Topical apigenin improves epidermal permeability barrier homoeostasis in normal murine skin by divergent mechanisms

Article (PDF Available)inExperimental Dermatology 22(3):210-215 · March 2013with13 Reads
DOI: 10.1111/exd.12102 · Source: PubMed
Abstract
The beneficial effects of certain herbal medicines on cutaneous function have been appreciated for centuries. Among these agents, chrysanthemum extract, apigenin, has been used for skin care, particularly in China, for millennia. However, the underlying mechanisms by which apigenin benefits the skin are not known. In this study, we first determined whether topical apigenin positively influences permeability barrier homoeostasis, and then the basis thereof. Hairless mice were treated topically with either 0.1% apigenin or vehicle alone twice daily for 9 days. At the end of the treatments, permeability barrier function was assessed with either an electrolytic water analyzer or a Tewameter. Our results show that topical apigenin significantly enhanced permeability barrier homoeostasis after tape stripping, although basal permeability barrier function remained unchanged. Improved barrier function correlated with enhanced filaggrin expression and lamellar body production, which was paralleled by elevated mRNA levels for the epidermal ABCA12. The mRNA levels for key lipid synthetic enzymes also were upregulated by apigenin. Finally, both cathelicidin-related peptide and mouse beta-defensin 3 immunostaining were increased by apigenin. We conclude that topical apigenin improves epidermal permeability barrier function by stimulating epidermal differentiation, lipid synthesis and secretion, as well as cutaneous antimicrobial peptide production. Apigenin could be useful for the prevention and treatment of skin disorders characterized by permeability barrier dysfunction, associated with reduced filaggrin levels and impaired antimicrobial defenses, such as atopic dermatitis.
Topical apigenin improves epidermal permeability barrier
homoeostasis in normal murine skin by divergent mechanisms
Maihua Hou
1,3
*, Richard Sun
1
*, Melanie Hupe
1
, Peggy L. Kim
1
, Kyungho Park
1
, Debra Crumrine
1
,
Tzu-Kai Lin
1,2
, Juan Luis Santiago
1
, Theodora M. Mauro
1
, Peter M. Elias
1
and Mao-Qiang Man
1
1
Dermatology Service, Veterans Affairs Medical Center, and University of California San Francisco, San Francisco, CA, USA;
2
Department of Dermatology,
National Cheng Kung University Hospital, and Graduate Institute of Clinical Medicine, College of Medicine, National Cheng Kung University,
Tainan, Taiwan;
3
Department of Dermatology, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
Correspondence: Mao-Qiang Man, MD, Dermatology (190), 4150 Clement Street, San Francisco, CA 94121, USA, Tel.: (415)750-2091, Fax: (415)
750-2106, e-mail: mqman@hotmail.com
*Both authors contributed equally to this work.
Abstract: The beneficial effects of certain herbal medicines on
cutaneous function have been appreciated for centuries. Among
these agents, chrysanthemum extract, apigenin, has been used for
skin care, particularly in China, for millennia. However, the
underlying mechanisms by which apigenin benefits the skin are
not known. In this study, we first determined whether topical
apigenin positively influences permeability barrier homoeostasis,
and then the basis thereof. Hairless mice were treated topically
with either 0.1% apigenin or vehicle alone twice daily for 9 days.
At the end of the treatments, permeability barrier function was
assessed with either an electrolytic water analyzer or a Tewameter.
Our results show that topical apigenin significantly enhanced
permeability barrier homoeostasis after tape stripping, although
basal permeability barrier function remained unchanged.
Improved barrier function correlated with enhanced filaggrin
expression and lamellar body production, which was paralleled by
elevated mRNA levels for the epidermal ABCA12. The mRNA
levels for key lipid synthetic enzymes also were upregulated by
apigenin. Finally, both cathelicidin-related peptide and mouse
beta-defensin 3 immunostaining were increased by apigenin. We
conclude that topical apigenin improves epidermal permeability
barrier function by stimulating epidermal differentiation, lipid
synthesis and secretion, as well as cutaneous antimicrobial peptide
production. Apigenin could be useful for the prevention and
treatment of skin disorders characterized by permeability barrier
dysfunction, associated with reduced filaggrin levels and impaired
antimicrobial defenses, such as atopic dermatitis.
Abbreviations: ABCA12, ATP-binding cassette transporter 12; CAMP,
cathelicidin-related peptide; mBD3, mouse beta-defensin 3.
