dysfunction: involvement of nuclear factor-kappaB signaling. J Agric Food Chem 2010;58: 12048–12054. 29. Guo H, Ling W, Wang Q, Liu C, Hu Y, Xia M. Cyanidin 3-glucoside protects 3T3-L1 adipocytes against H2O2- or TNF-alpha-induced insulin resistance by inhibiting c-Jun NH2-terminal kinase activation. Biochem Pharmacol 2008; 75:1393–1401.
ORIGINAL ARTICLE
Antiapoptotic Effects of Anthocyanin from the Seed Coat of Black Soybean Against Oxidative Damage of Human Lens Epithelial Cell Induced by H2O2 Jee Won Mok1, Dong-Jin Chang2,3 and Choun-Ki Joo1,2,3
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Catholic Institute for Visual Science, The Catholic University of Korea, Seoul, Korea, Department of Ophthalmology and Visual Science, The Catholic University of Korea, Seoul, Korea, and 3Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
ABSTRACT Purpose: To describe the protective effect of anthocyanin from black soybean in human lens epithelial cell line (HLE-B3) under H2O2-induced oxidative stress. Methods: Cytotoxicity of anthocyanin and H2O2 were determined by Cell Counting Kit-8 test. Viability of HLE-B3 cells under various H2O2 concentration (0, 50 and 100 mM) with or without pretreatment of anthocyanin (0, 50, 100 and 200 mg/ml) was measured. After quantifying the percentage of the apoptosis by Annexin V assay and APO-BrdU TUNEL assay, we conducted western blot and immunostaining of apoptosis-related molecules; Bcl2, BAD, BAX, p53 and caspase-3. To confirm the effect of anthocyanin on an ex vivo model, its effect on cultures of the lenses of porcine were examined. Results: Anthocyanin reduced cell death of HLE-B3 under H2O2-induced oxidative stress in a dose-dependent manner. In Annexin V analysis, anthocyanin protected HLE-B3 cells from apoptosis. H2O2 increased the expression of BAX, BAD, p53 and caspase-3 in a time-dependent manner, those of which anthocyanin significantly decreased. On the other hand, Bcl2 was increased from anthocyanin-treated lens cells. And in anthocyanin-treated lens organ culture, transparency was maintained. Conclusions: This study showed that anthocyanin protects HLE-B3 cells under oxidative stress from apoptosis, and the mechanism of the effect is related to the intrinsic pathway of apoptosis. Anthocyanin has a potential in prevention of cataract. Keywords: Anthocyanin, apoptosis, cataract, human lens epithelial cells, oxidative stress
INTRODUCTION
Anthocyanin contents in various food source were investigated, and plants rich in anthocyanins are Vaccinium species, such as blueberry, cranberry and bilberry, Rubus berries including black raspberry, red raspberry and blackberry, blackcurrant, cherry, eggplant peel, black rice, Concord grape and muscadine grape, red cabbage and violet petals.3 The highest recorded amount is specifically in the seed coat of black soybean (Glycine max L. Merr.) containing over 2000 mg per 100 g.4
Anthocyanins (Greek a´0& (anthos), flower and Greek 0& (kyanos) blue), used originally to describe the blue pigment of the cornflower Centaurea cyanus, is one of the subclasses of flavonoids which occur widely in the fruits and vegetables.1,2 By the year 2000, Over 500 of anthocyanin structures were reported and they occur in 27 families of plants.1
Received 30 October 2013; revised 2 March 2014; accepted 4 March 2014; published online 18 April 2014 Correspondence: Choun-Ki Joo, MD, PhD, Department of Ophthalmology, Catholic Institute for Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, #505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea. Tel: 82-2-2258-7621. Fax: 82-2-5333801. E-mail: [email protected]
