- Email: [email protected]
3,4-Dicaffeoylquinic acid protects human keratinocytes against environmental oxidative damage
Journal of Functional Foods 52 (2019) 430–441
Contents lists available at ScienceDirect
Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
3,4-Dicaffeoylquinic acid protects human keratinocytes against environmental oxidative damage
T
Yu Jae Hyuna, Mei Jing Piaoa, Kyoung Ah Kanga, Yea Seong Ryua, Ao Xuan Zhena, Suk Ju Choa, ⁎ Hee Kyoung Kanga, Young Sang Koha, Mee Jung Ahnb, Tae Hoon Kimc, Jin Won Hyuna, a
Jeju National University, School of Medicine and Jeju Research Center for Natural Medicine, Jeju 63243, Republic of Korea Laboratory of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju 63243, Republic of Korea c Department of Food Science and Biotechnology, Daegu University, Gyeongsan 38453, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: 3,4-Dicaffeoylquinic acid Antioxidant ROS Human keratinocyte UVB PM2.5
Skin is exposed to several harmful environmental effects including ultraviolet B (UVB) and air pollution, the most harmful component of which is particulate matter (PM). Damaging effects of UVB and PM include the generation of cellular reactive oxygen species (ROS), lipid peroxidation, protein carbonylation, DNA damage, and apoptosis. A compound with the potential to protect the skin against environmental oxidative damage is 3,4dicaffeoylquinic acid (DQA), an antioxidant found in plant matter, including the coffee bean. In this study, we investigated the protective effects of DQA against UVB- and PM-induced oxidative cell damage in cultured human keratinocytes (HaCaT). We demonstrated that UVB, and PM with a diameter < 2.5 µm (PM2.5), induced cellular damage via oxidative stress, and this was mitigated by the antioxidative action of DQA.
1. Introduction Reactive oxygen species (ROS) are generated by cellular metabolism and environmental factors. They include hydrogen peroxide (H2O2), superoxide anion (O2−), and hydroxyl radical (OH%), all of which induce oxidative stress and cell damage at a high concentration (Schieber & Chandel, 2014). Generation of ROS by UV radiation can lead to harmful effects including photo-aging, inflammation, and skin cancer (Park, Sim, & Kim, 2017; Yunfeng et al., 2016), and many efforts have been made to prevent or treat these effects. Antioxidants provide treatment and prevention of UVB-induced oxidative stress and damage, for example, polyphenol and flavonoids, such as luteolin, rosmarinic acid, and caffeic acid, protect against UVB-induced cell damage (PérezSánchez, Barrajón-Catalán, Herranz-López, Castillo, & Micol, 2016). The harmful environmental effects to which the skin is exposed include air pollution as well as UVB. Air pollution involves chemicals, and also PM, which is the most the most harmful component of air pollution to human health (Soeur et al., 2017). Studies have shown that PM2.5 (PM with particle size less than 2.5 µm) can induce oxidative stress and inflammation in skin cells (Lin et al., 2016; Kim, Cho, & Park, 2016). Recently, alongside UVB, PM2.5 has become the focus of public health research, including research on skin hazards. PM2.5 represents
outdoor air pollution and mainly consists of metals, allergens, toxic products of combustion of fossil fuels, and endotoxins (He et al., 2016). Recently we demonstrated that human keratinocytes can internalize PM2.5 and the internalization triggered oxidative stress, leading to subcellular dysfunction and apoptotic cell death (Piao et al., 2018). As skin and keratinocytes form the outermost barrier directly facing harmful PM2.5, the combined effects of UVB and PM2.5 on the skin are worth investigating. Several other have demonstrated that PM2.5 induces oxidative stress via production of ROS, and subsequently antioxidants protect skin against PM2.5-induced cell damage (Krutmann et al., 2014; Jin et al., 2018). An antioxidant compound, 3,4-dicaffeoylquinic acid (DQA), found in plant material such as coffee bean, fruits, and vegetables, (Liang & Kitts, 2015) has been shown to decrease oxidative stress in vitro and in vivo, and thus has cytoprotective potential (Budryn et al., 2017). However, the mechanism of the protective effect of DQA against UVB and/or PM-induced skin cell damage is poorly understood. In this study, we demonstrate that DQA protects against UVB- and PM-induced damage via an antioxidant effect in human keratinocyte HaCaT cells.
⁎
Corresponding author. E-mail addresses: [email protected] (Y.J. Hyun), [email protected] (M.J. Piao), [email protected] (K.A. Kang), [email protected] (Y.S. Ryu), [email protected] (S.J. Cho), [email protected] (H.K. Kang), [email protected] (Y.S. Koh), [email protected] (M.J. Ahn), [email protected] (T.H. Kim), [email protected] (J.W. Hyun). https://doi.org/10.1016/j.jff.2018.11.026 Received 2 August 2018; Received in revised form 9 November 2018; Accepted 13 November 2018 Available online 23 November 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
B
A
120
O OH
100
Cell viability (%)
HO O HO
O
OH
O
HO
O
80 60 40 20 0
HO
0
1
2.5
OH
10
20
DQA [ȝM]
D
C
Intracellular ROS scavenging (%)
5
H2O2 UVB
100 80
*
60
#
40
*
*
*
#
#
#
#
*
20 0
0
1
2.5
5
10
20
DQA [ȝM] Superoxide anion (Arbitrary unit)
3000
*
2000
#
1000
0
DQA Xan+Xanox
-
+
-
+
-
-
+
+
Fig. 1. The effects of DQA on cytotoxicity and ROS scavenging. (A) Structure of DQA. (B) Cell viability was measured by MTT assay. Cells were treated with 1, 2.5, 5, 10 and 20 μM DQA for 24 h. (C) Intracellular ROS was detected by specrofluormetry after DCF-DA treatment. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (D) The DMPO-OOH generated by superoxide anions and DMPO were detected by ESR spectrometry. *p < 0.05, #p < 0.05 compared to control and superoxide anion, respectively. (E) The DMPO-OH generated by hydroxyl radicals and DMPO were detected by ESR spectrometry. *p < 0.05, # p < 0.05 compared to control and hydroxyl radical, respectively. (F) Intracellular ROS generated were detected using confocal microscopy after DCF-DA staining and (G) detected by spectrofluorometry after DCF-DA treatment. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively.
2. Materials and methods
(MTT), 2′,7′-dichlorofluorescein diacetate (DCF-DA), 5,5-dimethyl-1pyrroline-N-oxide (DMPO), Hoechst 33342, caspase inhibitor (Z-VADFMK), p38 MAPK inhibitor (SB203580), primary antibodies against caspase-3 and caspase-9 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Diphenyl-1-pyrenylphosphine (DPPP) was purchased from Molecular Probes (Eugene, OR, USA). 5,5′,6,6′-Tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was provided by Invitrogen (Carlsbad, CA, USA). SP600125 and U0126 were purchased from Tocris (Bristol, UK) and Calbiochem (La Jolla, CA, USA), respectively. Primary antibodies against Bax, Bcl-2, p38, and PARP were
2.1. Reagents and chemicals Pure DQA was previously isolated from Ainsliaea acerifolia of Korean origin (Kim, Jo, et al., 2016). Bioassay-guided isolation of ethanol extract from Ainsliaea acerifolia led to isolation of DQA, and the structure of isolated DQA was elucidated on the basis of spectroscopic data (NMR, UV, MS) (Fig. 1A). Diesel particulate matter NIST SRM 1650b (PM2.5), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 431
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
E
F DQA +
+
UVB
-
-
Hydroxyl radicals (Arbitrary unit)
4000
*
3000
#
2000
1000
0
-
DQA H2O2+FeSO4
+ -
+
+ +
Intracellular ROS generation (%)
G 300
*
200
100
0
#
DQA
-
-
+
UVB
-
+
+ Fig. 1. (continued)
(100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 µg/mL amphotericin B; all from Thermo Fisher Scientific (Waltham, MA, USA) at 37 °C in an incubator with a humidified atmosphere of 5% CO2. Cell were exposed to 30 mJ/cm2 UVB (CL-1000 M UV Crosslinker, Upland, CA, USA) and/or 50 μg/mL diesel particulate matter NIST 1650b (PM with a diameter < 2.5, PM2.5) (Sigma-Aldrich Chemical Company, St. Louis, MO, USA). Preparation of PM2.5 is described in a previous study (Piao et al., 2018).
purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Primary antibodies against ERK and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-IgG secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals and reagents were of analytical grade. 2.2. Cell culture and treatment The human keratinocyte cell line HaCaT (CLS Cell Lines Service GmbH, Eppelheim, Germany) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies Co., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and antibiotics
2.3. Cell viability Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5432
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
A
B Index of protein carbonyl
2.5
*
2.0
#
1.5 1.0 0.5 0.0
C
+
+
-
+
-
-
+
+
+
UVB
-
Index of DNA fragmentation
2.0
* 1.5
#
1.0
0.5
0.0 DQA
-
+
-
+
UVB
-
-
+
+
100
% Fluorescence in tail
-
UVB
D
DQA -
DQA
E
80
*
60 40
DQA
-
+
-
+
UVB
-
-
+
+
Phospho-H2A.X
#
15 kDa
20
42 kDa
Actin 0
-
+
-
+
UVB
-
-
+
+
Relative expression level (Protein/Actin)
DQA
6
*
5 4
#
3 2 1 0
DQA
-
+
-
+
UVB
-
-
+
+
Fig. 2. The effect of DQA on UVB-induced lipid peroxidation and DNA damage. (A) Lipid peroxidation was detected using confocal microscopy after DPPP staining. (B) Protein oxidation was assayed by measuring carbonyl formation. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (C) DNA break was determined by the comet assay. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (D) DNA fragmentation was measured using DNA fragmentation ELISA kit. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (E) Expression level of phospho-H2A.X by western blot analysis. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively.
