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j.jff.2018.08.007

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Journal of Functional Foods 49 (2018) 12–19
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff
Protective effect of chitosan oligosaccharides on blue light light-emitting
diode induced retinal pigment epithelial cell damage
Chao-Wen Lina, Hsiao-Han Huangb, Chung-May Yanga, Chang-Hao Yanga,
a
b
T
⁎
Department of Ophthalmology, National Taiwan University Hospital, No. 7, Zhongshan South Road, Taipei 100, Taiwan
School of Medicine, Taipei Medical University, No. 250 Wuxing Street, Taipei 110, Taiwan
A R T I C LE I N FO
A B S T R A C T
Keywords:
Chitosan oligosaccharides
Blue light
Light-emitting diode (LED)
Photo-injury
Retinal pigment epithelial cell
NF-κB
Excessive blue light light-emitting diode (LED) exposure and consequent oxidative stress causes damage to
retinal pigment epithelial (RPE) cells. Chitosan oligosaccharides (COSs) are the hydrolyzed products of chitin,
which is abundant in the exoskeleton of crustaceans and cell walls of fungi. We investigated the protective effect
of COSs on blue light LED-induced RPE cell damage. These cells were treated with various concentrations of
COSs and then exposed to blue light LED. Our results confirmed that RPE cell apoptosis increased significantly
with longer light exposure. Treatment with COSs significantly reduced apoptosis in a dose-dependent manner.
These molecules also suppressed the production of reactive oxygen species and the expression of inflammationand apoptosis-related proteins. Moreover, COSs stabilized the mitochondrial membrane potential of cells and
down-regulated the NF-κB pathway. In this study, the protective effects of COSs on blue light LED-induced RPE
cell damage, as well as the associated mechanisms, were demonstrated.
1. Introduction
Artificial lighting is a common element of modern society; however,
the potential health risks caused by light pollution have increased with
the development of more sophisticated lighting technology (Chepesiuk,
2009). Among the wide variety of artificial lighting selections, lightemitting diodes (LEDs) emit higher levels of blue light than conventional light sources. These have provided humans with their first exposure to such extensive blue light (Behar-Cohen et al., 2011). Blue
light LED and white light LED are now widely used in illumination
light, flashlights, digital display, and the screens of computers, smartphones, or other consumer electronics. In the eye, wavelengths from the
spectral band of 400–1400 nm can reach the retina (Boettner & Wolter,
1962). The retina contains specific chromophores in the photoreceptor
that function to absorb visible radiation. The main sites of light energy
absorption are the melanin pigment granules in retinal pigment epithelial (RPE) cells, which can be injured by light exposure (Parver,
Auker, & Fine, 1983; Tso, 1973).
It is generally accepted that lights with shorter wavelengths are
significantly more hazardous than those with longer wavelengths; as
such, blue light-induced RPE cell damage has been reported (Boulton,
Rozanowska, & Rozanowski, 2001; Chamorro et al., 2013; Dorey,
Delori, & Akeo, 1990; Godley et al., 2005; Ham, Mueller, & Sliney,
1976). Epidemiological studies have suggested that short-wavelength
light exposure is a predisposing factor for age-related macular degeneration (AMD) (Wu, Seregard, & Algvere, 2006). Previous studies have
also demonstrated that exposure to blue and white light can induce
damage to photoreceptors and RPE cells of the rat retina (Hafezi, Marti,
Munz, & Reme, 1997; Shang, Wang, Sliney, Yang, & Lee, 2014). We also
previously found that the mechanism of retinal injury is related to
oxidative stress within the retina (Shang, Wang, Sliney, Yang, & Lee,
2017), which can induce the generation of reactive oxygen species
(ROS) and inflammatory reactions (Hollyfield et al., 2008; Zhou, Jang,
Kim, & Sparrow, 2006). Since RPE cells are the major ocular source of
pro-inflammatory mediators and the primary target of photo-oxidative
effects, oxidative damage to these cells might contribute to ocular inflammation and AMD-related lesions.