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Number of studies have investigated healthpromoting effects of the anthocyanin including antioxidant, anti-allergic, anti-inflammatory, anti-viral, anti-proliferative, anti-mutagenic, anti-microbial and anti-carcinogenic protection from cardiovascular damage and allergy, microcirculation improvement, peripheral capillary fragility prevention, diabetes prevention and vision improvement.2 Beneficial effect of the anthocyanins on the eye also has been studied. Orally administered black-currant (BC) extract significantly inhibited enlargement of vitreous-chamber depth, axial and ocular lengths in a dose-dependent manner, and intravenously administered BC anthocyanins inhibited elongation of vitreous-chamber depth and axial length in chick myopia model.5 It was also effective in the prevention of macular degeneration and cataract in senescenceaccelerated OXYS rats.6 The lens of the eye is a unique organ in that it is nonvascularized and noninnervated and contains a single layer of epithelial cells on its anterior surface. The epithelial cells remain quiescent in the central section, divide toward the equatorial area and terminally differentiate into fiber cells in the equatorial region.7,8 This single layer of lens epithelial cells is essential for maintaining the metabolic homeostasis and transparency of the entire lens. When the ocular lens faces environmental stress such as oxidative stress, UV, etc., it can detoxify such stress if a strong defense system exists and the level of insult is not overwhelming. However, with the defense system being weakened, moderate environmental stress can trigger lens epithelial cell apoptosis that probably initiates human cataract.9 For this reason, researchers try to find molecules that could block the apoptosis in lens epithelial cells to make them as a preventive agent of cataract.10 In this study, we investigated the effect of anthocyanin on apoptosis of human lens epithelial (HLE-B3) cells induced by H2O2 and the possible molecular mechanisms involved.
MATERIALS AND METHODS
FIGURE 1. The chromatogram of the crude anthocyanin obtained by HPLC. Anthocyanin was composed of the following three components: (1) delphinidin-3-O-glucoside (Dp3glc), (2) cyanidin-3-O-glucoside (Cy3glc) and (3) petunidine-3-Oglucoside (Pt3glc). The relative amount of the contents were 25.2%, 68.3% and 6.5%, respectively.
Crude anthocyanin from the coats of black soybean was composed of delphinidin-3-O-glucoside, Cy3glc and petunidine-3-O-glucoside. The relative amount of the contents were 25.2%, 68.3% and 6.5%, respectively (Figure 1).11,12
Cell Culture Conditions HLE-B3 cells were cultured in minimum essential medium (MEM; GIBCO, CA) with 20% fetal bovine serum (FBS; GIBCO, CA) and 50 mg/ml gentamicin (GIBCO, CA) at 37 C with 5% CO2. For experiments, the cells were grown to 70% confluence and then were treated with various concentrations of H2O2 (Sigma-Aldrich, St. Louis, MO) and anthocyanin in serum free media for the indicated times.13
Preparation of Anthocyanin Assay for Cell Viability Anthocyanin, purified cyanidin-3-O-glucoside (Cy3glc), was offered from Rural Developmental Administration, Republic of Korea. Brief explanation of the extraction procedure is as follows: Hand-peeled seed coats (500 g) from Cheongja 3 black soybeans were soaked in 80% ethanol (0.1% acetic acid), and the solution was filtered through a 0.45 mm filter. After the concentration in a rotary evaporator at 35 C, the freeze-drying technique was used to obtain crude anthocyanins. HPLC of the crude anthocyanins was performed to extract purified components.