433
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
A
B 120
Cell viability (%)
100 80
#
*
60 40 20 0
DQA
-
+
-
+
UVB
-
-
+
+ Index of apoptotic body
25
C
20
*
15
#
10 5 0
DQA
-
+
-
+
UVB
-
-
+
+
D +
-
+
-
-
+
+
Polarized
-
UVB
*
20
#
Depolarized
30
10
Merge
Depolarization of ¨ȥ (%)
DQA
0
DQA
-
+
-
+
UVB
-
-
+
+
Fig. 3. The effect of DQA against apoptosis. (A) Cell viability was determined by the MTT assay. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (B) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. * p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (C) The mitochondria membrane potential was assessed by flow cytometry after cells were stained with JC-1 and (D) was assessed by confocal microscopy after staining JC-1. *p < 0.05, #p < 0.05 compared to untreated cells and UVBtreated cells, respectively. (E) Protein level of Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP by western blot analysis. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (F) Cell viability was determined by the MTT assay. Z-VAD-FMK is caspase inhibitor. *p < 0.05, # p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively. (G) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. *p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively.
absorbance at 540 nm was measured on a scanning multi-well spectrophotometer. The cells were then visualized under a microscope and the proportions of viable and dead cells were determined.
diphenyl tetrazolium bromide (MTT) and trypan blue assays. HaCaT cells were seeded in plates at a density of 0.8 × 105 cells/mL, cultured for 16 h, and treated for 24 h with DQA (Chem Faces, Hubei, China) at various concentrations up to 20 µM, UVB at 30 mJ/cm2, and/or PM2.5 at 50 μg/mL. Then, MTT solution or 0.1% trypan blue solution was added. The formazan crystals were dissolved in DMSO and the 434
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
Bcl-2
26 kDa
Bax
23 kDa
DQA UVB Cleaved caspase-9 Cleaved caspase-3
Actin
42 kDa
Cleaved PARP
89 kDa
Actin
42 kDa
Relative expression level (Protein/Actin)
DQA UVB
-
+
+ +
10
Bax Bcl-2
*
8 6
#
4 2 #
*
0
DQA UVB
F
+ -
Relative expression level (Protein/Actin)
E
-
+ -
+
-
+ -
+
+ + 35 kDa 19 kDa 17 kDa
10
Cleaved caspase-9 Cleaved caspase-3 Cleaved PARP
8
*
*
6
#
4
*
#
2
#
0
+ +
DQA UVB
-
+ -
+
+ +
120
Cell viability (%)
100 #
80
## #
#
*
60 40 20 0
Z-VAD-FMK DQA UVB
G
-
+ +
+
+ +
+ + +
Z-VAD-FMK DQA
-
-
+ -
+
+ +
UVB
-
+
+
+
+
Index of apoptotic body
15
* 10 # # ## #
5
0
Z-VAD-FMK DQA UVB
-
+
+ +
+ +
+ + +
Fig. 3. (continued)
(H2O2 + FeSO4) were reacted with DMPO. The resultant DMPO/%OOH and DMPO/%OH adducts were measured using a JES-FA200 electron spin resonance (ESR) spectrometer (JEOL Ltd., Tokyo, Japan) (Kimura et al., 2016). The ESR spectrometer parameters were set as described in
2.4. Detection of superoxide anion and hydroxyl radical Superoxide anions generated by the xanthin-xanthin oxidase system and hydroxyl radicals generated by the Fenton reaction 435
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
A
UVB
UVB 0
15
30
45
0
60 min
1
3
12 h
44 kDa 42 kDa
p-ERK
44 kDa 42 kDa
p-JNK
54 kDa 46 kDa
p-JNK
54 kDa 46 kDa
p-p38
43 kDa
p-p38
43 kDa
Actin
42 kDa
Actin
42 kDa
p-ERK p-JNK p-p38
25
Relative expression level (Protein/Actin)
Relative expression level (Protein/Actin)
p-ERK
*
20 15
*
10 5
*
0
0
15
*
*
* 30
45
8
* 6
*
4
-
+
-
+
UVB
-
-
+
+
0
1
3
p-JNK
54 kDa 46 kDa
100
p-p38
43 kDa
Actin
Cell viability (%)
120
Relative expression level (Protein/Actin)
6
12 h
C 44 kDa 42 kDa
42 kDa p-Erk p-JNK p-p38
4
* *
##
80
##
# #
*
60
#
##
40 20 0
#
* #
1
*
0
60 min
p-ERK
2
* *
* * *
2
UVB
DQA
3
p-ERK p-JNK p-p38
*
UVB
B
6
#
-
U0126
-
-
+
-
-
+
-
SP600125
-
-
-
+
-
-
+
-
SB203580 DQA
-
-
-
-
+
-
-
+
-
-
-
-
-
+
+
+
UVB
-
+
+
+
+
+
+
+
0
DQA
-
+
-
+
UVB
-
-
+
+
Fig. 4. DQA inhibited UVB-induced activation of mitogen-activated protein kinase (phospho-MAPK). (A) Expression level of p-ERK, p-JNK and p-p38 by western blot analysis. *p < 0.05 compared to untreated cells. (B) Expression level of p-ERK, p-JNK and p-p38 by western blot analysis. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (C) Cell viability was determined by the MTT assay. U0126, SP600125 and SB203580 is MAPK inhibitor (ERK, JNK and p38 inhibitor, respectively). *p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively. (D) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. * p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively.
a previous study (Cha et al., 2014).
2.6. Lipid peroxidation assay
2.5. Intracellular ROS measurement
Cells were treated with 10 μM DQA, 30 mJ/cm2 UVB, and/or 50 μg/ mL PM2.5, incubated for 24 h, and then incubated with 20 μM DPPP. Images of DPPP fluorescence were collected using a FV1200 laser scanning confocal microscope (Olympus).
To measure intracellular ROS in HaCaT cells, cells were seeded in plates at a density of 1.0 × 105 cells/well, cultured for 16 h, and treated 10 μM DQA, 1 mM H2O2, 30 mJ/cm2 UVB, and/or 50 μg/mL PM2.5. Each treatment was carried out for 30 min, following which 50 μM 2′,7′dichlorofluorescein (DCF-DA) solution was added. DCF-DA fluorescence was measured using a LS-5B spectrofluorometer (PerkinElmer, Waltham, MA, USA) and images were collected using an FV1200 laser scanning confocal microscope (Olympus, Tokyo, Japan).
2.7. Protein carbonylation Protein carbonylation was measured using an OxiSelect™ protein carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA). All aspects of the protocol we followed, including the procedure for cell lysis and protein 436
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
D -
U0126
-
-
+
-
-
+
-
SP600125
-
-
-
+
-
-
+
-
SB203580
-
-
-
-
+
-
-
+
DQA
-
-
-
-
-
+
+
+
UVB
-
+
+
+
+
+
+
+
Index of apoptotic body
30
*
20
10
#
#
#
##
##
##
0
-
U0126
-
-
+
-
-
+
-
SP600125
-
-
-
+
-
-
+
-
SB203580 DQA
-
-
-
-
+
-
-
+
-
-
-
-
-
+
+
+
UVB
-
+
+
+
+
+
+
+
Fig. 4. (continued)
concentration used (10 μg/mL), were according to the manufacturer’s instructions.
2.11. Analysis of mitochondrial membrane potential (Δψ) Cells were seeded in plates at a density of 1 × 105 cells/mL, cultured for 16 h, and then treated with 10 μM DQA and PM2.5 (50 μg/mL) and/or UVB (30 mJ/cm2). Mitochondrial membrane potential was then analyzed by BD LSRFortessa flow cytometry and FV1200 laser scanning confocal microscopy after staining with membrane-permeant JC-1 dye (Invitrogen, Carlsbad, CA, USA).
2.8. Single cell gel electrophoresis (Comet assay) Cell suspensions were mixed with 1% low-melting agarose at 37 °C and then spread onto a 1% normal-melting-point agarose-coated slide. Cell-coated slide lysis solution (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Triton X-100, and 10% DMSO, pH 10) was applied for 1 h at 4 °C. After lysis, the slide was electrophoresed (300 mA, 25 V) for 30 min at 25 °C, stained with ethidium bromide, and observed using a fluorescence microscope and image analyzer (Komet 5.5, Kinetic Imaging Ltd, Wirral, UK). Tail length and percentage of total fluorescence in the comet tails were recorded for 50 cells per slide.
2.12. Western blot analysis The protein lysates (30 μg per lane) were electrophoresed on 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane which was incubated with the primary antibodies and then with HRPconjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (Invitrogen) for 1 h at room temperature. The protein bands were detected using a western blotting detection kit (GE Healthcare Life Sciences, Little Chalfont, UK) and then exposed to X-ray film. Primary antibodies used in this study were as follows: phosphor-H2A.X (Cell Signaling Technology, Beverly, MA, USA), Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (Santa Cruz Biotechnology), caspase-9 (Cell Signaling Technology), caspase-3 (Cell Signaling Technology), PARP (Santa Cruz Biotechnology), phospho-ERK (Santa Cruz Biotechnology), phospho-JNK (Cell Signaling Technology), phospho-p38 (Cell Signaling Technology), and actin (Sigma-Aldrich Chemical Company). The protein expression bands were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
2.9. DNA fragmentation Cells were seeded in plates at a density of 1.0 × 105 cells/well. Cellular DNA fragmentation was then measured using a kit from Roche Diagnostics (Portland, OR, USA) according to the manufacturer’s instructions.
2.10. Nuclear staining with Hoechst 33342 Cells were treated with 10 μM DQA, then with PM2.5 (50 μg/mL) and/or UVB (30 mJ/cm2) 1 h later. After a 24 h incubation at 37 °C, 20 µM Hoechst 33342, the DNA- specific fluorescent dye, was added to each well. After 10 min incubation at 37 °C, the stained cells were observed using a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera. The degree of nuclear condensation was evaluated, and apoptotic cells were counted.
