DOI: 10.1111/exd.

12964
www.wileyonlinelibrary.com/journal/EXD
Original Article

Glycyrrhizic acid prevents ultraviolet-B-induced photodamage: a

role for mitogen-activated protein kinases, nuclear factor kappa B
and mitochondrial apoptotic pathway
Quadri Afnan1,2, Peerzada J. Kaiser1, Rather A. Rafiq1, Lone A. Nazir1,3, Shashi Bhushan3,4,
Subhash C. Bhardwaj5, Rajat Sandhir2 and Sheikh A. Tasduq1,3
1
PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Jammu Tawi, Jammu and Kashmir, India; 2Department of
Biochemistry, Panjab University, Chandigarh, India; 3Academy of Scientific and Innovative Research (AcSIR), New Delhi, India; 4Cancer
Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu Tawi, Jammu and Kashmir, India; 5Department of Pathology,
Government Medical College, Jammu, Jammu and Kashmir, India
Correspondence: Sheikh A. Tasduq, PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Council of Scientific and
Industrial Research (CSIR), Canal Road, Jammu Tawi, Jammu and Kashmir, India, Tel.: +91-191-2569000-10-Ext-332, Fax: +91-191-2569333,
e-mails: stabdullah@iiim.ac.in; tasduq11@gmail.com

Abstract: Glycyrrhizic acid (GA), a natural triterpene, has received
attention as an agent that has protective effects against chronic
diseases including ultraviolet UV-B-induced skin photodamage.
However, the mechanism of its protective effect remains elusive.
Here, we used an immortalized human keratinocyte cell line
(HaCaT) and a small animal model (BALB/c mice), to investigate
the protective effects of GA against UV-B-induced oxidative
damage, and additionally, delineated the molecular mechanisms
involved in the UV-B-mediated inflammatory and apoptotic
response. In the HaCaT cells, GA inhibited the UV-B-mediated
increase in intracellular reactive oxygen species (ROS) and down-
regulated the release of pro-inflammatory cytokines interleukin
(IL)-1a, -1b and -6, tumor necrosis factor (TNF)-a and
prostaglandin E2 (PGE2). GA inhibited UV-B-mediated activation
of p38 and JNK MAP kinases, COX-2 expression and nuclear
translocation of NF-jB. Furthermore, GA inhibited UV-B-
mediated apoptosis by attenuating translocation of Bax from the
cytosol to mitochondria, thus preserving mitochondrial integrity.
GA-treated HaCaT cells also exhibited elevated antiapoptotic Bcl-2
protein, concomitant with reduced caspase-3 cleavage and
decreased PARP-1 protein. In BALB/c mice, topical application of
GA on dorsal skin exposed to UV-B irradiation protected against
epidermal hyperplasia, lymphocyte infiltration and expression of
several inflammatory proteins, p38, JNK, COX-2, NF-jB and
ICAM-1. Based on the above findings, we conclude that GA
protects against UV-B-mediated photodamage by inhibiting the
signalling cascades triggered by oxidative stress, including MAPK/
NF-jB activation, as well as apoptosis. Thus, GA has strong
potential to be used as a therapeutic/cosmeceutical agent against
photodamage.
Key words: apoptosis glycyrrhizic acid inflammation
mitogen-activated protein kinases reactive oxygen species ultraviolet-B
radiation
Accepted for publication 18 January 2016

Introduction
Exposure to ultraviolet (UV) radiation, particularly UV-B (k,
290320 nm), can lead to injurious consequences including the
formation of sunburn cells, induction of cyclobutane pyrimidine
dimmers (CPDs), photoaging and skin cancer (1,2). The primary
cell type in the epidermis directly affected by the detrimental
effects of UV-B radiation are the keratinocytes (3) which undergo
apoptosis consequent to erythema following acute exposure to
UV-B irradiation with maximum damage occurring at 2448 h
(4,5). The primary response to UV radiation comprises mainly the
production of inflammatory mediators, alterations in vascularity
and infiltration of inflammatory cells (6). While these initial
events of inflammation and apoptosis may offer a protective
response against the induction of tumorigenesis, acute and chronic
UV-B irradiation may result in photodamage due to the loss of
epidermal integrity (7).
In skin cells, UV-B irradiation stimulates the production of
reactive oxygen species (ROS) (8). Excessive generation of ROS
overwhelms the defense mechanism of the cell, making endoge-
nous cytoprotectants inadequate, resulting in oxidative damage
(9). ROS triggers the activation of cell surface receptors, stimulat-
ing various intracellular signalling pathways including mitogen-
activated protein kinases. The MAP kinase pathway comprises
three main subpathways extracellular signal-regulated kinase 1/2
(ERK 1/2), c-Jun NH2-terminal kinase/stress-activated protein
kinase (JNK) and p38 (10,11), of which latter two are the known
responders to stress signals like UV irradiations (12).
Substantial evidence suggests that human keratinocytes exposed
to UV-B irradiation show higher p38 activity than JNK (13). Acti-
vation of p38 during inflammation leads to the production of
pro-inflammatory cytokines such as IL-1, -6, TNF-a (14) and
induction of cyclooxygenase (COX)-2 expression (15). UV-B irra-
diation is also known to regulate the activation of transcription
factor NF-jB (16). Activated NF-jB translocates to the nucleus
and mediates the expression of several inflammatory cytokines,
exacerbating UV-B-induced inflammation (17). UV-B impinges
mainly on the mitochondrial pathway by causing redistribution of
Bax, a pro-apoptotic protein, from the cytosol to mitochondria
with concomitant cytochrome c release and effector caspase activa-
tion leading to cell death (18,19).

