Syringaresinol inhibits UVA-induced MMP-1 expression by suppression of mapk/ap-1 signaling in hacat keratinocytes and human dermal fibroblasts

Article abstract of DOI:10.3390/ijms21113981

Ultraviolet (UV) irradiation induces detrimental changes in human skin which result in photoaging. UV-induced intracellular changes cause degradation of extracellular matrix (ECM). UV-stimulated cleavage of collagen in ECM occurs via matrix metalloproteinases (MMPs). (±)syringaresinol (SYR), a phytochemical which belongs to the lignan group of polyphenols, was investigated for its ability to reverse the UVA-induced changes in human HaCaT keratinocytes and dermal fibroblasts (HDFs) in vitro. Effect of SYR on UVA-induced changes was investigated by production and activation of MMPs and its transcriptional upstream effectors; mitogen-activated protein kinases (MAPKs) and pro-inflammatory mediators. Levels of expression were determined using ELISA, RT-PCR and immunoblotting. UVA irradiation stimulated the production of MMP-1 and inhibited collagen production. SYR treatment suppressed MMP-1 and enhanced collagen production in UVA-irradiated HaCaT keratinocytes and HDFs. SYR repressed the UV-induced phosphorylation of p38, ERK and JNK MAPKs in HaCaT keratinocytes while only suppressing JNK phosphorylation in HDFs. In addition, SYR was able to inhibit UVA-induced production of inflammatory cytokines; TNF-α, COX-2, IL-1β and IL-6. Moreover, SYR suppressed the activator protein-1 (AP-1), a heterodimer of phosphorylated transcription factors c-Jun and c-Fos. SYRtreatment decreased nuclear levels of activated c-Fos and c-Jun as a mechanism to inhibit UVAinduced transcriptional activities leading to MMP-1 production. In conclusion, current results demonstrated that SYR could inhibit UVA-induced upregulation of MMP-1 by suppressing MAPK/AP-1 signaling in HaCaT keratinocytes and HDFs. Therefore, SYR was suggested as a potential compound with antiphotoaging properties against UVA-induced skin aging.

Full text of DOI:10.3390/ijms21113981

International Journal of
Molecular Sciences
Article
Syringaresinol Inhibits UVA-Induced MMP-1
Expression by Suppression of MAPK/AP-1 Signaling
in HaCaT Keratinocytes and Human
Dermal Fibroblasts
Jung Hwan Oh 1, Yung Hyup Joo 2, Fatih Karadeniz 1, Jaeyoung Ko 2,
*
and Chang-Suk Kong 1,3,
*
1
Marine Biotechnology Center for Pharmaceuticals and Foods, College of Medical and Life Sciences,
Silla University, Busan 46958, Korea; wjdghks0171@naver.com (J.H.O.); karadenizf@outlook.com (F.K.)
AMOREPACIFIC Research and Development Center, Yongin 17074, Korea; yunghyupjoo@hanmail.net
Department of Food and Nutrition, College of Medical and Life Sciences, Silla University, Busan 46958, Korea
Correspondence: jaeyoungko@amorepacific.com (J.K.); cskong@silla.ac.kr (C.-S.K.);
Tel.: +82-31-280-5928 (J.K.); +82-51-999-5429 (C.-S.K.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 14 May 2020; Accepted: 29 May 2020; Published: 1 June 2020
Abstract: Ultraviolet (UV) irradiation induces detrimental changes in human skin which result
in photoaging. UV-induced intracellular changes cause degradation of extracellular matrix
(ECM). UV-stimulated cleavage of collagen in ECM occurs via matrix metalloproteinases (MMPs).
( )-syringaresinol (SYR), a phytochemical which belongs to the lignan group of polyphenols,
±
was investigated for its ability to reverse the UVA-induced changes in human HaCaT keratinocytes
and dermal fibroblasts (HDFs) in vitro. Effect of SYR on UVA-induced changes was investigated by
production and activation of MMPs and its transcriptional upstream effectors; mitogen-activated
protein kinases (MAPKs) and pro-inflammatory mediators. Levels of expression were determined
using ELISA, RT-PCR and immunoblotting. UVA irradiation stimulated the production of MMP-1 and
inhibited collagen production. SYR treatment suppressed MMP-1 and enhanced collagen production
in UVA-irradiated HaCaT keratinocytes and HDFs. SYR repressed the UV-induced phosphorylation
of p38, ERK and JNK MAPKs in HaCaT keratinocytes while only suppressing JNK phosphorylation
in HDFs. In addition, SYR was able to inhibit UVA-induced production of inflammatory cytokines;
TNF-α, COX-2, IL-1β and IL-6. Moreover, SYR suppressed the activator protein-1 (AP-1), a heterodimer
of phosphorylated transcription factors c-Jun and c-Fos. SYR-treatment decreased nuclear levels of
activated c-Fos and c-Jun as a mechanism to inhibit UVA-induced transcriptional activities leading to
MMP-1 production. In conclusion, current results demonstrated that SYR could inhibit UVA-induced
upregulation of MMP-1 by suppressing MAPK/AP-1 signaling in HaCaT keratinocytes and HDFs.
Therefore, SYR was suggested as a potential compound with antiphotoaging properties against
UVA-induced skin aging.