2.13. Detection of 8-oxoguanine Cells were treated with 10 μM DQA, 50 µg/ml PM2.5, and/or 30 mJ/ cm2 UVB for 24 h and then incubated with avidin-TRITC (SigmaAldrich) for 30 min in the dark. Images of avidin-TRITC fluorescence 437
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
B
Control
PM2.5
DQA+PM2.5
+
UVB
-
A
C PM2.5
DQA+PM2.5
+
UVB
-
Control
PM2.5
30
DQA+PM2.5 Index of apoptotic body
Control
+
UVB
-
D
**# *
20
*
*
10
##
** 0
E
Control
PM2.5
DQA+PM2.5
DQA
-
-
+
-
-
+
UVB
-
-
-
+
+
+
PM2.5
-
+
+
-
+
+
120
**
UVB
-
Cell viability (%)
100
*
80
##
*
60
**# *
40
+
20 0
DQA
-
-
+
-
-
+
UVB
-
-
-
+
+
+
PM2.5
-
+
+
-
+
+
Fig. 5. The effect of DQA on PM2.5 and UVB-induced ROS and apoptosis. (A) Intracellular ROS was observed under confocal microscopy after DCF-DA staining. (B) Lipid peroxidation was observed under g confocal microscopy after DPPP staining. (C) 8-OxoG observed under confocal microscopy after avidin-TRITC staining. (D) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. *p < 0.05, **p < 0.05, # p < 0.05, ##p < 0.05 compared to untreated cells, PM2.5-treated cells, UVB-treated cells and PM2.5 with UVB-treated cells, respectively. (E) Cell viability was measured by trypan blue assay. *p < 0.05, **p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, PM2.5-treated cells, UVB-treated cells and PM with UVB-treated cells, respectively.
438
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
UVB
PM2.5
DQA
ROS
Fig. 2A, UVB induced lipid peroxidation, and this was reduced in DQA pretreated cells. Pretreatment DQA also significantly reduced protein carbonylation (Fig. 2B). Next, we aimed to determine whether DQA reduced UVB-induced DNA damage. Using comet assay and DNA fragmentation analysis, we found that DQA significantly reduced the number of DNA breaks induced by UVB (Fig. 2C and D). Additionally, western blotting revealed that the phosphorylation of nuclear histone H2A.X, a well-known DNA double strand break marker (Namas, Renauer, Ognenovski, Tsou, & Sawalha, 2016), increased in UVBtreated cells but decreased in DQA pretreated cells (Fig. 2E). Taken together, these results indicate that DQA has a protective effect against UVB-induced oxidative cellular damage to lipids, proteins, and DNA.
UVB
Phospho-ERK, JNK, p-38
Protein carbonylation
¨ȥ
Lipid peroxidation
Bax
DNA damage
Bcl-2
3.3. The effect of DQA against apoptosis Oxidative damage
Apoptosis is triggered by UVB radiation in HaCaT cells. The effect of DQA against UVB-induced cell death is shown in Fig. 3A. We confirmed that UVB-induced apoptotic bodies among HaCaT cells were significantly reduced by DQA (Fig. 3B). Given that apoptosis produces changes in mitochondrial membrane potential, we further confirmed the antiapoptotic effect of DQA using fluorescence dye JC-1 staining to examine mitochondrial membrane potential. As shown in Fig. 3C, fluorescence was increased in UVB-irradiated cells compared with controls, and DQA reduced UVB-induced fluorescence (Fig. 3C). Furthermore, confocal microscopy analysis confirmed that UVB-induced depolarization was reduced by DQA pretreatment (Fig. 3D). Examining the expression of apoptosis-related proteins we found that expression of Bax, the pro-apoptotic protein, was increased by UVB, and restored nearly to control levels in cells treated with both UVB and DQA. Irradiation with UVB decreased expression of the anti-apoptotic protein Bcl-2, and this was partially restored by the DQA. Given that induction of intracellular ROS leads to activation of the caspase-mediated apoptotic pathway (Wang et al., 2016), we also assayed the protein levels of active (cleaved) forms of caspase-9, caspase-3, and poly ADP ribose polymerase (PARP) (Fig. 3E). These were increased in UVB-treated cells and significantly reduced by DQA pretreatment. To further investigate the protective effect of DQA against UVB-induced activation of the caspase-mediated apoptotic pathway, we evaluated cell viability and apoptotic bodies. The number of apoptotic bodies induced by UVB was markedly reduced by DQA, both alone and in conjunction with the caspase inhibitor Z-VAD-FMK (Fig. 3F and 3G). These results indicated that DQA acts against UVB-induced apoptosis through the mitochondrial/caspase-mediated pathway.
Cleaved caspase 9, caspase 3, PARP
Apoptotic cell death Fig. 6. Schematic diagram of protective effect of DQA. Exposure of UVB or PM2.5 increased intracellular ROS and induced oxidative stress in HaCaT cells. DQA protects against UVB or PM2.5-indeced oxidative damage. In addition, it protects against UVB-induced apoptosis through inhibiting activation MAPKs.
were collected using a FV1200 laser scanning confocal microscope (Olympus). 2.14. Statistical analysis All analyses were performed in triplicate and all values are expressed as mean ± standard error of the mean. We used Tukey’s test to determine the statistical significance of differences between means. Statistical significance was inferred where p < 0.05. 3. Results 3.1. The effect of DQA on cytotoxicity and ROS scavenging We found that DQA has no cytotoxicity to HaCaT cells at any concentration up to 20 μM (Fig. 1B). The H2O2- and UVB-induced intracellular ROS scavenging effects of DQA, by comparison to the control group, were seen at concentrations ranging from 1 to 20 μM (Fig. 1C). We found that DQA concentrations of 10 μM and 20 μM have similar intracellular ROS scavenging effects in HaCaT cells, so we decided to use 10 μM DQA for all subsequent experiments. Next, we used ESR spectrometry to measure the superoxide anion- and hydroxyl radicalscavenging abilities of DQA. We observed that superoxide anion signals (1922 value) from a xanthine/xanthine oxidase system were significantly increased compared to the control group (222 value). However, DQA treatment significantly reduced the signal to a value of 1390 (Fig. 1D). In addition, hydroxyl radical signals (3375 value) from a H2O2 + FeSO4 system were significantly increased compared to the control group (32 value), and DQA treatment significantly reduced the value to 2278 (Fig. 1E). We also detected UVB-induced intracellular ROS generation using confocal microscopy and spectrofluorometry after DCF-DA staining. As shown Fig. 1F and G, UVB-induced intracellular ROS were reduced by DQA.
3.4. DQA inhibited UVB-induced activation of mitogen-activated protein kinase (phospho-MAPK) Next, we investigated the expression levels of the apoptosis-related signaling proteins phospho-extracellular signal regulated kinase (pERK), phospho-c-Jun N-terminal kinase (p-JNK), and phospho-p38 (pp38). These were significantly increased after 15 min of UVB irradiation for p-ERK, and after 60 min for p-JNK and p-p38 (Fig. 4A). Therefore, we investigated the effect of DQA on UVB-induced activation of these MAPKs. As shown Fig. 4B, their UVB-induced expression was decreased by DQA pretreatment. To further investigate the effect of DQA on UVBinduced activation of MAPK-mediated apoptosis, we pretreated cells with specific inhibitors and observed cell viability. Pretreatment with pERK, p-JNK, or p-p38 inhibitors (U0126, SP600125, and SB203580, respectively) decreased UVB-induced apoptosis. This was decreased further with DQA pretreatment (Fig. 4C and D).
3.2. The effect of DQA on UVB-induced oxidative cellular damage
3.5. The effect of DQA on PM- and UVB-induced ROS and apoptosis
Irradiation with UVB induces lipid peroxidation, protein carbonylation and cell damage. First, we detected lipid peroxidation in HaCaT cells using confocal microscopy after DPPP treatment. As shown in
Induction of intracellular ROS and cell damage has also been shown for combined PM and UVB treatment (Soeur et al., 2017). In our HaCaT cells, PM2.5 combined with UVB generated higher levels of ROS and 439
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
via scavenging ROS, and is associated with suppression of pro-apoptotic factors including caspase and MAPKs. Furthermore, its antioxidative effects also mitigate PM2.5-induced oxidative stress (Fig. 6).
lipid peroxidation than UVB treatment alone (Fig. 5A and B). This was also true of 8-oxoG, a marker of oxidative DNA damage (Fig. 5C). In all cases DQA significantly reduced the levels of intracellular ROS, lipid peroxidation, and 8-oxoG. We also measured apoptotic bodies and cell viability in response to combined UVB and PM2.5 treatment. As shown in Fig. 5D and E, PM2.5 combined with UVB generated more damage than PM2.5 or UVB alone, and the damage was clearly reduced by DQA pretreatment.