Cytotoxicity assays were performed using the Cell Counting Kit-8 (CCK-8) as described by the manufacturer (Dojindo Laboratory, Kumamoto, Japan). Cells were seeded onto 96-well plates and grown in a final volume of 100 ml media per well. After treating as indicated in the text, 10 ml of kit reagent was added and incubated for an additional 4 h. Absorbance was measured at a wavelength of 450 nm using a microplate reader (Synergy HT, BioTek, Winooski, VT). Current Eye Research
Effect of Anthocyanin on HLE
Migration Assay The HLE-B3 cells were seeded at a density of 1 105 cells in six-well plate chambers with MEM including 20% FBS. Confluent cells in six-well plates were wounded by manual scraping four times with a blue pipette tip. After the cells were carefully washed, they were exposed to 2 ml of 100, 200 and 400 mg/ml anthocyanin and serum-containing culture reagent (positive control). After 24 h, cells stained in 0.5% crystal violet and observed by microscope with digital image system. (Carl Zeiss AG, Oberkochen, Germany).14
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Annexin V Assay and TUNEL Assay The HLE-B3 cells were seeded at a density of 1 106 cells in MEM including 20% FBS. After reaching confluence, cells were treated with H2O2 (0, 50, 100 and 200 mM), anthocyanin (0, 100, 200 and 400 mg/ml) and both. Quantification of oxidative stress-induced apoptotic and/or necrotic cells was determined by FITCconjugated annexin V/PI (Annexin V-FITC Apoptosis Kit; Invitrogen, CA) and Alexa FluorÕ 488 dye-labeled anti-BrdU antibody (APO-BrdU TUNEL assay kit; Invitrogen, CA). For annexin V apoptosis assay, after treatment, cells were washed in phosphate-buffered saline (PBS) and centrifuged at 1000 g for 5 min at 4 C. Cellular pellets were resuspended in annexin V labeling solution and incubated for 15 min in the dark according to manufacturer’s instructions. Stained cells were dispersed with a pipette and apoptosis was carried out using a FACS LSR Fortessa (BD Biosciences Immunocytometry Systems, San Jose, CA). For APO-BrdU TUNEL assay, after treatment, cells were washed in PBS and centrifuged at 1000g for 5 min at 4 C. Cellular pellets were resuspended in DNA labeling solution included TdT enzyme and BrdUTP and incubated Alexa FluorÕ 488 dye-labeled anti-BrdU antibody in the dark according to manufacturer’s instructions. Stained cells were dispersed with a pipette, and apoptosis was carried out using a FACS LSR Fortessa (BD Biosciences Immunocytometry Systems).
Protein Extraction and BCA Assay Cells were washed with ice-cooled PBS. After incubation with RIPA buffer (Pierce, Rockford, IL), the supernatant was centrifuged at 12,000 rpm, 4 C for 10 min. The pellet was collected and vigorously vortexed in a 2 ml tube for 30 min in a 4 C-cold room. Centrifugation was done at 14,000 rpm, 4 C for 30 min, and the supernatant was saved for further procedures. Bovine serum albumin (BSA) was serially diluted form the concentration of 2 mg/ml to !
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0.03125 mg/ml to be used as standards. Standard curve, based on the protein standards, was plotted to determine the obtained protein samples according to the BCA Protein Assay Kit instructions (Pierce).
Western Blot Analysis and Immunostaining Antibodies used in this study were obtained as follows: Bcl2 (Santa Cruz, TX), BAX (Santa Cruz, TX), BAD (Santa Cruz, TX), p53 (Santa Cruz, TX), and caspase-3 (Santa Cruz, TX). For western blotting, treated HLE cells were washed in ice-cold PBS (pH 7.4) and collected in cell lysis buffer. Crude proteins were extracted, quantified and separated by electrophoresis (100 V, 2.5 h). After appropriate primary and secondary antibody incubations, protein signals were developed using chemiluminescence detection reagents (Bio-Rad, Hercules, CA). Membranes were exposed to Kodak X-OMAT AR film (Rochester, NY) for an appropriate length of time and developed according to manufacturer’s recommendations. Prestained molecular weight standards were purchased from Elpis-Biotech (Daejeon, Korea). Cells were cultured in four-well multi chamber slides (Invitrogen, CA) and then treated with anthocyanin and H2O2. Treated cells were rinsed in PBS, fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton-X 100 in PBS. After blocking, the cells were incubated with Bcl2, BAX and caspase-3. After blocking, the cells were incubated with Bcl2, Bax and Caspase-3. Primary antibodies detection were with the appropriate Alexa 488 or Alexa 594- conjugated secondary antibodies (Abcam, USA) and counterstaining with Hoechst33342 and images were acquired on confocal microscopy (Leica TSC 4D, Nussulock, Germany).