2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
440 Experimental Dermatology, 2016, 25, 440446

Because the generation of ROS by UV-B irradiation activates
several signalling cascades in the epidermis, therefore blocking
ROS-induced signalling pathways could prevent epidermal cells
from sunburn response. Hitherto, many plant-based agents have
been evaluated for their protective actions against the adverse
effects of UV irradiation (20). Licorice, the grandfather of herbs
and one of the most widely known medicines since ancient times,
contains glycyrrhizic acid (GA), a triterpene, as its primary bioac-
tive constituent (21). Studies have shown that GA exhibits cardio-
protective (22), neuroprotective (23), anti-inflammatory (24),
antiviral (25), antioxidant (26) and hepatoprotective (27) proper-
ties. Previously, we have demonstrated antiphotoaging properties
of GA in human dermal fibroblast (28). GA has also been shown
to inhibit UV-B-induced skin tumorigenesis in hairless mice (29).
In the present study, we used human HaCaT keratinocytes as
in vitro and BALB/c mice as in vivo models to examine the protec-
tive effect of GA against UV-B-induced inflammation and apopto-
sis. Our results demonstrate that GA attenuates detrimental effects
of UV-B by quenching ROS. We have also provided insights into
the molecular basis for the photoprotective effect of GA.
Materials and methods
Materials
GA (C42H62O16.NH3, FW = 840.0), dimethyl sulphoxide
(DMSO), Dulbeccos modified Eagles medium (DMEM),
penicillin, streptomycin, trypsinEDTA, D,L-buthionine-S,
R-sulphoximine (BSO), ascorbic acid, 3-(4,5-dimethylthiazol-2-yl)
-2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorodihydro-
fluorescein diacetate (H2DCFDA), propidium iodide, ribonuclease
A (RNase A), RIPA buffer, protease inhibitor cocktail, phosphatase
inhibitor cocktail, Bradford reagent and TBST were purchased
from Sigma-Aldrich Chemicals Private Limited (St. Louis, MO,
USA). Antibodies against p38, JNK, phospho p38, phospho JNK,
COX-2, NF-jB, ICAM, Bcl-2, Bim, Bax, GAPDH, PARP-1 and
secondary antibodies were purchased from Santa Cruz
Biotechnologies (Santa Cruz, CA, USA). Antibodies against
cleaved caspase 3, b-actin, VDAC and lamin were obtained from
SigmaAldrich.
Cell culture and UV-B light source
Human immortalized keratinocyte cell line HaCaT was obtained
from Cell Lines Service (GmbH Dr Eckener-Str. Eppelheim, Ger-
many) and maintained in DMEM, which was supplemented with
10% foetal bovine serum (FBS) and 100 U/ml of penicillinstrep-
tomycin. The cells were pretreated with GA for 24 h, washed with
phosphate-buffered saline and irradiated with UV-B. After UV-B
exposure, the cells were rinsed twice with PBS and treated with
GA in fresh culture medium for further 24 h. The source of UV-B
irradiation was a band of four UV-B lamps (Daavlin, UVA/UVB
Research Irradiation Unit, Bryan, OH, USA) equipped with a digi-
tal controller to regulate UV dosage, fixed at a distance of 24 cm.
The majority of resulting wavelengths (>90%) were in UV-B range
(290320 nm) with peak emission at 313 nm, and UV-A was less
than 10%.
Cell viability
The effect of UV-B irradiation on the viability of cells was deter-
mined by MTT assay as described earlier (28). Briefly, HaCaT cells
were incubated with MTT reagent (0.25 mg/ml) for 3 h at 37C.
The violet crystal (formazan), the end product of MTT conversion
