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European Journal of Histochemistry 2015; volume 59:2467
[page 98] [European Journal of Histochemistry 2015; 59:2467]
Apigenin induces dermal
collagen synthesis via smad2/3
signaling pathway
Y. Zhang,1J. Wang,1X. Cheng,2B. Yi,3
X. Zhang,4Q. Li1
1Department of Plastic and
Reconstructive Surgery, Shanghai 9th
People’s Hospital, School of Medicine,
Shanghai Jiao Tong University
2Department of Urology, Renji Hospital,
School of Medicine, Shanghai Jiao Tong
University
3Clinical College of General Hospital of
Beijing Military Region, Anhui Medical
University, Hefei
4The Key Laboratory of Stem Cell
Biology, Institute of Health Sciences,
Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences
and Shanghai Jiao Tong University School
of Medicine, Shanghai, China
Abstract
Decrease in fibroblast-produced collagen
has been proven to be the pivotal cause of skin
aging, but there is no satisfactory drug which
directly increases dermal thickness and col-
lage density. Here we found that a flavonoid
natural product, apigenin, could significantly
increase collagen synthesis. NIH/3T3 and pri-
mary human dermal fibroblasts (HDFs) were
incubated with various concentrations of api-
genin, with dimethyl sulfoxide (DMSO) serv-
ing as the negative control. Real-time reverse-
transcription polymerase chain reaction
(PCR), Western Blot, and Toluidine blue stain-
ing demonstrated that apigenin stimulated
type-I and type-III collagen synthesis of fibrob-
lasts on the mRNA and protein levels.
Meanwhile, apigenin did not induce expres-
sion of alpha smooth muscle actin (α-SMA) in
vitro and in vivo, a fibrotic marker in living tis-
sues. Then the production of collagen was con-
firmed by Masson’s trichrome stain,
Picrosirius red stain and immunohistochem-
istry in mouse models. We also clarified that
this compound induced collagen synthesis by
activating smad2/3 signaling pathway. Taken
together, without obvious influence on fibrob-
lasts’ apoptosis and viability, apigenin could
promote the type-I and type-III collagen synthe-
sis of dermal fibroblasts in vitro and in vivo,
thus suggesting that apigenin may serve as a
potential agent for esthetic and reconstructive
skin rejuvenation.
Introduction
Dermis consists of several structural compo-
nents, and collagen takes the major part. In
addition to glycosaminoglycans and elastin
fibers, dermal matrix in adult skin are com-
posed of type I (80-85%) and type III collagen
(10-15%).1It is of great importance that colla-
gen plays a main role in the texture and
appearance of skin. Skin aging is inevitably
associated with a disturbance in collagen
metabolism2(due to the decreased activity of
fibroblasts and their collagen synthesis), as
well as elastin. Increasing the collagen content
of dermis has been regarded as a well-effective
way for anti-aging in skin.
Collagen is a kind of biomacromolecule and
it cannot be absorbed through the stratum
corneum. Recently, some researches demon-
strated that collagen hydrolysate ingestion
might be beneficial to slow chronological skin
aging3and photoaging4in rats, and the density
of collagen fibrils increased compared with lac-
talbumin and water controls.5But the clinical
effect of oral collagen hydrolysate still lacks
convincing evidences. Up to now, injectable
collagen1or analogous composition6filling
implants are recognized as the well-accepted
treatment modality for cosmetic purposes.
However, maintaining skin appearance relies
on expansive and complex treatment repeated-
ly because of the short-term duration of exo-
genic injected collagen.