Keywords: HaCaT keratinocytes; human dermal fibroblasts; MMP-1; photoaging; UVA
1. Introduction
Ultraviolet (UV) radiation exposure leads to several skin problems, such as wrinkles, loss of
elasticity, sagging, and deformation [
to be the root cause of extrinsic skin aging, therefore referred to as photoaging [
1
,
2
]. Chronic exposure to UV-induced changes in skin is considered
]. Photoaging skin
3
was observed to exhibit UV-induced deterioration, mainly in dermal connective tissues: epidermis and
dermis layers. Ultraviolet A (UVA) is one of the light rays that are emitted by sun together with UVB
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and UVC. UVB has the most detrimental and broad harmful effects and affects mainly the top layer of
skin, causing sunburn, redness and cancer. UVA irradiation, on the other hand, can penetrate deep
layers of skin and is the main cause of premature skin aging and wrinkling [3,4]. Further, it has been
shown that exposure to UV radiation also affect the neuroendocrine system through skin exposure [
5].
This interaction between UVR and the neuroendocrine system shows itself in different ways including
opioidergic effects, immunosuppression, and brain stimulation, all of which open ways for UVR to be
used in a therapeutic approach. However, it was also shown that premature aging of the skin could
also be enhanced by UVR-mediated stimulation of neuroendocrine system by activating internal aging
factors in addition to its extrinsic aging stimulatory effects [6]. Research has targeted the molecular
changes after UVA exposure in skin. It was shown that, after a short time of exposure, UV radiation
activated growth factors, increased oxidative stress and induced pro-inflammatory response in
different types of skin cells [7,8]. Several of these outcomes are known to enhance the activation of
mitogen-activated protein kinase (MAPK) cascade, which is followed by an increase in transcriptional
activity of the activator protein-1 (AP-1) complex formed by heterodimerization of c-Fos and c-Jun
proteins [9]. Activity of the AP-1 complex is dependent on the activation of c-Fos and c-Jun proteins
by MAPK cascade and responsible for the upregulation of several extracellular-matrix-degrading
(ECM) enzymes, such as matrix metalloproteinase-1 (MMP-1), MMP-3 and MMP-9 [10]. Therefore,
UV-induced degradation of ECM, especially its collagen fragments, is one of the main causes of
photoaged skin.
Studies during the past decades showed an increasing interest in naturally occurring compounds,
especially phytochemicals, given their broad-spectrum bioactivities, high biodegradability, low cost
and low toxicity [11]. Lignans belong to a group of such phytochemicals found in various plants
and include numerous biologically active compounds with pharmaceutical value [12]. Syringaresinol
belongs to the lignan group of phytochemicals and exists as (R, R)-(+)-form and (S, S)-(-)-form. It can be
obtained by extracting various parts of plants, such as cortex, Amelia seed and flax seed. This substance
has been reported to show various physiological activities, such as antibacterial, antifungal activity [13
]
and anti-inflammatory [14] activity, and recently, research results related to skin, such as whitening,
anti-aging, improvement of immune disease, and improvement of metabolic disorders [15] are known.
Therefore, the current study was carried out to investigate the effects of ( )-syringaresinol (SYR)
±
(Figure 1A) in UVA-irradiated human keratinocyte and dermal fibroblast in order to provide insights
regarding its antiphotoaging properties.

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Figure 1. Synthesis of (
and procollagen I 1 in HaCaT keratinocytes. Cells were treated with given concentrations of SYR
for 24 h after UVA irradiation (10 J/cm2), and levels of MMP-1 (
) and procollagen I 1 ( ) release
along with MMP-1 activation ( ) were measured. Retinoic acid was used as a positive control.
Values are means SD of three independent experiments run in triplicate. Different letters (a–e) indicate
statistically significant difference at p < 0.05 level by Duncan’s multiple range test.
±)-syringaresinol (SYR) (A) and its effect on UVA-induced release of MMP-1
α
B
α
C
D
±
2. Results
2.1. SYR Reversed UVA-Induced Changes of MMP-1 and Procollagen I
To examine the effects of UVA irradiation on the production of MMP-1 and procollagen I
keratinocytes and whether SYR could counter these effects, HaCaT keratinocytes were irradiated with
UVA and treated with different concentrations (1, 5 and 20 M) of SYR. UVA exposure significantly
induced MMP-1 production in keratinocytes (Figure 1B). On the other hand, release of procollagen I
was inhibited by UVA exposure (Figure 1C). Treating keratinocytes with SYR significantly inhibited
MMP-1 production in a dose-dependent manner while increasing the release of procollagen I 1 from
α
1 Production in HaCaT Keratinocytes
α
1 in
µ
α
1
α
keratinocytes compared to UVA-irradiated non-treated cells. SYR was also shown to decrease the
activation of MMP-1 in cell culture medium (Figure 1D). A UVA-induced increase in active MMP-1
levels (8.16 ng/mL) was significantly decreased (2.56 ng/mL) by 20 µM SYR treatment. Similar effects
were observed in the expression of MMP-1 and type 1 procollagen in both mRNA and protein level.