by live cells, was solubilized in DMSO, and the optical density was
2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2016, 25, 440446
GA prevents UV-B -induced photodamage
recorded at 570 nm using ELISA plate reader (Multiskan Spec-
trum; Thermo Electron Corporation, USA Waltham, Massachu-
setts, USA).
Intracellular ROS detection
The intracellular ROS production was assayed using 20 ,70 -dichloro-
fluorescein diacetate (H2DCFDA) (30). Briefly, the cells were incu-
bated with 10 lM H2DCFDA for 30 min at 37C in a
CO2 incubator and then the cells were irradiated with UV-B
(10 mJ/cm2) under a thin layer of PBS. Fluorescence was detected
immediately after irradiation using fluorescence microscope (Nikon
Eclipse TE 2000-U Nikon Instruments, Melville, NY, USA).
Mitochondrial membrane potential (Dm) measurement
and detection of Apoptosis
Mitochondrial membrane potential was measured by JC-1 staining
cells (Molecular Probes, Eugene, OR, USA) by either flow cytome-
try or confocal microscopy (30,31). Briefly, the cells were har-
vested, washed and resuspended in PBS containing 5 lg/ml of
membrane-permeant dye JC-1. The cells were incubated with dye
for 15mins at 37C and analysed by flow cytometry (Becton Dick-
inson, Franklin Lakes, New Jersey, USA). For confocal microscopy,
the cells grown on glass coverslips were stained with JC-1 dye and
the images were captured and processed using a laser scan confo-
cal microscope (Olympus Fluoview FV-1000, Shinjuku, Tokyo,
Japan). Apoptosis was detected using Annexin V/PI double stain
as per the manufacturers instructions (BD PharmingenTM San
Diego, CA, USA). The cells were analysed for apoptosis by flow
cytometry.
DNA fragmentation
Cell pellets were suspended in lysis buffer [100 mM NaCl, 5 mM
EDTA, 10 mM TrisHCl (pH 8), 5% Triton X-100, 0.25% SDS]
containing 400 lg/ml DNase-free RNase and incubated at 37C
for 90 min. Samples were incubated for an additional 1 h at 50C
with 200 lg/ml proteinase K. DNA was extracted with phenol/
chloroform/isoamyl alcohol (25:24:1), precipitated with chilled
alcohol and resuspended in TrisEDTA and electrophoresed on a
1.8% agarose gel containing ethidium bromide. The DNA frag-
ments were visualized by Molecular Image ChemiDocTM XRS+
(Bio-Rad, Hercules, CA, USA).
Enzyme-linked immunosorbent assay
The cytokines IL-1a, IL-1b, IL-6, TNF-a and PGE2 were quanti-
fied using commercially available Enzyme-linked immunosorbent
assay (ELISA) kits (R and D Systems Minneapolis, MN, USA)
according to the manufacturers protocol.
Cell fractionation and immunoblotting
For the preparation of whole-cell lysate, the cells were lysed in ice-
cold RIPA buffer with freshly added protease and phosphatase inhi-
bitor cocktail and incubated on ice for 45 min. The homogenates
were centrifuged at 14 000 g at 4C for 30 min, and the super-
natants were collected as whole-cell lysates. The cytosolic-, nuclear-
and mitochondria-enriched fractions were prepared as described
previously (32,33). Proteins were estimated by Bradford assay.
About 4060 lg of protein samples was electrophoresed using
715% SDSPAGE gel and transferred to PVDF membrane (Milli-
pore, Billerica, MA, USA). The PVDF membranes were probed
with appropriate primary antibodies followed by a secondary
horseradish peroxidase (HRP)-conjugated antibody. The blots
were then analysed using a chemiluminescent HRP substrate (Mil-
lipore) and visualized by ChemiDocTM XRS+. Densitometric
441
 Afnan et al.

