1
2
3
4
5
6
7
8
9
10
11
Fisetin ameliorated photodamage by suppressing the MAP kinase/MMP
pathway and NF-κB pathways
12
13
14
Department of Cosmeceutics, China Medical University, Taichung, Taiwan
Hsiu-Mei Chiang *, Shih-Yun Chan, Yin Chu, Kuo-Ching Wen *
15
16
17
18
19
20
Correspondence to:
Professor Kuo-Ching Wen
21
22
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24
25
26
27
28
29
30
Department of Cosmeceutics, China Medical University, Taichung, Taiwan 404
E-mail: kcwen0520@mail.cmu.edu.tw, Telephone: 886-4-22053366 ext. 5302
Fax: 886-4-22078083
Associate Professor Hsiu-Mei Chiang
Department of Cosmeceutics, China Medical University, Taichung, Taiwan 404
E-mail: hmchiang@mail.cmu.edu.tw, Telephone: 886-4-22053366 ext. 5302
Fax: 886-4-22078083
31
32
33
34
35
36
37
Abbreviations: AP-1, activator protein-1; COX-2; cyclooxygenase-2;CREB, cAMP
response element-binding protein; ECM, extracellular matrix; IκB, inhibitor κB;
iNOS, inducible nitrite oxide synthase; MMP, matrix metalloproteinase; MAP kinase,
mitogen-activated protein kinase; NO, nitric oxide; PG, prostaglandins; UV,
ultraviolet.
1
38
ABSTRACT
39
Ultraviolet (UV) irradiation is one of the most important extrinsic factors
40
contributing to skin photodamage. After UV irradiation, a series of signal transduction
41
in skin will be activated, and leads to inflammatory response and photoaged skin. In
42
this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for
43
its photoprotective effects. The results revealed that 5-25 μM of fisetin inhibits
44
cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, -3, -9 expression
45
induced by ultraviolet B (UVB) irradiation in human fibroblasts. In addition, fisetin
46
suppressed UVB-induced collagen degradation. Regarding its effect on upper-stream
47
signal transduction, we found that fisetin reduced the expression of ultraviolet
48
(UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein
49
kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB)
50
degradation and increased the amount of p65, which is a major subunit of NF-κB, in
51
cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP
52
response element-binding protein (CREB) Ser-133 phosphorylation level in the
53
phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway.
54
Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS),
55
prostaglandin E2 (PGE2) and nitric oxide (NO) generation. The mentioned effects and
56
mechanisms suggest that fisetin can be used in the development of photoprotective
57
agents.
2
58
KEY WORDS: fisetin, photodamage, matrix metalloproteinase, MAP kinase, NF-κB,
59
CREB
3
60
INTRODUCTION
61
Ultraviolet (UV) irradiation is one of the most noxious environmental hazards
62
and can induce inflammation and oxidative stress in human skin. UV irradiation that
63
induces skin damage principally manifests as a degradation of extracellular matrix
64
(ECM) proteins, including collagen, elastin, proteoglycans, and fibronectin, which are
65
the main building blocks of the skin. 1, 2 Collagen, the most abundant ECM protein in
66
the dermis, is derived from dermal fibroblasts and regulated by mitogen-activated
67
protein (MAP) kinase. MAP kinase induces activator protein-1 (AP-1; a transcription
68
factor) and promotes collagen breakdown by upregulating enzymes called matrix
69
metalloproteinases (MMPs), especially MMP-1, the major collagen-degrading
70
enzyme in the skin. 3 MMPs are a family of structurally related matrix-degrading
71
enzymes that play crucial roles in various destructive processes, including skin aging
72
and photo-damage. MMP-1, also known as interstitial collagenase, initiates the
73
degradation of collagen types I, II, and III in the skin. 4 In addition, MMP-1, MMP-3,
74
and MMP-9 play vital roles in photodamage by degrading ECM in the dermis. 3, 5, 6In
75
addition to regulating MMPs and collagen, excessive UV irradiation can cause acute
76
skin inflammation and lead to the development of skin cancer. Prostaglandins (PGs)
77
and nitric oxide (NO) play crucial roles in the inflammatory process. 1,7 UVB induces
78
cyclooxygenase-2 (COX-2) production, which is the rate-limiting enzyme in PGs
4
79
generation. 8 UVB irradiation induces erythema in the skin (sunburn), which is related
80
to nitric oxide synthase (NOS) and the up-expression of cyclooxygenase (COX). UV
81
irradiation upregulates the expression of inducible nitric oxide synthase (iNOS) to
82
generate NO, which reacts with superoxide. This leads to the production of
83
peroxynitrite and other reactive oxygen species (ROS), 9 which up-regulate COX-2
84
expression to stimulate the inflammation process.