SYR treatment was able to reverse the effects of UVA exposure on mRNA expression (Figure 2A) and
protein levels (Figure 2B) of both MMP-1 and type 1 procollagen. Dose-dependently, MMP-1 expression
was inhibited by SYR after notable UVA-induced upregulation (Figure S1). Likewise, UVA-induced
suppression of type I procollagen expression was reverted by SYR. In addition, to determine the effects
of SYR on the UVA-induced expression of other MMPs with the same transcriptional regulation (AP-1),
MMP-9 protein levels were analyzed (Figure 2B). Expectedly, UVA irradiation upregulated MMP-9

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levels, which were decreased by the presence of SYR, suggesting that the effect of SYR on UVA-induced
MMP production was not MMP-type specific.
Figure 2. Effect of SYR on UVA-induced mRNA and protein expressions of MMPs, type I procollagen,
and pro-inflammatory mediators in HaCaT keratinocytes. Cells were exposed to UVA radiation
(10 J/cm2) and treated with given concentrations of SYR for 24 h. (
A
) mRNA expressions of MMP-1 and
) Protein levels of MMP-1, -9 and type I procollagen
) and pro-inflammatory mediators (COX-2, TNF- , iNOS, IL-1 and IL-6) were determined by
Western blotting. -Actin was used as an internal control for RT-PCR and Western blotting. Retinoic
type I procollagen were analyzed with RT-PCR. (
B
(
C
α
β
β
acid was used as a positive control.
2.2. UVA-Induced Inflammation in HaCaT Keratinocytes is Ameliorated by SYR Treatment
As TNF-
α mediated inflammatory cytokines can also stimulate MMP-1 production and collagen
degradation [16]; effects of SYR treatment against UVA-induced inflammatory response in keratinocytes
were analyzed via protein expression of pro-inflammatory mediators and cytokines (TNF-
α
, COX-2,
iNOS, IL-1β and IL-6). Keratinocytes exhibited increased levels of TNF-α, COX-2, iNOS, IL-1β and IL-6
following UVA exposure (10 J/cm2) (Figure 2C). UVA-induced stimulation of inflammatory cytokine
production was inhibited by SYR in a dose-dependent manner except for IL-6.
2.3. SYR Suppresses the UVA-Induced Activation of MAPK/AP-1 Signaling in Keratinocytes
As activation of p38, ERK and JNK MAPKs are required for upregulating MMP-1 expression [17
]
via UVA-induced ROS production, the effects of SYR on the phosphorylation levels of MAPKs were
investigated (Figure S2). HaCaT keratinocytes were irradiated by UVA (10 J/cm2) followed by SYR
treatment. Levels of phosphorylated (p-) p38, p-ERK and p-JNK were significantly increased by UVA
exposure (Figure 3A). Treatment with SYR inhibited the p- levels of all three MAPKs without affecting
their total protein levels, indicating that SYR downregulated the UVA-stimulated phosphorylation of
MAPKs, not their expression. Time-dependent activation of MAPK was also investigated after 6 and
12 h of UVA irradiation and treatment. SYR treatment (20 µM) showed decreased phosphorylation of
p38, ERK and JNK during the early stages of UVA irradiation, further suggesting the effect of SYR on
MAPK phosphorylation (Figure 3B).

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Figure 3. Effect of SYR on the total and phosphorylated (p-) protein levels of MAPK/AP-1 signaling
effectors in UVA-irradiated HaCaT keratinocytes. Total cell lysates and nuclear extracts of the cells
were used to determine the protein levels of phosphorylated (p-) and inactive forms of (
(p38, ERK and JNK), ( ) c-Fos and c-Jun proteins by Western blotting. Cells were exposed to UVA
radiation (10 J/cm2) and treated with or without given concentrations of SYR for 24 h (
), 6 h and 12
). -Actin was used as an internal control for Western blotting. Lamin B1 was used as an internal
control for nuclear fraction Western blotting.
A,B) MAPKs
C
A,C
(
B β
UVA-induced AP-1 activation and nuclear translocation were shown to result in increased MMP-1
expression due to AP-1 binding site presence on the MMP-1 promoter region [18]. Given that AP-1
is formed by Jun and Fos proteins and its activation is regulated by MAPKs, the effects of SYR on
UVA-mediated c-Jun and c-Fos phosphorylation were determined. UVA irradiation (10 J/cm2) resulted
in elevated phosphorylation of c-Jun and c-Fos (Figure 3C) both in the cytoplasm and nucleus of
keratinocytes. Treating UVA-irradiated HaCaT keratinocytes with 20 µM SYR inhibited UVA-induced
phosphorylation of c-Fos and c-Jun, as well as suppressing their nuclear translocation, as shown
by inhibited levels of p-c-Fos and p-c-Jun in the nuclear fraction of UVA-irradiated keratinocytes.
Coupled with suppressed MAPK activation, inhibition of c-Fos and c-Jun phosphorylation suggested
that SYR inhibited MMP-1 expression and, thus, MMP-1 production via downregulated AP-1
transcriptional activity.
2.4. SYR Inhibits the UVA-Induced MMP-1 Production in Human Dermal Fibroblasts (HDFs) by Inhibiting
MAPK-Mediated Pro-Inflammatory Response and AP-1 Activation
To investigate whether attenuation of UVA-induced MMP-1 production and collagen degradation
by SYR was cell-type-specific, HDFs were treated with SYR following UVA irradiation (10 J/cm2).