analysis of the blots was performed using Image LabTM software, in cell viability by 14%; 10 mJ/cm2, being physiological dose, with
version 3.0 (Hercules, CA, USA). reversible photodamage in HaCaT cells, was selected to study the
Zymography cytoprotective effect of GA and study its underlying mechanism of
MMP-2 and MMP-9 activities were measured by gelatin zymogra- protection against UV-B irradiation. GA pretreatment, to HaCaT
phy as described earlier (34) with the following modification. cells, at 10, 25, 50 and 100 lM restored the cell viability by 34, 50,
Briefly, the conditioned media collected from treated cells after 78 and 66%, respectively (Figure S1c.). Likewise, pretreatment of
72 h of respective treatments were electrophoretically separated on HaCaT cells with GA at different doses protected against UV-B-
10% SDSpolyacrylamide gel (SDSPAGE) with 1% gelatin. The mediated ROS formation as determined by H2DCFDA staining
gels were washed with denaturation buffer (50 mM Tris, pH 7.4, (Figs 1a and S1d).
5 mM CaCl2, 1 lM ZnCl2, 2.5% Triton X-100), followed by over- GA treatment prevented HaCaT cells against UV-B-induced
night incubation at 37C in incubation buffer (50 mM Tris, pH apoptosis
7.4, 5 mM CaCl2, 1 lM ZnCl2). The gel was stained with 0.5% Keratinocyte apoptosis is a characteristic feature of photodamage
Coomassie and destained with 30% ethanol, 10% acetic acid. The induced by UV-B irradiation. We evaluated the protective efficacy
bands were imaged with Molecular Image ChemiDocTM XRS+
(Bio-Rad).
UV-B+
Animals and treatment (a) Control UV-B(10 mJ/cm2) GA10 M
Female BALB/c mice (6 weeks old) were obtained from the Institu-
tional Animal House. The experimental procedures and animal care
protocols were approved by the Animals Ethics Committee of the
institute. The mice were acclimatized in a temperature-controlled
room with a 12-h light/dark illumination cycle for a week before the
start of the experiment. The animals were randomly divided into
three groups of six animals each and shaved dorsally each week for
16 weeks. The group, one of the mice, did not receive any treatment
and served as control. The mice in the group two received UV-B
(180 mJ/cm2) irradiation alone, three times a week, for 16 weeks.
The mice in the group three received GA 100 lM topically three
times a week, 1 h before UV-B irradiation, for 16 weeks. The ani-
mals were sacrificed 24-h postlast UV-B irradiation, and skin biop-
sies were harvested for histopathology and Western blotting.
UV-B+ UV-B+ UV-B+
Histology and tissue lysate preparation GA25 M GA50 M GA100 M
The skin specimens were fixed in 10% buffered formalin and
embedded in paraffin. The specimens were sectioned, deparaf-
(b)
finized and stained with haematoxylin and eosin for microscopic Bcl-2
analysis. Epidermal thickness was measured using ProgRes Cap- GAPDH
turePro v2.8.0 software Jena, Germany.
For lysate preparation, excised skin tissues were homogenized Bax cytosolic
in ice-cold lysis buffer (20 mM TrisHCl, 100 mM NaCl, 10 mM -Actin
EDTA, 1% NP-40) with freshly added protease and phosphatase
Bax mit
inhibitor cocktail and DTT (10 mM). The homogenates were cen-
trifuged at 14 000 g for 30 mins at 4C. The supernatant collected VDAC
was aliquoted and stored at 80C for Western blot analysis. Bim
Statistical analysis
-Actin
Data were expressed as the mean  standard deviation (SD). Sta-
tistical analysis was performed using one-way analysis of variance Cleaved caspase-3
(ANOVA) with post hoc Tukeys multiple comparison, and a P-
GAPDH
value of <0.05 was considered statistically significant.
Results PARP
GA treatment prevented cytotoxicity and generation of ROS Cleaved
triggered by UV-B
GAPDH
We assessed cytotoxic effects of UV-B irradiation at different doses
in HaCaT cells by MTT assay. UV-B irradiation reduced the cell GA(M) 10 25 50 100
viability in a dose-dependent manner from 5 to 40 mJ/cm2. UV-B(10 mJ/cm) + + + + +
An increase in the intensity of the UV-B irradiation from 5 to Figure 1. Protective effect of GA against UV-B-induced ROS generation and
40 mJ/cm2 reduced the cell viability from 6 to 67%, respectively, apoptosis. (a) Protective effect of GA against UV-B (10 mJ/cm2)-induced ROS
generation using H2DCFDA dye. The fluorescence was visualized immediately after
as compared to control cells (Figure S1a). Treatment with GA UV-B exposure under fluorescence microscope (magnification 20x). (b) Effect of GA
(5 to 100 lM) restored the cell viability from 6 to 18% on UV-B-mediated changes in Bax translocation, Bcl-2, Bim and caspase-3, PARP
cleavage. GAPDH, actin and VDAC served as loading control.
(Figure S1b). At 10 mJ/cm2, UV-B irradiation caused a decrease