Apigenin (4,5,7-trihydroxyflavone), a
flavone subclass of flavonoid widely distributed
in many herbs, fruits, and vegetables, is a sub-
stantial component of the human diet and has
been shown to possess a variety of biological
characteristics, including anti-oxidative,7anti-
inflammatory effect,8tumor growth inhibition9
and promoting neurogenesis.10 It has been
shown that apigenin could enhance wound
healing and tissue repair in diabetic rat skin.11
In the process of wound healing, fibroblasts
secreted collagen and the formation of colla-
gen-rich granulation tissue are vital patho-
physiological mechanisms for wound closure.12
Given this, we wonder what effect would
apigenin have on fibroblasts and whether api-
genin could induce collagen synthesis in nor-
mal human dermal fibroblasts. Consequently,
we examined its effects on collagen synthesis
in normal human dermal fibroblasts in vitro
and tested its function in the skin aging mouse
model induced by D-Galactose. Furthermore,
we investigated the potential mechanism
involved in the positive effects of apigenin on
collagen expression in fibroblasts.
Materials and Methods
Cell culture
Primary human dermal fibroblasts were
obtained from adolescent foreskin tissue of ten
people (aged 8-12 years), and none of them
had a history with skin diseases. Skin tissue
was obtained after obtaining informed consent
from the patients, with the approval of the
ethics committee of Shanghai 9th People’s
Hospital and in conformity with the Helsinki
guidelines. NIH/3T3 and human dermal fibrob-
lasts (HDFs) were maintained in DMEM
(Hyclone, Thermo Fisher Scientific, Waltham,
MA, USA) supplemented with 10% fetal bovine
Correspondence: Xiaoling Zhang and Qingfeng
Li, Department of Plastic and Reconstructive
Surgery, Shanghai 9th People’s Hospital,
Shanghai Jiao Tong University School of
Medicine, 639 Zhizaoju Road, Shanghai 200011,
China.
Tel. +86.21.23271699-5615 - Fax: +86.21.63089567.
E-mails: (X. Zhang) xlzhang@sibs.ac.cn; (Q. Li)
dr.liqingfeng@yahoo.com
Key words: Apigenin, flavonoid, collagen I/III,
fibroblasts, smad2/3.
Contributions: YZ and JW contributed equally to
this work; YZ, JW, experimental work, data collec-
tion and interpretation; XC, participation in
experimental work design and coordination, data
acquisition; BY, participation in study design,
data collection, analysis of data and manuscript
preparation; QL, XZ, study design, data analysis
and interpretation, manuscript drafting.
Conflict of interest: the authors declare no con-
flict of interest.
Funding: this study was supported by grants from
the key project of the National Natural Science
Foundation (No. 81230042), the National Key
Project of Scientific and Technical Supporting
Programs Funded by Ministry of Science &
Technology of China (No. 2012BAI11B03) (Q. Li)
and the Chinese Academy of Sciences (No.
XDA01030102), Shanghai Municipal Commission
of Health and Family Planning (No.
2013ZYJB0501) (X. Zhang).
Received for publication: 28 November 2014.
Accepted for publication: 9 March 2015.
This work is licensed under a Creative Commons
Attribution NonCommercial 3.0 License (CC BY-
NC 3.0).
©Copyright Y. Zhang et al., 2015
Licensee PAGEPress, Italy
European Journal of Histochemistry 2015; 59:2467
doi:10.4081/ejh.2015.2467
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[European Journal of Histochemistry 2015; 59:2467] [page 99]
serum (Hyclone, Thermo Fisher Scientific),
100 U/mL penicillin, and 100 mg/L strepto-
mycin. Both HDFs and NIH/3T3 were incubated
at 37°C in a humidified atmosphere with 5%
CO2. Primary fibroblasts of passages 6-8 were
used. Toluidine blue (Sigma-Aldrich, St. Louis,
MO, USA) staining was used to assess extra-
cellular matrix synthesis.13
Real-time PCR analysis
The total RNA of cells was isolated using
TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
and subjected to reverse transcription with
Oligo (dT) and M-MLV Reverse Transcriptase
(Thermo Fisher Scientific). Synthesized com-
plementary DNA (cDNA) was analysed with
quantitative real-time PCR using SYBR®
Premix (Takara, Dalian, China) and Roche480
system. Glyceraldehyde 3-phospate dehydroge-
nase (GAPDH) was used as a reference gene.