Porcine Lens Organ Culture Porcine eyes were obtained from a local abattoir and were stored at 4 C until used. Whole lenses from porcine were incubated in Medium 199 (SigmaAldrich, St. Louis, MO) containing 0.1% BSA (GIBCO, CA) and 50 mg/ml gentamicin at 37 C with 5% CO2. The next day, the medium was changed and 100 mM of H2O2, 200 mg/ml anthocyanin and both was added for one day and were observed daily under a stereomicroscope and photographed.13
Statistical Analysis The data were analyzed with the paired t-test and a one-way analysis of variance followed by Tukey’s multiple comparison test. Statistical analysis was performed by SigamPlotÕ 11.0 (Systat Software, Inc., Chicago, IL).
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FIGURE 2. Cell viability by CCK-8 test. Cell viability was significantly decreased from the concentration of 75 mM of H2O2 (A) and 500 mg/ml of anthocyanin (B). *p50.05.
FIGURE 3. Migration assay at different concentrations anthocyanin treated HLE-B3 cells. Wounds in the serum containing positive control (A); 100, 200 and 400 mg/ml anthocyanin (B–D) healed after 24 h.
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cell was significantly higher than that in the 100 mg/ ml or 400 mg/ml anthocyanin-treated HLE-B3 cell.
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Cell Culture and Migration Assay Figure 2 shows the absorbance of culture media in CCK-8 assay, which is believed to be proportional to the number of living cells. H2O2 treatment elicited dose-dependent cytotoxicity on HLE-B3 cells. H2O2 mildly reduced cell survival at a concentration of 100 mM. Marked reduction of cell survival occurred at the concentration of 125 mM. At a concentration of 500 mg/ml, anthocyanin showed significant toxicity than those at lower concentrations. Treatment concentrations of H2O2 were determined as 0, 50 and 100 mM, and anthocyanin as 0, 100, 200 and 400 mg/ml. Figure 3 compares the migration assay rates in various concentration of anthocyanin. The migration rate in the 200 mg/ml anthocyanin-treated HLE-B3
Prevention of Apoptosis in HLE-B3 Cell Under H2O2-Induced Oxidative Stress Examination of cell viability, apoptosis and morphologic change of HLE-B3 cells treated with H2O2 in the presence or absence of anthocyanin or only with anthocyanin were performed. As shown in Figure 4, the pretreatment of anthocyanin inhibited cell death and morphologic change. Morphologic observation of HLE cells treated with H2O2 in the presence or absence of anthocyanin or only with anthocyanin was performed. After H2O2 treatment, a high fraction of cells exhibited apoptosis-like morphologic changes, such as detachment, and cytoplasmic-condensation
FIGURE 4. Morphologic change of HLE-B3 cells under H2O2-induced oxidative stress with or without anthocyanin pretreatment. Anthocyanin inhibited morphological changes of HLE cells induced by H2O2. When treated by H2O2 (50 mM and 100 mM) for 24 h, a large fraction of cells demonstrated apoptosis signs such as detachment and cytoplasmic condensation leading to rounding. However, the proportion of cells with abnormal morphology decreased in parallel with the concentrations of anthocyanin. !
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FIGURE 5. Anti-apoptosis effects of anthocyanin on H2O2 treated HLE-B3 cells by (A) Annexin V assay and (B) APO-BrdU TUNEL assay. Anthocyanin inhibited apoptosis of HLE-B3 cells induced by H2O2. When treated by H2O2 (100 mM) for 24 h, 52.4% of cells went apoptosis, respectively. However, the proportion of the cells decreased in parallel with the concentrations of anthocyanin.