Conflict of interest statement The authors state no conflict of interest. Ethics statement
4. Discussion This work does not use the animals or human subjects. Damage induced by UVB includes skin inflammation, edema, erythema, and sunburn, while chronic UVB exposure leads to aging and carcinogenesis in the skin (Ivan et al., 2014). These harmful effects are linked to generation of first-stage intracellular ROS, including H2O2, superoxide anions, and hydroxyl radicals, either directly or through enzyme activation (Pérez-Sánchez et al., 2016). High concentrations of intracellular ROS damage lipids, proteins, and DNA (Schieber & Chandel, 2014). In many studies, antioxidants, including plant-derived flavonoids and polyphenols, have been shown to protect against UVB-induced cell damage (Choi, Kundu, Chun, Na, & Surh, 2014; Yunfeng et al., 2016). The antioxidant DQA studied here, is an isomer of the well-known antioxidant chlorogenic acid found in coffee bean and fruits (Liang, Xue, Kennepohl, & Kitts, 2016). We previously demonstrated that chlorogenic acid protects cells from UVB-induced oxidative stress (Cha et al., 2014). It has also been shown previously that dicaffeoylquinic acids are better antioxidants than caffeoylquinic acids including chlorogenic acid (Xu, Hu, & Liu, 2012). Here we report that DQA is not cytotoxic in HaCaT cells, and is effective at scavenging ROS, including the superoxide anion and the hydroxyl radical. Using DCF-DA, we showed that DQA scavenges UVBinduced intracellular ROS. We also demonstrated the protective effect of DQA against UVB-induced lipid peroxidation, protein carbonylation, DNA breakage, and apoptosis in HaCaT cells. Examining apoptosis, we showed that the effects of DQA to increase viability in the presence of oxidative stress are consistently reflected in the levels of apoptotic protein markers. These included Bcl-2 an antiapoptotic protein, and Bax, which is pro-apoptotic. We also investigated caspase-9, caspase-3, and PARP (Jia et al., 2015; Wang et al., 2016). The levels of these factors were increased by UVB and restored by DQA pretreatment. Given that the proteins we examined regulate apoptosis through the mitochondrial pathway (Jia et al., 2015), we also confirmed the effects of UVB on mitochondrial membrane potential, using fluorescence dye JC-1 staining. Similarly to the other markers of cell damage and death we examined, this effect was blocked by DQA. The MAPK pathway is mainly activated through environmental stresses such as oxidative stress and is involved in apoptosis (Che, Ma, & Xin, 2017; Jeayeng et al., 2017). We showed that its constituent proteins p-Erk, p-JNK, and p-p38 were significantly increased 6 h after irradiation with UVB, and this effect was reduced by pretreatment with DQA. Specific inhibition of these factors reduced UVB-induced cell death, an effect which was enhanced by DQA. We therefore suggest that the protective effects of DQA are mediated by the inhibition of MAPK in UVB-induced apoptosis. Skin can be damaged by air pollution such as PM as well as by UVB. Recent studies have shown that a combination of PM and UVA induces cell damage in keratinocytes (Soeur et al., 2017). We therefore investigated intracellular ROS level, lipid peroxidation, and apoptosis following exposure to PM2.5 and UVB. The group given a combination of PM2.5 and UVB generated more intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptosis than groups given only PM2.5 or UVB. Pretreatment with DQA significantly recovered from intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptosis induced by the combination of PM2.5 and UVB. In conclusion, DQA protects against UVB-induced oxidative stress
Acknowledgement This work was supported by grant from the Basic Research Laboratory Program (NRF-2017R1A4A1014512) by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT and future Planning (MSIP) of Korea government. References Budryn, G., Zakłos-Szyda, M., Zaczyńska, D., Żyżelewicz, D., Grzelczyk, J., Zduńczyk, Z., & Juśkiewicz, J. (2017). Green and roasted coffee extracts as antioxidants in βTC3 cells with induced oxidative stress and lipid accumulation inhibitors in 3T3L1 cells, and their bioactivity in rats fed high fat diet. European Food Research and Technology, 243, 1323–1334. https://doi.org/10.1007/s00217-017-2843-0. Cha, J. W., Piao, M. J., Kim, K. C., Yao, C. W., Zheng, J., Kim, S. M., ... Hyun, J. W. (2014). The polyphenol chlorogenic acid attenuates UVB-mediated oxidative stress in human HaCaT keratinocytes. Biomolecules & Therapeutics, 22, 136–142. https://doi.org/10. 4062/biomolther.2014.006. Che, N., Ma, Y., & Xin, Y. (2017). Protective role of fucoidan in cerebral ischemia-reperfusion injury through inhibition of MAPK signaling pathway. Biomolecules & Therapeutics, 25, 272–278. https://doi.org/10.4062/biomolther.2016.098. Choi, K. S., Kundu, J. K., Chun, K. S., Na, H. K., & Surh, Y. J. (2014). Rutin inhibits UVB radiation-induced expression of COX-2 and iNOS in hairless mouse skin: p38 MAP kinase and JNK as potential targets. Archives of Biochemistry and Biophysics, 559, 38–45. https://doi.org/10.1016/j.abb.2014.05.016. He, M., Ichinose, T., Yoshida, S., Shiba, F., Arashidani, K., Takano, H., ... Shibamoto, T. (2016). Differences in allergic inflammatory responses in murine lungs: Comparison of PM2.5 and coarse PM collected during the hazy events in a Chinese city. Inhalation Toxicology, 28, 706–718. https://doi.org/10.1080/08958378.2016.1260185. Ivan, A. L., Campanini, M. Z., Martinez, R. M., Ferreira, V. S., Steffen, V. S., Vicentini, F. T., ... Casagrande, R. (2014). Pyrrolidine dithiocarbamate inhibits UVB-induced skin inflammation and oxidative stress in hairless mice and exhibits antioxidant activity in vitro. Journal of Photochemistry and Photobiology B, Biology, 138, 124–133. https:// doi.org/10.1016/j.jphotobiol.2014.05.010. Jeayeng, S., Wongkajornsilp, A., Slominski, A. T., Jirawatnotai, S., Sampattavanich, S., & Panich, U. (2017). Nrf2 in keratinocytes modulates UVB-induced DNA damage and apoptosis in melanocytes through MAPK signaling. Free Radical Biology & Medicine, 108, 918–928. https://doi.org/10.1016/j.freeradbiomed.2017.05.009. Jia, G., Wang, Q., Wang, R., Deng, D., Xue, L., Shao, N., ... Yang, Y. (2015). Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway. OncoTargets and Therapy, 8, 303–311. https://doi.org/10. 2147/OTT.S76063. Jin, S. P., Li, Z., Choi, E. K., Lee, S., Kim, Y. K., Seo, E. Y., ... Cho, S. (2018). Urban particulate matter in air pollution penetrates into the barrier-disrupted skin and produces ROS-dependent cutaneous inflammatory response in vivo. Journal of Dermatological Science, 91, 175–183. https://doi.org/10.1016/j.jdermsci.2018.04. 015. Kim, K. E., Cho, D., & Park, H. J. (2016). Air pollution and skin diseases: Adverse effects of airborne particulate matter on various skin diseases. Life Sciences, 152, 126–134. https://doi.org/10.1016/j.lfs.2016.03.039. Kim, T. W., Jo, C., Kim, H. S., Park, Y. M., Wu, Y. X., Cho, J. H., & Kim, T. H. (2016). Chemical constituents from Ainsliaea acerifolia as potential anti-obesity agents. Phytochemistry Letters, 16, 146–151. https://doi.org/10.1016/j.phytol.2016.04.005. Kimura, S., Inoguchi, T., Yamasaki, T., Yamato, M., Ide, M., Sonoda, N., ... Takayanagi, R. (2016). A novel DPP-4 inhibitor teneligliptin scavenges hydroxyl radicals: In vitro study evaluated by electron spin resonance spectroscopy and in vivo study using DPP4 deficient rats. Metabolism, 65, 138–145. https://doi.org/10.1016/j.metabol.2015. 10.030. Krutmann, J., Liu, W., Li, L., Pan, X., Crawford, M., Sore, G., & Seite, S. (2014). Pollution and skin: From epidemiological and mechanistic studies to clinical implications. Journal of Dermatological Science, 76, 163–168. Liang, N., Xue, W., Kennepohl, P., & Kitts, D. D. (2016). Interactions between major chlorogenic acid isomers and chemical changes in coffee brew that affect antioxidant activities. Food Chemistry, 213, 251–259. https://doi.org/10.1016/j.foodchem.2016. 06.041. Liang, N., & Kitts, D. D. (2015). Role of chlorogenic acids in controlling oxidative and inflammatory stress conditions. Nutrients, 8, 16. https://doi.org/10.3390/
440
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
subcellular organelle dysfunction, and apoptosis. Archives of Toxicology, 92, 2077–2091. https://doi.org/10.1007/s00204-018-2197-9. Schieber, M., & Chandel, N. S. (2014). ROS function in redox signaling and oxidative stress. Current Biology, 24, R453–R462. https://doi.org/10.1016/j.cub.2014.03.034. Soeur, J., Belaïdi, J. P., Chollet, C., Denat, L., Dimitrov, A., Jones, C., ... Marrot, L. (2017). Photo-pollution stress in skin: Traces of pollutants (PAH and particulate matter) impair redox homeostasis in keratinocytes exposed to UVA1. Journal of Dermatological Science, 86, 162–169. https://doi.org/10.1016/j.jdermsci.2017.01.007. Yunfeng, H., Yuetang, M., Shi, W., Tianfeng, C., Yong, H., Jianxia, S., ... Weibin, B. (2016). Protective effect of cyanidin-3-O-glucoside against ultraviolet B radiationinduced cell damage in human HaCaT keratinocytes. Frontiers in Pharmacology, 7, 301. https://doi.org/10.3389/fphar.2016.00301. Wang, D., Qu, X., Zhuang, X., Geng, G., Hou, J., Xu, N., ... Chen, Y. S. (2016). Seed oil of Brucea javanica induces cell cycle arrest and apoptosis via reactive oxygen speciesmediated mitochondrial dysfunction in human lung cancer cells. Nutrition and Cancer, 68, 1394–1403. https://doi.org/10.1080/01635581.2016.1224362. Xu, J. G., Hu, Q. P., & Liu, Y. (2012). Antioxidant and DNA-protective activities of chlorogenic acid isomers. Journal of Agricultural and Food Chemistry, 60, 11625–11630. https://doi.org/10.1021/jf303771s.
nu8010016. Lin, Z. C., Lee, C. W., Tsai, M. H., Ko, H. H., Fang, J. Y., Chiang, Y. C., ... Yen, F. L. (2016). Eupafolin nanoparticles protect HaCaT keratinocytes from particulate matter-induced inflammation and oxidative stress. International Journal of Nanomedicine, 11, 3907–13626. https://doi.org/10.2147/IJN.S109062. Namas, R., Renauer, P., Ognenovski, M., Tsou, P. S., & Sawalha, A. H. (2016). Histone H2AX phosphorylation as a measure of DNA double-strand breaks and a marker of environmental stress and disease activity in lupus. Lupus Science & Medicine, 3, e000148. https://doi.org/10.1136/lupus-2016-000148. Park, M. A., Sim, M. J., & Kim, Y. C. (2017). Anti-photoaging effects of angelica acutiloba root ethanol extract in human dermal fibroblasts. Toxicological Research, 33, 125–134. https://doi.org/10.5487/TR.2017.33.2.125. Pérez-Sánchez, A., Barrajón-Catalán, E., Herranz-López, M., Castillo, J., & Micol, V. (2016). Lemon balm extract (Melissa officinalis, L.) promotes melanogenesis and prevents UVB-induced oxidative stress and DNA damage in a skin cell model. Journal of Dermatological Science, 84, 169–177. https://doi.org/10.1016/j.jdermsci.2016.08. 004. Piao, M. J., Ahn, M. J., Kang, K. A., Ryu, Y. S., Hyun, Y. J., Shilnikova, K., ... Hyun, J. W. (2018). Particulate matter 2.5 damages skin cells by inducing oxidative stress,
441
Contents lists available at ScienceDirect
Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
3,4-Dicaffeoylquinic acid protects human keratinocytes against environmental oxidative damage
T
Yu Jae Hyuna, Mei Jing Piaoa, Kyoung Ah Kanga, Yea Seong Ryua, Ao Xuan Zhena, Suk Ju Choa, ⁎ Hee Kyoung Kanga, Young Sang Koha, Mee Jung Ahnb, Tae Hoon Kimc, Jin Won Hyuna, a
Jeju National University, School of Medicine and Jeju Research Center for Natural Medicine, Jeju 63243, Republic of Korea Laboratory of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju 63243, Republic of Korea c Department of Food Science and Biotechnology, Daegu University, Gyeongsan 38453, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: 3,4-Dicaffeoylquinic acid Antioxidant ROS Human keratinocyte UVB PM2.5
Skin is exposed to several harmful environmental effects including ultraviolet B (UVB) and air pollution, the most harmful component of which is particulate matter (PM). Damaging effects of UVB and PM include the generation of cellular reactive oxygen species (ROS), lipid peroxidation, protein carbonylation, DNA damage, and apoptosis. A compound with the potential to protect the skin against environmental oxidative damage is 3,4dicaffeoylquinic acid (DQA), an antioxidant found in plant matter, including the coffee bean. In this study, we investigated the protective effects of DQA against UVB- and PM-induced oxidative cell damage in cultured human keratinocytes (HaCaT). We demonstrated that UVB, and PM with a diameter < 2.5 µm (PM2.5), induced cellular damage via oxidative stress, and this was mitigated by the antioxidative action of DQA.