Key words: antioxidant apigenin barrier differentiation
transepidermal water loss
Accepted for publication 28 January 2013
Introduction
Herbal medicines have been used widely, especially in Asia, for the
prevention and treatment of a variety of disorders, including
inflammatory dermatoses, largely due to their considerable efficacy
and improved side effect profiles in comparison with Western
medicines. Well-controlled clinical studies have demonstrated that
herbal medicines are effective in treating cardiovascular disease
(1,2), renal disorders (3,4), respiratory disease (5,6), as well as
some cancers (7,8). Similarly, herbal medicines have shown benefi-
cial effects in inflammatory dermatoses, such as psoriasis, acne,
contact dermatitis and atopic dermatitis, not only in murine dis-
ease models, but also in humans (913). Our recent studies show
that topical applications of several herbal extracts improve epider-
mal permeability barrier function, in part by stimulating keratino-
cyte differentiation and lipid production, in addition to
upregulating epidermal antimicrobial peptide expression in mur-
ine models (14,15), perhaps accounting for the clinical impact of
some of these preparations.
Chrysanthemum, also known as Bracteantha bracteata,isa
common ingredient in some of these herbal medicines. It has
proved effective in treating skin disorders in China for millennia.
For instance, topical chrysanthemum extract alleviated diaper der-
matitis in infants and newborns with erythema venenatum
(16,17). Improvement of certain cutaneous drug reactions also has
been reported with chrysanthemum (18). Commercialized chry-
santhemum tea, as well as chrysanthemum extracts contained in
skin care products, is claimed to increase skin hydration and to
provide a protective barrier (http://fr.provitalgroup.com/en/
products_botanical/moisturizing/chrysanthemum-oil).
Apigenin is an active constituent that is present in large
quantity in chrysanthemum extract. Certain benefits of apigenin
on cutaneous function have already been documented. For
example, apigenin exhibits preventive activity against UVB-
induced skin tumors, apparently through inhibition of cyclooxy-
genase-2 (COX-2) expression (1921). In a murine model, an
apigenin-enriched diet attenuated the development of atopic
dermatitis-like lesions (22), and one clinical study showed that
an apigenin-containing cream inhibited cutaneous inflammation
(23). Some of these benefits of apigenin, such as UVB protec-
tion and cancer prevention, could be due to the antioxidant
properties of apigenin (24,25).
Recent studies suggest that antioxidant defense and epidermal
permeability barrier could be interrelated functions. Oxidative
stress inevitably occurs in skin disorders with epidermal perme-
ability barrier abnormalities (2628), while conversely, applica-
tions of antioxidants improve epidermal permeability barrier
210
ª 2013 John Wiley & Sons A/S
Experimental Dermatology, 2013, 22, 210–215
DOI: 10.1111/exd.12102
www.blackwellpublishing.com/EXD
Original Article
function (15,2931). However, whether the antioxidant, apigenin,
improves epidermal permeability barrier function and the underly-
ing mechanisms responsible for such changes remain undefined.
In this study, we evaluated the effects of apigenin on epidermal
permeability barrier function in normal murine skin. After first
finding that apigenin improved barrier function, we assessed the
responsible mechanisms, including the impact of apigenin on
keratinocyte differentiation, lipid secretion and antimicrobial
peptide expression.
Materials and methods
Materials
Six- to eight-week-ol d female hairless mice (h/h) were purchased
from Charles River Laboratories (Wilmington, MA, USA) and fed
mouse diet (Ralston-Purina Co., St Louis, MO, USA) and water
ad libitum. Apigenin powder was purchased from Sigma Chemical
Co (St Louis, MO, USA). Affinity-purified, rabbit anti-mouse
antibodies to loricrin, involucrin and filaggrin were purchased
from Covance (Emeryville CA, USA) for immunohistochemistry.
Experimental protocols and functional studies
All animal procedures were approved by the Animal Studies Sub-
committee (IACUC) of the San Francisco Veterans Administration
Medical Center and performed in accordance with their guide-
lines. Both flanks of mice were treated topically with 60 llof
0.1% apigenin or ethanol twice daily for 9 days. Basal epidermal
permeability barrier function was assessed by measuring transepi-
dermal water loss (TEWL) using TM300 connected to MPA5
(C&K, Cologne, Germany). For barrier recovery, TEWL was mea-
sured using an electrolytic water analyzer (Meeco, Warrington,
PA) at 0, 2 and 4 h after tape stripping (10-fold increase in
TEWL), and per cent barrier recovery was calculated as described
earlier (32).
Cell culture
Second-passage keratinocytes isolated from newborn foreskins
were cultured in serum-free keratinocyte growth medium contain-
ing 0.07 m
M calcium (Clonetics, San Diego, CA, USA). Cells at
8090% confluence were switched to a medium containing
1.2 m
M calcium and treated with either 10 lM apigenin or vehicle
alone (0.05% ethanol). After 24 and 48 h of treatment, keratino-
cytes were collected for Western blot and Q-PCR analysis.