Primers sequences were as follows: collagen,
type I, alpha 2 (Col1a2; mouse), 5-GGTGAGC-
CTGGTCAAACGG-3 (forward) and 5- ACTGT-
GTCCTTTCACGCCTTT-3 (reverse); Col1a2
(human), 5-GGCCCTCAAGGTTTCCAAGG-3
(forward) and 5-CACCCTGTGGTCCAA-
CAACTC-3 (reverse); collagen, type III, alpha 1
(Col3a1; mouse), 5- CTGTAACATG-
GAAACTGGGGAAA-3 (forward) and 5-
CCATAGCTGAACTGAAAACCACC-3 (reverse);
Col3a1 (human), 5-TTGAAGGAGGATGTTCC-
CATCT-3 (forward) and 5- ACAGACA-
CATATTTGGCATGGTT-3 (reverse); matrix
metalloproteinases 1 (MMP1; human), 5-
AAAATTACACGCCAGATTTGCC-3 (forward)
and 5-GGTGTGACATTACTCCAGAGTTG-3
(reverse); matrix metalloproteinases 2
(MMP2; human), 5-TACAGGATCATTGGCTA-
CACACC-3 (forward) and 5-GGTCA-
CATCGCTCCAGACT-3 (reverse); matrix metal-
loproteinases 9 (MMP9; human), 5-TGTAC-
CGCTATGGTTACACTCG-3 (forward) and 5-
GGCAGGGACAGTTGCTTCT-3 (reverse); tissue
inhibitor of metalloproteinases1 (TIMP1;
human), 5-CTTCTGCAATTCCGACCTCGT-3
(forward) and 5-ACGCTGGTATAAGGTG-
GTCTG-3 (reverse); α-SMA (human), 5-
AAAAGACAGCTACGTGGGTGA-3 (forward) and
5-GCCATGTTCTATCGGGTACTTC-3 (reverse).
Cell viability assay
For the cell viability assay, HDFs were seed-
ed on 96-well plates (100 mL per well), fol-
lowed by apigenin (cat. no. 42251; Sigma-
Aldrich) or DMSO (Sigma-Aldrich) treatment.
After 3 or 5 days, cell culture medium was
replaced by Thiazolyl Blue Tetrazolium
Bromide (MTT) working solution, followed by
a 4-hour incubation at 37°C in a 5% CO2incu-
bator. After MTT working solution was
removed and DMSO added, the absorbances at
490 nm were detected.
Flow cytometric analysis
Cell apoptosis was assessed by flow cytome-
try using the Alexa Fluor®488 Annexin V/Dead
Cell Apoptosis Kit (Invitrogen, Carlsbad, CA,
USA). Following apigenin (5 μmol/L or 1
μmol/L) or dimethyl sulfoxide (DMSO) treat-
ment, harvested cells were suspended in 100
μL Annexin-binding buffer. Then, 5 μL Alexa
Fluor® 488 Annexin V and 1 μL PI working
solution were added and incubated with the
cells for 15 min in the dark. After the incuba-
tion period, 400 μL 1X Annexin-binding buffer
was added and mixed gently. The stained cells
were analysed directly by flow cytometry using
the Cell Quest program (Becton Dickinson, CA,
USA). Data were analysed using FlowJo soft-
ware.
Colony formation assay
Anchorage-dependent growth of HDFs were
investigated by monolayer colony formation
assay.14 Cells were cultured in a 6-well plate
(500 per well) and treated with 5 μmol/L or 1
μmol/L apigenin or DMSO. After cultured for
14 days, surviving colonies were stained 5 min
with Gentian Violet (Sigma-Aldrich) after 4%
paraformaldehyde fixation.