Nutritional supplements are widely taken by the general population
and several of these products are marketed specifically for the improvement of eye health. The Age-Related Eye Disease Study (AREDS), a
randomized clinical trial, showed a significantly lower incidence of late
AMD in a cohort of patients with drusen maculopathy treated with high
Abbreviations: LED, light-emitting diode; RPE, retinal pigment epithelia; COS, chitosan oligosaccharides; AMD, age-related macular degeneration; ROS, reactive
oxygen species; AREDS, Age-Related Eye Disease Study; PI, propidium iodide; DCFH, dichlorodihydrofluorescein; HO-1, heme oxygenase-1; iNOS, inducible nitric
oxide synthase; Bcl-2, B-cell lymphoma 2; HRP, horseradish peroxidase; MCP-1, monocyte chemoattractant protein-1
⁎
Corresponding author.
E-mail address: chyangoph@ntu.edu.tw (C.-H. Yang).
https://doi.org/10.1016/j.jff.2018.08.007
Received 25 July 2018; Received in revised form 31 July 2018; Accepted 6 August 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 49 (2018) 12–19
C.-W. Lin et al.
V and propidium iodide (PI) staining. The staining solution contained
5 μL Annexin-V-FITC in 250 μL binding buffer and 5 μL PI (Strong
Biotech, Taiwan). The ARPE-19 cells were washed with PBS and centrifuged at 200g for 5 min. Then, the cell pellet was resuspended in
100 μL of staining solution and incubated for 10 min at 20 °C. Finally,
the sample was added to 900 μL binding buffer and analyzed using a
FACScan cytometer (BD Bioscience, NJ, USA).
doses of antioxidants, compared to that in a placebo group (Chiu &
Taylor, 2007; Robman et al., 2007). In addition, sulforaphane, a compound found in cruciferous vegetables, has been shown to protect RPE
cells from photo-oxidative damage (Gao & Talalay, 2004). Polyphenols
extracted from bilberry and lingonberry also protect retinal photoreceptor cells from blue light LED-induced damage (Ogawa et al., 2014).
Chitosan oligosaccharides (COSs), the hydrolyzed products of chitin,
consist of a mixture of oligomers of β-1,4-linked D-glucosamine residues.
Chitin is abundant in the exoskeleton of crustaceans and the cell walls of
fungi and insects (Pae et al., 2001). COSs are known to exert various
biological effects including anti-tumor, anti-bacterial, anti-inflammation,
anti-oxidative, and anti-apoptotic activities (Chen, Liu, Du, Peng, & Sun,
2006; Joodi, Ansari, & Khodagholi, 2011; Pangestuti & Kim, 2010; Qin,
Du, Xiao, Li, & Gao, 2002). Importantly, COSs are non-toxic and biodegradable and have thus been used as bioactive material. In addition,
they have good solubility in water and are easily absorbed in the intestine, which makes them an attractive ingredient in many healthy
foods or dietary supplements. We have previously shown that COSs exert
their anti-oxidative effects by inhibiting NF-κB activation and by attenuating retinal oxidative stress-related retinal degeneration in rats
(Fang, Yang, Yang, & Chen, 2013). Recently, we also demonstrated the
protective effect of COSs on retinal ischemia and reperfusion injury,
which was shown to occur via the inhibition of oxidative stress and inflammatory mediators (Fang, Yang, & Yang, 2015).
The increasing popularity of the consumer electronics brings convenience to our lives; however, most of these products use LED as their
light source and thus emit blue light. Since COSs have anti-apoptotic
and anti-oxidative effects, we investigated the protective effects of these
molecules on blue light LED-induced RPE cell damage and evaluated
the potential of COSs to be used as a nutrient supplement for the prevention of blue light-mediated damage to the human eye.
2.4. Cell viability assay
Cell viability was determined using the alamar blue assay (Thermo
Fisher Scientific, MA, USA). ARPE-19 cells were cultured in 96-well
plates, and then incubated for 24 h in a humidified atmosphere of 5%
CO2 at 37 °C. We added 500 μL of alamar blue to each well. Cells were
treated with COSs at various concentrations (0, 50, 100, 200 μM) for
1 h. Then, the cells in the exposure group were exposed to blue light
LED for 24 h. The absorbance was measured at a wavelength of 570 nm
and 600 nm using microplate reader (Bio-Rad Laboratories Inc., CA,
USA).