Western blot analysis of epidermal differentiation proteins
Epidermis from mice treated topically with 0.1% apigenin or
vehicle alone for 9 days was obtained by EDTA separation (15).
Epidermis was prepared in radioimmunoprecipitation assay buffer
and was resolved by electrophoresis on 412% Bis-Tris gel (Invi-
trogen, Carlsbad, CA, USA). Resultant bands were blotted onto
polyvinylidene fluoride membranes and were subsequently probed
with monoclonal anti-mouse b-actin antibody (Sigma), polyclonal
antifilaggrin (against the 37 kD, monomer; Covance, Emeryville
CA, USA), polyclonal anti-involucrin (against the 56 kD;
Covance) or polyclonal antiloricrin (against the 57 kD; Covance),
and the corresponding bands were detected by enhanced chemilu-
minescence (Thermo Fisher Sci., Rockford, IL, USA) and quanti-
tated by scanning densitometry. For keratinocyte cultures,
polyclonal antifilaggrin (against the 37 kD, monomer), monoclo-
nal anti-involucrin and polyclonal antiloricrin were purchased
from Abcam, Cambridge, MA. Results were presented as percent-
age of vehicle-treated control, setting vehicle treated as 100%.
b-actin was used to normalize changes in expression levels. Results
were presented as percentage of vehicle-treated control, setting
vehicle treated as 100%.
Q-PCR for mRNA expression of filaggrin and lipid synthetic
enzymes
Total RNA was isolated from cultured human keratinocytes using
TRI Reagent (Sigma). First-strand cDNA was synthesized from
1 lg of total RNA with the iScript cDNA Synthesis kit (Bio-Rad,
Hercules, CA). The real-time PCR contained 20 ng of reversed
transcribed total RNA, 450 n
M forward and reverse primers, and
10 llof29 LightCycler 480 SYBR Green I Master in a final vol-
ume of 20 ll in 96-well plates using M93000P
TM
real-time PCR
system (Stratagene, La Jolla, CA, USA). Quantification was per-
formed by the comparative C
T
method with cyclophilin A used
for normalization. The primers for filaggrin and lipid synthetic
enzymes, including 3-hydroxy-3-methyl-glutaryl-CoA reductase
(HMGCoA), serine palmitoyltransferase 1 (SPT1), fatty acid syn-
thase (FAs), the lipid transporter [ATP-binding cassette A12
(ABCA12)], as well as cyclophilin A are listed in Table S1. Relative
expression of the mRNAs compared to control mRNA was calcu-
lated. Data are expressed as percentage of control (as 100%).
Immunohistochemistry
Immunohistochemical staining for changes in epidermal differenti-
ation was performed as described previously (33). Briefly, 5-lm
paraffin sections were incubated with the primary antibodies at
the dilutions of 1:2000 for filaggrin, 1:1000 for involucrin and
1:500 for loricrin overnight at 4°C. After washes 93, sections were
incubated with the secondary antibody for 30 min. Staining was
detected with ABC-peroxidase kit from Vector Lab (Burlingame,
CA, USA), and sections were then counterstained with haematoxy-
lin. All antibodies for differentiation markers were from Covance.
For assessment of changes in two antimicrobial peptides, mouse
beta-defensin 3 (mBD3) and cathelicidin-related peptide (CAMP),
expression was performed in 5-lm frozen and paraffin sections,
respectively [primary antibodies to mBD3 from Alpha Diagnostics;
mouse cathelicidin (CAMP) antibody was a gift from Dr. Richard
Gallo (UCSD)] (14). Sections were examined with a Zeiss fluores-
cence microscope (Jena, Germany), and digital images were cap-
tured with AxioVision software (Carl Zeiss Vision, Munich,
Germany). All pictures were taken with the same exposure times.
Electron microscopy
Skin biopsies from both vehicle and apigenin-treated mice were
taken for electron microscopy (15). Briefly, samples were minced
to <0.5 mm
3
, fixed in modified Karnovsky’s fixative overnight and
postfixed in either 0.2% ruthenium tetroxide or 1% aqueous
osmium tetroxide, containing 1.5% potassium ferrocyanide. After
fixation, all samples were dehydrated in a graded ethanol series
and embedded in an Epon-epoxy mixture. Ultrathin sections were
examined, with or without further contrasting with lead citrate, in
a Zeiss 10A electron microscope (Carl Zeiss, Thornwood, NJ,
USA), operated at 60 kV. Number of lamellar body was counted
in every 30 cm
2
area in the first layer of stratum granulosum on
micrograph with magnification of 25 000 times. The data were
expressed as number of lamellar body per 30 cm
2
area.
