Food & Function View Article Online PAPER View Journal | View Issue Cyanidin-3-glucoside attenuates 4-hydroxynonenal- and visible light-induced Cite this: Food Funct., 2019, 10, 2871 retinal damage in vitro and in vivo Yong Wang, *†a Wentao Qi,†a Yazhen Huo,b Ge Song,a Hui Sun,a Xiaoxuan Guoc and Chengtao Wang*d 4-Hydroxynonenal (HNE) is a highly reactive end-product of lipid peroxidation reaction that leads to retinal pigment epithelial (RPE) cell damage. Cyanidin-3-glucoside (C3G), the most abundant anthocyanin in the edible parts of plants, is a nutritional supplement used for preventing retinal damage. However, the protective effect of C3G against HNE-induced RPE cell damage remains to be elucidated. The protective mechanisms of C3G on ARPE-19 cells after HNE exposure were investigated in this study. Results showed that compared with HNE-treated cells, the viability of ARPE-19 cells was significantly (P < 0.05) increased after 1 and 5 μM C3G treatment. C3G exhibited a significant (P < 0.05) inhibitory effect on the expression of senescence-associated β-galactosidase in ARPE-19 cells. VEGF levels in the C3G groups were signifi- cantly (P < 0.05) decreased relative to those of the HNE-treated group. C3G also regulated the release of Received 10th February 2019, two inflammatory mediators, namely monocyte chemoattractant protein 1 and interleukine-8, in ARPE-19 Accepted 16th April 2019 cells after HNE treatment. Furthermore, C3G attenuated retinal cell apoptosis in pigmented rabbits DOI: 10.1039/c9fo00273a induced by visible light. Therefore, our data showed that C3G has efficient protective effects on HNE- rsc.li/food-function induced apoptosis, angiogenesis, and dysregulated cytokine production in ARPE-19 cells. Introduction light, and phagocytosis of the shed photoreceptor outer seg- ments.4 Senescence and inflammation disturb the function of The retina is particularly vulnerable to photooxidative damage RPE, eventually leading to retinal degeneration.5 Dysfunction caused by lipid peroxidation because it has a high polyun- or progressive degeneration of RPE plays a central role in the saturated fatty acid (PUFA) content, high oxygen tension, and pathogenesis of age-related macular degeneration (AMD), high levels of light exposure.1 Lipid peroxidation products, which is one of the major causes of blindness in the elderly such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), people worldwide.6 can attack proteins, DNA, and phospholipids, resulting in the HNE is the major reactive aldehyde derived from the none- dysfunction of retinal pigment epithelium (RPE) and photo- nzymatic oxidation of n-6 PUFA, such as linoleic acid and ara- receptor cell damage.2,3 RPE is a single-cell layer in the eye chidonic acid.7 Proteomic studies showed the presence of oxi- located between the photoreceptors and vascular choroid. It dative modifications of proteins with lipid peroxidation pro- plays critical roles in the maintenance of the underlying photo- ducts namely, HNE and MDA, in RPE cells from human donor receptors by transporting nutrients from the vascular choroid, eyes.8 HNE is a highly reactive end-product of lipid peroxi- forming the blood-retinal barrier, absorbing the scattered dation reaction that can reduce lysosomal degradation and eventually induce RPE permeability and cell death.9 Anthocyanins (ACNs) are beneficial for vision because they a Academy of State Administration of Grain, Beijing 100037, stimulate regeneration of rhodopsin, inhibit lens opacities, People’s Republic of China. E-mail: [email protected] improve night vision, and inhibit photooxidative-induced bState Key Laboratory of Biomacromolecules, Institute of Biophysics, 10–12 Chinese Academy of Sciences, Beijing 100101, People’s Republic of China apoptosis in RPE cells. Dietary ACNs are widely regarded cInstitute of Quality Standard and Testing Technology for Agro-products, to possess protective effects on light-induced retinal degener- Chinese Academy of Agricultural Sciences, Beijing 100081, ation. Bilberry ACNs can effectively protect against blue light- ’ People s Republic of China induced N-retinylidene-N-retinylethanolamine (A2E) photo- dBeijing Engineering and Technology Research Center of Food Additives, oxidation and membrane permeabilization in RPE cells.11 The Beijing Technology & Business University (BTBU), Beijing 100048, People’s Republic of China. E-mail: [email protected] protective function of bilberry ACNs may be caused by increas- †These authors contributed equally to this work. ing the antioxidant defense mechanisms, suppressing proin- This journal is © The Royal Society of Chemistry 2019 Food Funct.,2019,10,2871–2880 | 2871 View Article Online Paper Food & Function flammatory cytokines and inhibiting retinal cell