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442 Experimental Dermatology, 2016, 25, 440446
 GA prevents UV-B -induced photodamage

of GA against UV-B irradiation in HaCaT cells by assessing apop- (a)

tosis and the loss of mitochondrial membrane potential (Dm) by pp38
flow cytometry and DNA damage by gel electrophoresis. UV-B
irradiation induced early apoptosis in 9.3% cells and late apopto- p38
sis in 13.8% cells (Figure S1e,f). GA treatment reduced the per-
centage of apoptotic cells as compared to the cells subjected to pJNK
UV-B irradiation alone. GA treatment markedly reduced UV-B-
induced DNA fragmentation (Figure S2a) and loss in cellular JNK
Dm. UV-B irradiation caused a loss of Dm in 32% of cells and
treatment with 10, 25, 50 and 100 lM GA reduced the percentage COX-2
of Dm dissipated cells to 18.5, 17.1, 15.7 and 11.4%, respectively
(Figure S2b). GAPDH
Treatment with GA modulated the expression of proteins
involved in UV-B-mediated apoptosis GA(M) 10 25 50 100
UV-B irradiation caused an increased translocation of Bax from UV-B(10 mJ/cm) + + + + +
cytosol to the mitochondria. GA pretreatment significantly pre-
vented this translocation (Fig. 1b). Further, UV-B irradiation (b)
decreased the expression of antiapoptotic protein Bcl-2 and NFB p50
increased the expression of pro-apoptotic protein Bim. GA pre- (cytosolic)
treatment reversed these effects (Figs 1b and S2c). To confirm the
inhibitory effect of GA on UV-B irradiation-induced apoptosis, we -Actin
studied the cleavage of apoptotic marker proteins caspase-3 and
PARP-1. UV-B irradiation of HaCaT cells resulted in an increased
NFB p50
cleavage of caspase-3 and PARP-1 proteins and treatment with
(Nuclear)
GA significantly decreased this cleavage (Figs 1b and S2c). These Lamin A
results clearly indicate that GA inhibits UV-B irradiation-induced
apoptotic signalling in UV-B-irradiated HaCaT cells.
IkB
GA treatment protected against UV-B-induced inflammation
in HaCaT cells
To analyse the anti-inflammatory effect of GA treatment against GAPDH
UV-B irradiation-induced inflammatory responses, we investigated
the effect of GA treatment on cytokine release in HaCaT cells. GA(M) 10 25 50 100
Treatment of UV-B-irradiated HaCaT cells with different concen- UV-B(10 mJ/cm) + + + + +
trations of GA significantly reduced the production of pro-inflam- Figure 2. Protective effect of GA against inflammatory markers. (a) Immunoblot
matory cytokines IL-1a, IL-1b, IL-6 and TNF-a (Figure S3a). analysis of various inflammation-related proteins. The cells were harvested and
whole-cell lysates were prepared, and protein expression was determined. (b)
Because Mitogen-activated protein kinases (MAPKs) are known to Effect of GA on nuclear translocation of NF-jB (p50) and Ij-Ba expression in
enhance the synthesis of pro-inflammatory cytokines, we studied HaCaT cells exposed to UV-B. Lamin-A and GAPDH were used as loading controls.
the protective effect of GA on phosphorylation of p38 and JNK
MAPKs. Treatment of HaCaT cells with increasing doses of GA
(10, 25, 50 and 100 lM) decreased UV-B-induced phosphorylation MMP-2/9 that was significantly higher in cells exposed to UV-B
of p38 by 2.2-, 4-, 6.6- and 6.5-fold, respectively, and that of JNK irradiation compared to non-irradiated cells (Figure S3e). These
by 1.1-, 1.7-, 1.8- and 2.1-fold, respectively (Figs 2a and S3d). results collectively indicate that GA treatment strongly inhibits the
Treatment of UV-B-irradiated HaCaT cells with GA also markedly UV-B-induced pro-inflammatory responses in HaCaT cells.
reduced COX-2 expression (Figs 2a and S3d). Treatment of GA treatment ameliorated D, L-buthionine sulphoximine-
HaCaT cells with GA also suppressed the increase in PGE2 release enhanced effects in UV-B-irradiated HaCaT cells
after UV-B exposure (Figure S3b). NF-jB, a transcription factor, The results obtained were indicative of the fact that GA treatment
regulates the transcription of various genes involved in inflamma- suppressed the inflammatory and apoptotic signalling by inhibit-
tion. As shown in Fig. 2b, exposure of HaCaT cells to UV-B irra- ing UV-B irradiation-induced oxidative stress. To prove this
diation resulted in nuclear translocation of NF-jB. This hypothesis, we investigated the inhibitory action of GA treatment
translocation was inhibited by GA pretreatment in a dose-depen- on UV-B-exposed HaCaT cells in the presence/absence of BSO.
dent manner (Figure S3c). UV-B irradiation caused the depletion Treatment of BSO (20 lM) alone showed no cytotoxic effect in
of IkB-a protein, an inhibitor of NF-jB, and treatment with GA HaCaT cells. However, the addition of BSO to cells, 24 h prior to
prevented the reduction in IkB-a levels in UV-B-irradiated HaCaT UV-B exposure, exacerbated a decrease in cell viability by 12% as
cells (Fig. 2b). compared to cells exposed to UV-B irradiation alone. GA treat-
UV-B irradiation leads to the release of various MMPs, culmi- ment, in this setting of the experiment (BSO + UV-B irradiation),
nating in epidermal destruction. On culturing HaCaT cells for resulted in the restoration of the cell viability (Figure S4a). BSO
72 h in the presence or absence of GA after UV-B exposure, we treatment caused an enhanced ROS generation in UV-B-irradiated
found that GA treatment effectively reduced the activity of HaCaT cells as determined by fluorescent microscopy (H2DCFDA