Similar to HaCaT keratinocytes, HDFs showed increased MMP-1 production coupled with decreased
procollagen Iα1 release (Figure 4A,B). Treating UVA-irradiated HDFs with SYR resulted in reverted
production levels for both MMP-1 and procollagen. SYR-treated HDFs released less MMP-1 and
more procollagen compared to that of the UVA-irradiated-only group. Similar to its effect on HaCaT
keratinocytes, SYR suppressed UVA-induced MMP activation (Figure 4C). At the concentration of 20 µM,
SYR treatment decreased the active MMP-1 levels from 2.28 ng/mL (untreated UVA-irradiated group) to
1.37 ng/mL. Parallel results were obtained from investigating mRNA and protein expression of MMP-1
and type I procollagen. SYR ameliorated the change in MMP-1 and type I procollagen mRNA expression
(Figure 5A) in UVA-irradiated HDFs. Likewise, MMP-1 and collagen protein levels were reversed to
non-irradiated non-treated HDF group levels by SYR compared to the UVA-irradiated-only group
(Figure 5B). In addition, MMP-9 protein levels were also slightly inhibited by SYR in a dose-dependent
manner. The protein levels of pro-inflammatory mediators in UVA-irradiated HDFs were also analyzed
with or without SYR treatment to determine whether SYR could inhibit UVA-induced inflammation in
HDFs as well. Treating HDFs with SYR following UVA irradiation inhibited the protein levels of TNF-
α,
COX-2, iNOS and IL-1 in HDFs (Figure 5B). As in keratinocytes, dermal fibroblasts were shown to
β

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exhibit overactivated p38, ERK and JNK MAPKs following UV exposure as a mechanism to express
AP-1-regulated MMP-1. Presence of SYR (20 M) inhibited the phosphorylated levels of JNK but did
µ
not suppress the p38 and ERK phosphorylation levels (Figure 5C). Analysis of MAPK activation at the
early stages of UVA irradiation showed that SYR treatment was able to decrease the phosphorylation
of ERK at the 6 and 12 h periods after UVA irradiation (Figure 5D). However, treatment with SYR
enhanced the activation of ERK in HDFs at the 24 h period. Nevertheless, SYR was able to hinder
the phosphorylation of c-Fos and c-Jun and nuclear translocation of activated c-Jun but not c-Fos
(Figure 5C) in nuclear fractions of UVA-irradiated HDFs. Results indicated that SYR reversed the
UVA-induced production of MMP-1 in HDFs via a similar mechanism to that of HaCaT keratinocytes:
repression of inflammation and MAPK-activated AP-1 transcriptional activity (Figure S3).
Figure 4. Effect of SYR on UVA-induced release of MMP-1 (A) and procollagen Iα1 (B) along with
MMP-1 activation (C) in human dermal fibroblasts (HDFs). Cells were treated with given concentrations
of SYR for 24 h after UVA irradiation (10 J/cm2), and the levels of MMP-1 and procollagen Iα1 release
were measured by ELISA using cell culture medium. Retinoic acid was used as a positive control.
Values are means
±
SD of three independent experiment run in triplicate. Different letters (a–d) indicate
statistically significant differences at the p < 0.05 level by Duncan’s multiple range test.

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Figure 5. Effect of SYR on the expression of MMPs, type I procollagen, pro-inflammatory cytokines
and MAPK/AP-1 phosphorylation in UVA-irradiated HDFs. ( ) mRNA expressions of MMP-1 and
type I procollagen were determined by RT-PCR. ( ) Protein levels of MMP-1, MMP-9, collagen, COX-2,
TNF- , and IL-1 were analyzed with Western blotting using total cell lysates. ( ) Phosphorylated
A
B
α
β
C,D
(p-) and total levels of p38, ERK, JNK, c-Fos and c-Jun were analyzed with Western blotting using total
cell lysates and nuclear extracts. Cells were exposed to UVA radiation (10 J/cm2) and treated with given
concentrations of SYR. After 24 h (
A
–
C) or 6 and 12 h (D) incubation cells were harvested for RT-PCR
and Western blotting. Retinoic acid was used as a positive control.
β-Actin was used as an internal
control for RT-PCR and Western blotting. Lamin B1 was used as an internal control for nuclear fraction
Western blots.
3. Discussion
UV-irradiation-induced skin aging concerns the deterioration of the balance between MMP
production and ECM formation in favor of MMPs, which results in increasing degradation of ECM
by various cell types including dermal fibroblasts and keratinocytes [2]. UVA is known to induce
the production and release of MMP-1 and MMP-9 in epidermal and dermal layers of skin, resulting
in degradation of collagen. Considering the importance of collagen in skin function and elasticity,
UVA-induced collagen degradation can be regarded as the main path for the progression of photoaging.
In present study, it was shown that SYR treatment dose-dependently inhibited the UVA-induced
production of MMP-1 both in keratinocytes and HDFs. Results also showed that UVA irradiation
inhibited the procollagen release from cells, which might be the reason behind chronic collagen
deficiency in photodamaged skin, along with increased collagen degradation by MMPs. Present results
demonstrated that UVA-induced decreases in procollagen release from HaCaT keratinocytes and HDFs
were also attenuated by SYR. Immortalized HaCaT keratinocytes were chosen for the current study
in order to minimize the problems and variations that come with primary keratinocyte cultures due
to donor to donor differences and different characteristics for cells with different passage numbers.