85
(including ERK, JNK, and p38) activation, which transfer c-Fos and c-Jun to the
86
nucleus, induces NF-κB activation, and subsequently upregulates the gene related to
87
pro-inflammatory reactions.
88
complex with an inhibitor κB (IκB), and the degradation of IκB causes translocation
89
of NF-κB to the nucleus. UV irradiation activates NF-κB, which increases MMP-1 in
90
the dermis. 14 UV irradiation is absorbed by skin molecules, which results in ROS
91
generation and, subsequently, causes oxidative stress to cellular components
92
involving cell walls, lipid membranes, mitochondria, and DNA. 4, 15
11-13
10
ROS drive MAP kinase
NF-κB existed in the cytoplasm as an inactive
93
Fisetin (3,7,3’,4’-tetrahydroxyflavone) (Figure 1) is a bioactive flavonol
94
molecule that exists in fruits and vegetables such as strawberries, apples, grapes, and
95
onions. 16 It has been reported that fisetin exhibits neuroprotective, anti-tumor, 17, 18
96
anti-oxidative,
97
attenuate inflammation through the reduction of COX-2, iNOS, and NO levels in
19
and anti-inflammatory activities.
5
20,21
In addition, fisetin can
98
RAW 264.7 cells and mice. 20, 22 Fisetin possesses chemotherapeutic potential against
99
human epidermoid carcinoma A431 cells and may be developed as treatment for
23
100
non-melanoma skin cancers.
It has been reported that a wide variety of new
101
cosmeceuticals and formulas can accelerate collagen synthesis, prevent aging and
102
photoaging, and facilitate the reparation of wrinkles in the skin. 24 Development of
103
MMP inhibitors is a potential strategy for photo-aging therapy. 5, 25, 26 In literature, it
104
had been reported that fisetin inhibited UV-induced ROS and activation of NF-κB and
105
MAP kinase in lens epithelial cells. 27 However, few studies exist on the effect of
106
fisetin against UV-induced photodamage in human skin and the related mechanisms.
107
Therefore, this study investigates the potential mechanisms by which fisetin
108
counteracts UVB-induced overexpression of MMPs/MAP kinases, COX-2, AP-1, and
109
MMPs; the degradation of IκB and translocation of NF-κB; and the reduction in type I
110
procollagen levels in human skin fibroblasts.
111
112
6
113
MATERIALS AND METHODS
114
Materials.
115
Fisetin, DL-dithiothreitol, phenylmethylsulfonyl fluoride (PMSF), and Triton
116
X-100 were purchased from Sigma-Aldrich Chemicals Corporation (St. Louis, MO,
117
USA). Collagenase was purchased from Calbiochem, Merck (Darmstadt, Germany).
118
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS),
119
penicillin-streptomycin, and trypsin-EDTA were purchased from Gibco, Invitrogen
120
(Carlsbad, CA, USA). Coomassie blue R-250, dibasic sodium phosphate, lgepalTM
121
CA-630, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and
122
tris and sodium dodecyl sulfate (SDS) were purchased from USB Corporation
123
(Cleveland, OH, USA). C-Jun, phosphor-c-Jun, NF-κB p65, and IκB antibodies were
124
purchased from Cell Signaling Technology (Beverly, MA, USA). Donkey anti-goat
125
p38, p-p38, IgG-HRP, CREB, phosphor-CREB, ERK1, p-ERK 1/2, JNK1, p-JNK,
126
MMP-1, MMP-3 and MMP-9 antibodies were purchased from Santa Cruz
127
Biotechnology, Inc. (Santa Cruz, CA, USA).
128
Cell Culture.
129
Human foreskin fibroblasts (Hs68) were purchased from the Bioresources
130
Collection and Research Center, Food Industry Research and Development Institute,
131
Hsinchu, Taiwan. The cells were cultured in DMEM, which contained 10% FBS, 100
7
132
U/mL of penicillin, and 100 U/mL of streptomycin, and were maintained at 37°C in a
133
humidified, 5% CO2 incubator.
134
UVB Irradiation Dose.