Statistics
GraphPad Prism 4 software was used for all statistical analyses
(GraphPad Software, Inc., La Jolla, CA, USA). An unpaired t-test
with Welch’s correction was used for comparisons between two
groups. Data are expressed as mean SEM.
ª 2013 John Wiley & Sons A/S
Experimental Dermatology, 2013, 22, 210–215
211
Apigenin improves epidermal permeability barrier and differentiation
Results
Topical apigenin improves epidermal permeability
homoeostasis in normal murine skin
We first assessed whether topical apigenin improves epidermal
permeability barrier function in normal murine skin. After 9 days
of topical apigenin treatment, there were no changes in gross
appearance of mouse skin. Baseline skin surface pH and TEWL
rates did not differ significantly in apigenin- versus vehicle-treated
mice (Fig. 1a,c; P = 0.3411 for Fig. 1a and P = 0.0963 for Fig. 1c).
But stratum corneum (SC) hydration was slightly but significantly
lower in apigenin-treated as compared to vehicle-treated mice
(Fig. 1b). In contrast to basal TEWL, topical apigenin accelerated
barrier recovery, a change that was highly significant at 4 h after
acute barrier disruption (Fig. 1d). These results demonstrate that
topical apigenin improves epidermal permeability barrier homoeo-
stasis in normal murine skin.
Topical apigenin stimulates filaggrin expression in the
murine model
Because differentiation-related structural proteins are a key deter-
minant of normal barrier function, we next assessed whether
topical apigenin influences the expression of epidermal differentia-
tion-related proteins, potentially providing one potential mecha-
nism whereby apigenin could improve barrier function. As
showed in Fig. 2, epidermal filaggrin immunostaining became
much more prominent following topical apigenin treatment
(Fig. 2d versus Fig.2a), but neither involucrin nor loricrin immu-
nostaining differed in apigenin- versus vehicle-treated skin
(Fig. 2b,c versus Fig. 2e,f). To further confirm the immunohisto-
chemical results, Western blotting was employed to quantitate the
changes in expression of the differentiation-related proteins.
Apigenin again significantly and selectively stimulated epidermal
filaggrin expression, without significantly altering either involucrin
or loricrin expression (Fig. 2g and Fig. S1).
To further validate the in vivo data, the effect of exogenous
apigenin (10 l
M) on keratinocyte differentiation was also assessed
in cultured human keratinocytes. Filaggrin expression was slightly
elevated at 24 h following exogenous apigenin treatments (Fig. 2h
and Fig. S2) and significantly elevated at 48 h (Fig. 2h and Fig.
S3). In contrast to the in vivo results, apigenin induced a signifi-
cant reduction in loricrin expression at both 24 and 48 h
(Fig. 2h), while involucrin expression was reduced at 24 h, but
increased at 48 h (Fig. 2h). To determine whether the apigenin-
induced elevation in filaggrin occurs at transcriptional levels, filag-
grin mRNA expression was assessed in human keratinocyte
cultures. The results showed that apigenin induced a significant
increase in filaggrin mRNA expression at both 24 and 48 h (vehi-
cle 100 + 9.22 vs apigenin 204 + 7.52, P < 0.0001, for 24 h; vehi-
cle 100 + 10.20 vs apigenin 160 + 16, P < 0.05, for 48 h).
Together, these results indicate that topical apigenin selectively
upregulates epidermal filaggrin expression, thereby providing one
mechanism by which apigenin could improve epidermal perme-
ability barrier homoeostasis.
Apigenin stimulates the mRNA expression of three key
barrier-related lipid synthetic enzymes
Epidermal permeability barrier function requires synthesis of three
key lipids, ceramides, cholesterol and fatty acids. We next quanti-
tated changes in mRNA levels of the three rate-limiting enzymes
for synthesis each of these key lipids, that is, HMGCoA, SPT1 as
well as FAs, after addition of exogenous apigenin to cultured
(a) (b)
(c)
(d)
Figure 1. Topical apigenin improves epidermal permeability barrier homoeostasis
in normal murine skin: hairless mice were treated topically with 60 ll of 0.1%
apigenin in 100% ethanol or ethanol alone twice daily for 9 days. Basal epidermal
permeability barrier function, skin surface pH and stratum corneum (SC) hydration
were assessed with a MPA5 (CK electronic GmbH, Cologne, Germany) connected
to TM 300, pH905 and Corneometer 825. Two readings were taken from each
mouse for basal transepidermal water loss (TEWL), hydration, as well as pH. For
barrier recovery, TEWL was measured with an electrolytic water analyzer (Meeco,
Warrington, PA, USA) at 0, 2 and 4 h after tape stripping, which results in a 10-
fold increase in TEWL, and per cent barrier recovery rates were calculated as
described earlier (32). Figure (a) is basal TEWL; (b), SC hydration; (c), skin surface
pH; (d), barrier recovery. Numbers and significances are indicated in the figures.
