Pleiotropic Effects of White Willow Bark and 1,2-Decanediol on Human Adult Keratinocytes

Abstract

Background:
Acne vulgaris is a common skin defect, usually occurring during adolescence, but often it can persist in adults leaving permanent face scarring. Acne is usually treated with topical drugs, oral antibiotics, retinoids, and hormonal therapies, but medicinal plants are increasingly employed.

Objective:
To investigate the protective role of white willow bark (WWB) and 1,2-decanediol (DD) on the damage caused by lipopolysaccharides (LPS) on human adult keratinocytes (HaCaT).

Methods:
HaCaT were exposed to LPS alone or in association with WWB and DD. Epidermal viability, metabolic modulation, inflammatory activity, and cell migration were assessed with both common standardized protocols or high-throughput screening systems.

Results:
The preincubation of HaCaT with WWB and DD (used separately or in combination) differently prevented the alterations induced by LPS on HaCaT in terms of growth factor release (IGF, EGF, VEGF), cytokine production (IL-1α, IL-6, IL-8), or expression of the transcription factor FOXO-I. Moreover, they partially restore wound repair lowered by LPS.

Conclusions:
These results suggest that both natural compounds were able to differently affect several functions of LPS-stressed keratinocytes suggesting their potential role for the prevention of acne vulgaris, without adverse effects.

Full text

E-Mail karger@karger.com
Original Paper
Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
Pleiotropic Effects of White Willow
Bark and 1,2-Decanediol on Human
Adult Keratinocytes
Eleonora Bassino a Franco Gasparri b Luca Munaron a
a Department of Life Sciences and Systems Biology, University of Turin, Turin , and
b Department of
Pharmacy (DIFARMA), University of Salerno, Salerno , Italy
ered by LPS. Conclusions: These results suggest that both
natural compounds were able to differently affect several
functions of LPS-stressed keratinocytes suggesting their
potential role for the prevention of acne vulgaris, without
adverse effects.
© 2017 S. Karger AG, Basel
Introduction
The human skin is a complex organ which has many
functions such as regulation of water/electrolyte homeo-
stasis, defense against physical, chemical, and biological
factors, and secretion
[1, 2] . The skin is formed by differ-
ent cell types in a well-structured interplay among epider-
mal and follicular keratinocytes, sebocytes, melanocytes,
dermal papilla cells, fibroblasts, endothelial cells, and
sweat gland cells
[3] . It is a target for hormones and con-
sidered an endocrine gland, whose physiology is influ-
enced by environmental, genetic, or nutritional factors.
The primary mechanism of skin cell alteration is based on
oxidative stress processes
[4] .
Acne vulgaris is a very common defect, typically asso-
ciated with adolescence, but sometimes leading to perma-
nent scarring on the face in adults. Its complex develop-
Keywords
Natural products · Keratinocytes · 1,2-Decanediol · White
willow bark
Abstract
Background: Acne vulgaris is a common skin defect, usu-
ally occurring during adolescence, but often it can persist
in adults leaving permanent face scarring. Acne is usually
treated with topical drugs, oral antibiotics, retinoids, and
hormonal therapies, but medicinal plants are increasingly
employed. Objective: To investigate the protective role of
white willow bark (WWB) and 1,2-decanediol (DD) on the
damage caused by lipopolysaccharides (LPS) on human
adult keratinocytes (HaCaT). Methods: HaCaT were ex-
posed to LPS alone or in association with WWB and DD.
Epidermal viability, metabolic modulation, inflammatory
activity, and cell migration were assessed with both com-
mon standardized protocols or high-throughput screening
systems. Results: The preincubation of HaCaT with WWB
and DD (used separately or in combination) differently pre-
vented the alterations induced by LPS on HaCaT in terms of
growth factor release (IGF, EGF, VEGF), cytokine production
(IL-1α, IL-6, IL-8), or expression of the transcription factor
FOXO-I. Moreover, they partially restore wound repair low-
Received: May 12, 2017
Accepted after revision: September 15, 2017
Published online: November 8, 2017
Luca Munaron, PhD
Department of Life Sciences and Systems Biology, University of Turin
Via Accademia Albertina 13
IT–10123 Turin (Italy)
E-Mail luca.munaron  @  unito.it
© 2017 S. Karger AG, Basel
www.karger.com/spp
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Effects of White Willow Bark and
1,2-Decanediol on Keratinocytes
Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
11
ment involves androgen-mediated stimulation of seba-
ceous gland activity, follicular hyperkeratinization and
colonization of Propionibacterium acnes . Acne is current-
ly treated by the use of local applications, oral antibiotics,
retinoids and hormonal therapies: nonetheless, increas-
ing interest is focused on natural plant derivatives
[5] .