2.5. Detection of intracellular ROS
Intracellular ROS levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (2′,7′-DCFDA, Sigma-Aldrich, St. Louis, MO, USA)
oxidation. ARPE-19 cells were exposed to 2500 lx of blue light LED for
48 h after being treated with different concentrations of COSs (0, 25,
100 μM) for 1 h. Then, 10 mM of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), a free
radical probe, was added to the cell and incubation was continued for 1 h
at 37 °C. The radical probe was converted to 2′,7′-dichlorodihydrofluorescein (DCFH) by the action of intracellular esterase. Intracellular
DCFH (non-fluorescent) was oxidized to 2′,7′-dichlorfluorescein (DCF,
fluorescent) by intracellular ROS. Fluorescence was measured using a
microplate reader (Bio-Rad Laboratories Inc., CA, USA) at 485 nm (excitation) and 535 nm (emission).
2. Material and methods
2.1. Human ARPE-19 cell culture
ARPE-19 cells were purchased from the American Type Culture
Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) with 10%
fetal bovine serum, 4.5 mg/mL glucose, 100 units/mL penicillin, and
100 μg/mL streptomycin in a humidified incubator at 37 °C in an atmosphere of 5% CO2. ARPE-19 cells were seeded at a density of 1 × 104
cells per well in plates. These cells were passaged by trypsinization
every 3–4 d. Cells were used at the third to fifth passages.
2.6. Determination of mitochondrial dysfunction
The mitochondrial membrane potential was measured using the JC1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical
Company, Ann Arbor, MI). Different concentrations of COSs (0, 50, 100,
200 μM) were added to the cells exposed to blue light LED. After 24 h of
2500-lux blue LED light exposure, 50 μL of JC-1 staining buffer was
added to 1 mL of culture medium. JC-1 formed J-aggregates in healthy
cells and remained in its monomeric form in apoptotic or unhealthy
cells. J-aggregates and JC-1 monomers were detected with settings
designed to detect Texas Red and FITC, respectively. The images were
captured using a fluorescence microscope, which was used to detect
healthy cells with mainly JC-1 J-aggregates (excitation/emission, 540/
605 nm) and apoptotic or unhealthy cells with mainly JC-1 monomers
(excitation/emission, 480/510 nm). The number of cells (red or green
stained cells) were counted in a blind manner using image-processing
software (Image-J).
2.2. Blue light LED and COSs treatment of RPE cells
ARPE-19 cells were exposed to blue light LED with an intensity of
2500 lx; cells were cultured for 24–48 h after exposure. A medical blue
light LED tube (450 ± 20 nm, 20 W) was placed on a special framework, and the illuminometer was used to measure light intensity at the
cell surface. The distance between the tube and the cell surface was
adjusted according to light intensity. To ensure a stable growth environment for RPE cells, the blue light device was placed inside the CO2
incubator. Temperature was maintained between 36.5 and 37.5 °C.
COSs were purchased from Sigma-Aldrich (catalog number 523682, St.
Louis, MO, USA). The average molecular weight of COSs was 5000 and
the degree of N-deacetylation was more than 90%. Cells were treated
with COSs at various concentrations (ranging from 50 to 200 μM) and
then exposed to blue light LED. COSs remained in the medium during
LED exposure.
2.7. Western blot analysis
After treatment with different concentrations of COSs (0, 50, 100,
200 μM), ARPE-19 cells were incubated for 1 h. The cells were exposed
to 2500 lx of blue light LED for 24 h. Then, the cells were washed with
PBS, lysed in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor
cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA), and harvested.
Lysates were centrifuged at 12,000g for 15 min at 4 °C. Protein concentrations were measured using a BCA Protein Assay Kit with bovine
serum albumin as the standard. Thereafter, an equal volume of protein
sample and sample buffer were mixed, and the samples were boiled for
2.3. Analysis of apoptosis by flow cytometry
The proportion of ARPE-19 cells undergoing apoptosis was determined at 24 or 48 h after exposure by flow cytometry using Annexin13
Journal of Functional Foods 49 (2018) 12–19
C.-W. Lin et al.