135
Cells were washed with phosphate-buffered saline (PBS) and covered with PBS
136
for UV irradiation by using a UV lighter (302 nm, CL-1000M, UVP, Upland, CA,
137
USA). The UVB irradiation dose was 40 mJ/cm2 (exposure time was 15 seconds)
138
according to the previous studies. 6, 28 This dose equates to about 23 seconds exposure
139
at the noontime on July in Middle Taiwan measured by a UV meter (UVP, Upland,
140
CA, USA). At this UV dose, the cell viability does not reduce considerably, whereas
141
the protein expression is induced. After UVB irradiation, PBS was replaced with a
142
serum-free medium and then incubated for 24 h for the MTT and various assays.
143
Cell Viability Test.
144
An MTT assay was performed to examine the viability of the cells as previously
145
studies described. 25, 29 After being irradiated with UVB, the cells were incubated with
146
various concentrations of fisetin for 24 h, and the culture medium was replaced with
147
MTT solution (0.5 mg/mL) and incubated. The MTT solution was then removed and
148
10% SDS-HCl was added. The absorbance of the dissolved formazan crystals was
149
then determined at 570 nm by using a spectrophotometer (Tecan, Grodig, Austria).
150
Measuring Intracellular ROS.
8
151
Intracellular ROS generation was measured by using a DCFH-DA fluorescence
152
dye assay as a previous study described with slightly modification. 6, 30 Fibroblasts
153
were seeded into 24-well plates and irradiated with UVB. Subsequently, various
154
concentrations of fisetin (5, 10, and 25 μM) were added and then incubated for 2 h.
155
The cells were incubated with 10 μM DCFHDA for 30 min. The fluorescence
156
intensity was determined at 488 nm excitation and 520 nm emission by using a
157
fluorescence microplate reader (Thermo Electron Corporation, Ratastie, Vantaa,
158
Finland), and images were observed under a fluorescence microscope (Leica DMIL,
159
Wetzla, Germany).
160
Measurement of Total Collagen Synthesis.
161
Total collagen synthesis in fibroblasts after UVB exposure was measured using
162
the SircolTM soluble collagen assay kit and modified based on a previous study. 6, 25
163
Briefly, a cell culture medium was collected and mixed with Sircol dye reagent and
164
incubated. After centrifugation, ice-cold acid-salt washing reagent was added to the
165
precipitate and then the mixture was centrifuged. The precipitate was dissolved using
166
alkali reagent and the absorbance was determined at 555 nm by using an ELISA
167
reader (Tecan, Grodig, Austria).
168
Western Blot Analysis.
169
The cells were harvested at the indicated time and washed twice with ice-cold
9
170
PBS for western blot analysis. The cells were lysed in a cold lysis buffer, the lysates
171
were centrifuged, and an aliquot of the lysate was used to determine the protein
172
content by conducting a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).
173
Equal amounts of proteins (30 μg) were separated using SDS-polyacrylamide gel
174
(SDS-PAGE) electrophoresis and blotted onto polyvinyl difluoride membranes. The
175
membranes were blocked with non-fat milk in a TBST buffer and then incubated
176
overnight with specific antibodies. These were goat polyclonal antibodies, which are
177
used against MMP-1 and type I procollagen, and mouse polyclonal antibodies, which
178
are used against phosphor-CREB, CREB, MMP-3, MMP-9, ERK, p-ERK, JNK,
179
p-JNK, p38, p-p38, c-Jun, phosphor-c-Jun, and NF-κB p65. The membranes were
180
incubated with the corresponding horseradish peroxidase-conjugated secondary
181
antibody after wash with PBS buffer. Immunoreactive proteins were detected using
182
the ECL western blotting detection system (Fujifilm, LAS-4000, Tokyo, Japan), and
183
the signal intensity of each band was quantified using a densitometer system (multi
184
Gauge V2.2) and then normalized with the internal control (actin).
185
Immunofluorescence Staining.
186
For immunofluorescence staining, 1 × 105 cells were cultivated on glass
187
coverslips overnight. The medium was removed and the cells were washed twice with
188
a PBS buffer. The cells were fixed with 4% paraformaldehyde in PBS for 30 min. The
10
189
fixed cells were blocked with 5% non-fat milk with 0.3% Triton X-100/PBS-buffer
190
for 40 min and incubated overnight, separately, with the primary antibody. After being
191
washed, cells were incubated with the Alexa Fluor 488 anti-rabbit IgG secondary
192
antibody for 2 h (Invitrogen, Carlsbad, CA, USA). The unbound secondary antibody
193
was removed by washing the cells 3 times with a PBS buffer. Thereafter, the samples
194
were counterstained with ProLong® Gold antifade reagent and DAPI and observed
195
using a microscope.