As current study is the first report of SYR UVA protective effects on keratinocytes, a standardized and
long-lived HaCaT keratinocytes were used to obtain preliminary results. However, due to possible
limitations and transcriptional differences between immortalized HaCaT keratinocytes and primary
keratinocyte cultures, further studies using primary cultures and in vivo models are needed to fully
evaluate the potential of SYR.
The effect of SYR on the production of MMP-1 and procollagen was also observed in expressional
level. SYR-treated keratinocytes and HDFs showed decreased MMP-1 and increased collagen mRNA

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and protein levels compared to UVA-irradiated-only cells. SYR treatment was also observed to result
in significantly decreased amounts of active MMP-1, indicating suppressed MMP-1 activation.
UVA-induced expression of MMP-1 is mainly governed by MAPK-regulated activation of AP-1 and
NF-κB, the former as a result of UVA-induced inflammatory response in skin [19,20]. On the other hand,
AP-1 transcriptional activity is regulated by p38, ERK and JNK-mediated phosphorylation of c-Fos and
c-Jun, which form the AP-1 complex upon phosphorylation. Therefore, the MAPK signaling cascade
plays pivotal roles in UVA-induced MMP expression. This study showed that treating keratinocytes
and HDFs with SYR inhibited the phosphorylation of p38, JNK and ERK MAPKs, with the exception
of ERK in HDFs. Inhibition of MAPK activation by SYR was also shown to occur, along with inhibition
of production of inflammatory cytokines such as TNF-α, COX-2, IL-1β and IL-6, all of which are part
of the UVA-induced inflammatory response in skin, leading to production of MMPs. SYR treatment
also significantly inhibited the phosphorylation of c-Jun and c-Fos and the levels of active c-Fos and
c-Jun in nuclear fractions in UVA-irradiated keratinocytes and HDFs. Suppression of c-Fos and c-Jun
activation indicated that SYR might inhibit MMP-1 production via blocking MAPK-cascade-regulated
AP-1 transcriptional activity. Considering SYR inhibited the inflammatory cytokine production along
MMP-1 expression, it was suggested that the effects of SYR targeted upstream activators of AP-1 and
inflammation response, suggestive of phosphorylation of p38 and JNK. The stimulatory effects of SYR
on collagen production in HDFs might be attributed to enhanced ERK activation, as some studies
reported that dermal fibroblast collagen production is dependent on ERK1/2 signaling and negatively
regulated by p38 [21,22]. However, SYR treatment was shown to suppress ERK phosphorylation at
the early stages of treatment following UVA irradiation. Time-dependent opposite effects of SYR on
ERK phosphorylation also suggest that the SYR-mediated stimulation of collagen synthesis via ERK
activation occurred after downregulation of MMP-1 expression. Results indicated that SYR repressed
MAPK activation independent of the stimuli, as both inflammatory and AP-1-mediated transcriptional
activities were suppressed. SYR also showed its MAPK inhibitory effects in both keratinocytes
and HDFs, demonstrating that inhibition of MMP-1 production via MAPK suppression was not
cell-type-specific. On the other hand, SYR stimulated ERK phosphorylation in collagen-producing
HDFs but not in keratinocytes, suggesting that SYR-mediated amelioration of UVA-induced changes
in collagen production might be linked with ERK activation.
Lignans are important phytochemicals with various health benefits, including skin protection and
skin whitening [23,24]. Syringaresinol has already been reported to be an important lead molecule
with tyrosinase inhibitory [15] and antiaging [25] activities along with age-related skin atrophy
reversion [26]. These reports showed that compositions containing syringaresinol and its derivatives
as main ingredients possessed skin-improving abilities with antioxidant properties. In this context,
the current study provided insights into the anti-photoaging properties of syringaresinol against
UVA-induced changes in MMP and collagen production in the skin. In addition, syringaresinol contains
several hydroxyl groups which are prone to forming glycosides. Several lignan glycosides have been
studied as promising cosmeceuticals [27]. Overall, the present study showed that syringaresinol has
UVA-protective effects in keratinocytes and dermal fibroblasts via suppression of MMP expression.
4. Materials and Methods
4.1. Synthesis of (±)-Syringaresinol (SYR)
A pale green solution of sinapyl alcohol (0.321 g, 1.53 mmol), H2O (32 mL) and CuSO4 5H2O
·
(0.381 g, 1.00 eq) was stirred at room temperature (20 ◦C) in a photo-reactor. After 48 h, the reaction
mixture was extracted with ethyl acetate (5 mL) twice. The combined organic layer was dried over
MgSO4 and concentrated under reduced pressure. The residue was crystallized in EtOH to give a
white solid (0.131 g, 41.0%).
1H NMR: (500 MHz, CDCl3)
δ 6.57 (s, 4H), 5.53 (s, 2H), 4.72 (d, J = 3.0 Hz, 2H), 4.29–4.26 (m, 2H),
3.91–3.89 (m, 14H), 3.09 (s, 2H).