2.10. Statistical analysis
5 min at 100 °C. The protein samples were separated by 10% SDS-PAGE
gradient electrophoresis and then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp. Billerica, MA, USA).
For immunoblotting, the following primary antibodies were used:
rabbit anti-heme oxygenase-1 (HO-1) (1:2500, Abcam, Hong Kong,
China), rabbit anti-inducible nitric oxide synthase (iNOS) (1:400,
Abcam, Hong Kong, China), rabbit anti-cleaved caspase-3 (1:1000, Cell
Signaling Technology, MA, USA), rabbit anti-cleaved caspase-9
(1:2000, Abcam, Hong Kong, China), rabbit anti-B-cell lymphoma 2
(Bcl-2) (1:1000, Cell Signaling Technology, MA, USA), and mouse antiβ-actin mouse monoclonal antibodies (1:5000, Sigma-Aldrich, St. Louis,
MO, USA). A horseradish peroxidase (HRP)-conjugated goat anti-rabbit
antibody and an HRP-conjugated goat anti-mouse antibody were used
as secondary antibodies. Immunodetection was performed by enhanced
chemiluminescence (Pierce Biotechnology Waltham, MA, USA). Protein
levels were determined using densitometry analysis of the protein
bands. The optical densities were calculated and standardized based on
the density of the β-actin band.
The values are shown as mean ± SD. Statistical analysis for multiple comparisons in each study was determined by performing an
analysis of variance (one-way ANOVA) followed by post hoc Least
Significant Difference (LSD) test. The Student’s t-test was used for
comparisons of the means of two independent groups. A P value of 0.05
or less was considered significant. Statistical analysis was performed
using SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA).
3. Results
3.1. Blue light LED exposure significantly increases apoptosis in ARPE-19
cells
We first investigated the damage to ARPE-19 cells with different
durations (24 and 48 h) of blue light LED exposure by flow cytometry.
With an illumination of 2500 lx of blue light LED, a remarkable increase
in apoptosis was noted in ARPE-19 cells. Moreover, the percentage of
apoptotic cells increased significantly with longer light exposure
(Fig. 1).
2.8. RNA extraction and quantitative reverse transcription polymerase
chain reaction
3.2. COSs prevent blue light LED-induced cell death in ARPE-19 cells
After treatment with different concentrations of COSs (0, 50, 100,
200 μM), ARPE-19 cells were incubated for 1 h. Cells were exposed to
2500 lx blue light LED for 24 h. Total RNA was extracted from cultured
cells using TRIzol reagent (Invitrogen-Life Technologies, Inc.,
Gaithersburg, MD, USA). One microgram of total RNA from each
sample was annealed with 300 ng oligo(dT) (Promega, Madison, WI,
USA) for 5 min at 65 °C and reverse transcribed into cDNA using
Moloney murine leukemia virus reverse transcriptase (80 U/50 μg) for
1 h at 37 °C, which was followed by incubation at 90 °C for 5 min to
terminate the reaction. The resultant cDNA product from each sample
was subjected to PCR with specific primers. The primer sequences used
for PCR were as follows: iNOS: 5-TATCTGCAGACACATACTTTACGC-3
(forward); 5-TCCTGGAACCACTCGTACTTG-3 (reverse); monocyte chemoattractant protein-1 (MCP-1): 5-GCTCATAGCAGCCACCTTCATTC-3
(forward) and 5-GTCTTCGGAGTTTGGGTTTGC-3 (reverse). The 50 μL
reaction mixture contained 5 μL cDNA, 1 μL sense and antisense primers, 200 μM of each deoxynucleotide, 5 μL of 10× Taq polymerase
buffer, and 1.25 U Taq polymerase (Promega Madison, WI, USA). A
thermocycler (MJ Research, Waltham, MA, USA) was used for amplification. Amplification was performed as follows: 1 min denaturation at
94 °C and 3 min extension at 72 °C. The annealing temperature was
between 42 and 62 °C, and the temperature was decreased by 1 °C increments, which was followed by 21 cycles at 55 °C. Finally, the temperature was elevated to 72 °C for 10 min and then reduced to 4 °C. A
10-μL sample of each PCR product was obtained to perform electrophoresis using a 2% agarose gel containing ethidium bromide (SigmaAldrich, St. Louis, MO, USA). DNA molecular weight markers were used
to analyze the results under ultra-violet light. The intensity was determined using image analysis software (Photoshop, version 7.0; Adobe
Systems, San Jose, CA, USA), and the results were standardized based
on the intensity of β-actin.