196
NO Measurement.
197
The measurement of NO generation in human skin fibroblasts was followed
198
previous study with slight modification. 25 Cells were grown in 6-well plate for 24 h
199
before treatment. After UVB irradiation, cells were treated with different
200
concentrations of fisetin for 24 h. Cell culture medium was then collected and mixed
201
with Griess reagent (Promega, Madison, WI, USA), the absorbance of azo compound
202
was determined at 540 nm by using a spectrophotometer (Tecan, Grodig, Austria).
203
Quantitation of PGE2.
204
PGE2 levels in cultured medium were determined using the protocol provided by
205
the kit’s manufacturer (Cayman, Ann Arbor, MI, USA). The cell culture and
206
treatments were as NO measurement described.
207
208
Statistical Analysis.
11
209
All measurements in the present study were obtained as averages of experiments
210
that were at least three independent experiments performed in triplicate and are
211
expressed as means ± the standard deviation (SD). Differences between groups in
212
experiments were analyzed for statistical significance by using ANOVA with LSD
213
post hoc tests or the Student’s t test. P < 0.05 was considered statistically significant.
214
215
12
216
RESULTS and DICUSSION
217
Effect of Fisetin on the Cell Viability and UVB-Induced Phototoxicity.
218
Safety is the primary criterion for skin-care products. Hs68 cells were treated
219
with various concentrations of fisetin and cell viability was measured using the MTT
220
assay. The resulting survival curve indicated that fisetin (5-50 μM) did not exhibit
221
cytotoxic effects on the proliferation of cells (Figure 2). After UVB irradiation (40
222
mJ/cm2), the cell viability was significantly decreased to 82.7 ± 1.9% (P < 0.001).
223
Fifteen to twenty five micro molar fisetin did not significantly influence the cell
224
viability of fibroblasts after UVB irradiation, but decreased the cell viability to 64.8 ±
225
0.9% at 50 μM (Figure 2). Fisetin does not recover UVB-induced fibroblast mortality;
226
indeed, the highest dose of fisetin (50 μM) even increases UVB damage. Therefore,
227
5-25 μM of fisetin was used in the study of its anti-photoaging activity and
228
mechanism.
229
Fisetin Reduced UVB-Induced Intracellular ROS.
230
DCFDA staining and fluorescence microscopy were used to qualitatively
231
characterize the degree of ROS generation. ROS are potential inducers of
232
skin-photoaging-related proteins and cause intracellular oxidative damage in human
233
skin fibroblasts. Fibroblasts were exposed to UVB and then treated with various
234
concentrations of fisetin for 2 h in a 24-well plate. After removing the
235
fisetin-containing medium, the cells were washed with PBS and incubated with 10
13
236
μM of DCFDA for 30 min. In this study, treatment with UVB significantly increased
237
ROS generation 1.4-fold compared with control cells, whereas treatment with 5, 10,
238
and 25 μM of fisetin reduced UVB-induced ROS generation to 0.9-, 0.8-, and 0.7-fold,
239
respectively (Figure 3). The results of fluorescence and immunofluorence staining
240
indicate that fisetin protected Hs68 cells from the damage of UVB-induced ROS.
241
UV irradiation induced ROS generation in living organisms, causing oxidative
242
stress, especially when ROS is not scavenged by an antioxidant defense system.
243
Antioxidants such as N-acetyl cysteine scavenged ROS to protect skin from
244
UVB-induced oxidative stress and cell death. 31, 32 In this study, fisetin suppressed
245
UV-induced intracellular ROS formation protecting skin cells from oxidative damage
246
and
247
1,1-diphenyl-2-picrylhydrazyl radical scavenging activity.33 It has been reported that
248
the presence of a catechol group in the C-2 position, and the number of hydroxyl
249
substituents and their location in the molecule (especially at C-3, C-5, and/or C-7), are
250
determinant structural factors for their ability to scavenge free radicals. 33, 34 Fisetin,
251
which has C-2 catechol and hydroxyl groups at C3 and C7, is an excellent free-radical
252
scavenger. Therefore, its protective effect on skin against UV damage may contribute
253
to its ability to scavenge UV-induced radical generation.
