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Blue Light–Induced Oxidative Stress in Human Corneal Epithelial Cells: Protective Effects of Ethanol Extracts of Various Medicinal Plant Mixtures

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Blue Light–Induced Oxidative Stress in Human Corneal Epithelial Cells: Protective Effects of Ethanol Extracts of Various Medicinal Plant Mixtures Cornea Blue Light–Induced Oxidative Stress in Human Corneal Epithelial Cells: Protective Effects of Ethanol Extracts of Various Medicinal Plant Mixtures Jee-Bum Lee,1 Soo-Hyun Kim,1 Seung-Chul Lee,1 Hee-Gu Kim,2 Ho-Geun Ahn,2 Zhengri Li,3 and Kyung Chul Yoon3 1Department of Dermatology, Chonnam National University Medical School and Hospital, Gwangju, Korea 2Department of Chemical Engineering, Sunchon National University, Chonnam, Korea 3Department of Ophthalmology, Chonnam National University Medical School and Hospital and Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, Korea Correspondence: Kyung Chul Yoon, PURPOSE. To investigate the effects of visible light on human corneal epithelial cells and the Department of Ophthalmology, impact of natural antioxidants on oxidative stress produced by overexposure to light. Chonnam National University Medi- calSchoolandHospital,42Jebong- METHODS. Light-emitting diodes with various wavelengths (410–830 nm) were used to irradiate ro, Dong-gu, Gwangju 501-757, human corneal epithelial cells, and cell viability was assessed. The production of reactive oxygen South Korea; species (ROS) was analyzed using 20,70-dichlorodihydrofluorescein diacetate (DCF-DA). Ethyl [email protected]. alcohol (EtOH) extracts were prepared from mixtures of medicinal plants. After application of Submitted: October 15, 2013 the EtOH extracts, the free radical scavenging activity was measured using a 2,2-diphenyl-1- Accepted: June 2, 2014 picrylhydrazyl (DPPH) radical scavenging assay. The induction of antioxidant enzymes including heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), catalase (CAT), and superoxide dismutase-2 Citation: Lee J-B, Kim S-H, Lee S-C, et al. Blue light–induced oxidative stress (SOD-2) by the extracts was evaluated by reverse transcription-polymerase chain reaction and in human corneal epithelial cells: Western blotting. The ability of the extracts to inhibit ROS was also analyzed using DCF-DA. protective effects of ethanol extracts RESULTS. The viability of corneal epithelial cells was diminished after irradiation of blue light of various medicinal plant mixtures. (above 10 J at 410 nm and 50 J at 480 nm). Reactive oxygen species production was induced Invest Ophthalmol Vis Sci. by irradiation at 410 and 480 nm at doses of 5 J/cm2 and higher. Ethyl alcohol extracts had 2014;55:4119–4127. DOI:10.1167/ iovs.13-13441 potent radical scavenging activity. Application of the extracts not only increased the expression of HO-1, Prx-1, CAT, and SOD-2, but it also attenuated the ROS production induced by blue light in a dose-dependent manner. CONCLUSIONS. Overexposure to blue light (410–480 nm) may have a harmful effect on human corneal epithelial cells compared with other visible light wavelengths. Medicinal plant extracts can have potent protective effects on blue light–induced oxidative stress. Keywords: corneal epithelial cells, light emitting diode, visible light, reactive oxygen species, oxidative stress, medicinal plants ight-emitting diodes (LEDs) are complex semiconductors induced by UV-B light has been shown to upregulate the L that convert electrical current into incoherent narrow- expression of proinflammatory cytokines, growth factors, and spectrum light. These diodes are available at wavelengths enzymes mediating prostaglandin and leukotriene biosynthesis, ranging from UV to visible to near infrared bandwidths (247– as well as antioxidant enzymes in corneal epithelial cells, 1300 nm).1 The visible spectrum is the portion of the suggesting that UV-B light can induce inflammation and tissue electromagnetic spectrum that is visible to the human eye. A damage in the ocular surface epithelium.10–16 typical human eye responds to wavelengths ranging from Many colors can be reproduced by current technologies, approximately 400 to 700 nm.2 including smartphones, computers, and televisions. The Oxidative stress leads to damage to lipids and DNA and the spectrum of visible light as well as UV light may have an inhibition and deactivation of proteins with a consequent impact on normal naked eyes. However, although UV light has disruption of overall biological function.3 Ocular UV radiation- been demonstrated to produce harmful effects on eyes, it is induced DNA and protein damage has been associated with a uncertain whether a certain spectrum of visible light can also number of eye pathologies, including photokeratitis, pterygium, produce harmful effects on human eyes. We hypothesized that eyelid malignancies (basal cell carcinoma and squamous cell blue light with short wavelengths may also induce oxidative carcinoma), cataract, and corneal and retinal degeneration.4–9 It stress on the corneal epithelium. It is thought that medicinal is well known that human corneal epithelial cells are responsive plants may protect the eyes against oxidative stress induced by to UV light. Ultraviolet-induced corneal injury can result from UV or visual light. In this study, ethyl alcohol (EtOH) extracts oxidative stress by the localized generation of reactive oxygen were prepared from a mixture of various medicinal plants species (ROS), including superoxide anion, hydrogen peroxide, (XERO-M; BM Biotechnology, Sunchon, South Korea) that have and hydroxyl radical by corneal epithelial cells. Oxidative stress antioxidant and anti-inflammatory effects. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 4119 Downloaded from iovs.arvojournals.org on 09/29/2021 Blue Light-Induced Oxidative Stress in the Cornea IOVS j July 2014 j Vol. 55 j No. 7 j 4120 TABLE 1. Irradiance of LED Lamps With Various Wavelengths cytometer (FACSCalibur; BD Biosciences, Fullerton, CA, USA), with an excitation wavelength of 488 nm and emission 2 LED Wavelength, nm Irradiance, mW/cm wavelength of 530 nm. Data analysis was based on 10,000 850 6 3 7.28 detected events using flow and image cytometry analysis 630 6 8 17.34 software (Cell Quest; BD Biosciences). Alternatively, changes in 595 6 2 6.66 the fluorescence at 488/538 nm in the cells were quantified by 580 6 4 8.4 a plate reader (SPECTRAmax Gemini; Molecular Devices). 525 6 2 21.68 To analyze the intracellular ROS production, after reacting with DCF-DA, the cell suspension was secured on a micro- 480 6 7 25.6 0 0 410 6 10 10.75 scope slide and counter-stained with 4 ,6 -diamidino-2-phenyl- indole (DAPI). Images were obtained using confocal microscopy with a laser scanning microscope (LMS 510; Carl In the present study, we aimed to investigate which Zeiss, Jena, Germany) and analyzed using the browser imaging wavelengths of visible light from LEDs influenced human software (LSM 5; Carl Zeiss). corneal epithelial cells by measuring cell viability and ROS production, and we also investigated whether EtOH extracts of Ethanol Extracts of Mixed Medicinal Plants various medicinal plants could reverse oxidative stress induced Mixed medicinal plants (BM Biotechnology), which consist of by visible light in human corneal epithelial cells in vitro. Schizonepeta tenuifolia var. japonica Kitagawa, Angelica dahurica Bentham ET hooker, Rehmannia glutinosa Libo- schitz var. purpurea Makino, and Cassia tora L., were used in ATERIALS AND ETHODS M M this experiment. All plant samples were air-dried and ground LED Light Source into powder using a plant milling machine. A 100 g sample of the powder was extracted with 1000 mL of 95% aqueous EtOH Seven LED lamps with various wavelengths, including 410, under continuous shaking at room temperature for 12 hours 480, 525, 580, 595, 630, and 850 nm, were used (Table 1). The and filtered through Whatman No. 4 qualitative filter paper. irradiance of each LED was measured using a quantum photo- The extraction was repeated three times to ensure complete radiometer (Delta OHM, Padova, Italy) connected to a visual extraction of the plant material. The extracts were concentrat- probe (Sonda LP 9021 RAD; Delta OHM). The distance from ed by evaporation under reduced pressure in a rotary the LED module to the cells was 5 cm. evaporator at 408C and freeze-dried. Culture of Human Corneal Epithelial Cells and Cell 2,2-Diphenyl-1-Picrylhydrazyl Radical Scavenging Viability Assay Epithelial adenovirus 12-SV40 hybrid transformed human The free radical-scavenging activity of the mixed plant extracts corneal epithelial cells (HCE-2) were received at passage 28 was evaluated using a modification of a previously published from American Type Culture Collection (Cat No. CRL-11135, method.18 Aliquots of sample extracts at various concentra- Manassas, VA, USA) and were maintained in liquid tissue tions were each mixed with ethanol and then with 100 lLof culture medium (EpiLife; Cascade Biologics, Inc., Portland, 2,2-diphenyl-1-picrylhydrazyl (DPPH) in EtOH to a final OR, USA) supplemented with human corneal growth supple- concentration of 100 lM. The mixture was vigorously shaken ment (Cascade Biologics, Inc.), 100 U/mL penicillin, and 100 and left to stand at room temperature for 30 minutes in the lg/mL streptomycin at 378C in a humidified incubator with dark. The absorbance of the reaction solution was spectro- 5% CO2. photometrically measured at 517 nm with an ELISA reader Cell viability was measured using a colorimetric assay (3- (VERSAMAX; Molecular Devices). The percentages of DPPH (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sig- decolorization of the samples were calculated according to the ma-Aldrich, St. Louis, MO, USA) according to the manufactur- equation: % decolorization ¼ [1 – (ABSsample/ABScontrol)] 3 100. 17 5 er’s instructions. Human corneal epithelial cells (2 3 10 The IC50 value was the effective concentration at which 50% of cells/well) were seeded in 35-mm tissue culture dishes and the DPPH radicals were scavenged, and butylated hydroxyto- were irradiated with the LEDs. The cells were maintained in luene (BHT) was used as a positive control. All tests were serum-free conditions for 24 hours. Two hundred microliters of performed in triplicate. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT: 5 mg/mL) was added to each dish.
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