apoptosis and the protective effect of C3G pretreatment. After the in vivo.13 Cyanidin-3-glucoside (C3G) is the most abundant designed treatments, 10 μL of CCK-8 solution was added into ACN found in blackberry and purple rice with great beneficial 96-well plates and OD 450 nm was measured by using a 2300 potential for preventing retinal degeneration.14,15 EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, The direct oxidant H2O2 had been used in most previous USA). The LDH activity was determined by using a commercial studies that induced RPE cell oxidative damage models.16,17 kit according to the manufacturer’s instructions. We preferred HNE because RPE cells are susceptible to the type of photooxidative damage induced by lipid peroxidation.18 Cell apoptosis measurement In our previous studies, the HNE levels were significantly ARPE-19 cell apoptosis was detected by using the Annexin- increased in bisretinoid-burdened rod outer segments after V-PI/FITC apoptosis detection kit according to the manufac- blue light irradiation in vitro and in the retinal cells after turer’s instructions. In brief, after the indicated treatments, visible light exposure in vivo.14,19 Therefore, the present work the cells were washed, harvested, and resuspended in 100 μL investigated the protective effects of C3G against HNE-induced Annexin binding buffer. The cells were stained with 5 μL damage in ARPE-19 cells and visible light-induced retinal Annexin V-FITC and 5 μL propidium iodide solution, and then damage in vivo. the reaction system was incubated at room temperature in the dark. Flow cytometry analysis was performed by using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, Materials and methods NJ, USA). Chemicals and reagents Senescence-associated β-galactosidase activity C3G was obtained from Nanjing Jingzhu Bio-technology, Ltd Senescence was assayed by using the senescence-associated (Nanjing, Jiangsu, China). HNE was obtained from BioVision β-galactosidase staining kit according to our previous study.15 ’ Research Products (Mountain View, CA, USA). Dulbecco s The treated ARPE-19 cells were washed twice with PBS and ’ ’ modified Eagle s/Ham s F12 medium (DMEM/F12) was then fixed with 2% formaldehyde and 0.2% glutaraldehyde in obtained from Invitrogen (Carlsbad, CA, USA). Fetal bovine PBS at room temperature for 4 min. Afterward, the cells were serum (FBS) was obtained from Biological Industries Co. washed with PBS and incubated in the dark for 8 h at 37 °C (BioInd, Israel). Penicillin and streptomycin were obtained with the fresh β-galactosidase staining solution and photo- from Gibco Life Technologies (Grand Island, NY, USA). Cell graphed under a light microscope. The senescent cell percen- Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular tage was calculated as the number of blue-stained cells divided Technologies, Inc. (Kumamoto, Japan). Lactate dehydrogenase by the number of total cells. (LDH) kit and senescence-associated β-galactosidase staining kit were obtained from Beyotime (Shanghai, China). Annexin Enzyme-linked immunosorbent assay (ELISA) V-FITC apoptosis detection kit was obtained from BD After C3G and HNE treatments, the cell culture supernatants Biosciences Pharmingen (San Diego, CA, USA). Deionized were collected and subsequently centrifuged at 1000g for water was produced by using a Milli-Q water-purification 5 min. The expression levels of monocyte chemoattractant system (Millipore, Billerica, MA, USA). All other chemicals and protein 1 (MCP-1), interleukine-8 (IL-8), and vascular endo- reagents were obtained from Sigma-Aldrich (St Louis, MO, thelial growth factor (VEGF) in the supernatants were USA). measured via the corresponding commercial ELISA kits (CUSABIO, Wuhan, Hubei, China), following the manufac- Cell culture and treatment turer’s instructions. The absorbance was measured at 450 nm The ARPE-19 cells were obtained from the American Type by using a 2300 EnSpire Multimode Plate Reader. Culture Collection (Manassas, VA, USA) and cultured according to our previous studies.14,15 They were cultured in DMEM/F12 Real time quantitative polymerase chain reaction (RT-qPCR) with 10% FBS in a humidified incubator at 37 °C in 5% CO2, Total RNA was extracted with Trizol (Tiangen Biotech, Beijing, −1 −1 supplemented with 100 U mL penicillin and 100 μgmL China). Reverse transcription was carried out by using the streptomycin. The cells were seeded in 6-well or 96-well plates High Capacity cDNA Reverse Transcription Kit (Applied and cultivated with serum-containing DMEM/F12 until conflu- Biosystems, Foster City, CA, USA). Real-time PCR was per- ence. Then, they were washed once with the serum-free formed with the QuantStudio 3D Digital PCR (Thermo Fisher medium before C3G and HNE were added in the