Immunofluorescence cell staining
Human dermal fibroblasts at a density of
2×103cells per well were seeded on cover
slides in 24-well plates and incubated
overnight. Cells were fixed with 4%
paraformaldehyde and blocked with 5% goat
serum in PBST (0.1% TritonX-100 in phos-
phate buffered saline) for 1 h. For F-actin
staining, cells were incubated with Alexa Fluor
488 Phalloidin (Cytoskeleton Inc., Denver, CO,
USA; 1:200) for 1 h at room temperature. For
α-SMA staining, cells were incubated with pri-
mary antibodies against α-SMA (Abcam,
Cambridge, UK, 1:200) for 2 h at room temper-
ature, followed by an Alexa Fluor 555-conjugat-
ed secondary antibody. For smad3 staining,
cells were incubated with primary antibodies
against smad3 (Cell Signaling Technology,
Beverly, MA, USA; 1:200) for 2 h at room tem-
perature, followed by an Alexa Fluor 488-conju-
gated secondary antibody. Immunofluore -
scence signals were captured using confocal
microscopy (LSM 510, META Laser Scanning
Microscope; Zeiss, Jena, Germany).
Smad2/3 knockdown by siRNA
RNA interference was performed using
smad2/3 siRNA (human) (sc-37238; Santa
Cruz Biotechnology, Dallas, TX, USA), target-
ing human smad2/3 and control siRNA (sc-
37007) as negative control. Transfection for
HDFs was conducted using Lipofectamine
RNAiMAX reagent (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer’s protocol.
Western blot
Cultured cells were lysed using radioim-
munoprecipitation assay (RIPA) lysis buffer.
Protein concentrations were determined using
a micro bicinchoninic acid (BCA) assay
(Thermo Fisher Scientific). Twenty micro-
grams total protein extract was separated by
8% or 10% sodium dodecyl sulfate-polyacry-
lamide gel electrophoresis (SDS-PAGE) under
reducing conditions and electroblotted onto
polyvinylidene difluoride membranes
(Millipore, Bedford, MA, USA). The membrane
was then blocked and then incubated with pri-
mary antibodies overnight at 4°C.
The primary antibodies used included the
followings: anti-Col1a2, anti-Col3a1, anti-α-
SMA (Abcam; 1:1000), anti-cyclin-dependent
kinase (CDK) family, anti-cyclin D1, anti-
cyclin E1, anti-smads, anti-MAPK family (Cell
Signaling Technology, Beverly, MA, 1:1000),
anti-GAPDH (Sigma-Aldrich; 1:10,000).
Immunoreactive bands were quantitatively
analyzed with ImageJ software.
D-galactose-induced skin aging
mouse model and animal
experiments
Six-week-old female C57BL/6 mice were
purchased from the Shanghai Slac Laboratory
Animal (Slac, Shanghai, China). All animal
studies have been approved by the Animal
Care and Use Committee of Shanghai Jiao
Tong University. All efforts were made to mini-
mize animal suffering.
A total of 18 mice were randomly assigned to
three groups (n=6). Two groups of animals
received daily subcutaneous injection of D-
galactose (D-gal; 1000 mg/kg) for 8 weeks.15
The third group received phosphate buffered
saline (PBS) as a negative control. Two weeks
later, DMSO and apigenin (5 μmol/L) was
delivered by microneedles16 [MTS-Roller
Model: CR2 (0.2 mm)] to the dermis of D-
galactose treated mice once a day for 4 weeks,
respectively. Mice were sacrificed at the end of
treatment, and skin tissue was harvested for
further analyses.
Histology and immunohistochemistry
Paraformaldehyde-fixed paraffin-embedded
tissue sections (5 μm) were stained with
hematoxylin and eosin (H&E), Masson’s
trichrome (Trichrome stain LG solution,
HT10316; Sigma-Aldrich) and Picrosirius red
(Fluka, Buchs, Switzerland). For immunohis-
tochemical staining, the sections were detect-
ed with primary antibodies against collagen
I/III (Millipore; 1:1,000) and α-SMA (Abcam;
1:200) overnight at 4°C. After incubation with
the appropriate secondary antibodies, the sec-
tions were developed with diaminobenzidine
and counterstained with hematoxylin.