(a) (b) (c)
(d) (e) (f)
(g)
(h)
Figure 2. Topical selectively apigenin stimulates filaggrin expression in normal
mouse epidermis and keratinocyte cultures: 5 lm paraffin sections were incubated
with respective antibodies. The sections were visualized with a Zeiss microscope.
Figure (a) and (d) are filaggrin staining; (b) and (e) are involucrin staining; c and f
are loricrin staining. Figure (a)(c) are vehicle-treated samples and (d)(f) are
apigenin-treated samples. Magnifications are the same for all figures. Magnification
bars represent 20 lm(af). For Western blot analysis in vivo, differentiation-related
protein from mouse epidermis was isolated and quantitated by scanning
densitometry, as described in Materials and methods. (n = 5 for each group). For
Western blot analysis in human keratinocyte cultures, second-passage human
keratinocytes isolated from newborn foreskins were cultured in serum-free
keratinocyte growth medium containing 0.07 m
M calcium. Cells at 8090%
confluence were switched to medium containing 1.2 m
M calcium and treated with
either 10 l
M apigenin or vehicle alone (0.05% ethanol). After 24 and 48 h of
treatment, keratinocytes were collected for Western blotting. The corresponding
protein bands were detected by enhanced chemiluminescence and quantitated by
scanning densitometry. Figure (g) and (h) displays the quantitative changes of
expression in mouse epidermis and human keratinocyte cultures, respectively.
Results were presented as percentage of vehicle-treated control, setting vehicle
treated as 100% as indicated by dotted line. Significances are indicated in the
figures.
212
ª 2013 John Wiley & Sons A/S
Experimental Dermatology, 2013, 22, 210–215
Hou et al.
human keratinocytes. As seen in Fig. 3, apigenin treatment signifi-
cantly elevated the mRNA levels of HMGCoA, SPT1 and FAS.
Together, these results suggest that apigenin stimulates epidermal
lipid production, which could provide another mechanism by
which apigenin improves epidermal permeability barrier homoeo-
stasis.
Topical apigenin increases production of lamellar bodies
Formation of the epidermal permeability barrier function requires
not only the synthesis of lipids, but also production and secretion
of lamellar bodies as a means to deliver lipids to the SC (34,35).
Hence, we next evaluated whether topical apigenin accelerates
lamellar body formation and secretion. Indeed, the density of
lamellar bodies in intact epidermis significantly increased after
topical apigenin treatment (Fig. 4ac). As these ultrastructural
studies strongly suggested that topical apigenin treatment increases
lamellar body production, we next assessed changes in mRNA
levels of the ATP-binding cassette transporter 12 (ABCA12), a
transmembrane glycosylceramide transporter, required for lamellar
body formation (3639). As predicted, topical apigenin induced a
marked increase in ABCA12 mRNA expression in human kerati-
nocytes cultured in 1.2 m
M calcium (Fig. 4d, P = 0.0186). Thus,
apigenin appears to accelerate the delivery of newly synthesized
lipids into nascent lamellar bodies.
Topical apigenin increases immunostaining for epidermal
CAMP and mBD3
Previous studies demonstrated that two antimicrobial peptides,
cathelicidin-related peptide (CAMP) and mBD3, are packaged
within and secreted by lamellar bodies, and that CAMP is crucial
not only for antimicrobial defense, but also for epidermal perme-
ability barrier function (4043). As our recent studies showed that
topical applications of another herbal extract improved epidermal
permeability barrier function in parallel with increased epidermal
antimicrobial peptide expression (14), we next asked whether
apigenin treatment also could increase epidermal CAMP and/or
mBD3 expression. As shown in Fig. S4, immunostaining for both
CAMP and mBD3 markedly increased following 9 days of topical
apigenin treatment. These results suggest that topical apigenin
enhances epidermal CAMP and mBD3 expression.