leading to rounding. However, the proportion of cells with abnormal morphology suggestive of apoptosis decreased with the increasing anthocyanin doses, indicating a dose-dependent prevention effect by anthocyanin. The viability of cells incubated with H2O2 alone was 41.61% and 7.95% at the concentration of 100 mM and 200 mM, compared to that of controls, whereas anthocyanin significantly suppressed H2O2-induced cell death. The percentage of apoptotic cells was detected by Annexin V-FITC/PI double staining and APO-BrdU TUNEL Assay to obtain a definitive quantification of the effect of different concentrations of anthocyanin (Figure 5). A significant increase of apoptosis was observed in HLE-B3 cells treated with 50 and 100 mM H2O2 compared with the control (control versus 50 mM versus 100 mM = 13.2% versus 38.5% and 67.4%, respectively). The percentages of apoptotic cells treated together with H2O2 and anthocyanin of 100 and 200 mg/ml were 56.2% and 33.3%, respectively. Anthocyanin significantly reduced the percentage of apoptotic cells compared with the H2O2-treated cells in a dose-dependent manner (p50.01 at all three concentrations).
Anthocyanin Reduced Pro-Apoptotic Protein Expression Induced by H2O2 After confirming that anthocyanin can protect HLE-B3 cells from H2O2-induced apoptosis, an effort to
investigate the potential molecular mechanisms involved was made. The expression of Bcl2, BAX, BAD, P53 and caspase-3 at the protein levels were measured. Western blot analysis revealed in increase in BAX, BAD, p53 and caspase-3 after treatment with 100 mM H2O2. The data in Figure 5 demonstrate increase in Bcl2 at 200 mm anthocyanin, whereas significantly inhibited the protein expression of BAX, BAD, p53 and caspase-3. And also anthocyanin at concentrations of 200 mM significantly inhibited the protein expression of BAX, BAD, p53 and caspase-3 and promoted that of Bcl2 in HLE-B3 cells after 24 h treatment of H2O2, as shown in Figure 6. In addition, immunofluorescence staining of H2O2 or anthocyanin-treated HLE-B3 cells showed that the levels of Bcl2 increased, where BAX and caspase-3 declined in the HLE-B3 cells treated with 200 mg/ml anthocyanin (Figure 7). To confirm these conclusions ex vivo, we used the intact porcine lens system; when porcine lenses were incubated in the presence of 100 mM H2O2, opacification was observed. But in exposure to 200 mg/ml anthocyanin, transparency of porcine lens was maintained (Figure 8).
DISCUSSION Apoptosis of lens epithelial cells is generally accepted as a causal factor of the development of cataracts, and oxidative stress is regarded as a major cause of Current Eye Research
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FIGURE 6. Western blot analysis of the expression of Bcl2, BAX, BAD, p53 and caspase-3. H2O2 increased the expression of BAX, BAD, p53 and caspase-3. Anthocyanin significantly decreased the expression of BAX, BAD, p53 and caspase-3 in H2O2-treated HLE-B3 cells.
FIGURE 7. Immunostaining of Bcl2, BAX and caspase-3 in cultured HLE-B3 cells. Cells were cocultured anthocyanin and H2O2 for 24 h. Treated HLE cells were stained with Hoechst (blue), Bcl2 (red), BAX (green) and caspase-3 (green). !
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FIGURE 8. Effects of anthocyanin on lens transparency. Whole porcine lenses were cultured for seven days with H2O2 and anthocyanin. With anthocyanin, lenses remained clear.