254
The Anti-Inflammatory Effect of Fisetin.
related
diseases.
In
a
previous
14
study,
fisetin
exhibited
255
Fisetin on UVB-Induced COX-2 Expression.
256
The levels of COX-2 were 8.31-fold higher in fibroblasts exposed to UVB (40
257
mJ/cm2) than in control cells (Figure 4 (a)). In addition, fisetin (5, 10, 25 μM)
258
exhibited a dose-dependent reduction in UVB-induced COX-2 expression and a
259
reduction of the UVB-induced expression of COX-2, which were 3.57-, 2.94-, and
260
1.06-fold compared with the control, respectively. The effect was significant when the
261
dose was higher than 5 μM (Figure 4 (a)). EGCG (1 μM; positive control)
262
significantly suppressed UVB-induced COX-2 expression (from 8.31-fold to
263
2.66-fold of the control).
264
Fisetin on UVB-Induced iNOS Expression.
265
The result of fisetin on iNOS expression in fibroblasts is shown in Figure 4 (b).
266
iNOS expression was increased to 1.6-fold after exposure to UVB; however, fisetin
267
treatment (5, 10, and 25 μM) did not considerably reduce the levels of iNOS.
268
Effect of Fisetin on NO and PGE2 Generation.
269
As shown in Figure 5 (a), UVB exposure induced NO to 3-fold of control and
270
fisetin treatment reduced UVB-induced NO generation in human skin fibroblasts.
271
UVB elevated PGE2 in human skin fibroblasts and fisetin at 10 and 25 μM would
272
significant reduce PGE2 production induced by UVB (Figure 5 (b)).
273
UV irradiation leads to direct or indirect DNA damage and the formation of
15
274
ROS, which induces an inflammatory response (induced COX-2 and iNOS) and
275
damages the integrity of the extracellular matrix.
276
fisetin possesses anti-inflammatory activities by inhibiting UVB-induced PGE2 and
277
NO. The results of this study indicated that fisetin reduced UVB-induced COX-2
278
expression, but not iNOS. The suppressive effect on COX-2 induction might be an
279
independent process or an indirect phenomenon via reduced NO production. In
280
addition, NO was controversially reported to affect the level of COX activity and/or
281
COX-2 expression.
282
COX-2 activity and/or expression, for example, wogonin, which was a direct COX-2
283
inhibitor but not an iNOS inhibitor. 37 Therefore, fisetin might direct inhibit COX-2,
284
but not iNOS. The effect mechanism of fisetin on iNOS and COX-2 needs further
285
study.
286
Effects of Fisetin on UVB-Induced Photo-Damage and Photo-Damage Related
287
Protein Expression.
288
Fisetin Attenuated UVB-Induced Reduction in Total Collagen Synthesis.
36
35
Our studies have shown that
It had been reported that, flavonoids might directly affect
289
Fibroblasts were pretreated with fisetin (5-25 μM) for 1 h, exposed to UVB, and
290
then treated with fisetin for 24 h. As shown in Figure 6 (a), fisetin treatment resulted
291
in a dose-dependent restoration of collagen and fisetin at 25 μM would significant
292
increase total collagen synthesis.
16
293
Effect of Fisetin on MMPs Expression
294
To examine the anti-damage effects of fisetin on UVB-irradiated human skin
295
fibroblasts, we measured cellular MMP-1, -3, and -9 protein expressions. As Figures 6
296
(b) shown, UVB caused significant elevation of MMP-1, -3, and -9 protein expression
297
(1.33-, 1.42-, and 1.53-fold compared with the control, respectively), whereas fisetin
298
attenuated MMP expression. As shown in Figure 6 (b), the UVB-induced MMP-1
299
expression was reduced to 0.64-, 0.51-, and 0.42-fold when using 5, 10, and 25 μM of
300
fisetin, respectively, and 1-μM EGCG reduced MMP-1 0.54-fold compared with the
301
control. Fisetin (5, 10, and 25 μM) suppressed UVB-induced MMP-3 expression
302
1.22-, 1.19-, and 0.92-fold compared with the control, respectively, and EGCG
303
decreased 0.93-fold (Figure 6 (b)). Furthermore, fisetin (5, 10, and 25 μM) inhibited
304
UVB-induced MMP-9 expression 1.41-, 1.38-, and 1.43-fold, respectively, and EGCG
305
decreased 0.8-fold compared with the control (Figure 6 (b)). These results provide
306
evidence that fisetin prevents the UVB-induced elevation of MMP-1, -3 and -9 levels,
307
thus protecting UVB-induced skin damage and photoaging.