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13C NMR: (125 MHz, CDCl3) δ 147.3, 134.5, 132.2, 102.9, 86.2, 72.0, 56.5, 54.5.
HRMS (ESI positive): [M+Na]+ calculated for C22H26O8Na 441.1526 found 441.1514.
4.2. Cell Culture
HaCaT keratinocytes (300493; Cell Line Service, Eppelheim, Germany) were cultured in Dulbecco’s
modified Eagle medium with 10% fetal bovine serum (FBS), and cells were kept in 37 ◦C incubators
with an atmosphere containing 5% CO2 between the experiments. Human dermal fibroblasts
(HDFs) (C-12302; PromoCell, Heidelberg, Germany) were cultured in Fibroblast Growth Medium
(C-23020, PromoCell) and kept in 37 ◦C incubators with an atmosphere containing 5% CO2 between
the experiments.
4.3. UVA Irradiation of HaCaT Keratinocytes and HDFs
HaCaT keratinocytes and HDFs were irradiated by UVA using a Bio-Sun UV Irradiation System
(Vilber Lourmat, Marine, France) fitted with a UVA source designed for microplates. Cells grown in
microplates were irradiated at a 10 J/cm2 UVA dose. Cells were irradiated in phosphate-buffered saline
(PBS) without the plastic lid. When the irradiation received matched the desired programmed energy,
the UVA irradiation stopped automatically, and, subsequently, the cells were incubated with their own
growth medium without FBS until analysis.
4.4. Enzyme-Linked Immunosorbent Assay (ELISA)
Releases of MMP-1 and procollagen Iα1 in UVA-irradiated HaCaT keratinocytes and HDFs were
analyzed by ELISA. Cells were pre-incubated in 6-well plates for 24 h and washed with PBS prior to
UVA (10 J/cm2) exposure. Following UVA irradiation, the cells were treated with or without different
concentrations (1, 5, 20
µM) of SYR for 24 h. Cell culture medium from each well were analyzed for its
MMP-1 and type I 1 procollagen contents per manufacturer’s instructions for the ELISA kit (R&D
α
systems, Inc., Minneapolis, MN, USA).
4.5. Determination of MMP-1 Activation
MMP-1 activation was assessed using a quenched fluorogenic-substrate-based enzyme activity
assay kit (Fluorokine E human active MMP-1 fluorescent assay kit; F1M00, R&D Systems,
Minneapolis, MN, USA) according to the manufacturer’s protocol. MMP-1 activation was given
as the active MMP-1 levels (ng/mL) in the cell culture medium.
4.6. Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from nonirradiated and irradiated (UVA, 10 J/cm2) HaCaT keratinocytes
and HDFs using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA (2
µg) and oligo(dT)
were mixed in RNase-free water for the cDNA synthesis. Synthesis was performed in a thermo cycler
(T100; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with an initial denaturation of the mix at 70 ◦C
for 5 min and cooing immediately followed by the preparation of a master mix containing 1X RT buffer,
1 mM dNTPs, 500 ng oligo(dT), 140 units M-MLV reserve transcriptase and 40 units RNase inhibitor.
The remaining cycles were 42 ◦C for 60 min and 72 ◦C for 5 min.
For RT-PCR analysis, the target cDNA was amplified using the sense and antisense primers as
previously noted [28]. The amplification was carried out with 30 cycles; each cycle consisted of 95 ◦C
for 45 s, 60 ◦C for 1 min and 72 ◦C for 45 s. The final PCR products were separated by agarose gel
(1.5%) electrophoresis for 30 min at 100 V. Staining of gels were carried out with 1 mg/mL ethidium
bromide and the images of the stained gels were taken under UV light using a Davinch-Chemi imager
(CAS-400SM, Seoul, Korea).
™

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4.7. Western Blotting
Protein levels were investigated using immunoblotting according to common Western blot
protocols. HaCaT keratinocytes and HDFs were cultured in 6-well plates up to 80% confluence and
exposed to UVA (10 J/cm2) irradiation. Next, SYR (1, 5, 20
µM) was added to the wells and incubated
for 6, 12 or 24 h. Following incubation, wells were aspirated, and cells were lysed by vigorous pipetting
in 1 mL of RIPA buffer (Sigma–Aldrich, St. Louis, MO, USA) at 4 ◦C. The nuclear fraction extraction
was carried out using NE-PERTM Nuclear Extraction Kit (Catalog No. #78835; Thermo Fisher Scientific,
Waltham, MA, USA) according to manufacturer’s instructions. Protein content of the lysates was
measured with a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) following kit’s
protocol. The same amount (20 µg) of protein from each well was loaded onto 10% SDS-polyacrylamide
gel and run at 100 V. Proteins on gel were then transferred onto a polyvinylidene fluoride membrane
◦
(Amersham, GE Healthcare, Little Chalfont, UK) using a wet system run at 100 V for 1 h at 4 C.