lens opacity.9,15,16 This study examined the effect of anthocyanin against H2O2-induced apoptosis of HLE-B3 cells and the possible molecular mechanisms involved. This study demonstrates that anthocyanin could suppress H2O2-induced HLE cells apoptosis in a dose-dependent manner in vitro. Under the oxidative stress which the treatment of H2O2 produced, a high fraction of the HLE-B3 cells showed apoptotic signs, which were significantly decreased in the presence of anthocyanin.2 With the increasing appreciation of the potential benefits on human health, a number of studies have researched on anthocyanin recently. Effects such as antioxidant, anti-allergic, anti-inflammatory, anti-viral, anti-proliferative, anti-mutagenic, antimicrobial, anti-carcinogenic, protection from cardiovascular damage and allergy, microcirculation improvement, peripheral capillary fragility prevention, diabetes prevention and vision improvement have been presented.17–26 In the point of view toward antioxidant effect, Zhang et al. suggested that anthocyanin in Chinese bayberry extract protect pancreatic b cells from H2O2-induced cell injury via ERK1/2 - and PI3K/Akt-mediated HO-1 upregulation.27 Speciale et al. demonstrate that Cy3glc, which comprised 68.3% of anthocyanins of our study, was able to
protect human endothelial cells against alterations induced by tumor necrosis factor alpha (TNF-a), including the activation of NF-kB, increased gene expression of adhesion molecules, leukocyte adhesion to endothelium and intracellular accumulation of H2O2 and lipid peroxidation byproducts.28 Guo et al. described that Cy3glc exerts a protective role against H2O2- or TNF-a-induced insulin resistance in 3T3-L1 adipocytes by inhibiting the JNK signal pathway.29 But there was no report about the antioxidative effect of anthocyanin in HLE-B3. In this in vitro work, anthocyanin increased the expression of Bcl2, an antiapoptotic factor. On the other hand, it reduced the expression of BAD and BAX, which are pro-apoptotic members of the Bcl-2 protein family, and also reduced that of p53 tumor suppressor, a sequence-specific transcription factor which promotes apoptotic pathway, and caspase-3, a protease which is associated with the activation of cell death. The change of expression of apoptosis-associated proteins implies that the anthocyanin suppress the extrinsic apoptotic pathways in HLE cells under H2O2-induced oxidative stress. This study showed the flavonoids from the coat of black soybean, anthocyanin, can protect HLE cells under H2O2-induced oxidative stress from apoptosis by promoting the expression of Bcl2 and inhibiting Current Eye Research
Effect of Anthocyanin on HLE the activation of BAD, BAX and caspase-3. Our study suggests that anthocyanin have potential in the prevention of cataract.
DECLARATION OF INTEREST Author has no conflict of interests to be disclosed. This research was supported by the Biogreen21 Program (Code#PJ007186), Rural Development Administration, Korea.
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REFERENCES 1. Pietta PG. Flavonoids as antioxidants. J Nat Prod 2000;63: 1035–1042. 2. Ghosh D, Konishi T. Anthocyanins and anthocyanin-rich extracts: role in diabetes and eye function. Asia Pac J Clin Nutr 2007;16:200–208. 3. Wu X, Gu L, Prior RL, McKay S. Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity. J Agric Food Chem 2004;52:7846–7856. 4. Choung MG, Baek IY, Kang ST, Han WY, Shin DC, Moon HP, et al. Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.). J Agric Food Chem 2001;49:5848–5851. 5. Iida H, Nakamura Y, Matsumoto H, Takeuchi Y, Harano S, Ishihara M, et al. Effect of black-currant extract on negative lens-induced ocular growth in chicks. Ophthalmic Res 2010;44:242–250. 6. Fursova A, Gesarevich OG, Gonchar AM, Trofimova NA, Kolosova NG. [Dietary supplementation with bilberry extract prevents macular degeneration and cataracts in senesce-accelerated OXYS rats]. Adv Gerontol 2005;16: 76–79. 7. Papaconstantinou J. Molecular aspects of lens cell differentiation. Science 1967;156:338–346. 8. Piatigorsky J. Lens differentiation in vertebrates. A review of cellular and molecular features. Differentiation 1981;19: 134–153. 9. Li WC, Kuszak JR, Dunn K, Wang RR, Ma W, Wang GM, et al. Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals. J Cell Biol 1995;130:169–181. 10. Yao H, Tang X, Shao X, Feng L, Wu N, Yao K. Parthenolide protects human lens epithelial cells from oxidative stressinduced apoptosis via inhibition of activation of caspase-3 and caspase-9. Cell Res 2007;17:565–571. 11. Jang H, Ha US, Kim SJ, Yoon BI, Han DS, Yuk SM, et al. Anthocyanin extracted from black soybean reduces prostate weight and promotes apoptosis in the prostatic hyperplasia-induced rat model. J Agric Food Chem 2010; 58:12686–12691. 12. Ha YA, Lee MH, Park CH, Pae SB, Shim KB, Ko JM, et al. Identification and characterization of anthocyanins in yard-long beans (Vigna unguiculata ssp. sesquipedalis L.) by high-performance liquid chromatography with diode array detection and electrospray ionization/mass spectrometry (HPLC-DAD-ESI/MS) analysis. J Agric Food Chem 2010;58:2571–2576.