Original Paper
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[page 100] [European Journal of Histochemistry 2015; 59:2467]
Statistical analysis
Statistical differences were calculated using
Friedman’s analysis of variance (ANOVA), with
post-hoc least significant difference (LSD) test
as appropriate. A significant difference among
groups was set at P<0.05.
Results
Apigenin stimulated collagen
synthesis but had no effect on
matrix metalloproteinases
in vitro
Fibroblasts are the predominant mesenchy-
mal cells in the dermis, and their function is
strongly implicated in dermatology. To study
apigenin’s effect on fibroblasts (Figure 1A),
NIH/3T3 and HDFs were administered with
apigenin at the concentrations of 5 μmol/L. As
shown in Figure 1B, apigenin could potently
increase Toluidine blue staining after api-
genin treatment for 5 days, which mean the
Original Paper
Figure 1. Apigenin stimulated collagen synthesis of fibroblasts. A) The molecular structure of apigenin. B) Toluidine blue staining in
NIH/3T3 and HDFs for 5 days. C) Dose-dependent effects of apigenin on mRNA expression of Col1a2 and Col3a1 in NIH/3T3 and
HDFs for 3 days. D) The protein level of Col1a2 and Col3a1 was measured by Western Blot at 5 days after apigenin was applied at
concentrations of 0.1 μmol/L to 10 μmol/L. E) The expression of MMP1, MMP2, MMP9 and TIMP1 were also assessed by real-time
PCR. Data are presented as mean ± SD, n≥3; NS, not significant; *P<0.05; **P< 0.01; ***P<0.001.
EJH_2015_02-article.qxp_Hrev_master 22/06/15 12:54 Pagina 100
[European Journal of Histochemistry 2015; 59:2467] [page 101]
synthesis of extracellular matrix was
increased.13 Further real-time PCR analysis
showed that, in NIH/3T3 and HDFs, apigenin
(0.1 μmol/L - 10 μmol/L) dose-dependently
stimulated endogenous expression of Col1a2
and Col3a1. The most significant changes
were observed when NIH/3T3 and HDFs were
treated with apigenin at the concentration of 5
μmol/L, and the increase of Col3a1 was more
obvious than Col1a2 (Figure 1C). These upreg-
ulation effect of apigenin on collagen expres-
sion were then confirmed by Western blot
analysis. When HDFs were treated with api-
genin for 5 days, the protein level of Col1a2
and Col3a1 were higher than that of cells treat-
ed with DMSO (Figure 1D). In addition, we
next examined the effect of apigenin on
matrix metalloproteinases (MMPs) and
Original Paper
Figure 2. No obvious cytotoxicity exerted by apigenin on fibroblasts viability, apoptosis, proliferation, cell cycle and activation. A) Cell
viability was examined by MTT assays at 3 or 5 days after apigenin was applied in HDFs. B) Apoptosis was evaluated after treating
HDFs with 5 μmol/L or 1 μmol/L apigenin or DMSO; flow cytometry profile represents Alexa Fluor® 488 Annexin V staining in X
axis and PI in Y axis. C) The effect of apigenin on fibroblasts growth was investigated by monolayer colony formation assay. D) The
expression of cyclin E1, CDK4, cyclin D1, CDK2 and p-CDK2 proteins was analysed using Western blot in HDFs. E-F) The levels of
α-SMA mRNA and protein expression were measured by real-time PCR and Western blot. G) Immunofluorescence cell staining for α-
SMA and F-actin in cultured HDFs after incubation with apigenin or DMSO for 72 h; F-actin is shown by green fluorescence and α-
SMA is shown by red fluorescence; nucleus (blue) was stained with DAPI; scale bar: 50 μm. Data are presented as mean ± SD, n≥3;
NS, not significant; **P<0.01; ***P<0.001.
EJH_2015_02-article.qxp_Hrev_master 22/06/15 12:55 Pagina 101
TIMP1, well-known proteases that degrade col-
lagen proteins. The expression of MMP1,
MMP2, MMP9, and their inhibitor TIMP1 were
unchanged (Figure 1E).