Next, membrane blocking was carried out by incubating membranes for 1 h at room temperature
in 5% skimmed milk for blocking. Blocked membranes were washed with 1
with primary antibodies of specific proteins (diluted 1:1000) in primary antibody dilution buffer
containing 1
TBST with 5% bovine serum albumin overnight at 4 ◦C. Membranes were then incubated
×
TBST and incubated
×
with horseradish-peroxidase-conjugated secondary antibodies (diluted 1:1000) specific to the primary
antibody source organism at room temperature for 1 h. Detection of proteins on blotted membranes
was achieved using a ECL Western blot detection kit (Amersham) according to the manufacturer’s
instructions. Protein bands were imaged with CAS-400SM Davinch-Chemi imager™ (Davinch-K).
4.8. Statistical Analysis
All numerical data were given as the mean
±
standard deviation of three separate experiments
carried out in triplicates. Statistical differences between the means of the sample groups were calculated
by the analysis of variance (ANOVA) followed by Duncan’s multiple range test using SAS v9.1 software
(SAS Institute, Cary, NC, USA). Any statistically significant difference between the groups were
determined at p < 0.05 level.
5. Conclusions
In conclusion, the current study demonstrated that SYR attenuated UVA-induced changes in
MMP-1 and collagen production as well as MMP-1 activation in HaCaT keratinocytes and HDFs. Results
indicated that SYR inhibited MMP-1 expression by suppressing UVA-stimulated phosphorylation of p38
and JNK MAPKs and their downstream effectors c-Fos and c-Jun, which regulates the AP-1-activated
MMP-1 expression. In addition, SYR was also suppressed the UVA-induced increase in inflammatory
cytokine levels. Therefore, the present study suggests that SYR is a potential lead compound for
cosmeceutical development against UVA-induced photoaging. Considering previous reports on
beneficial and protective effects of syringaresinol on human skin, futures studies that focus on the
in-vivo efficiency of SYR and its detailed action mechanisms are urged for the development of
SYR-based cosmeceuticals.
Supplementary Materials: Supplementary materials are available online at http://www.mdpi.com/1422-0067/21/
11/3981/s1.
Author Contributions: Conceptualization and methodology, J.H.O., Y.H.J. and J.K.; validation, formal analysis
and investigation, J.H.O., Y.H.J. and F.K.; resources, C.-S.K.; data curation, J.K. and C.-S.K.; writing—original draft
preparation, visualization, F.K.; supervision, and project administration, J.K. and C.-S.K.; funding acquisition, J.K.
and C.-S.K. All authors have read and agreed to the published version of the manuscript.
Funding: This work was performed within the program of the AMOREPACIFIC Open Research
(SRT-01-R19E80012) supported by a grant from AMOREPACIFIC.
Conflicts of Interest: The authors declare no conflict of interest.

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References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Rittié, L.; Fisher, G.J. UV-light-induced signal cascades and skin aging. Ageing Res. Rev. 2002, 1, 705–720.
[CrossRef]
Fisher, G.J.; Kang, S.; Varani, J.; Bata-Csorgo, Z.; Wan, Y.; Datta, S.; Voorhees, J.J. Mechanisms of photoaging
and chronological skin aging. Arch. Dermatol. 2002, 138, 1462–1470. [CrossRef] [PubMed]
Krutmann, J.; Bouloc, A.; Sore, G.; Bernard, B.A.; Passeron, T. The skin aging exposome. J. Dermatol. Sci.
2017, 85, 152–161. [CrossRef] [PubMed]
Gallagher, R.P.; Lee, T.K. Adverse effects of ultraviolet radiation: A brief review. Prog. Biophys. Mol. Biol.
2006, 92, 119–131. [CrossRef]
Slominski, A.T.; Zmijewski, M.A.; Plonka, P.M.; Szaflarski, J.P.; Paus, R. How UV light touches the brain and
endocrine system through skin, and why. Endocrinology 2018, 159, 1992–2007. [CrossRef] [PubMed]
Bocheva, G.; Slominski, R.M.; Slominski, A.T. Neuroendocrine aspects of skin aging. Int. J. Mol. Sci.
2019, 20, 2798. [CrossRef] [PubMed]
Yaar, M.; Gilchrest, B.A. Photoageing: Mechanism, prevention and therapy. Br. J. Dermatol. 2007, 157, 874–887.
[CrossRef]
Rabe, J.H.; Mamelak, A.J.; McElgunn, P.J.S.; Morison, W.L.; Sauder, D.N. Photoaging: Mechanisms and repair.
J. Am. Acad. Dermatol. 2006, 55, 1–19. [CrossRef]
Silvers, A.L.; Bachelor, M.A.; Bowden, G.T. The role of JNK and p38 MAPK activities in UVA-induced
signaling pathways leading to AP-1 activation and c-Fos expression. Neoplasia 2003, 5, 319–329. [CrossRef]
10. Pardo, A.; Selman, M. MMP-1: The elder of the family. Int. J. Biochem. Cell Biol. 2005, 37, 283–288. [CrossRef]
11. Altemimi, A.; Lakhssassi, N.; Baharlouei, A.; Watson, D.; Lightfoot, D. Phytochemicals: Extraction, isolation,
and identification of bioactive compounds from plant extracts. Plants 2017, 6, 42. [CrossRef] [PubMed]
12. Teponno, R.B.; Kusari, S.; Spiteller, M. Recent advances in research on lignans and neolignans. Nat. Prod. Rep.
2016, 33, 1044–1092. [CrossRef] [PubMed]
13. Kim, Y.-G.; Watanabe, N.; Sano, Y.; Uraki, Y.; Sano, Y.O. Extractives of kitakobusi Magnolia kobus DC.
var. borealis Sarg. III.: Antibacterial and antifungal activity of extractives. Bull. Hokkaido Univ. For.