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Experimental Dermatology, 2016, 25, 440446 443
 Afnan et al.
(a)
Control UV-B(10 mj/cm2) UV-B+BSO
UV-B+BSO+ UV-B+BSO UV-B+BSO
GA25 M +GA50 M +A.A20
+Trolox200
UV-B+
UV-B+
(b) UV-B BSO+ BSO+ Campt
Control UV-B +BSO GA 25 M GA50 M 3 M
Figure 3. GA reduced cytotoxic effects augmented by BSO in UV-B-exposed
HaCaT cells. (a) BSO (20 lM) enhanced ROS production stimulated by UV-B (10 mJ/
cm2), while GA significantly reduced fluorescence. Antioxidant combination of
ascorbic acid (20 lM) and Trolox (200 lM) served as positive control. (b)
Mitochondrial membrane potential loss by confocal microscopy.
fluorescence intensity). The increased ROS production was signifi-
cantly reduced by the treatment with GA (Figs 3a and S4b).
BSO-treated, HaCaT cells exposed to UV-B irradiation showed
an increased loss of Dm, enhanced phosphorylation of p38 and
JNK MAPKs, elevated translocation of NF-jB from cytosol to
nucleus and increased expression of Bim and PARP cleavage as
compared to cells exposed to UV-B irradiation alone. Treatment
with GA to BSO-treated + UV-B-exposed cells reversed all these
changes in a significant manner (Figs 3b, S4c,d and S5a,b).
GA markedly reduced UV-B-induced hyperplasia and skin
inflammation in BALB/c mice
Finally, using an in vivo model, we demonstrated the protective
effect of GA against UV-B-induced skin damage (Fig. 4a). Haema-
toxylin and eosin staining showed detrimental effects of UV-B
irradiation as epidermal hyperplasia and leucocyte infiltration. As
shown in Fig. 4b, UV-B irradiation thrice a week for 16 weeks to
mice resulted in an increase in epidermal thickness along with
aggregates of chronic lymph mononuclear cells as compared to
the control group that showed normal epidermis and dermal
appendages. Topical application of GA (100 lM) 1 h prior to
UV-B irradiation reversed these effects (Figs 4b and S5c). Infiltra-
tion of leucocytes was also markedly reduced by GA. Paralleling
our earlier observations in HaCaT cells, UV-B irradiation to the
mouse skin resulted in a distinct increase in phosphorylation of
p38 and JNK and increased expression of COX-2 and p50 NF-jB
444
(a)
(b)
Figure 4. Protection of GA against UV-B-mediated damage in BALB/c mice. (a)
Representative photographs of the mice either unexposed (control) or exposed to
UV-B radiation (180 mJ/cm2) only, and treated topically with GA (100 lM) on
dorsal side 1 h before UV-B irradiation. (b) Histological sections showing the effect
of GA (100 lM) on UV-B (180 mJ/cm2)-induced epidermal thickness and leucocyte
infiltration. Leucocyte infiltration has been represented by arrows. Representative
sections shown from each group (n = 6).
protein levels. These effects were markedly reversed by the topical
treatment of GA (100 lM) (Figures S6a, S3c, S6c). We also studied
the inhibitory effect of GA treatment on the expression of intracel-
lular adhesion molecule (ICAM-1) in the mouse skin irradiated
with UV-B. Exposure of mouse to UV-B irradiation resulted in a
distinct increase in the expression of ICAM-1, which was inhibited
by the topical treatment of GA (Figure S6a, S6c).
Discussion
Solar ultraviolet (UV)-B irradiation is the primary cause of
environmentally acquired cutaneous pathologies that can culmi-
nate in severe morbidity and even mortality. The primary rea-
son for the loss of skin barrier integrity and tissue erosion,
associated with acute exposure to UV-B irradiation, is the gen-
eration of ROS (35,36). Elevated ROS disturbs equilibrium
between pro- and antioxidants in the cell and activates various
intracellular signalling pathways, leading to photodamage
(37,38). Therefore, new strategies to ameliorate UV-B-induced
damage deserve examination. One such strategy to reduce UV-
2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2016, 25, 440446