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13. Seomun Y, Kim JT, Kim HS, Park JY, Joo CK. Induction of p21Cip1-mediated G2/M arrest in H2O2-treated lens epithelial cells. Mol Vis 2005;11:764–774. 14. Choi JA, Oh TH, Choi JS, Chang DJ, Joo CK. Impact of b-1,3-glucan Isolated from Euglena gracilis on corneal epithelial cell migration and on wound healing in a rat alkali burn model. Curr Eye Res 2013;38:1207–1213. 15. Li WC, Spector A. Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract. Free Radic Biol Med 1996;20:301–311. 16. Li WC, Kuszak JR, Wang GM, Wu ZQ, Spector A. Calcimycin-induced lens epithelial cell apoptosis contributes to cataract formation. Exp Eye Res 1995;61:91–98. 17. Al-Awwadi NA, Araiz C, Bornet A, Delbosc S, Cristol JP, Linck N, et al. Extracts enriched in different polyphenolic families normalize increased cardiac NADPH oxidase expression while having differential effects on insulin resistance, hypertension, and cardiac hypertrophy in high-fructose-fed rats. J Agric Food Chem 2005;53: 151–157. 18. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 1993;90:7915–7922. 19. Cohen-Boulakia F, Valensi PE, Boulahdour H, Lestrade R, Dufour-Lamartinie JF, Hort-Legrand C, et al. In vivo sequential study of skeletal muscle capillary permeability in diabetic rats: effect of anthocyanosides. Metabolism 2000;49:880–885. 20. Duthie GG, Duthie SJ, Kyle JA. Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr Res Rev 2000;13:79–106. 21. Galvano F, La Fauci L, Lazzarino G, Fogliano V, Ritieni A, Ciappellano S, et al. Cyanidins: metabolism and biological properties. J Nutr Biochem 2004;15:2–11. 22. Hollman PC, Katan MB. Dietary flavonoids: intake, health effects and bioavailability. Food Chem Toxicol 1999;37: 937–942. 23. Jayaprakasam B, Vareed SK, Olson LK, Nair MG. Insulin secretion by bioactive anthocyanins and anthocyanidins present in fruits. J Agric Food Chem 2005;53:28–31. 24. Lee J, Lee HK, Kim CY, Hong YJ, Choe CM, You TW, et al. Purified high-dose anthocyanoside oligomer administration improves nocturnal vision and clinical symptoms in myopia subjects. Br J Nutr 2005;93:895–899. 25. Levy Y, Glovinsky Y. The effect of anthocyanosides on night vision. Eye (Lond) 1998;12:967–969. 26. Morimitsu Y, Sugihara N, Furuno K. Inhibitory effect of flavonoids on sulfo- and glucurono-conjugation of acetaminophen in rat cultured hepatocytes and liver subcellular preparations. Biol Pharm Bull 2004;27:714–717. 27. Zhang B, Kang M, Xie Q, Xu B, Sun C, Chen K, et al. Anthocyanins from Chinese bayberry extract protect beta cells from oxidative stress-mediated injury via HO-1 upregulation. J Agric Food Chem 2011;59:537–545. 28. Speciale A, Canali R, Chirafisi J, Saija A, Virgili F, Cimino F. Cyanidin-3-O-glucoside protection against TNF-alphainduced endothelial dysfunction: involvement of nuclear factor-kappaB signaling. J Agric Food Chem 2010;58: 12048–12054. 29. Guo H, Ling W, Wang Q, Liu C, Hu Y, Xia M. Cyanidin 3-glucoside protects 3T3-L1 adipocytes against H2O2- or TNF-alpha-induced insulin resistance by inhibiting c-Jun NH2-terminal kinase activation. Biochem Pharmacol 2008; 75:1393–1401.