Apigenin did not effect fibroblasts
viability and activity
Afterwards, we studied the effect of api-
genin on fibroblasts viability, apoptosis, prolif-
eration and activation. In vitro, MTT assays
showed that the viability of HDFs was similar
with those incubated with DMSO, when incu-
bated with apigenin (1 to 10 μmol/L) for 3 or 5
days, respectively (Figure 2A). In addition, no
significant differences in percentages of apop-
totic cells was observed after the exposure of
fibroblasts to apigenin (Figure 2B). To investi-
gate the effects of apigenin on proliferation
and cell cycle, colony formation assay and
Western Blot analysis of cell cycle related pro-
teins were performed. Colony forming ability
of HDFs was similar in apigenin-treated group
to DMSO-treated group (Figure 2C). The
expression levels of cell cycle associated pro-
teins remained unchanged between apigenin
and DMSO treated groups (Figure 2D). These
results suggested that apigenin had no obvi-
ous cytotoxicity on fibroblasts’ viability, apop-
tosis and proliferation.
Fibroblast overactivation leads to pathologi-
cal collagen deposition or scar formation.17
Myofibroblasts, known as activated fibroblasts,
are marked by α-SMA expression. To deter-
mine the effects of apigenin on fibroblasts’
activation, we evaluated the levels of α-SMA
mRNA and protein expression (Figure 2 E,F)
in cultured HDFs treated with apigenin. We
found that α-SMA mRNA expression had no
obvious change in apigenin-treated cells com-
pared with DMSO-treated cells. We also
showed that apigenin did not affect α-SMA
expression in vitro by immunofluorescence
staining (Figure 2G). These findings suggest-
ed that apigenin did not cause fibroblasts to
overactivate into myofibroblasts while colla-
gen synthesis was increasing.
Induction of collagen synthesis was
mediated by smad2/3 activation
To further explore the underlying mecha-
nism of how apigenin activated type-I and
type-III collagen gene expression, transform-
ing growth factor beta 1 (TGF-β1) and mito-
gen-activated protein kinase (MAPK) signal-
ing pathway were analysed. TGF-β1 is a proto-
typic fibrogenic cytokine, enhancing extracel-
lular matrix gene expression. Previous studies
proved that Col1a2 and Col3a1 were direct
TGF-β1/smad3 targets in human dermal
fibroblasts.18 As shown in Figure 3A, when
HDFs were treated for 12 h, apigenin (1
μmol/L or 5 μmol/L) markedly increased the
expression of phosphorylated smad2 and
smad3 in a dose-dependent manner, whereas
total smad2, smad3 and smad4 did not obvi-
ously alter. It also showed that apigenin had
sustained effect on promoting phosphorylation
of smad2 and smad3 after a 3-day treatment
(Figure 3B). Yoon et al.19 revealed that MAPK
pathway was involved with peptide-induced
collagen synthesis of fibroblasts. However,
when fibroblasts were treated with apigenin
for 12 h, the expression of total and phospho-
rylated JNK, ERK and p38 protein remained
unchanged, compared with DMSO (Figure
3C). Immunofluorescence experiments de -
monstrated that after treatment with apigenin
for 12 h, smad3 protein (labeled by green) was
significantly increased and mostly translocat-
ed into the nucleus (labeled by blue) (Figure
3D). By contrast, in the DMSO groups, smad3
were retained in the cytoplasm. Once targeted
knockdown smad2/3 by specific siRNA, the up-
regulation effect of apigenin on the expres-
sion of collagen type-I and type-III protein was
obviously reduced (Figure 3E), which con-
firmed that smad2/3 is required for the trans-
duction of apigenin effect on collagen expres-
sions.