Some natural compounds (e.g., mixtures of phenolic
compounds) have recently come under investigation as
promising drugs for new dermal cosmetics that possess
the ability to maintain skin cell renewal (elastin and col-
lagen stimulation)
[6] . Mixed formulations (kaempferol
and either erythromycin or clindamycin; quercetin and
either erythromycin or clindamycin) synergically inhibit
antibiotic-resistant P. acnes growth
[7] . The European
Pharmacopeia defines white willow bark (WWB) as the
whole or fragmented dried bark of young branches or
dried pieces of current year twigs from various species of
the genus Salix [8] . WWB has been used as a traditional
medicine for the treatment of fever, pain, and inflamma-
tion
[9] . Salicin, the major constituent of WWB extract,
is metabolized to salicylic acid in vivo and shows anti-
inflammatory effects; moreover, other ingredients in the
extracts include other salicylates as well as polyphenols
and flavonoids, which could play prominent roles in the
therapeutic efficacy
[10] . WWB suppresses inflammatory
molecules and reduces oxidative stress in human endo-
thelial cells
[11, 12] . In vitro and in vivo studies evidenced
that the anti-inflammatory activity of WWB is associated
with the downregulation of the inflammatory mediators
TNF-α and NF-κB
[13] . In 2010, a paper by Gopaul et al.
[14] described the ability of salicin to reduce the visible
signs of skin aging when applied topically, thus showing
anti-aging capabilities.
Among natural compounds involved in the regulation
of skin homeostasis, 1,2-alkanediols display several prop-
erties (high water solubility and effectiveness as a solvent)
that allow their use as moisturizing compounds in the
dermal cosmetic field
[15–17] . Indeed, their bacteriostat-
ic and fungistatic activity decreased the amount of con-
ventional preservatives inside cosmetic formulations.
Generally, the antimicrobial effect of 1,2-alkanediols in-
creases with the length of their carbon chain, with 1,2-dec-
anediol (DD) having a minimal inhibitory concentration
of <1%, which is 10-fold lower than that of 1,2-octane-
diol
[18] . A recent study reported the effects of five 1,2-al-
kanediols on skin irritation potentials
[19] that represent
an important factor for determining the usefulness of
1,2-alkanediols.
Here we tested the effects of WWB and DD, used
alone or in combination, on human adult keratinocytes
(HaCaT) stressed by lipopolysaccharides (LPS). More-
over, we investigated a possible role of DD not only as a
moisturizing agent, but also as bioactive molecule on epi-
dermal layers.
Table 1. Natural compounds and relative concentrations used in this study
Compound Concentration
WWB A: 520 g/mL B: 260 g/mL C: 130 g/mL
DD A: 5.2 mg/mL B: 26 g/mL C: 13 g/mL
WWB/DD complex C/C: 130 g/mL + 13 g/mL
WWB, white willow bark; DD, 1,2-decanediol.
Table 2. Schematic configuration of HaCaT treatment: experimental time course
Conditions Preincubation (2 h) Treatment (24 h)
CTRL positive Only DMEM 10%
CTRL negative DMEM 10% DMEM 10% + LPS
DD + LPS DMEM 10% + DD DMEM 10% + DD + 10 g/mL LPS
WWB + LPS DMEM 10% + WWB DMEM 10% + WWB + 10 g/mL LPS
WWB/DD + LPS DMEM 10% + WWB/DD DMEM 10% + WWB/DD + 10 g/mL LPS
As indicated, cells were preincubated (2 h) with natural compounds before a prolonged treatment with LPS
(24 h). CTRL, control; DMEM, Dulbecco modified Eagle medium; DD, 1,2-decanediol; LPS, lipopolysaccharides;
WWB, white willow bark.