308
Effect of Fisetin on MAP Kinases Phosphorylation.
309
In Figure 6 (c), the phosphorylation of ERK was increased after UVB irradiation,
310
and pretreatment with a high dose (25 μM) of fisetin diminished the effect (from
311
2.2-fold to 1.7-fold compared with the control), but pretreatment with lower doses (5
17
312
and 10 μM) did not. The results of the phosphorylation of JNK resembled those of the
313
phosphorylation of ERK, whereas fisetin (10 and 25 μM) significantly reduced the
314
phosphorylation of p-38 expression (Figure 6 (c)).
315
Effect of Fisetin on c-Jun and p-c-Jun Expression.
316
As Figure 7 (a) shows, UV induced p-c-Jun expression (3.28-fold compared with
317
the control), whereas fisetin treatment reduced c-Jun phosphorylation level in a
318
dose-dependent manner. C-Jun expression was increased after UV irradiation,
319
whereas fisetin inhibited the effect at 25 μM, but not at 5 and 10 μM.
320
Effect of Fisetin on the IκB /NF-κB Pathway.
321
Western blot analysis revealed that the protein levels of IκB were inhibited after
322
the cells were treated with UVB irradiation, whereas fisetin significantly increased
323
IκB expression in a dose-dependent manner (Figure 7 (b)). In addition, the level of the
324
NF-κB subunit, p65, was reduced 0.66-fold compared with control in cytoplasma,
325
whereas fisetin treatment significantly increased the expression of p65 in a
326
dose-dependent manner (Figure 7 (b)).
327
Immunohistochemistry Assay of NF-κB.
328
The immunohistochemistry staining assay of NF-κB was conducted in fibroblast
329
cells to determine the degree of NF-κB activation. As shown in Figure 7 (c), UVB
330
induced the translocation of NF-κB to the nucleus, whereas fisetin suppressed the
18
331
translocation of NF-κB induced by UVB.
332
Effect of Fisetin on the PI3K/AKT/CREB Pathway.
333
Treatment of skin fibroblasts with UVB irradiation resulted in a 2.21-fold
334
increase in p-CREB Ser-133 levels (Figure 8). This showed the intense effect of UVB
335
irradiation on the content of p-CREB Ser-133 in skin fibroblasts. This increase in
336
p-CREB Ser-133 expression was significantly suppressed by fisetin treatment at 25
337
μM (P < 0.001). CREB expression was unchanged after either UVB or fisetin
338
treatment, and, similarly, was obtained after co-treatment of these two factors.
339
UV irradiation has been shown to upregulate the expression of iNOS, producing
340
NO, and to react with superoxide, resulting in peroxynitrite and other ROS formations.
341
11
342
of IκB, and then degradation, subsequently activating NF-κB and inducing COX-2
343
expression.
344
pathways, leading to upregulation of CREB Ser-133 and the subsequent
345
transcriptional activation of the COX-2 gene 40. In previous studies, the activation of
346
p-38 resulted in AP-1 transcription, NF-κB expression and activation in the
347
cytoplasma, and p-CREB Ser-133 expression. 41 Results from this study indicated that
348
fisetin attenuated the UV-induced overexpression of COX-2, p-CREB Ser-133, and
349
MAP kinases, and restored UV-induced IκB degradation, resulting in NF-κB
These ROS then induce an MAP kinase transcription factor, AP-1, phosphorylation
38, 39
Furthermore, UV exposure activates MAP kinase and PI3K⁄AKT
19
350
down-regulation. In addition, fisetin inhibited UVB-induced NF-κB activation by
351
inhibiting the translocation of NF-κB into the nucleus. Fisetin also inhibited
352
UVB-induced NO and PGE2 production. The inhibition of the phosphorylation of
353
p-38, NF-κB transcription, and the reduction of p-CREB Ser-133 by fisetin may
354
contribute to the suppression of COX-2 expression. Inhibition of the MAP kinase
355
pathway, therefore, prevents the phosphorylation of ERK, thereby enhancing the
356
expression of type I procollagen.