1. Introduction Reactive oxygen species (ROS) are generated by cellular metabolism and environmental factors. They include hydrogen peroxide (H2O2), superoxide anion (O2−), and hydroxyl radical (OH%), all of which induce oxidative stress and cell damage at a high concentration (Schieber & Chandel, 2014). Generation of ROS by UV radiation can lead to harmful effects including photo-aging, inflammation, and skin cancer (Park, Sim, & Kim, 2017; Yunfeng et al., 2016), and many efforts have been made to prevent or treat these effects. Antioxidants provide treatment and prevention of UVB-induced oxidative stress and damage, for example, polyphenol and flavonoids, such as luteolin, rosmarinic acid, and caffeic acid, protect against UVB-induced cell damage (PérezSánchez, Barrajón-Catalán, Herranz-López, Castillo, & Micol, 2016). The harmful environmental effects to which the skin is exposed include air pollution as well as UVB. Air pollution involves chemicals, and also PM, which is the most the most harmful component of air pollution to human health (Soeur et al., 2017). Studies have shown that PM2.5 (PM with particle size less than 2.5 µm) can induce oxidative stress and inflammation in skin cells (Lin et al., 2016; Kim, Cho, & Park, 2016). Recently, alongside UVB, PM2.5 has become the focus of public health research, including research on skin hazards. PM2.5 represents
outdoor air pollution and mainly consists of metals, allergens, toxic products of combustion of fossil fuels, and endotoxins (He et al., 2016). Recently we demonstrated that human keratinocytes can internalize PM2.5 and the internalization triggered oxidative stress, leading to subcellular dysfunction and apoptotic cell death (Piao et al., 2018). As skin and keratinocytes form the outermost barrier directly facing harmful PM2.5, the combined effects of UVB and PM2.5 on the skin are worth investigating. Several other have demonstrated that PM2.5 induces oxidative stress via production of ROS, and subsequently antioxidants protect skin against PM2.5-induced cell damage (Krutmann et al., 2014; Jin et al., 2018). An antioxidant compound, 3,4-dicaffeoylquinic acid (DQA), found in plant material such as coffee bean, fruits, and vegetables, (Liang & Kitts, 2015) has been shown to decrease oxidative stress in vitro and in vivo, and thus has cytoprotective potential (Budryn et al., 2017). However, the mechanism of the protective effect of DQA against UVB and/or PM-induced skin cell damage is poorly understood. In this study, we demonstrate that DQA protects against UVB- and PM-induced damage via an antioxidant effect in human keratinocyte HaCaT cells.
⁎
Corresponding author. E-mail addresses: [email protected] (Y.J. Hyun), [email protected] (M.J. Piao), [email protected] (K.A. Kang), [email protected] (Y.S. Ryu), [email protected] (S.J. Cho), [email protected] (H.K. Kang), [email protected] (Y.S. Koh), [email protected] (M.J. Ahn), [email protected] (T.H. Kim), [email protected] (J.W. Hyun). https://doi.org/10.1016/j.jff.2018.11.026 Received 2 August 2018; Received in revised form 9 November 2018; Accepted 13 November 2018 Available online 23 November 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
B
A
120
O OH
100
Cell viability (%)
HO O HO
O
OH
O
HO
O
80 60 40 20 0
HO
0
1
2.5
OH
10
20
DQA [ȝM]
D
C
Intracellular ROS scavenging (%)
5
H2O2 UVB
100 80
*
60
#
40
*
*
*
#
#
#
#
*
20 0
0
1
2.5
5
10
20
DQA [ȝM] Superoxide anion (Arbitrary unit)
3000
*
2000
#
1000
0
DQA Xan+Xanox
-
+
-
+
-
-
+
+
Fig. 1. The effects of DQA on cytotoxicity and ROS scavenging. (A) Structure of DQA. (B) Cell viability was measured by MTT assay. Cells were treated with 1, 2.5, 5, 10 and 20 μM DQA for 24 h. (C) Intracellular ROS was detected by specrofluormetry after DCF-DA treatment. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (D) The DMPO-OOH generated by superoxide anions and DMPO were detected by ESR spectrometry. *p < 0.05, #p < 0.05 compared to control and superoxide anion, respectively. (E) The DMPO-OH generated by hydroxyl radicals and DMPO were detected by ESR spectrometry. *p < 0.05, # p < 0.05 compared to control and hydroxyl radical, respectively. (F) Intracellular ROS generated were detected using confocal microscopy after DCF-DA staining and (G) detected by spectrofluorometry after DCF-DA treatment. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively.
2. Materials and methods
(MTT), 2′,7′-dichlorofluorescein diacetate (DCF-DA), 5,5-dimethyl-1pyrroline-N-oxide (DMPO), Hoechst 33342, caspase inhibitor (Z-VADFMK), p38 MAPK inhibitor (SB203580), primary antibodies against caspase-3 and caspase-9 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Diphenyl-1-pyrenylphosphine (DPPP) was purchased from Molecular Probes (Eugene, OR, USA). 5,5′,6,6′-Tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was provided by Invitrogen (Carlsbad, CA, USA). SP600125 and U0126 were purchased from Tocris (Bristol, UK) and Calbiochem (La Jolla, CA, USA), respectively. Primary antibodies against Bax, Bcl-2, p38, and PARP were
2.1. Reagents and chemicals Pure DQA was previously isolated from Ainsliaea acerifolia of Korean origin (Kim, Jo, et al., 2016). Bioassay-guided isolation of ethanol extract from Ainsliaea acerifolia led to isolation of DQA, and the structure of isolated DQA was elucidated on the basis of spectroscopic data (NMR, UV, MS) (Fig. 1A). Diesel particulate matter NIST SRM 1650b (PM2.5), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 431
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
E
F DQA +
+
UVB
-
-
Hydroxyl radicals (Arbitrary unit)
4000
*
3000
#
2000
1000
0
-
DQA H2O2+FeSO4
+ -
+
+ +
Intracellular ROS generation (%)
G 300
*
200
100
0
#
DQA
-
-
+
UVB
-
+
+ Fig. 1. (continued)
(100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 µg/mL amphotericin B; all from Thermo Fisher Scientific (Waltham, MA, USA) at 37 °C in an incubator with a humidified atmosphere of 5% CO2. Cell were exposed to 30 mJ/cm2 UVB (CL-1000 M UV Crosslinker, Upland, CA, USA) and/or 50 μg/mL diesel particulate matter NIST 1650b (PM with a diameter < 2.5, PM2.5) (Sigma-Aldrich Chemical Company, St. Louis, MO, USA). Preparation of PM2.5 is described in a previous study (Piao et al., 2018).
purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Primary antibodies against ERK and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-IgG secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals and reagents were of analytical grade. 2.2. Cell culture and treatment The human keratinocyte cell line HaCaT (CLS Cell Lines Service GmbH, Eppelheim, Germany) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies Co., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and antibiotics
2.3. Cell viability Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5432
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
A
B Index of protein carbonyl
2.5
*
2.0
#
1.5 1.0 0.5 0.0
C
+
+
-
+
-
-
+
+
+
UVB
-
Index of DNA fragmentation
2.0
* 1.5
#
1.0
0.5
0.0 DQA
-
+
-
+
UVB
-
-
+
+
100
% Fluorescence in tail
-
UVB
D
DQA -
DQA
E
80
*
60 40
DQA
-
+
-
+
UVB
-
-
+
+
Phospho-H2A.X
#
15 kDa
20
42 kDa
Actin 0
-
+
-
+
UVB
-
-
+
+
Relative expression level (Protein/Actin)
DQA
6
*
5 4
#
3 2 1 0
DQA
-
+
-
+
UVB
-
-
+
+
Fig. 2. The effect of DQA on UVB-induced lipid peroxidation and DNA damage. (A) Lipid peroxidation was detected using confocal microscopy after DPPP staining. (B) Protein oxidation was assayed by measuring carbonyl formation. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (C) DNA break was determined by the comet assay. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (D) DNA fragmentation was measured using DNA fragmentation ELISA kit. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (E) Expression level of phospho-H2A.X by western blot analysis. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively.