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Bassino/Gasparri/Munaron
Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
12
Materials and Methods
Drugs
LPS from Escherichia coli were purchased from Sigma-Aldrich.
DD is a diol monomer, solid and soluble only in cosmetic esters,
and acts as an anti-acne agent and antiperspirant (Symrise, Holz-
minden, Germany). WWB is an extract of the white bark of Salix
alba that is standardized for its salicin content (Euromed, Barce-
lona, Spain). DD and WWB were prepared in 0.001% DMSO (see
Table1 for concentrations).
Cell Cultures
A human adult keratinocyte cell line (HaCaT) was obtained
from Cell Line Services (CLS Cell Lines Service, Germany). HaCaT
were grown in Dulbecco modified Eagle medium (DMEM) with
10% fetal calf serum and 1% antibiotic/antimycotic (Invitrogen,
Grand Island, NY, USA).
Cell Viability
HaCaT were seeded in 96-well plates (5,000 cells/well). Cells
were treated as indicated in Table2 . After 24 h cell viability was
evaluated by the CellTiter 96
® AQueous Non-Radioactive Cell
Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-di-
methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H tetrazolium, inner salt. Its conversion into the aque-
ous soluble formazan product is accomplished by dehydrogenase
enzymes found in metabolically active cells. Formazan product
was measured with a FilterMax F5 Microplate reader (Molecular
Devices, USA) at 490 nm, as absorbance is directly proportional to
the number of viable cells.
Enzyme-Linked Immunosorbent Assay
Proinflammatory cytokines (IL-1α, IL-6 and IL-8), growth fac-
tors (VEGF, EGF, IGF-I), metabolic markers (keratin 16, KRT-16)
and transcriptional factors (FOXO-I) were quantified in cell ly-
sates or medium by enzyme-linked immunosorbent assay (ELISA)
using commercially available kits (Sigma ELISA kit, Invitrogen
ELISA kit). Briefly, 100 µL of medium or cell lysate were incubated
into an antibody-coated 96-well plate at room temperature for
2.5 h. The wells were washed 4 times with wash buffer solution.
Then 100 µL of primary anti-human antibody was added, and the
samples were again incubated for 1 h at room temperature. The
plate was washed 4 times, 100 µL of streptavidin-peroxidase con-
jugate was applied for 1 h at room temperature. After a final wash-
ing, 100 µL tetramethylbenzidine substrate was added and allowed
to develop for 30 min in the dark at room temperature. After stop-
ping the reaction with 50 µL stop solution containing citric acid
2.0 mmol/L, absorbance was read at 450 nm with an F5 FilterMax
microplate reader 550 (Molecular Devices, USA). Sample concen-
tration was calculated from the standard curve.
Cytosolic and Nuclear Protein Extraction for FOXO-I
Quantification
HaCaT were grown to 80% confluence. Afterwards, cells were
scraped using fresh PBS, collected into an appropriate conical tube
and centrifuged (5 min at 450 g ). Then the supernatant was dis-
carded, and 1 mL of lysis buffer (10 m
M Tris HCl, pH 7.5, 2 m M
MgCl
2 , 3 m M CaCl 2 , 0.3 M sucrose, including DTT and protease
inhibitors) was added to 200 µL of packed cell volume for 15 min.
Suspended cells were centrifuged for 5 min at 420 g . The pellet of
packed cells was resuspended in 400 µL (2× packed cell volume)
lysis buffer and fragmented using a syringe with a narrow gauge.
The disrupted cells in suspension were centrifuged for 20 min at
10,000 g . The supernatant was transferred into a fresh tube, and
this fraction corresponds to the cytoplasmic fraction. The pellet
was resuspended in 140 µL extraction buffer (20 m
M HEPES, pH
7.9, with 1.5 m
M MgCl 2 , 0.42 M NaCl, 0.2 m M EDTA, 25% (v/v)
glycerol added with 1.5 µL of the 0.1
M DTT solution and 1.5 µL
of the protease inhibitor cocktail) and centrifuged for 5 min at
20,000 g . The resulting supernatant is the nuclear protein extract.