357
The scavenging of ROS by fisetin may lead to a blockage of the MAP kinase
358
pathway, which, in turn, may inhibit the activation of NF-κB and AP-1 and, therefore,
359
inhibit the expression of MMP-1, -3, -9 and COX-2. MMPs play a vital role in
360
UV-induced skin aging and degrade ECM in the dermis, resulting in the structural
361
dysfunction of skin. MMP-1 is a major enzyme that degrades collagen, MMP-3
362
activates proMMP-1, and MMP-9 degrades collagen fragments already degraded by
363
MMP-1. 3 It has been shown that MMP-1 is expressed when the skin is exposed to UV,
364
which induces a decrease of collagen and wrinkle formation.5,
365
fisetin-inhibited MMP-1, -3, and -9 attenuated collagen degradation, thereby protect
366
skin from aging and photo-aging. Regarding MMP expression, MAP kinase signaling
367
cascades activate AP-1, a transcription factor and a complex containing c-Fos and
368
c-Jun, and activate AP-1-dependent gene expression. The results indicate that fisetin
20
6, 42
Therefore,
369
suppressed the overexpression of UVB-induced MAP kinase/AP-1/MMPs and
370
increased collagen content in human fibroblasts.
371
In summary, our results demonstrate that fisetin attenuated UVB-induced
372
oxidative stress, photodamage, and inflammation by modulating the expression of
373
MMPs, MAP kinases, AP-1, COX-2, and p-CREB Ser-133 (Figure 9). In addition,
374
fisetin effectively restored UV-induced IκB degradation, resulting in NF-κB
375
inhibition. Therefore, fisetin is a potential agent used on UV-induced skin damage.
376
377
ACKNOWLEDGMENTS
378
This
379
(NSC99-2320-B-039-012-MY3, NSC99-2622-B-039-001-CC3), Taipei, Taiwan and
380
China Medical University (CMU99-S-39), Taichung, Taiwan.
study
was
sponsored
by
the
National
381
382
CONFLICTS OF INTEREST
383
The authors declare no conflicts of interest in this work.
384
21
Science
Council
385
386
387
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Biochem. Pharmacol. 2001, 61, 1195-203.
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39. Cooper, S. J.; Bowden, G. T., Ultraviolet B regulation of transcription factor
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40. Rundhaug, J. E.; Fischer, S. M., Cyclo-oxygenase-2 plays a critical role in
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UV-induced skin carcinogenesis. Photochem. Photobiol. 2008, 84, 322-9.
41. Terazawa, S.; Nakajima, H.; Shingo, M.; Niwano, T.; Imokawa, G., Astaxanthin
attenuates the UVB-induced secretion of prostaglandin E2 and interleukin-8 in human
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518
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42. Kim, S.; Chung, J. H., Berberine prevents UV-induced MMP-1 and reduction of
520
521
522
type I procollagen expression in human dermal fibroblasts. Phytomedicine 2008, 15,
749-53.
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524
25
525
Figure Legends
526
Figure 1. Structure of fisetin (3,7,3’,4’-tetrahydroxyflavone).
527
Figure 2. Cell viability of fisetin with or without UVB exposure on human fibroblasts.
528
Significant difference versus control: ###, P < 0.001; Significant difference
529
versus UV-exposed control: ***, P < 0.001.
530
Figure 3. Repressive effect of fisetin on UVB-induced intracellular oxidative stress in
531
human fibroblasts. Groups sharing the same superscript letter are not
532
significantly different (p > 0.05) as reveaed by LSD post hoc tests.
533
Figure 4. (a) Effect of fisetin on the UV-induced expression of COX-2 in human
534
fibroblasts. (b) Effect of fisetin on the UVB-induced expression of iNOS in
535
human fibroblasts. Groups sharing the same superscript letter are not
536
significantly different (p > 0.05) as reveaed by LSD post hoc tests.
537
Figure 5. (a) Effect of fisetin on NO generation in human skin fibroblasts. Human
538
fibroblasts were treated with/without UVB 40 mJ/cm2 and fisetin of 5, 10, 25
539
µM. Groups sharing the same superscript letter are not significantly different
540
(p > 0.05) as reveaed by LSD post hoc tests. (b) Effect of fisetin on PGE2
541
generation in human skin fibroblasts. Human fibroblasts were treated
542
with/without UVB 40 mJ/cm2 and fisetin of 5, 10, 25 µM. Groups sharing the
543
same superscript letter are not significantly different (p > 0.05) as reveaed by
26
544
LSD post hoc tests.