Without light treatment, cell viability did not change significantly
after the addition of COSs. (Fig. 2a), demonstrating that COSs are not
toxic to ARPE-19 cells. After exposure of blue light LED, cell viability
was significantly higher with COSs treatment, as compared to that in
untreated cells (Fig. 2b). Moreover, COSs treatment reduced apoptosis in
a dose-dependent manner, and had a protective effect on blue light LEDinduced ARPE-19 cell death; this effect was concentration-dependent.
3.3. COSs inhibit ROS production in ARPE-19 cells
To detect the anti-oxidative effects of COSs on ARPE-19 cells, an
ROS assay was used to assess ROS production. ROS production increased considerably after exposure to blue light LED. However,
treating cells with COSs before exposure led to a significant decline in
ROS production (Fig. 3). The anti-oxidative effects were similar in both
concentrations (25 and 100 μM).
3.4. COSs suppress blue light LED-induced mitochondrial damage in ARPE19 cells
The extent of mitochondrial dysfunction was analyzed by JC-1
staining. JC-1 forms J-aggregates in healthy cells, but is maintained in
its monomeric form in apoptotic or unhealthy cells. Here, exposure to
blue light LED markedly decreased JC-1 aggregation in ARPE-19 cells.
However, with COSs treatment, mitochondrial damage decreased, with
a higher portion of JC-1 aggregation, after blue light LED exposure
(Fig. 4). Further, the extent of JC-1 aggregation was correlated with
COSs concentration.
2.9. Immunostaining
3.5. COSs reduce the expression of enzymes associated with apoptosis and
the inflammatory response
ARPE-19 cells were seeded onto glass chamber slides and incubated
for 24 h. The cells were then exposed to 2500 lx blue light LED for 24 h
after treatment with different concentrations of COSs (0, 50, 100,
200 μM) for 1 h. Thereafter, cells were fixed with 4% paraformaldehyde
for 15 min, blocked in 3% horse serum for 30 min, and incubated
overnight at 4 °C with primary antibodies (rabbit anti-NF-κB, Abcam,
Hong Kong, China). After washing, the cells were incubated for 1 h with
secondary antibodies. Counter-staining with DAPI was conducted to
verify the location and integrity of the nuclei, and the stained cells were
then washed and observed under a confocal fluorescence microscope.
COSs are known to have a detoxification effect against oxidative
stress. To elucidate the mechanism through which COSs mitigate blue
light LED-induced cell damage, we examined both the protein expression and mRNA profiles that are associated with apoptosis and the inflammatory response. Exposure to blue light LED induced a modest
increase in the protein expression of HO-1, iNOS, cleaved caspase-3,
and cleaved caspase-9. However, COSs treatment tended to suppress the
expression of these proteins (Fig. 5). These reductions also occurred in a
dose-dependent manner. Bcl-2 is known to regulate mitochondrial dynamics and is considered an important anti-apoptotic protein. Although
14
Journal of Functional Foods 49 (2018) 12–19
C.-W. Lin et al.
Fig. 1. Blue light light-emitting diode (LED) induces apoptosis in retinal pigment epithelial cells. Apoptosis in ARPE-19 cells was analyzed by flow cytometry with
different durations of exposure to 2500 lx blue light LED as follows: (a) cells without blue light exposure, (b) cells with 24 h exposure, and (c) cells with 48 h
exposure. The X-axis is Annexin V-FITC staining and the Y-axis is propidium iodide staining. (d) The relative percentage of apoptotic cells compared to the control
group after exposure to different durations of blue light LED. C: control group, no blue light LED exposure.
Fig. 2. Chitosan oligosaccharides (COSs) suppressed blue light light-emitting diode (LED)induced cell death. Cell viability was analyzed
by alamar blue assays. (a) Cells were incubated
with different concentrations of COSs for 24 h.