433
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
A
B 120
Cell viability (%)
100 80
#
*
60 40 20 0
DQA
-
+
-
+
UVB
-
-
+
+ Index of apoptotic body
25
C
20
*
15
#
10 5 0
DQA
-
+
-
+
UVB
-
-
+
+
D +
-
+
-
-
+
+
Polarized
-
UVB
*
20
#
Depolarized
30
10
Merge
Depolarization of ¨ȥ (%)
DQA
0
DQA
-
+
-
+
UVB
-
-
+
+
Fig. 3. The effect of DQA against apoptosis. (A) Cell viability was determined by the MTT assay. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (B) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. * p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (C) The mitochondria membrane potential was assessed by flow cytometry after cells were stained with JC-1 and (D) was assessed by confocal microscopy after staining JC-1. *p < 0.05, #p < 0.05 compared to untreated cells and UVBtreated cells, respectively. (E) Protein level of Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP by western blot analysis. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (F) Cell viability was determined by the MTT assay. Z-VAD-FMK is caspase inhibitor. *p < 0.05, # p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively. (G) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. *p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively.
absorbance at 540 nm was measured on a scanning multi-well spectrophotometer. The cells were then visualized under a microscope and the proportions of viable and dead cells were determined.
diphenyl tetrazolium bromide (MTT) and trypan blue assays. HaCaT cells were seeded in plates at a density of 0.8 × 105 cells/mL, cultured for 16 h, and treated for 24 h with DQA (Chem Faces, Hubei, China) at various concentrations up to 20 µM, UVB at 30 mJ/cm2, and/or PM2.5 at 50 μg/mL. Then, MTT solution or 0.1% trypan blue solution was added. The formazan crystals were dissolved in DMSO and the 434
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
Bcl-2
26 kDa
Bax
23 kDa
DQA UVB Cleaved caspase-9 Cleaved caspase-3
Actin
42 kDa
Cleaved PARP
89 kDa
Actin
42 kDa
Relative expression level (Protein/Actin)
DQA UVB
-
+
+ +
10
Bax Bcl-2
*
8 6
#
4 2 #
*
0
DQA UVB
F
+ -
Relative expression level (Protein/Actin)
E
-
+ -
+
-
+ -
+
+ + 35 kDa 19 kDa 17 kDa
10
Cleaved caspase-9 Cleaved caspase-3 Cleaved PARP
8
*
*
6
#
4
*
#
2
#
0
+ +
DQA UVB
-
+ -
+
+ +
120
Cell viability (%)
100 #
80
## #
#
*
60 40 20 0
Z-VAD-FMK DQA UVB
G
-
+ +
+
+ +
+ + +
Z-VAD-FMK DQA
-
-
+ -
+
+ +
UVB
-
+
+
+
+
Index of apoptotic body
15
* 10 # # ## #
5
0
Z-VAD-FMK DQA UVB
-
+
+ +
+ +
+ + +
Fig. 3. (continued)
(H2O2 + FeSO4) were reacted with DMPO. The resultant DMPO/%OOH and DMPO/%OH adducts were measured using a JES-FA200 electron spin resonance (ESR) spectrometer (JEOL Ltd., Tokyo, Japan) (Kimura et al., 2016). The ESR spectrometer parameters were set as described in
2.4. Detection of superoxide anion and hydroxyl radical Superoxide anions generated by the xanthin-xanthin oxidase system and hydroxyl radicals generated by the Fenton reaction 435
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
A
UVB
UVB 0
15
30
45
0
60 min
1
3
12 h
44 kDa 42 kDa
p-ERK
44 kDa 42 kDa
p-JNK
54 kDa 46 kDa
p-JNK
54 kDa 46 kDa
p-p38
43 kDa
p-p38
43 kDa
Actin
42 kDa
Actin
42 kDa
p-ERK p-JNK p-p38
25
Relative expression level (Protein/Actin)
Relative expression level (Protein/Actin)
p-ERK
*
20 15
*
10 5
*
0
0
15
*
*
* 30
45
8
* 6
*
4
-
+
-
+
UVB
-
-
+
+
0
1
3
p-JNK
54 kDa 46 kDa
100
p-p38
43 kDa
Actin
Cell viability (%)
120
Relative expression level (Protein/Actin)
6
12 h
C 44 kDa 42 kDa
42 kDa p-Erk p-JNK p-p38
4
* *
##
80
##
# #
*
60
#
##
40 20 0
#
* #
1
*
0
60 min
p-ERK
2
* *
* * *
2
UVB
DQA
3
p-ERK p-JNK p-p38
*
UVB
B
6
#
-
U0126
-
-
+
-
-
+
-
SP600125
-
-
-
+
-
-
+
-
SB203580 DQA
-
-
-
-
+
-
-
+
-
-
-
-
-
+
+
+
UVB
-
+
+
+
+
+
+
+
0
DQA
-
+
-
+
UVB
-
-
+
+
Fig. 4. DQA inhibited UVB-induced activation of mitogen-activated protein kinase (phospho-MAPK). (A) Expression level of p-ERK, p-JNK and p-p38 by western blot analysis. *p < 0.05 compared to untreated cells. (B) Expression level of p-ERK, p-JNK and p-p38 by western blot analysis. *p < 0.05, #p < 0.05 compared to untreated cells and UVB-treated cells, respectively. (C) Cell viability was determined by the MTT assay. U0126, SP600125 and SB203580 is MAPK inhibitor (ERK, JNK and p38 inhibitor, respectively). *p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively. (D) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. * p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, UVB-treated cells and inhibitor with UVB-treated cells, respectively.
a previous study (Cha et al., 2014).
2.6. Lipid peroxidation assay
2.5. Intracellular ROS measurement
Cells were treated with 10 μM DQA, 30 mJ/cm2 UVB, and/or 50 μg/ mL PM2.5, incubated for 24 h, and then incubated with 20 μM DPPP. Images of DPPP fluorescence were collected using a FV1200 laser scanning confocal microscope (Olympus).
To measure intracellular ROS in HaCaT cells, cells were seeded in plates at a density of 1.0 × 105 cells/well, cultured for 16 h, and treated 10 μM DQA, 1 mM H2O2, 30 mJ/cm2 UVB, and/or 50 μg/mL PM2.5. Each treatment was carried out for 30 min, following which 50 μM 2′,7′dichlorofluorescein (DCF-DA) solution was added. DCF-DA fluorescence was measured using a LS-5B spectrofluorometer (PerkinElmer, Waltham, MA, USA) and images were collected using an FV1200 laser scanning confocal microscope (Olympus, Tokyo, Japan).
2.7. Protein carbonylation Protein carbonylation was measured using an OxiSelect™ protein carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA). All aspects of the protocol we followed, including the procedure for cell lysis and protein 436
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
D -
U0126
-
-
+
-
-
+
-
SP600125
-
-
-
+
-
-
+
-
SB203580
-
-
-
-
+
-
-
+
DQA
-
-
-
-
-
+
+
+
UVB
-
+
+
+
+
+
+
+
Index of apoptotic body
30
*
20
10
#
#
#
##
##
##
0
-
U0126
-
-
+
-
-
+
-
SP600125
-
-
-
+
-
-
+
-
SB203580 DQA
-
-
-
-
+
-
-
+
-
-
-
-
-
+
+
+
UVB
-
+
+
+
+
+
+
+
Fig. 4. (continued)
concentration used (10 μg/mL), were according to the manufacturer’s instructions.
2.11. Analysis of mitochondrial membrane potential (Δψ) Cells were seeded in plates at a density of 1 × 105 cells/mL, cultured for 16 h, and then treated with 10 μM DQA and PM2.5 (50 μg/mL) and/or UVB (30 mJ/cm2). Mitochondrial membrane potential was then analyzed by BD LSRFortessa flow cytometry and FV1200 laser scanning confocal microscopy after staining with membrane-permeant JC-1 dye (Invitrogen, Carlsbad, CA, USA).
2.8. Single cell gel electrophoresis (Comet assay) Cell suspensions were mixed with 1% low-melting agarose at 37 °C and then spread onto a 1% normal-melting-point agarose-coated slide. Cell-coated slide lysis solution (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Triton X-100, and 10% DMSO, pH 10) was applied for 1 h at 4 °C. After lysis, the slide was electrophoresed (300 mA, 25 V) for 30 min at 25 °C, stained with ethidium bromide, and observed using a fluorescence microscope and image analyzer (Komet 5.5, Kinetic Imaging Ltd, Wirral, UK). Tail length and percentage of total fluorescence in the comet tails were recorded for 50 cells per slide.
2.12. Western blot analysis The protein lysates (30 μg per lane) were electrophoresed on 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane which was incubated with the primary antibodies and then with HRPconjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (Invitrogen) for 1 h at room temperature. The protein bands were detected using a western blotting detection kit (GE Healthcare Life Sciences, Little Chalfont, UK) and then exposed to X-ray film. Primary antibodies used in this study were as follows: phosphor-H2A.X (Cell Signaling Technology, Beverly, MA, USA), Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (Santa Cruz Biotechnology), caspase-9 (Cell Signaling Technology), caspase-3 (Cell Signaling Technology), PARP (Santa Cruz Biotechnology), phospho-ERK (Santa Cruz Biotechnology), phospho-JNK (Cell Signaling Technology), phospho-p38 (Cell Signaling Technology), and actin (Sigma-Aldrich Chemical Company). The protein expression bands were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
2.9. DNA fragmentation Cells were seeded in plates at a density of 1.0 × 105 cells/well. Cellular DNA fragmentation was then measured using a kit from Roche Diagnostics (Portland, OR, USA) according to the manufacturer’s instructions.
2.10. Nuclear staining with Hoechst 33342 Cells were treated with 10 μM DQA, then with PM2.5 (50 μg/mL) and/or UVB (30 mJ/cm2) 1 h later. After a 24 h incubation at 37 °C, 20 µM Hoechst 33342, the DNA- specific fluorescent dye, was added to each well. After 10 min incubation at 37 °C, the stained cells were observed using a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera. The degree of nuclear condensation was evaluated, and apoptotic cells were counted.