1998, 55, 63–73.
14. Salleh, W.M.N.H.W.; Ahmad, F.; Heng Yen, K.; Zulkifli, R.M. Anticholinesterase and anti-inflammatory
constituents from Beilschmiedia pulverulenta kosterm. Nat. Prod. Sci. 2016, 22, 225–230. [CrossRef]
15. Wang, H.M.; Chou, Y.T.; Hong, Z.L.; Chen, H.A.; Chang, Y.C.; Yang, W.L.; Chang, H.C.; Mai, C.T.;
Chen, C.Y. Bioconstituents from stems of Synsepalum dulcificum Daniell (Sapotaceae) inhibit human melanoma
proliferation, reduce mushroom tyrosinase activity and have antioxidant properties. J. Taiwan Inst. Chem. Eng.
2011, 42, 204–211. [CrossRef]
16. Honda, A.; Abe, R.; Makino, T.; Norisugi, O.; Fujita, Y.; Watanabe, H.; Nishihira, J.; Iwakura, Y.; Yamagishi, S.-I.;
Shimizu, H.; et al. Interleukin-1
β and macrophage migration inhibitory factor (MIF) in dermal fibroblasts
mediate UVA-induced matrix metalloproteinase-1 expression. J. Dermatol. Sci. 2008, 49, 63–72. [CrossRef]
17. Muthusamy, V.; Piva, T.J. The UV response of the skin: A review of the MAPK, NFκB and TNFα signal
transduction pathways. Arch. Dermatol. Res. 2010, 302, 5–17. [CrossRef]
18. Benbow, U.; Brinckerhoff, C.E. The AP-1 site and MMP gene regulation: What is all the fuss about? Matrix Biol.
1997, 15, 519–526. [CrossRef]
19. de Jager, T.L.; Cockrell, A.E.; Du Plessis, S.S. Ultraviolet light induced generation of reactive oxygen species.
In Advances in Experimental Medicine and Biology; Springer: New York, NY, USA, 2017; Volume 996, pp. 15–23.
20. Lee, B.; Moon, K.M.; Son, S.; Yun, H.Y.; Han, Y.K.; Ha, Y.M.; Kim, D.H.; Chung, K.W.; Lee, E.K.; An, H.J.; et al.
(2R/S,4R)-2-(2,4-Dihydroxyphenyl)thiazolidine-4-carboxylic acid prevents UV-induced wrinkle formation
through inhibiting NF-κB-mediated inflammation. J. Dermatol. Sci. 2015, 79, 313–316. [CrossRef]
21. Yang, Y.; Kim, H.J.; Woo, K.-J.; Cho, D.; Bang, S.I. Lipo-PGE1 suppresses collagen production in human
dermal fibroblasts via the ERK/Ets-1 signaling pathway. PLoS ONE 2017, 12, e0179614. [CrossRef]
22. Bhogal, R.K.; Bona, C.A. Regulatory effect of extracellular signal-regulated kinases (ERK) on type I collagen
synthesis in human dermal fibroblasts stimulated by IL-4 and IL-13. Int. Rev. Immunol. 2008, 27, 472–496.
[CrossRef] [PubMed]
23. Rodr
íguez-García, C.; Sánchez-Quesada, C.; Toledo, E.; Delgado-Rodríguez, M.; Gaforio, J.J. Naturally
lignan-rich foods: A dietary tool for health promotion? Molecules 2019, 24, 917. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2020, 21, 3981
12 of 12
24. Fang, X.; Hu, X. Advances in the synthesis of lignan natural products. Molecules 2018, 23, 3385. [CrossRef]
25. Kim, J.; Cho, S.Y.; Kim, S.H.; Cho, D.; Kim, S.; Park, C.W.; Shimizu, T.; Cho, J.Y.; Seo, D.B.; Shin, S.S. Effects
of Korean ginseng berry on skin antipigmentation and antiaging via FoxO3a activation. J. Ginseng Res.
2017, 41, 277–283. [CrossRef]
26. Kim, J.; Toda, T.; Watanabe, K.; Shibuya, S.; Ozawa, Y.; Izuo, N.; Cho, S.; Seo, D.B.; Yokote, K.;
Shimizu, T.J. Syringaresinol Reverses Age-Related Skin Atrophy by Suppressing FoxO3a-Mediated Matrix
Metalloproteinase—2 Activation in Copper/Zinc Superoxide Dismutase—Deficient Mice. J. Investig. Dermatol.
2019, 139, 648–655.
27. Kun, N.; Tsuchiya, K.; Nakajima, S. New anti-aging cosmetic ingredients. J. Soc. Cosmet. Chem. Jpn.
1999, 33, 245–253. [CrossRef]
28. Oh, J.H.; Seo, Y.; Kong, C. Anti-photoaging effects of solvent-partitioned fractions from Portulaca oleracea L.
on UVB-stressed human keratinocytes. J. Food Biochem. 2019, 43, e12814. [CrossRef]
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