(b) Cells were exposed to 2500 lx blue light LED
for 24 h after treatment with different concentrations of COSs for 1 h. (C: control group,
no COSs or blue light LED exposure;
All data represent mean ± SD. *p < 0.05,
***
p < 0.001 compared to the blue light LED
alone exposed group; ANOVA with post hoc
Least Significant Difference (LSD) test; N = 5
per group).
15
Journal of Functional Foods 49 (2018) 12–19
C.-W. Lin et al.
Fig.
3. Chitosan
oligosaccharides
(COSs) inhibit the production of reactive oxygen species (ROS). (a) ROS
production was analyzed by flow cytometry after ARPE-19 cells were exposed to 2500 lx blue light light-emitting diode (LED) for 48 h; cells were
pre-treated with different COSs concentrations for 1 h. The X-axis is 2′,7′dichlorfluorescein (DCF) fluorescence
and the Y-axis is cell number. (b) Foldchange in ROS production, as compared to that in the control group (C:
control group, no COSs or blue light
LED exposure).
the interaction between COSs and NF-κB. For this, immunofluorescence
staining was used. When ARPE-19 cells were exposed to blue light LED,
NF-κB p65 translocated from the cytoplasm to the nucleus. However,
COSs treatment was found to decrease this translocation. Moreover,
p65 translocation declined as the concentration of COSs increased
(Fig. 7).
the differences did not reach statistical significance, COSs treatment
tended to enhance the expression of Bcl-2, and this effect was also
concentration dependent (Fig. 5f).
The expression of mRNA encoding pro-inflammatory mediators
iNOS and MCP-1 was significantly elevated after exposure to blue light
LED. However, COSs treatment significantly down-regulated the expression of iNOS and MCP-1, which was also concentration-dependent
(Fig. 6).
4. Discussion
COSs can be prepared through the deacetylation and hydrolysis of
chitins, which are abundant in the exoskeleton of crustaceans and the
cell walls of fungi. These compounds have various biological activities
including anti-inflammatory, anti-tumor, anti-oxidative, anti-microbial,
and anti-apoptotic effects. Chitosans have also been proven to induce a
3.6. COSs suppress the blue light LED-induced nuclear translocation of NFκB
Since COSs were shown to down-regulate the expression of iNOS
and MCP-1, which are NF-κB-dependent genes, we further investigated
Fig. 4. Chitosan oligosaccharides (COSs) protect retinal pigment epithelial cells from mitochondrial damage. (a) ARPE-19 cells were subjected to JC-1 staining,
which was analyzed by florescence microscopy. J-aggregates and JC-1 monomers were detected with settings designed to detect Texas Red and FITC, respectively.
Treatment with COSs facilitated JC-1 aggregation and decreased JC-1 monomers in ARPE-19 cells after exposure to 2500 lx blue light light-emitting diode (LED) for
24 h. The protective effect of COSs was also concentration-dependent. (Ctrl: control group, no COSs or blue light LED exposure). (b) Green/Red-fluorescence (+) cells
ratio (%). The number of cells (red or green stained cells) were counted using image-processing software (Image-J). (All data represent mean ± SD. ***p < 0.001
compared to the blue light LED alone exposed group by ANOVA with post hoc Least Significant Difference (LSD) test; N = 3 in each group). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
16
Journal of Functional Foods 49 (2018) 12–19
C.-W. Lin et al.
Fig. 5. Chitosan oligosaccharides (COSs) down-regulate the protein expression of pro-apoptotic and pro-inflammatory markers. (a) Evaluation of the protein expression of enzymes associated with apoptosis and the inflammatory response by western blot analysis. ARPE-19 cells were exposed to 2500 lx blue light lightemitting diode (LED) for 24 h after treatment with different concentrations of COSs for 1 h. β-actin was used as the internal control. (UT: untreated group, no COSs or
blue light LED exposure). (b) Relative expression of heme oxygenase-1 (HO-1); (c) Relative expression of inducible nitric oxide synthase (iNOS); (d) Relative
expression of cleaved caspase-3; (e) Relative expression of cleaved caspase-9; (f) Relative expression of B-cell lymphoma 2 (Bcl-2). (C: control group, no COSs or blue
light LED exposure; All data represent mean ± SD. **p < 0.01, *p < 0.05 compared to blue light LED alone exposed group by ANOVA with post hoc Least
Significant Difference (LSD) test; #p < 0.05 compared to blue light LED alone exposed group by Student’s t-test; N = 5 per group.)