2.13. Detection of 8-oxoguanine Cells were treated with 10 μM DQA, 50 µg/ml PM2.5, and/or 30 mJ/ cm2 UVB for 24 h and then incubated with avidin-TRITC (SigmaAldrich) for 30 min in the dark. Images of avidin-TRITC fluorescence 437
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
B
Control
PM2.5
DQA+PM2.5
+
UVB
-
A
C PM2.5
DQA+PM2.5
+
UVB
-
Control
PM2.5
30
DQA+PM2.5 Index of apoptotic body
Control
+
UVB
-
D
**# *
20
*
*
10
##
** 0
E
Control
PM2.5
DQA+PM2.5
DQA
-
-
+
-
-
+
UVB
-
-
-
+
+
+
PM2.5
-
+
+
-
+
+
120
**
UVB
-
Cell viability (%)
100
*
80
##
*
60
**# *
40
+
20 0
DQA
-
-
+
-
-
+
UVB
-
-
-
+
+
+
PM2.5
-
+
+
-
+
+
Fig. 5. The effect of DQA on PM2.5 and UVB-induced ROS and apoptosis. (A) Intracellular ROS was observed under confocal microscopy after DCF-DA staining. (B) Lipid peroxidation was observed under g confocal microscopy after DPPP staining. (C) 8-OxoG observed under confocal microscopy after avidin-TRITC staining. (D) Apoptotic body formation was observed using a fluorescence microscope after Hoechst 33342 staining. The arrows indicate apoptotic bodies. *p < 0.05, **p < 0.05, # p < 0.05, ##p < 0.05 compared to untreated cells, PM2.5-treated cells, UVB-treated cells and PM2.5 with UVB-treated cells, respectively. (E) Cell viability was measured by trypan blue assay. *p < 0.05, **p < 0.05, #p < 0.05, ##p < 0.05 compared to untreated cells, PM2.5-treated cells, UVB-treated cells and PM with UVB-treated cells, respectively.
438
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
UVB
PM2.5
DQA
ROS
Fig. 2A, UVB induced lipid peroxidation, and this was reduced in DQA pretreated cells. Pretreatment DQA also significantly reduced protein carbonylation (Fig. 2B). Next, we aimed to determine whether DQA reduced UVB-induced DNA damage. Using comet assay and DNA fragmentation analysis, we found that DQA significantly reduced the number of DNA breaks induced by UVB (Fig. 2C and D). Additionally, western blotting revealed that the phosphorylation of nuclear histone H2A.X, a well-known DNA double strand break marker (Namas, Renauer, Ognenovski, Tsou, & Sawalha, 2016), increased in UVBtreated cells but decreased in DQA pretreated cells (Fig. 2E). Taken together, these results indicate that DQA has a protective effect against UVB-induced oxidative cellular damage to lipids, proteins, and DNA.
UVB
Phospho-ERK, JNK, p-38
Protein carbonylation
¨ȥ
Lipid peroxidation
Bax
DNA damage
Bcl-2
3.3. The effect of DQA against apoptosis Oxidative damage
Apoptosis is triggered by UVB radiation in HaCaT cells. The effect of DQA against UVB-induced cell death is shown in Fig. 3A. We confirmed that UVB-induced apoptotic bodies among HaCaT cells were significantly reduced by DQA (Fig. 3B). Given that apoptosis produces changes in mitochondrial membrane potential, we further confirmed the antiapoptotic effect of DQA using fluorescence dye JC-1 staining to examine mitochondrial membrane potential. As shown in Fig. 3C, fluorescence was increased in UVB-irradiated cells compared with controls, and DQA reduced UVB-induced fluorescence (Fig. 3C). Furthermore, confocal microscopy analysis confirmed that UVB-induced depolarization was reduced by DQA pretreatment (Fig. 3D). Examining the expression of apoptosis-related proteins we found that expression of Bax, the pro-apoptotic protein, was increased by UVB, and restored nearly to control levels in cells treated with both UVB and DQA. Irradiation with UVB decreased expression of the anti-apoptotic protein Bcl-2, and this was partially restored by the DQA. Given that induction of intracellular ROS leads to activation of the caspase-mediated apoptotic pathway (Wang et al., 2016), we also assayed the protein levels of active (cleaved) forms of caspase-9, caspase-3, and poly ADP ribose polymerase (PARP) (Fig. 3E). These were increased in UVB-treated cells and significantly reduced by DQA pretreatment. To further investigate the protective effect of DQA against UVB-induced activation of the caspase-mediated apoptotic pathway, we evaluated cell viability and apoptotic bodies. The number of apoptotic bodies induced by UVB was markedly reduced by DQA, both alone and in conjunction with the caspase inhibitor Z-VAD-FMK (Fig. 3F and 3G). These results indicated that DQA acts against UVB-induced apoptosis through the mitochondrial/caspase-mediated pathway.
Cleaved caspase 9, caspase 3, PARP
Apoptotic cell death Fig. 6. Schematic diagram of protective effect of DQA. Exposure of UVB or PM2.5 increased intracellular ROS and induced oxidative stress in HaCaT cells. DQA protects against UVB or PM2.5-indeced oxidative damage. In addition, it protects against UVB-induced apoptosis through inhibiting activation MAPKs.
were collected using a FV1200 laser scanning confocal microscope (Olympus). 2.14. Statistical analysis All analyses were performed in triplicate and all values are expressed as mean ± standard error of the mean. We used Tukey’s test to determine the statistical significance of differences between means. Statistical significance was inferred where p < 0.05. 3. Results 3.1. The effect of DQA on cytotoxicity and ROS scavenging We found that DQA has no cytotoxicity to HaCaT cells at any concentration up to 20 μM (Fig. 1B). The H2O2- and UVB-induced intracellular ROS scavenging effects of DQA, by comparison to the control group, were seen at concentrations ranging from 1 to 20 μM (Fig. 1C). We found that DQA concentrations of 10 μM and 20 μM have similar intracellular ROS scavenging effects in HaCaT cells, so we decided to use 10 μM DQA for all subsequent experiments. Next, we used ESR spectrometry to measure the superoxide anion- and hydroxyl radicalscavenging abilities of DQA. We observed that superoxide anion signals (1922 value) from a xanthine/xanthine oxidase system were significantly increased compared to the control group (222 value). However, DQA treatment significantly reduced the signal to a value of 1390 (Fig. 1D). In addition, hydroxyl radical signals (3375 value) from a H2O2 + FeSO4 system were significantly increased compared to the control group (32 value), and DQA treatment significantly reduced the value to 2278 (Fig. 1E). We also detected UVB-induced intracellular ROS generation using confocal microscopy and spectrofluorometry after DCF-DA staining. As shown Fig. 1F and G, UVB-induced intracellular ROS were reduced by DQA.
3.4. DQA inhibited UVB-induced activation of mitogen-activated protein kinase (phospho-MAPK) Next, we investigated the expression levels of the apoptosis-related signaling proteins phospho-extracellular signal regulated kinase (pERK), phospho-c-Jun N-terminal kinase (p-JNK), and phospho-p38 (pp38). These were significantly increased after 15 min of UVB irradiation for p-ERK, and after 60 min for p-JNK and p-p38 (Fig. 4A). Therefore, we investigated the effect of DQA on UVB-induced activation of these MAPKs. As shown Fig. 4B, their UVB-induced expression was decreased by DQA pretreatment. To further investigate the effect of DQA on UVBinduced activation of MAPK-mediated apoptosis, we pretreated cells with specific inhibitors and observed cell viability. Pretreatment with pERK, p-JNK, or p-p38 inhibitors (U0126, SP600125, and SB203580, respectively) decreased UVB-induced apoptosis. This was decreased further with DQA pretreatment (Fig. 4C and D).
3.2. The effect of DQA on UVB-induced oxidative cellular damage
3.5. The effect of DQA on PM- and UVB-induced ROS and apoptosis
Irradiation with UVB induces lipid peroxidation, protein carbonylation and cell damage. First, we detected lipid peroxidation in HaCaT cells using confocal microscopy after DPPP treatment. As shown in
Induction of intracellular ROS and cell damage has also been shown for combined PM and UVB treatment (Soeur et al., 2017). In our HaCaT cells, PM2.5 combined with UVB generated higher levels of ROS and 439
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
via scavenging ROS, and is associated with suppression of pro-apoptotic factors including caspase and MAPKs. Furthermore, its antioxidative effects also mitigate PM2.5-induced oxidative stress (Fig. 6).
lipid peroxidation than UVB treatment alone (Fig. 5A and B). This was also true of 8-oxoG, a marker of oxidative DNA damage (Fig. 5C). In all cases DQA significantly reduced the levels of intracellular ROS, lipid peroxidation, and 8-oxoG. We also measured apoptotic bodies and cell viability in response to combined UVB and PM2.5 treatment. As shown in Fig. 5D and E, PM2.5 combined with UVB generated more damage than PM2.5 or UVB alone, and the damage was clearly reduced by DQA pretreatment.