It is finally collected into a clean tube and analyzed with FOXO-I
ELISA assay.
Scratch Wound Healing
HaCaT were seeded in 24 multiwell plates and cultured to con-
fluence. A scratch was made in the confluent monolayer with a
plastic disposable pipette tip (10 L). Debris was removed from the
culture by gently washing with sterile PBS. Hereafter, HaCaT were
cultured in DMEM 10% and treated with 10 g/mL LPS, DD,
WWB alone or in association for 24 h. Experiments were per-
formed using a Nikon T-E microscope (4× objective). Cells were
kept at 37
 °  C and 5% CO
2 for all experiments. Photos were taken
every 4 h using Metamorph software. Cell migration was measured
with ImageJ software (Rasband W.S., ImageJ, US National Insti-
tutes of Health, Bethesda, MD, USA). At least 3 fields for each con-
dition were analyzed in each independent experiment.
Statistical Analysis
Statistical significance of all experiments was evaluated by
GraphPad software (Synergy Software, USA). The Dunnet multi-
comparison test was chosen because 5 biological replicates were
done for each condition in each experiment, and they were not
normally distributed. Five technical replicates were performed for
each experimental condition; 3 biological replicates were per-
formed for each experimental condition.
Results with p values <0.05 were considered statistically sig-
nificant.
Fig. 1. WWB and DD exert protective effects on HaCaT treated
with LPS. *  p < 0.05.
A 25 g/mL LPS significantly reduced cell vi-
ability compared to untreated cells.
B The highest dose of DD (A)
significantly reduced cell viability of HaCaT treated with 10 g/mL
LPS. The other concentrations slightly but not significantly re-
duced cell viability.
C The intermediate dose of WWB (B) signifi-
cantly reduced cell viability of HaCaT treated with 10 g/mL LPS.
The other concentrations were ineffective.
D The highest dose of
DD (A) significantly reduced cell viability of HaCaT treated with
25 g/mL LPS. The other concentrations were ineffective.
E All the
concentrations of WWB (doses A–C) were ineffective on cell via-
bility of HaCaT treated with 25 g/mL LPS.
F The combination of
the highest doses of WWB/DD (A) significantly reduced cell via-
bility of HaCaT treated with 10 g/mL LPS.
G All the concentra-
tions of WWB/DD were ineffective on cell viability of HaCaT
treated with 25 g/mL LPS. DD doses: A = 5.2 mg/mL, B = 26 g/
mL, C = 13 g/mL; WWB doses: A = 520 g/mL, B = 260 g/mL,
C = 130 g/mL; WWB/DD doses: A = 5.2 mg/mL + 520 g/mL,
B = 26 g/mL + 260 g/mL, C = 13 g/mL + 130 g/mL.
(For figure see next page.)
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Effects of White Willow Bark and
1,2-Decanediol on Keratinocytes
Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
13
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Bassino/Gasparri/Munaron
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DOI: 10.1159/000481690
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Fig. 2. The lowest concentrations of WWB and DD differently af-
fect EGF, IGF-I and KRT-16 production and FOXO-I transloca-
tion. *  p < 0.05. CTRL, control.
A HaCaT were stimulated with 10
g/mL LPS alone (
i ) or in association with the lowest concentra-
tions (C) of WWB and DD (
ii ). Supernatant was collected after
24 h, and EGF secretion was quantified with ELISA. 10 g/mL LPS
increased EGF production (
i ). WWB and DD (alone or in associa-
tion) significantly reduced EGF production increased by LPS treat-
ment (24 h) (
ii ). B HaCaT were stimulated with 10 g/mL LPS used
alone (
i ) or in association with the lowest concentrations (C) of
WWB and DD (
ii ). Supernatant was collected after 24 h, and IGF-
I secretion was quantified with ELISA. 10 g/mL LPS increased
IGF-I production after 24 h of treatment (
i ). WWB/DD complex
significantly reduced IGF-I production affected by LPS (24 h) (
ii ).