Fig. 6. Chitosan oligosaccharides (COSs) downregulate the mRNA expression of pro-inflammatory
mediators. Evaluation of the relative expression of
pro-inflammatory mediators by quantitative reverse transcription polymerase chain reaction
(qRT-PCR). ARPE-19 cells were exposed to 2500 lx
blue light-emitting diode (LED) for 24 h after being
treated with different concentrations of COSs for
1 h. (a) Relative expression of inducible nitric oxide
synthase (iNOS); (b) Relative expression of
monocyte chemoattractant protein-1 (MCP-1).
(C: control group, no COSs or blue light LED
exposure; All data represent mean ± SD.
*
p < 0.05, **p < 0.01, ***p < 0.001 compared to
the blue light LED alone exposed group by ANOVA
with post hoc Least Significant Difference (LSD)
test; N = 5 in each group.)
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Journal of Functional Foods 49 (2018) 12–19
C.-W. Lin et al.
Fig. 7. Chitosan oligosaccharides (COSs) inhibit the nuclear translocatibon of NF-κB p65. Immunofluorescence staining of ARPE-19 cells was analyzed by a florescence microscope. Nuclear translocation of NF-κB p65 subunit was assessed in ARPE-19 cells exposed to 2500 lx blue light light-emitting diode (LED) for 24 h after
treatment with different concentrations of COSs for 1 h. (Green: NF-κB p65 subunit, Blue: nucleus, counterstained with DAPI. C: control group, no COSs or blue light
LED exposure.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(caspase-3, caspase-9) increase in ARPE-19 cells exposed to blue light
LED. COSs treatment was shown to reduce the production of these
proteins and down-regulate genes encoding MCP-1 and iNOS. MCP-1 is
a chemokine that can attract monocytes/macrophages to target tissues
(Jo, Wu, & Rao, 2003). Both MCP-1 and iNOS are NF-κB downstream
genes. Our results demonstrated that COSs can inhibit the activation of
NF-κB by inhibiting its translocation to the nucleus. NF-κB is a wellknown transcription factor that regulates the expression of genes involved in inflammation and apoptosis. Previous studies showed that
ROS is an inducer of NF-κB (Aggarwal, 2000; Schreck, Albermann, &
Baeuerle, 1992). Our findings indicated that the protective effect of
COSs in inhibiting NF-κB activation might be mediated by a decrease in
ROS production. In our study, we also found that COSs can up-regulate
the expression of Bcl-2 in a dose-dependent manner. Bcl-2 is an antiapoptotic protein that belongs to the Bcl-2 family, and this marker
might determine susceptibility to apoptosis (Ju, Lindsey, Angert, Patel,
& Weinreb, 2008). Taken together, the findings of the current study
suggest that COSs exert protective effects against oxidative stress by
down-regulating the NF-κB pathway and up-regulating the expression
of the anti-apoptotic protein Bcl-2. These findings are consistent with
those of previous studies, which demonstrated that COSs have anti-inflammatory activities that are mediated by the suppression of
NF-κB-dependent genes (Fang, Yang, & Yang, 2014; Khodagholi,
Eftekharzadeh, Maghsoudi, & Rezaei, 2010; Wei et al., 2012).
In the current study, we provided evidence that COSs have the
ability to protect ARPE-19 cells from blue light LED-induced damage by
inhibiting oxidative stress and the NF-κB signaling pathway. However,
there were some limitations of our study. First, our study is an in vitro
analysis. The protective effect of COSs thus needs to be demonstrated
using animal experiments. Second, the illuminance of light exposure in
our experiment was much higher than typical human daily exposure.