B-induced adverse effects is the use of botanicals that have the
ability to scavenge or quench ROS (39). Glycyrrhizic acid, a
botanical, found in the roots and rhizomes of liquorice plant
(Glycyrrhiza glabra) has been used as traditional medicine for
several chronic ailments (21). In this study, we found that
human skin keratinocytes (HaCaT) cells, irradiated with UV-B,
resulted in an enhanced ROS formation. This ROS formation
was markedly reduced by GA treatment (Fig. 1a). This study
highlights the protective effect of GA on HaCaT cells and
BALB/c mouse skin against UV-B irradiation-mediated photo-
damage and elucidates a clear role for mitogen-activated protein
kinases, nuclear factor kappa B and mitochondrial apoptotic
pathway in this damage.
UV-B-induced DNA damage (via p53), death receptor
activation (via TNF-a, Fas, activating caspase 8) and ROS genera-
tion initiate a cascade of events leading to apoptotic sunburn cells
in the epidermis of skin (18). Reactive oxygen species predomi-
nantly activate intrinsic or mitochondrial apoptotic pathway by
inducing Bax translocation to mitochondria that trigger the open-
ing of mitochondrial permeability transition pores (MPTP), caus-
ing uncoupling of the respiratory chain with reduction of Dm,
ATP depletion, mitochondrial release of apoptotic proteins and
activation of caspases (40,41). In the present study, we studied the
mechanistic basis for the antiapoptotic effects of GA treatment in
UV-B-irradiated HaCaT keratinocytes. Annexin V/PI staining of
UV-B-irradiated HaCaT cells indicated that GA treatment
inhibited UV-B irradiation-induced apoptotic process (Figure S1e).
GA treatment could prevent UV-B-induced apoptosis by inhibit-
ing the translocation of Bax from cytosol to mitochondria while
increasing Bcl-2 levels (Fig. 1b). The antiapoptotic effect of GA
was further confirmed by studying downstream cascade of
apoptotic events. GA treatment significantly inhibited UV-
B-induced alteration in mitochondrial membrane potential, DNA
fragmentation and cleavage of PARP-1 and caspase-3 (Figures
S2b,a and 1b).
Inflammation is an immediate response to UV-B-mediated
injury and photodamage, characterized by the development of
erythema, elevated levels of proinflammatory mediators and an
increase in inflammatory infiltrates into the dermis (42). Ker-
atinocytes are the main source of cytokines synthesis after UV expo-
sure and predominantly produce IL-1a, IL-1b, IL-6, and TNF-a
(4345). Besides these mediators, COX-2 plays a vital role in
inflammation as it is involved in the biosynthesis of PGE-2, a well-
recognized mediator of active inflammation (46). An increase in the
production of PGE2 has been observed in skin erythema 24 h after
UV-B exposure (47). Results of the present study indicate that GA
treatment attenuated UV-B-induced release of pro-inflammatory
cytokines (IL-1a, -1b,-6 and TNF-a) and inhibited the expression of
COX-2 and subsequent PGE-2 synthesis (Figures S3a, 2a and S3b).
To further characterize the mechanistic basis for the photoprotec-
tive effects of GA, we investigated its effect on the p38, JNK MAPK
and NF-jB subpathways. UV-B irradiation-mediated ROS produc-
tion is known to activate p38 and JNK MAPKs, but not ERK
(48,49). We have previously shown that UV-B-treated fibroblasts
did not show any significant change in ERK phosphorylation,
although GA reduced pERK significantly as compared to control
cells (50). In the present study, treatment of UV-B-irradiated
HaCaT cells with GA decreased phosphorylation of p38 and JNK
2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2016, 25, 440446
GA prevents UV-B -induced photodamage
MAPK in UV-B-irradiated cells (Fig. 2a). These results were also
confirmed in the mouse model of photodamage (Fig. 4a). UV-B
irradiation also activates NF-jB, a transcription factor that activates
multiple genes involved in the expression of various proinflamma-
tory mediators (51). The present study has shown that GA treat-
ment inhibited the translocation of NF-jB from cytosol to the
nucleus and reduced its protein content in UV-B-irradiated HaCaT
cells and BALB/c mouse skin (Figs 2b, S3d, S6a, S6c). Hence, it
seems that GA exerts its anti-inflammatory effect, at least in part, by
modulating MAPK, p38/JNK and NF-jB.
UV-B irradiation also leads to the release of various MMPs that
are involved in the degradation of epidermal basement membrane
(52). Our results on HaCaT cells demonstrate that GA treatment
could abrogate the activities of MMP-2 and MMP-9 (Figure S3e).
Histopathological analysis of skin of BALB/c mouse exposed to
UV-B irradiation suggests that chronic UV-B exposure alters epi-
dermal morphology by increasing epidermal thickness and induces
an inflammatory response with aggregates of chronic lymph
mononuclear cells (Fig. 4b). Topical treatment of GA to BALB/c
mice markedly decreased these signs of UV-B-induced inflamma-
tory response (Fig. 4b). Further, in the skin of UV-B irradiation-
exposed BALB/c mouse, GA treatment was shown to decrease the
expression of ICAM-1 (Figure S6a), an adhesion molecule
involved in the interaction between keratinocytes and T cells dur-
ing UV-B-induced pro-inflammatory responses (53). Overall, these
results suggest that GA exhibits potent anti-inflammatory activity
in UV-B-irradiated HaCaT keratinocytes and BALB/c mouse
exposed to chronic UV-B irradiation.
Our hypothesis that GA could inhibit oxidative stress-mediated
events was further confirmed by additional experiments, wherein
the levels of cellular antioxidants were chemically depleted. BSO is
known to deplete the cellular antioxidant, glutathione (GSH), and
thereby to increase ROS levels in keratinocytes (54). In our study,
GA could abolish the exacerbated oxidative stress and its down-
stream cytotoxic effects induced by BSO in UV-B-exposed cells
(Figure S4a), thus suggesting that quenching ROS is an important
cytoprotective mechanism mediated by GA.
In conclusion, our results demonstrate that treatment using GA
prevents UV-B-induced oxidative stress in HaCaT keratinocytes and
BALB/c mouse skin. GA ameliorated UV-B-induced photodamage
at least in part by inhibiting ROS-induced MAPKs and NF-jB sig-
nalling and via modulation of a Bcl-2 protein family. By inhibiting
the pathways involved in UV-B-induced photodamage, GA has a
strong potential to be developed as a therapeutic or cosmetic pro-
duct against various cutaneous disorders caused by UV-B irradia-
tions.
Contributions
QA, PKJ, LAN and RAR performed the experiments. QA, RS and
SAT analysed the data. SCB performed and analysed the
histopathology. SAT and PKJ designed the research study. QA, RS
and SAT wrote the manuscript. SAT and RAV supervised the
research and procured the funding.
Acknowledgements
This study was supported by Council of Scientific and Industrial Research
(CSIR), New Delhi, India, under its 12th Five Year Plan Project titled,
Towards Understanding Skin Cell Homeostasis (TOUCH), Project No.
BSC 0302. Financial assistance to QA and LAN by University Grants
Commission (UGC), New Delhi, India; RAR by Council of Scientific and
445
 Afnan et al.