C HaCaT were stimulated with 10 g/mL LPS used alone ( i ) or in
association with the lowest concentrations (C) of WWB and DD
(
ii ). Cytosolic extract was collected after 24 h, and KRT-16 secre-
tion was quantified with ELISA. 10 g/mL LPS slightly but not
significantly increased KRT-16 production (
i ). WWB or DD used
alone or in combination did not significantly modify KRT-16 pro-
duction (24 h) (
ii ). D HaCaT were stimulated with 10 g/mL LPS
used alone (
i ) or in association with the lowest concentrations (C)
of WWB and DD (
ii ). After 24 h cytosolic and nuclear FOXO-I
extracts were obtained and quantified with ELISA. 10 g/mL LPS
increased the cytosolic/nuclear (cyt/nu) ratio of FOXO-I (
i ). Both
WWB and DD used alone or in association (lowest dose) signifi-
cantly reduced the FOXO-I ratio (24 h) (
ii ). DD dose C: 13 g/mL;
WWB dose C: 130 g/mL; WWB/DD dose C: 13 g/mL + 130 g/
mL.
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Effects of White Willow Bark and
1,2-Decanediol on Keratinocytes
Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
15
Results
WWB and DD Affect Cell Viability of Human Adult
Keratinocytes Treated with LPS
In order to set a suitable pattern of LPS concentrations
to be tested, we performed a preliminary dose-response
curve of HaCaT viability. Lower concentrations of LPS
(up to 10 µg/mL for 24 h) did not affect HaCaT number,
while a higher dose (25 g/mL for 24 h) significantly re-
duced cell viability ( Fig. 1 A). Based on these results, we
first evaluated the effect of WWB and DD (separately or
as WWB/DD complex) on cells treated with 10 g/mL
LPS (ineffective dose). High concentrations of DD sig-
nificantly affected cell viability, while lower doses were
ineffective ( Fig.1 B). WWB (B concentration) significant-
ly affected cell viability ( Fig.1 C). The association of both
compounds (WWB/DD complex) significantly reduced
cell viability when added both at higher doses in HaCaT
treated with 10 g/mL LPS ( Fig. 1 F). Both compounds
failed to prevent the cytotoxicity induced by 25 µg/ml LPS
both separately ( Fig.1 D, E) and in complex ( Fig.1 G). For
the following tests we chose the lowest, noncytotoxic con-
centrations of the natural compounds, and we tested their
effects on 10 g/mL LPS.
Release of EGF, IGF-I, and KRT-16
10 g/mL LPS promoted a significant EGF release by
HaCaT (24 h) ( Fig.2 Ai). DD and WWB, both separately
and in complex, significantly prevented LPS-induced
EGF release ( Fig.2 Aii). The same trend was observed on
IGF-I release ( Fig.2 Bi, ii). Finally, the treatment with 10
g/mL LPS slightly, but not significantly, increased KRT-
16 production ( Fig.2 Ci), an effect slightly prevented by
DD ( Fig.2 Cii).
FOXO-I Distribution
Incubation with 10 g/mL LPS (24 h) drastically en-
hanced cytosolic expression of FOXO-I ( Fig. 2 Di); this
effect was prevented by incubation with DD and WWB,
both separately or in combination ( Fig.2 Dii).
Modulation of Wound Closure and
Anti-Inflammatory Role of WWB and DD
To evaluate the role of both compounds in the wound
healing rate of HaCaT, we first examined their ability to
modulate cell motility employing an established in vitro
scratch wound healing assay. Wound closure was evalu-
ated by observing the repopulated area between the wound
margins at different time intervals (0–24 h) after the le-
sion. The wound monitoring showed that untreated cells
(DMEM 10%) followed the physiological healing process,
reaching approximately 65% of closure at 24 h after injury
( Fig.3 A, Bi). 10 g/mL LPS (24 h) drastically reduced the
wound closure percentage, reaching approximately 35%.
This effect was prevented by both DD and WWB applied
separately ( Fig.3 A, Bii). Application of 10 g/mL LPS re-
duced VEGF production in scratched keratinocytes
( Fig.3 Ci). DD and WWB/DD complex prevented the LPS
effect, while WWB was found to be ineffective ( Fig.3 Cii).