Conflict of interest statement The authors state no conflict of interest. Ethics statement
4. Discussion This work does not use the animals or human subjects. Damage induced by UVB includes skin inflammation, edema, erythema, and sunburn, while chronic UVB exposure leads to aging and carcinogenesis in the skin (Ivan et al., 2014). These harmful effects are linked to generation of first-stage intracellular ROS, including H2O2, superoxide anions, and hydroxyl radicals, either directly or through enzyme activation (Pérez-Sánchez et al., 2016). High concentrations of intracellular ROS damage lipids, proteins, and DNA (Schieber & Chandel, 2014). In many studies, antioxidants, including plant-derived flavonoids and polyphenols, have been shown to protect against UVB-induced cell damage (Choi, Kundu, Chun, Na, & Surh, 2014; Yunfeng et al., 2016). The antioxidant DQA studied here, is an isomer of the well-known antioxidant chlorogenic acid found in coffee bean and fruits (Liang, Xue, Kennepohl, & Kitts, 2016). We previously demonstrated that chlorogenic acid protects cells from UVB-induced oxidative stress (Cha et al., 2014). It has also been shown previously that dicaffeoylquinic acids are better antioxidants than caffeoylquinic acids including chlorogenic acid (Xu, Hu, & Liu, 2012). Here we report that DQA is not cytotoxic in HaCaT cells, and is effective at scavenging ROS, including the superoxide anion and the hydroxyl radical. Using DCF-DA, we showed that DQA scavenges UVBinduced intracellular ROS. We also demonstrated the protective effect of DQA against UVB-induced lipid peroxidation, protein carbonylation, DNA breakage, and apoptosis in HaCaT cells. Examining apoptosis, we showed that the effects of DQA to increase viability in the presence of oxidative stress are consistently reflected in the levels of apoptotic protein markers. These included Bcl-2 an antiapoptotic protein, and Bax, which is pro-apoptotic. We also investigated caspase-9, caspase-3, and PARP (Jia et al., 2015; Wang et al., 2016). The levels of these factors were increased by UVB and restored by DQA pretreatment. Given that the proteins we examined regulate apoptosis through the mitochondrial pathway (Jia et al., 2015), we also confirmed the effects of UVB on mitochondrial membrane potential, using fluorescence dye JC-1 staining. Similarly to the other markers of cell damage and death we examined, this effect was blocked by DQA. The MAPK pathway is mainly activated through environmental stresses such as oxidative stress and is involved in apoptosis (Che, Ma, & Xin, 2017; Jeayeng et al., 2017). We showed that its constituent proteins p-Erk, p-JNK, and p-p38 were significantly increased 6 h after irradiation with UVB, and this effect was reduced by pretreatment with DQA. Specific inhibition of these factors reduced UVB-induced cell death, an effect which was enhanced by DQA. We therefore suggest that the protective effects of DQA are mediated by the inhibition of MAPK in UVB-induced apoptosis. Skin can be damaged by air pollution such as PM as well as by UVB. Recent studies have shown that a combination of PM and UVA induces cell damage in keratinocytes (Soeur et al., 2017). We therefore investigated intracellular ROS level, lipid peroxidation, and apoptosis following exposure to PM2.5 and UVB. The group given a combination of PM2.5 and UVB generated more intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptosis than groups given only PM2.5 or UVB. Pretreatment with DQA significantly recovered from intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptosis induced by the combination of PM2.5 and UVB. In conclusion, DQA protects against UVB-induced oxidative stress
Acknowledgement This work was supported by grant from the Basic Research Laboratory Program (NRF-2017R1A4A1014512) by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT and future Planning (MSIP) of Korea government. References Budryn, G., Zakłos-Szyda, M., Zaczyńska, D., Żyżelewicz, D., Grzelczyk, J., Zduńczyk, Z., & Juśkiewicz, J. (2017). Green and roasted coffee extracts as antioxidants in βTC3 cells with induced oxidative stress and lipid accumulation inhibitors in 3T3L1 cells, and their bioactivity in rats fed high fat diet. European Food Research and Technology, 243, 1323–1334. https://doi.org/10.1007/s00217-017-2843-0. Cha, J. W., Piao, M. J., Kim, K. C., Yao, C. W., Zheng, J., Kim, S. M., ... Hyun, J. W. (2014). The polyphenol chlorogenic acid attenuates UVB-mediated oxidative stress in human HaCaT keratinocytes. Biomolecules & Therapeutics, 22, 136–142. https://doi.org/10. 4062/biomolther.2014.006. Che, N., Ma, Y., & Xin, Y. (2017). Protective role of fucoidan in cerebral ischemia-reperfusion injury through inhibition of MAPK signaling pathway. Biomolecules & Therapeutics, 25, 272–278. https://doi.org/10.4062/biomolther.2016.098. Choi, K. S., Kundu, J. K., Chun, K. S., Na, H. K., & Surh, Y. J. (2014). Rutin inhibits UVB radiation-induced expression of COX-2 and iNOS in hairless mouse skin: p38 MAP kinase and JNK as potential targets. Archives of Biochemistry and Biophysics, 559, 38–45. https://doi.org/10.1016/j.abb.2014.05.016. He, M., Ichinose, T., Yoshida, S., Shiba, F., Arashidani, K., Takano, H., ... Shibamoto, T. (2016). Differences in allergic inflammatory responses in murine lungs: Comparison of PM2.5 and coarse PM collected during the hazy events in a Chinese city. Inhalation Toxicology, 28, 706–718. https://doi.org/10.1080/08958378.2016.1260185. Ivan, A. L., Campanini, M. Z., Martinez, R. M., Ferreira, V. S., Steffen, V. S., Vicentini, F. T., ... Casagrande, R. (2014). Pyrrolidine dithiocarbamate inhibits UVB-induced skin inflammation and oxidative stress in hairless mice and exhibits antioxidant activity in vitro. Journal of Photochemistry and Photobiology B, Biology, 138, 124–133. https:// doi.org/10.1016/j.jphotobiol.2014.05.010. Jeayeng, S., Wongkajornsilp, A., Slominski, A. T., Jirawatnotai, S., Sampattavanich, S., & Panich, U. (2017). Nrf2 in keratinocytes modulates UVB-induced DNA damage and apoptosis in melanocytes through MAPK signaling. Free Radical Biology & Medicine, 108, 918–928. https://doi.org/10.1016/j.freeradbiomed.2017.05.009. Jia, G., Wang, Q., Wang, R., Deng, D., Xue, L., Shao, N., ... Yang, Y. (2015). Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway. OncoTargets and Therapy, 8, 303–311. https://doi.org/10. 2147/OTT.S76063. Jin, S. P., Li, Z., Choi, E. K., Lee, S., Kim, Y. K., Seo, E. Y., ... Cho, S. (2018). Urban particulate matter in air pollution penetrates into the barrier-disrupted skin and produces ROS-dependent cutaneous inflammatory response in vivo. Journal of Dermatological Science, 91, 175–183. https://doi.org/10.1016/j.jdermsci.2018.04. 015. Kim, K. E., Cho, D., & Park, H. J. (2016). Air pollution and skin diseases: Adverse effects of airborne particulate matter on various skin diseases. Life Sciences, 152, 126–134. https://doi.org/10.1016/j.lfs.2016.03.039. Kim, T. W., Jo, C., Kim, H. S., Park, Y. M., Wu, Y. X., Cho, J. H., & Kim, T. H. (2016). Chemical constituents from Ainsliaea acerifolia as potential anti-obesity agents. Phytochemistry Letters, 16, 146–151. https://doi.org/10.1016/j.phytol.2016.04.005. Kimura, S., Inoguchi, T., Yamasaki, T., Yamato, M., Ide, M., Sonoda, N., ... Takayanagi, R. (2016). A novel DPP-4 inhibitor teneligliptin scavenges hydroxyl radicals: In vitro study evaluated by electron spin resonance spectroscopy and in vivo study using DPP4 deficient rats. Metabolism, 65, 138–145. https://doi.org/10.1016/j.metabol.2015. 10.030. Krutmann, J., Liu, W., Li, L., Pan, X., Crawford, M., Sore, G., & Seite, S. (2014). Pollution and skin: From epidemiological and mechanistic studies to clinical implications. Journal of Dermatological Science, 76, 163–168. Liang, N., Xue, W., Kennepohl, P., & Kitts, D. D. (2016). Interactions between major chlorogenic acid isomers and chemical changes in coffee brew that affect antioxidant activities. Food Chemistry, 213, 251–259. https://doi.org/10.1016/j.foodchem.2016. 06.041. Liang, N., & Kitts, D. D. (2015). Role of chlorogenic acids in controlling oxidative and inflammatory stress conditions. Nutrients, 8, 16. https://doi.org/10.3390/
440
Journal of Functional Foods 52 (2019) 430–441
Y.J. Hyun et al.
subcellular organelle dysfunction, and apoptosis. Archives of Toxicology, 92, 2077–2091. https://doi.org/10.1007/s00204-018-2197-9. Schieber, M., & Chandel, N. S. (2014). ROS function in redox signaling and oxidative stress. Current Biology, 24, R453–R462. https://doi.org/10.1016/j.cub.2014.03.034. Soeur, J., Belaïdi, J. P., Chollet, C., Denat, L., Dimitrov, A., Jones, C., ... Marrot, L. (2017). Photo-pollution stress in skin: Traces of pollutants (PAH and particulate matter) impair redox homeostasis in keratinocytes exposed to UVA1. Journal of Dermatological Science, 86, 162–169. https://doi.org/10.1016/j.jdermsci.2017.01.007. Yunfeng, H., Yuetang, M., Shi, W., Tianfeng, C., Yong, H., Jianxia, S., ... Weibin, B. (2016). Protective effect of cyanidin-3-O-glucoside against ultraviolet B radiationinduced cell damage in human HaCaT keratinocytes. Frontiers in Pharmacology, 7, 301. https://doi.org/10.3389/fphar.2016.00301. Wang, D., Qu, X., Zhuang, X., Geng, G., Hou, J., Xu, N., ... Chen, Y. S. (2016). Seed oil of Brucea javanica induces cell cycle arrest and apoptosis via reactive oxygen speciesmediated mitochondrial dysfunction in human lung cancer cells. Nutrition and Cancer, 68, 1394–1403. https://doi.org/10.1080/01635581.2016.1224362. Xu, J. G., Hu, Q. P., & Liu, Y. (2012). Antioxidant and DNA-protective activities of chlorogenic acid isomers. Journal of Agricultural and Food Chemistry, 60, 11625–11630. https://doi.org/10.1021/jf303771s.
nu8010016. Lin, Z. C., Lee, C. W., Tsai, M. H., Ko, H. H., Fang, J. Y., Chiang, Y. C., ... Yen, F. L. (2016). Eupafolin nanoparticles protect HaCaT keratinocytes from particulate matter-induced inflammation and oxidative stress. International Journal of Nanomedicine, 11, 3907–13626. https://doi.org/10.2147/IJN.S109062. Namas, R., Renauer, P., Ognenovski, M., Tsou, P. S., & Sawalha, A. H. (2016). Histone H2AX phosphorylation as a measure of DNA double-strand breaks and a marker of environmental stress and disease activity in lupus. Lupus Science & Medicine, 3, e000148. https://doi.org/10.1136/lupus-2016-000148. Park, M. A., Sim, M. J., & Kim, Y. C. (2017). Anti-photoaging effects of angelica acutiloba root ethanol extract in human dermal fibroblasts. Toxicological Research, 33, 125–134. https://doi.org/10.5487/TR.2017.33.2.125. Pérez-Sánchez, A., Barrajón-Catalán, E., Herranz-López, M., Castillo, J., & Micol, V. (2016). Lemon balm extract (Melissa officinalis, L.) promotes melanogenesis and prevents UVB-induced oxidative stress and DNA damage in a skin cell model. Journal of Dermatological Science, 84, 169–177. https://doi.org/10.1016/j.jdermsci.2016.08. 004. Piao, M. J., Ahn, M. J., Kang, K. A., Ryu, Y. S., Hyun, Y. J., Shilnikova, K., ... Hyun, J. W. (2018). Particulate matter 2.5 damages skin cells by inducing oxidative stress,
441