Moreover, blue light-induced damage to the human retina is a gradual
process. Therefore, the effect of nutrient supplements is very difficult to
determine. However, we have already proved the LED-induced retinal
injury by rat model with common domestic luminance level of 750 lx
for 28 days in our previous study (Shang et al., 2014). The results of this
study still provide strong evidence that COSs might be useful for the
prevention of blue light LED-induced retinal injury.
set of defense/stress responses and stimulate plant growth (Malerba &
Cerana, 2015, 2016). Based on a literature review, COSs can attenuate
hydrogen peroxide-induced oxidative damage to human umbilical vein
endothelial cells (Liu et al., 2009). They also have protective effects
against glucose deprivation-induced cell apoptosis in cultured cortical
neurons through the stabilization of mitochondrial membrane potential
(Xu, Zhang, Yu, Yang, & Ding, 2011). COSs have been shown to have
therapeutic effects on neurodegenerative diseases including Alzheimer's
disease and Parkinson's disease (Hao, Wang, Wang, Zhang, & Guo,
2017). In another study, COSs suppressed UVB irradiation-induced ROS
generation and DNA damage in human dermal fibroblasts via the
downregulation of mitogen-activated protein kinase-responsive
(MAPK) signaling pathway (Ahn et al., 2012). With the increasing popularity of consumer electronics, excessive blue light exposure can
easily lead to the damage of RPE cells. To the best of our knowledge,
there have not been any other studies on the protective effect of COSs
on light-induced retina damage. Our study confirmed the protective
effects of these compounds on blue light-induced RPE damage and
elucidated the associated molecular mechanisms.
In the present study, a blue light LED-induced photo-injury model
was established using the human ARPE-19 cell line. Blue light LED
causes damage to ARPE-19 cells by increasing ROS production and the
expression of genes associated with inflammation and apoptosis. With
longer exposure time, the percentage of apoptotic cells was also higher.
Exposure to blue light LED decreased cell viability; however, treatment
with COSs attenuated this decline. The protective effect of COSs was
also found to be concentration-dependent. The cellular survival advantage conferred by COSs was due to a decrease in cellular ROS formation and minor cellular mitochondrial membrane potential damage.
ROS are highly related to cell injury. When cellular ROS production
increases, oxygen radicals react with non-radicals and further amplify
the consequences of the initial event via a chain reaction; this causes
cellular dysfunction and results in cell death (Fridovich, 1998). COSs
have been reported to have ROS scavenging ability, as they can donate
hydrogen to free radicals to form stable molecules (Xie, Xu, & Liu,
2001). In this study, we confirmed that COSs treatment decreased
oxidative damage after light exposure. Mitochondria are responsible for
many cellular physiological functions including ion homeostasis and
the generation of adenosine triphosphate, and those functions are dependent on the membrane potential. Studies showed that mitochondrial
dysfunction participates in the induction of apoptosis; thus, the assessment of mitochondrial damage by JC-1 staining is also an important
indicator of cellular injury (Perry, Norman, Barbieri, Brown, & Gelbard,
2011; Solaini, Sgarbi, Lenaz, & Baracca, 2007). Our results demonstrated that COSs had the ability to decrease damage to mitochondrial
membrane potential. Taken together, our study revealed that COSs
treatment in ARPE-19 cells reduces ROS formation, decreases mitochondrial damage, inhibits apoptosis, and subsequently enhances cell
survival.
The results of our study also showed that the expression of proteins
associated with inflammation (HO-1, iNOS) and the apoptotic pathway
5. Conclusions
Our study demonstrated that COSs have a protective effect on RPE
cell damage mediated by blue light LED exposure. COSs can reduce the
production of ROS and suppress the expression of inflammation- and
apoptosis-related proteins. They stabilize the mitochondrial membrane
potential of cells and up-regulate the anti-apoptotic protein Bcl-2. These
compounds also down-regulate the NF-κB pathway by decreasing its
translocation to the nucleus and subsequently inhibiting expression of
downstream genes (iNOS, MCP-1). This study supports the idea that
COSs could be potentially used for the prevention of blue light-induced
damage to the human eye.
18
Journal of Functional Foods 49 (2018) 12–19
C.-W. Lin et al.
Funding
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This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declarations of interest
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Ethics statement
Our research did not include any human subjects and animal experiments.
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