Discussion
An increasingly long line of evidence has demonstrated that
certain herbal medicines benefit inflammatory dermatoses charac-
terized and driven by abnormalities in barrier function. Yet, until
recently, the influence of herbal medicines on epidermal perme-
ability barrier function has not been well studied. In the present
study, we demonstrated first that topical applications of apigenin,
an extract from chrysanthemum, improve epidermal permeability
barrier homoeostasis. Although the exact mechanisms by which
apigenin accelerates epidermal permeability barrier recovery are
not clear, it is a known antioxidant (25), and topical or systemic
administrations of other antioxidants, such as vitamin C, vitamin
E as well as epigallocatechin gallate, improve epidermal permeabil-
ity barrier function (25,29,44,45). Hence, it is possible that the
apigenin-induced improvement in epidermal permeability barrier
homoeostasis could result from its antioxidant properties.
Whether or not antioxidant mechanisms are involved, a most
(a)
(b)
(c)
Figure 3. Apigenin upregulates mRNA expression of lipid synthetic enzymes in
vitro: total RNA was isolated from cultured human keratinocytes (detailed in
Materials and methods) and further purified by RNeasy RNA purification kit. cDNA
was prepared using the reverse transcription kit. Levels of mRNA expression were
measured by PCR using SYBR Green Master Mix. Relative expression of the mRNAs
compared to GAPDH control mRNA was calculated. Data are normalized to vehicle
control (setting vehicle control as 100% indicated by dotted line on figures). Figure
(a) is the levels of HMGCoA mRNA expression, and Fig. 3b represents the levels of
FAS mRNA expression. Figure (c) is SPT1 mRNA expression. Significances are
indicated in the figures (n = 5 for each group).
(a) (b)
(c)
(d)
Figure 4. Topical apigenin stimulates lamellar body production in normal mouse
epidermis, paralleled by increase ABCA12 mRNA expression in cultured human
keratinocytes: skin biopsies from both vehicle- and apigenin-treated mice were
taken for electron microscopy and processed as described in Materials and
methods. Figure (a) and (b) display the density of lamellar bodies (see arrows) in
vehicle-(a) or apigenin (b)-treated epidermis. Figure (c) quantitatively displays
changes in lamellar body density in vehicle- and apigenin-treated epidermis. Figure
(d) exhibits changes in the levels of ABCA12 mRNA expression after addition of
apigenin (10 l
M) to cultured keratinocytes growing under differentiating conditions
(1.2 m
M calcium). Magnification bars, numbers (N) and significant differences are
indicated in the figures.
ª 2013 John Wiley & Sons A/S
Experimental Dermatology, 2013, 22, 210–215
213
Apigenin improves epidermal permeability barrier and differentiation
noteworthy finding in the present study was a selective, apigenin-
induced upregulation of filaggrin expression, shown both in vivo
and in vitro. Moreover, we show here that the increased filaggrin
is likely, at least in part, to be due to upregulation of its mRNA
expression. The importance of filaggrin in epidermal permeability
barrier function is shown by the observation that filaggrin muta-
tions compromise epidermal permeability barrier function (26,46).
Pertinently, increased epidermal filaggrin expression has been pro-
posed to account in part for the enhanced epidermal permeability
barrier function induced by topical peroxisome proliferator
activated receptor and liver X receptor activators (32,4749).
Although increased filaggrin expression could account in part
for the improved epidermal permeability barrier homoeostasis
induced by apigenin, we show there that apigenin also stimulated
epidermal lipid synthesis and lamellar body production. The
epidermal lamellar body is the only known organelle that delivers
lipids and their respective postsecretory processing enzymes to the
extracellular space of the SC. Previous studies showed that either
abrogation of lamellar body formation or blockade of their secre-
tion disrupts epidermal permeability barrier homoeostasis (34). In
the present study, we show that apigenin induced a marked
increase in mRNA expression of not only key lipid synthetic
enzymes, but also the lipid transporter protein, ABCA12, which is
required for lamellar body formation. Hence, enhanced lipid pro-
duction and lamellar body formation could further explain how
apigenin improves epidermal permeability barrier homoeostasis.
Finally, the present study revealed that topical apigenin causes a
marked elevation in two critical antimicrobial peptides, epidermal
CAMP and mBD3 protein expression. In addition, CAMP is cru-
cial for epidermal permeability barrier homoeostasis. Prior studies
have shown that CAMP-deficient mice display delayed barrier
recovery and expression of both CAMP and mBD3 is regulated in
parallel with changes in epidermal permeability barrier status
(42,50). Thus, the accelerated barrier recovery could also be due
in part to an upregulation of epidermal CAMP and mBD3 expres-
sion induced by apigenin. In the present study, a remarkable
reduction in SC hydration was observed following apigenin treat-
ment. While the exact mechanism for such a reduction is not
clear, it could reflect inhibition of epidermal proteasomes, induced
by apigenin (51,52). It is know that amino acid metabolites are
the natural moisturizers in SC (53), and higher activity of prote-
asomal enzymes (which largely account for the degradation of
proteins to amino acids) (Rev. in ref. 54) presents in the epider-
mis (55). Apigenin is a known proteasome inhibitor (51,52),
which could progressively lower the amino acid content in SC,
accounting for the low SC hydration in apigenin-treated skin.