545
Figure 6. (a) Effect of fisetin on the UV suppressed total collagen synthesis in human
546
fibroblasts. (b) Effect of fisetin on the UVB-induced expression of MMP-1,
547
MMP-3 and MMP-9 in human fibroblasts. (c) Effect of fisetin on the
548
UVB-induced ERK, JNK, p38 phosphorylation in human fibroblasts. Groups
549
sharing the same superscript letter are not significantly different (p > 0.05) as
550
reveaed by LSD post hoc tests.
551
Figure 7. (a) Effect of fisetin on the UVB-induced c-Jun and c-Jun phosphorylation in
552
human fibroblasts. (b) Effect of fisetin on the UVB-induced IκB degradation
553
and the amount of NF-κB/p65 in cytoplasm. Groups sharing the same
554
superscript letter are not significantly different (p > 0.05) as reveaed by LSD
555
post hoc tests. (c) Fisetin inhibits UVB-induced NF κB activation by FITC
556
immunofluorescence labeling.
557
Figure 8. Effect of fisetin on the UVB-induced CREB phosphorylation at Ser-133.
558
Groups sharing the same superscript letter are not significantly different (p >
559
0.05) as reveaed by LSD post hoc tests.
560
561
Figure 9. The schematic diagram showing inhibitory effects of fisetin in UVB induced
skin damage.
562
27
563
564
565
Figure1.
28
###
120
***
Cell viability (% of control)
100
80
60
40
20
0
566
567
568
569
UVB
Fisetin
0
5
10
25
50
Figure 2.
29
+
0
+
5
+
10
+
25
+
50
(40 mJ/cm 2)
(M)
570
571
572
573
574
575
576
577
578
579
580
control
UVB+ fisetin 5 μM
UVB 80 mJ/cm2
UVB+ fisetin 10 μM
581
582
583
584
585
586
587
588
589
590
591
592
Figure 3.
30
UVB+ fisetin 25 μM
593
594
Figure 4. (a)
595
596
597
Figure 4. (b)
598
31
4
b
Fold of control
3
c
2
a
a
a
1
0
599
600
601
-
+
+
+
+
0
0
5
10
25
UVB 40 mJ/cm2
Fisetin ( M)
Figure 5 (a)
1600
b
1400
PGE2 concentration (pg/mL)
bc
1200
1000
c
800
d
600
400
a
200
0
602
603
604
-
+
+
+
+
0
0
5
10
25
Figure 5 (b)
605
606
32
UVB 40 mJ/cm2
Fisetin ( M)
607
608
Figure 6. (a)
609
610
611
Figure 6. (b)
612
pMAP kinase relative density (of control)
8.0
7.0
c
p-ERK
p-JNK
p-p38
6.0
5.0
b
b
b
4.0
cd
c
3.0
c
bd
d
e
2.0
a a a
1.0
Figure 6 (c)
615
33
d
d
bd
0.0
613
614
b
UVB
-
b
+
0
+
+
5
10
Fisetin ( M)
+
25
+
1
(40 mJ/cm2)
EGCG ( M)
p-c-Jun and c-Jun relative density (of control)
3.5
p-c-Jun
c-Jun
b
b
3.0
b
d
2.5
e
e
2.0
c
c
**
**
1.5
**
**
a a
1.0
0.5
0.0
UVB
-
+
0
+
5
616
617
618
b
c
+
10
+
25
Fisetin ( M)
EGCG ( M)
Figure 7. (a)
I B
NF-kB/p65
I B and p65 relative density (of control)
1.2
e
d
0.8
b
b
0.6
c
d
c
0.4
e
cd
b
0.2
619
UVB
-
+
0
+
5
+
10
Fisetin ( M)
Figure 7. (b)
621
622
623
624
a a
1.0
0.0
620
(40 mJ/cm2)
+
1
Figure 7. (c)
34
+
25
+
1
(40 mJ/cm2)
EGCG ( M)
625
626
627
628
Figure 8.
35
Fisetin
(25 μM)
Fibroblast
ROS
Cytoplasm
NF-κB
MAPKs
Nucleus
AP-1
c-Jun c-Fos
MMP-1, -3, -9 ↑
NF-κB
COX-2 ↑
Photodamage
629
630
631
Figure 9.
36
IκB
P
632
TOC graphic
633
Fisetin
(25 μM)
Fibroblast
ROS
Cytoplasm
NF-κB
MAPKs
Nucleus
AP-1
c-Jun c-Fos
MMP-1, -3, -9 ↑
NF-κB
COX-2 ↑
Photodamage
634
635
636
37
IκB
P