Skin wounding and inflammatory responses involve cyto-
kines that exert inhibitory activity on human keratinocyte
growth. We investigated the effects of DD and WWB both
separately and in association on cytokine production (IL-
1 α, IL-6, and IL-8) by HaCaT upon treatment with LPS.
Incubation with 10 g/mL LPS (24 h) promoted the
release of IL-8, IL-1α, and IL-6 ( Fig.3 Di, Ei, Fi). The low-
est doses of DD and WWB, separately or in combination,
did not prevent IL-1α production ( Fig.3 Eii); conversely,
WWB and WWB/DD were significantly effective on IL-6
and IL-8 release, while DD induced a nonsignificant pro-
tective activity ( Fig.3 Dii, Fii).
Discussion
Acne is a multifactorial disease based on an alteration
in the pattern of keratinization within the pilosebaceous
follicles resulting in comedo formation, an increase in se-
bum production which is influenced by androgens, the
proliferation of P. acnes , and the development of perifol-
licular inflammation
[20, 21] . New commercially available
formulations and drugs for skin treatment are increasing-
ly introduced. Evidence suggests a potential beneficial ac-
tivity of plant-derived phenolic compounds obtained ei-
ther by the diet or through skin application; indeed, they
can alleviate symptoms and inhibit the development of
various skin disorders. Recently, a novel face compact
cream containing a new patented formulation (including
both WWB extract and DD) was developed to provide
acne patients with cosmetic camouflage for their lesions
and to have beneficial effects on the multifactorial compo-
nents of the disease
[22] . Here we tested the role of both
compounds on adult keratinocytes stressed with LPS.
Both compounds (used alone or in combination) counter-
acted LPS effects, reducing growth factor and cytokine
production. They reduced EGF expression altered by the
treatment with LPS. This is interesting because several
studies report the alteration of EGF signaling during acne
associated with the use of anticancer agents (e.g., EGF re-
ceptor inhibitors). Acneiform eruptions are a common
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Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
16
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3
(For legend see next page.)
Color version available online
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Effects of White Willow Bark and
1,2-Decanediol on Keratinocytes
Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
17
adverse reaction to EGF receptor inhibition and can be
treated with traditional acne therapy
[23] . Similarly,
WWB/DD complex significantly prevented IGF-I pro-
duction induced by LPS. During puberty, IGF-I signaling
may have a causal role in the pathogenesis of acne by in-
fluencing adrenal and gonadal androgen metabolism that
was reported to be an inducer of sebum production
through sterol response element-binding proteins
[24,
25] . Recently, the involvement of IGF-I, but not andro-
gens, has been suggested in acne pathogenesis [26] . A rel-
evant IGF-I-dependent mechanism that increases andro-
gen receptor signaling involves the metabolic transcrip-
tion factor FOXO-I. FOXO-I is proposed to be the key to
understand the link between genetic and environmental
factors in acne. Nuclear FOXO-I functions as an androgen
receptor cosuppressor which regulates the activity of most
important target genes involved in the pathogenesis of
acne [27] . All growth factors or acneigenic stimuli mim-
icking growth factor signaling might lead to the reduction
of the nuclear content of FOXO-I. In the present study, we
observed that WWB and DD, used separately or in com-
bination, significantly decrease the FOXO-I cytosolic/nu-
clear ratio. Both WWB and DD slightly but not signifi-
cantly prevented KRT-16 expression after LPS treatment.
KRT-16, as well as KRT-6 and KRT-17, is considered a
stress KRT chronically expressed in human premalignant
hyperproliferative epithelium
[28] . Finally, we tested the
role of natural compounds in the modulation of keratino-
cyte migration and in the production of proinflammatory
cytokines. Li et al.