Apigenin stimulated collagen syn-
thesis in the D-galactose-induced
skin aging mouse model
The in vivo effects of apigenin on collagen
synthesis was investigated in the D-galactose-
induced skin aging mouse model. The collagen
expression was showed by H&E, Masson’s
trichrome stain, Picrosirius red stain and
immunohistochemistry. Histology showed sig-
nificant changes in dermal thickness and den-
sity in samples obtained from D-gal-treated
mice compared with PBS control group
(Figure 4 A-D). After 1 month of apigenin
administration at the concentration of 5
μmol/L, the mice exhibited obviously
increased dermal thickness and collagen den-
sity compared with DMSO-treated mice
(Figure 4 A-D). Magnified images showed that
dermis in the apigenin-treated group exhibit-
ed compact and clearly evident staining,
whereas collagens were loosely distributed in
DMSO-treated dermis of the aging skin model
(Figure 4 A-D), in both Masson’s trichrome
stain, Picrosirius red stain and immunohisto-
chemistry examinations. Dermal collagen
could be subdivided into type I and type III col-
lagen after Picrosirius red staining under
polarized light. As shown in Figure 4F, api-
genin could significantly increase both colla-
gen type I and type III density in the dermis of
the skin aging mouse model. Quantitative data
of dermal thickness and collagen density
showed that mice subcutaneously injected
with D-gal showed thinner skin and less colla-
gen compared to control mice and apigenin-
treated mice demonstrated significantly thick-
er and compact dermis (Figure 4 E,F). The in
vivo study also demonstrates that apigenin
does not show any effect on activation of
fibroblasts (Figure 4G).
Discussion
Dermal atrophy is the major causes of aging
appearance.20 In vivo and in vitro studies show
that decline in the production of collagen in
aging fibroblasts is mainly responsible for
decreasing in dermal thickness seen in extrin-
sically aging skin, which reveals dermal atro-
phy, fragmentation, and irregular collagen
bundles.21 Since the ‘70s, animal and human
derived collagens have been studied for soft
tissue augmentation.22 Injectable filling
implants are now widely used for cosmetic pur-
poses. However, exogenic injectable collagen
often presented various complications such as
allergy, ecchymosis, local necrosis and infec-
tions of bacteria or virus. Scientists have tried
for decades to find other alternative to stimu-
late endogenous collagen synthesis. There are
several anti-oxidants, such as vitamins C and
E, co-enzyme Q10 and retinoids used for treat-
ing UV-induced skin aging and wrinkles.23, 24
However, only few compounds are able to
induce type I collagen synthesis25-27 and none
of them can stimulate both type I and type III
collagen synthesis according to the record in
literatures.
Apigenin, a plant flavone, has gained con-
siderable attention due to its health benefits,
chemopreventive properties and wide distribu-
tion in the plant kingdom.28 Many studies have
demonstrated that apigenin possesses a wide
range of biological activities to the skin. It has
been reported that apigenin can stimulate
nucleotide excision repair genes to protect
skin keratinocytes29 against UVB-induced skin
inflammation.30 Dietary apigenin attenuates
the development of atopic dermatitis-like skin
lesions in atopic dermatitis model.31 Apigenin
could also effectively reduce the incidence and
size of skin tumors caused by ultraviolet B
(UVB) exposure through the enhancement of
UVB-induced apoptosis.32 In this study, we
investigated the effect of apigenin on dermal
fibroblasts’ function.
At first, we found apigenin could increase
the mRNA expression of Col1a2 and Col3a1 in
NIH/3T3 and HDFs. With extracellular matrix
staining and Western Blot analysis, the stimu-
lative effect of collagen on protein level was
more significant. Although as reported in the
literature, basal levels of Col1a1 and α-SMA
mRNAs were reduced in fibroblasts treated
with high concentration of apigenin (20
μmol/L),33 our research confirmed that api-
genin of 25 μmol/L showed obvious cytotoxici-
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[European Journal of Histochemistry 2015; 59:2467] [page 103]
Original Paper
Figure 3. Apigenin-mediated collagen synthesis increase via smad2/3 signaling pathway. A,B) Western blot analysis and quantification
of phosphorylated and total smad2, smad3 and total smad4 in HDFs with or without apigenin stimulation for 12 h or 3 days. C)
Western Blot analysis of JNK, ERK and p38 in HDFs with or without apigenin stimulation for 12 h. D) Immunofluorescence experi-
ments: smad3 was labeled as green; nucleus (blue) was stained with DAPI; scale bar: 50 μm. E) Western blot showed the expression of
Col1a2 and Col3a1 in HDFs when cells were treated with DMSO, apigenin or apigenin with specific siRNA of smad2/3. Data are pre-
sented as mean ± SD, n≥3; NS, not significant; *P<0.05; **P<0.01; ***P<0.001.