Industrial Research (CSIR), New Delhi, India; and PKJ By Science and
Engineering Research Board, Department of Science and Technology, New
Delhi, India, is highly acknowledged. The English language editing by Dr.
Anuradha Krishnan, Division of Gastroenterology and Hepatology, Mayo
Clinic, Rochester, MN, USA, is acknowledged.
Conflict of interest
The authors found report no conflict of interest relevant to the content of
the article.
Supporting Information
Additional Supporting Information may be found online in the supporting
information tab for this article:
Figure S1. Protective effect of GA against UV-B-induced cytotoxicity. (a) Per cent cell
viability of HaCaT cells exposed to UV-B radiation. (b) Effect of GA on cell viability in
HaCaT cells. (c) Effect of GA on viability in HaCaT cells treated with UV-B (10 mJ/cm2)
radiation. Cell viability was assessed using MTT assay. (d) Quantitative analysis of the flu-
orescence by Image J software. (e) Effect of GA on UV-B (10 mJ/cm2)-mediated apopto-
sis as determined by Annexin V/PI staining by flow cytometry. The upper right quadrant
(Q2) contains non-viable cells, positive for annexin Vfluorescein isothiocyanate binding
and the uptake of propidium iodide, representing late-stage apoptotic cells. The lower
right quadrant (Q4) represents the early-stage apoptotic cells, which are positive for
annexin Vfluorescein isothiocyanate binding but exclude propidium iodide. (f) Quanti-
tative data of % total apoptotic cells. Data are expressed as mean  SD from three inde-
pendent experiments.*P 0.05, **P 0.01 and ***P 0.001 statistically different
compared with control group; #P 0.05, ##P 0.01, ###P 0.001 statistically different
when compared with UV-B alone group.
Figure S2. (a) GA protects HaCaT keratinocytes against UV-B-induced DNA frag-
mentation. DNA isolated was subjected to 1.8% agarose gel electrophoresis, followed
by visualization of bands. Camptothecin (3 lM) was used as positive control. (b)
Effect of GA on UV-B (10 mJ/cm2)-induced loss of mitochondrial membrane poten-
tial in HaCaT cells stained with JC-1 dye. (c) Densitometric analysis of indicated
proteins.
Figure S3. (a) Effect of GA on secretion of IL-1a, IL-1b, IL-6 and TNF-a in UV-
B-exposed HaCaT cells. The secretion of various cytokines was determined in condi-
tioned media by ELISA. (b) Effect of GA on UV-B-induced secretion of PGE2. Data
are expressed as mean  SD of three independent experiments. **P 0.01,
***P 0.001 vs control; #P 0.05, ##P 0.01, ###P 0.001 vs UV-B treated cells
only. (c) Represents nuclear to cytosolic ratio of NF-jB normalized with their
respective loading controls. (d) Densitometric analysis of indicated proteins. (e) Pro-
MMP2 and MMP9 activities were measured by gelatin zymography. The supernatant
was collected after 72 h of treatment and subjected to zymography.
Figure S4. (a) Effect of GA on cell viability in BSO (20 lM)-treated or BSO/UV-
B-treated cells. Confluent cells were left untreated or treated with BSO, GA for 24 h
followed by UV-B (10 mJ/cm2) exposure for additional 24 h. Viability of cells was
examined by MTT assay. (b) BSO (20 lM) enhanced ROS production stimulated by
UV-B (10 mJ/cm2), while GA significantly reduced fluorescence. GA restored loss
enhanced by BSO (20 lM) in UV-B (10 mJ/cm2)-treated cells. (c) Effect of GA on PARP
cleavage and Bim expression in BSO/UV-B cells. (d) GA reduced phosphorylation of p38,
JNk MAPKs, translocation of NF-jB enhanced by BSO in UV-B-treated HaCaT cells.
Data for (a) and (b) are represented as *P 0.05, **P 0.01***P 0.001, vs control
only; !P 0.05, !!0.01 vs UV-B treated cells only; #P 0.05, ###P 0.001 vs UV-
B + BSO-treated cells only.
Figure S5. (a, b) Densitometric analysis of indicated proteins. (c) GA inhibits UV-B-
induced epidermal thickness. Values are represented as mean  SD from six ani-
mals.*P 0.001 vs control group only; ###P 0.01 vs UV-B-treated group.
Figure S6. (a) Effect of topical administration of GA on expression of various
inflammatory mediators. Whole tissue lysates prepared were subjected to Western blot
analysis with primary antibody against p38MAPK, JNK MAPK, COX-2, ICAM-1 and
NF-jB p50. b-Actin and GAPDH were used as loading control. Two samples were
selected randomly from each group. (b, c) Densitometric analysis of the indicated
proteins.

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