[29] reported the ability of LPS to re-
duce keratinocyte migration in a diabetes-like microenvi-
ronment. Here we observed that both compounds mark-
edly promoted HaCaT migration and enhanced artificial
wound closure compromised by LPS treatment, an effect
related to their ability to sustain VEGF production. Cell-
free extracts of P. acnes significantly stimulate secretion of
interleukins (IL-8 and IL-6) in SZ95 sebocytes
[30] and
promote IL-8 secretion by interacting with Toll-like re-
ceptor 2
[31] . IL-6 is a pleiotropic cytokine that plays a
pivotal role in host defense, immune response regulation,
hematopoiesis, and inflammation
[32] . Single-nucleotide
polymorphisms in the IL-6 gene have been identified and
associated with several diseases
[33, 34] . However, there
are relatively few studies on IL-6 gene polymorphism in
acne patients. It has recently been reported that the IL-6
and IL-1α gene promoter polymorphisms are associated
with the pathogenesis of acne vulgaris
[35] . IL-1α has been
widely studied in inflammation and is considered to affect
the pathogenesis of acne
[36] ; it also contributes to para-
crine signaling between keratinocytes and fibroblasts
during wound healing. WWB and DD differently affect
cytokine production. Both compounds failed to counter-
act IL-1α release increased by LPS treatment. Conversely,
WWB alone or WWB/DD complex significantly reduced
IL-6 and IL-8 release, while only a slight effect could be
observed upon stimulation with DD alone.
In conclusion, taken together, our results show the
ability of both natural compounds to affect different func-
tions of LPS-stressed keratinocytes.
Acknowledgment
This study was funded by Rottapharm Madaus.
Statement of Ethics
No ethical conflicts exist, because only in vitro tests on cells
were performed.
Fig. 3. The lowest concentrations of WWB and DD differentially
regulate wound closure and proinflammatory cytokine produc-
tion. *  p < 0.05. CTRL, control.
A
Representative micrograph of
keratinocytes directly (0 h) or 24 h after scraping. Original mag-
nification ×4.
B
HaCaT cells were scratched and stimulated with
10 g/mL LPS used alone or in association with WWB and DD.
The percentage of wound closure is shown as means ± SEM of 3
independent experiments.
C
HaCaT were scratched and stimu-
lated with 10 g/mL LPS used alone or in the presence of WWB
and DD (alone or in complex, both at the lowest concentration).
Supernatant was collected 24 h after scraping, and VEGF produc-
tion was quantified with ELISA. 10 g/mL LPS significantly
reduced VEGF production (
i
). DD alone or in complex with
WWB complex significantly prevented the VEGF reduction (
ii
).
D
HaCaT were stimulated with 10 g/mL LPS used alone or in the
presence of WWB and DD (alone or in complex, both at the low-
est concentration). Supernatant was collected after 24 h, and IL-8
secretion was quantified with ELISA. 10 g/mL LPS significantly
increased IL-8 production (
i
). WWB alone or WWB/DD complex
significantly prevented this effect (
ii
).
E
HaCaT were stimulated
with 10 g/mL LPS used alone (
i
) or in the presence (
ii
) of WWB
and DD (used alone or in complex, both at the lowest concentra-
tion). Supernatant was collected after 24 h, and IL-1α secretion
was quantified with ELISA. 10 g/mL LPS significantly increased
Il-1α release (
i
). DD and WWB alone or in association did not
modify this effect (
ii
).
F
HaCaT were stimulated with 10 g/mL
LPS used alone (
i
) or in association (
ii
) with WWB and DD (alone
or in complex, both at the lowest concentration). Supernatant was
collected after 24 h, and IL-6 secretion was quantified with ELISA.
10 g/mL LPS significantly increased IL-6 release (
i
). WWB alone
or in association with DD significantly prevented this effect (
ii
).
DD dose C: 13 g/mL; WWB dose C: 130 g/mL; DD/WWB dose
C: 13 g/mL + 130 g/mL.
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Skin Pharmacol Physiol 2018;31:10–18
DOI: 10.1159/000481690
18
Disclosure Statement
The authors have declared that there is no conflict of interest.
Author Contributions
E.B. designed the research study, performed the research, ana-
lyzed the data, and wrote the paper. F.G. contributed essential re-
agents or tools and designed the research study. L.M. designed the
research study, analyzed the data, wrote the paper, and contrib-
uted essential reagents or tools.
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