Additionally, it is also shown that filaggrin is the source of natural
moisturizing factors (NMFs) and its degradation pathway to
NMFs includes at least caspase-14 and bleomycin hydrolase
(56,57). Caspase-14 degrades deiminated filaggrin into peptides,
which is further catalysed into NMFs by bleomycin hydrolase
(56). Filaggrin-deficient mice display dry skin with lower levels of
NMFs (58). Therefore, it is possible that apigenin also inhibits the
pathway breaking down filaggrin into NMFs. However, further
studies would be needed to elucidate the mechanisms by which
apigenin reduces SC hydration.
The influence of antioxidants on cutaneous structure and func-
tion varies dramatically, according to the types of agents deployed.
For example, vitamin E reduces lipid peroxidation and stimulates
keratinocyte differentiation (59,60), while vitamin C stimulates
both keratinocyte lipid synthesis and differentiation (61,62). Even
within the same class of antioxidants, keratinocyte function may
be regulated differently. Balasubramanian et al. showed that epig-
allocatechin gallate, a flavonoid, increases involucrin expression
(63), whereas another flavonoid, curcumin, inhibits involucrin
expression induced by epigallocatechin gallate (64). In contrast,
hesperidin, also a flavonoid, increases epidermal filaggrin expres-
sion, without changing involucrin and loricrin expression (15),
which is in agreement with data from the present study with
apigenin in vivo, whereas the present in vitro study showed that
apigenin reduced loricrin expression. These discrepant in vivo and
in vitro results may reflect the difference in keratinocyte differenti-
ation in vivo and in vitro (65). For example, treatment of kerati-
nocytes with apigenin for 48 h may not suffice to stimulate
loricrin, the later differentiation protein. Nevertheless, the present
study demonstrates that apigenin upregulates filaggrin expression.
Taken together, these results suggest further that antioxidants dif-
ferentially impact cutaneous structure and function by a variety of
mechanism. Accordingly, both the types of skin condition and the
specific characteristics of each antioxidant should be taken into
account when choosing the most appropriate agent for possible
clinical deployment in dermatology. As apigenin selectively upre-
gulates filaggrin expression and lipid production, it could be
particularly useful for the treatment atopic dermatitis.
In summary, the present study demonstrates that topical apige-
nin improves epidermal permeability barrier homoeostasis, stimu-
lates lamellar body formation and upregulates filaggrin and lipid
synthetic enzyme mRNA expression. Therefore, apigenin could be
useful in treating skin disorders with permeability barrier dysfunc-
tion, especially those accompanied by reduced filaggrin expression,
such as atopic dermatitis.
Acknowledgements
This work was supported by grants (AR19089, PEM; AR051930, TM) from
the National Institutes of Health.
Author contributions
MH performed keratinocyte culture, W estern blotting in vitro and prepared
the graphs and figures for publication; MH performed the immunohisto-
chemical staining; PLK performed qPCR; RS assessed barrier function and
prepared samples for morphology and Western blotting in vitro;KP
performed Western blotting in vivo; DC carried out the ultrastructural
studies; TKL and JLS assessed basal stratum corneum function and statisti-
cal analyses; TMM and PME designed the experiment, interpreted data,
critically reviewed the manuscript and approved the final version; MQM
designed the experiment, interpreted data and drafted the manuscript.
Conflict of interests
The authors have declared no conflicting interests.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Effects of apigenin on filaggrin expression
in vivo.
Figure S2. Effects of apigenin on filaggrin expression
in vitro at 24 h after apigenin treatment.
Figure S3. Effects of apigenin on filaggrin expression
in vitro at 48 h after apigenin treatment.
Figure S4. Topical apigenin increases immunostain-
ing for epidermal antimicrobial peptides.
Table S1. Primer sequences.