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[page 104] [European Journal of Histochemistry 2015; 59:2467]
Original Paper
Figure 4. Apigenin increased dermal thickness and collagen density in the D-galactose-induced skin aging mouse model. A-D) H&E,
Masson’s trichrome, Picrosirius red and immunohistochemistry stained dermis of control mice and D-gal-treated mice respectively
received apigenin and DMSO; scale bars: 100 μm; zoom scale bars: 20 μm. E) Quantification of dermal thickness. F) Picrosirius red
staining under polarized light and quantification of type I and type III collagen density; collagen type I is shown as red fibers and col-
lagen type III is shown as green fibers; scale bars: 100 μm. G) Immunohistochemistry staining of α-SMA of control mice and D-gal-
treated mice respectively received apigenin and DMSO; scale bars: 100 μm; zoom scale bars: 20 μm. Data are presented as mean ± SD,
n=6/6/6; *P<0.05; **P<0.01; ***P<0.001.
EJH_2015_02-article.qxp_Hrev_master 22/06/15 12:55 Pagina 104
[European Journal of Histochemistry 2015; 59:2467] [page 105]
ty in fibroblasts. We believed that the attenuat-
ing effect of high concentration of apigenin on
phenotypic transitions in the analyzed cell pop-
ulations were not independent of its cytotoxic
activity. We detected the markers related to
extracellular matrix degradation and found
that apigenin had no effect on the balance of
MMPs/TIMPs.
We further observed that apigenin directly
activated smad2/3-dependent signaling path-
way. This is not surprising since this flavonoid
displays considerable muti-effect. It targets a
number of secondary messengers, including
those potentially involved in TGF-β1 signaling
pathway, such as NF-kB,34 MAPK/ERK,35
FAK,36,37 PKC38 and PI3K-Akt39 in a cell context-
dependent manner. We observed that apigenin
markedly increased the expression of phos-
phorylated smad2 and smad3 protein, while
total smad2, smad3 and smad4 protein all
remained unaltered. So, a more meticulous
network may connect TGF-β1 signaling path-
way and the abovementioned secondary mes-
sengers.
A previous study showed the accelerated
aging effect of D-gal injection on mouse skin,
as well as changes in dermal thickness and col-
lagen content.15 In order to confirm the effect
of apigenin on collagen synthesis in vivo, the
D-galactose-induced skin aging mouse model
was established. Our data indicated that skin
aging mice treated with apigenin showed
markedly increasing dermal thickness and col-
lagen expression, compared with DMSO-treat-
ed mice. Hou et al.40 reported that topical api-
genin improved epidermal permeability barrier
function by stimulating epidermal differentia-
tion, lipid synthesis and secretion, as well as
cutaneous antimicrobial peptide production,
and our result showed that dermal injection of
apigenin significantly increased dermal thick-
ness and density. So we could conclude that
apigenin caused different biological functions
with two forms of drug administration by act-
ing on epidermis or derma, which might indi-
cate the importance of choosing suitable
administration methods to different skin dis-
eases, even for the same drug.
Our study demonstrates that apigenin could
induce both type I and type III collagen synthe-
sis of fibroblasts in vitro and could increase
dermal thickness and collagen deposition in
the dermis of mice. This compound is a poten-
tial target for drug design and development for
esthetic and reconstructive purpose.
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Original Paper
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