ª 2013 John Wiley & Sons A/S
Experimental Dermatology, 2013, 22, 210–215
215
Apigenin improves epidermal permeability barrier and differentiation
    • "Many chemicals can activate AhR. They include environmental polycyclic aromatic hydrocarbons, coal tar for medical use, phytochemicals, ultraviolet B (UVB) photoproducts, and products from commensal and pathogenic microorganisms such as skin-residing yeasts (Rannug et al., 1987;Fritsche et al., 2007;Gaitanis et al., 2008;Denison et al., 2011;Moura-Alves et al., 2014;van den Bogaard et al., 2013;Hou et al., 2013). The signaling outcome of AhR is additionally shaped by interference with other signaling pathways such as EGFR, MAPK, NFkB, β-catenin, or STATs in different inflammatory contexts (Haarmann Stemmann et al., 2015). "
    [Show abstract] [Hide abstract] ABSTRACT: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in adaptive cell functions, and is highly active in the epidermis. AhR ligands can accelerate keratinocyte differentiation, but the precise role of AhR in the skin barrier is unknown. Our study showed that transepidermal water loss (TEWL), a parameter of skin barrier integrity, is high in AhR-deficient (AhR-KO) mice. Experiments with conditionally AhR-deficient mouse lines identified keratinocytes as the primary cell population responsible for high TEWL. Electron microscopy showed weaker inter-cellular connectivity in the epidermis of keratinocytes in AhR-KO mice, and gene expression analysis identified many barrier-associated genes as AhR targets. Moreover, AhR-deficient mice had higher inter-individual differences in their microbiome. Interestingly, removing AhR ligands from the diet of wild-type mice mimicked AhR deficiency with respect to the impaired barrier; conversely, re-addition of the plant-derived ligand indole-3-carbinol (I3C) rescued the barrier deficiency even in aged mice. Our results suggested that functional AhR expression is critical for skin barrier integrity and that AhR represents a molecular target for the development of novel therapeutic approaches for skin barrier diseases, including by dietary intervention.
    Full-text · Article · Jul 2016
    • "Apigenin prevents colon and prostate cancer [21,22] and is also a potent inhibitor of skin carcinogenesis induced by UVB-light or the chemical co-carcinogens, 7, 12-dimethylbenz[a]anthracene (DMBA) and TPA [23,24]. The antitumor effects of apigenin may be mediated by its ability to modulate terminal differentiation [25], cell proliferation [26], and/ or apoptosis [27]. Moreover, in cultured keratinocytes apigenin inhibited UVB-and TPA-induced expression of pro-tumorigenic COX-2 [12,13,28] . "
    [Show abstract] [Hide abstract] ABSTRACT: Non-melanoma skin cancer (NMSC) is the most prevalent cancer in the United States. NMSC overexpresses cyclooxygenase-2 (COX-2). COX-2 synthesizes prostaglandins such as PGE2 which promote proliferation and tumorigenesis by engaging G-protein-coupled prostaglandin E receptors (EP). Apigenin is a bioflavonoid that blocks mouse skin tumorigenesis induced by the chemical carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the effect of apigenin on the COX-2 pathway has not been examined in the DMBA/TPA skin tumor model. In the present study, apigenin decreased tumor multiplicity and incidence in DMBA/TPA-treated SKH-1 mice. Analysis of the non-tumor epidermis revealed that apigenin reduced COX-2, PGE2, EP1, and EP2 synthesis and also increased terminal differentiation. In contrast, apigenin did not inhibit the COX-2 pathway or promote terminal differentiation in the tumors. Since fewer tumors developed in apigenin-treated animals which contained reduced epidermal COX-2 levels, our data suggest that apigenin may avert skin tumor development by blocking COX-2.
    Full-text · Article · Dec 2015
  • [Show abstract] [Hide abstract] ABSTRACT: More than 40 null mutations in the filaggrin (FLG) gene are described. It is therefore possible to find two different null mutations in one individual (compound heterozygosity). It has been generally perceived that homozygous and compound heterozygous individuals were genotypically comparable; however, this has not been scientifically investigated. Two different FLG null mutations in the same individual may be in trans position, meaning that each mutation locates to a different allele functionally equivalent to homozygosity, or may be in cis position, meaning that both mutations locate to the same allele functionally equivalent to heterozygosity. To experimentally investigate allelic in cis versus in trans configuration of the two most common filaggrin (FLG) mutations (R501X and 2282del4) in compound heterozygous individuals. Testing for in cis or in trans allele configuration was performed by means of allele-specific PCR amplification and analysis of PCR products by agarose gel electrophoresis. All R501X/2282del4 compound heterozygous samples collected over a 4-year period of routine FLG mutation testing were investigated. In total, 37 samples were tested. All thirty-seven R501X/2282del4 compound heterozygous individuals were found to carry the two mutations in trans position. FLG null mutation compound heterozygous individuals can be considered functionally equivalent to FLG null mutation homozygosity for any of the two mutations.
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