Sunteți pe pagina 1din 7

DOI: 10.1111/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
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,

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

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

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

2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2016, 25, 440446 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+
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.
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-
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
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-
value of <0.05 was considered statistically significant.
Results PARP
GA treatment prevented cytotoxicity and generation of ROS Cleaved
triggered by UV-B
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

2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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
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.
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

2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2016, 25, 440446 443
Afnan et al.


Control UV-B(10 mj/cm2) UV-B+BSO


GA25 M +GA50 M +A.A20
(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- 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
cantly reduced by the treatment with GA (Figs 3a and S4b). UV-B radiation (180 mJ/cm2) only, and treated topically with GA (100 lM) on
BSO-treated, HaCaT cells exposed to UV-B irradiation showed 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
an increased loss of Dm, enhanced phosphorylation of p38 and infiltration. Leucocyte infiltration has been represented by arrows. Representative
JNK MAPKs, elevated translocation of NF-jB from cytosol to sections shown from each group (n = 6).
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 protein levels. These effects were markedly reversed by the topical
changes in a significant manner (Figs 3b, S4c,d and S5a,b). treatment of GA (100 lM) (Figures S6a, S3c, S6c). We also studied
GA markedly reduced UV-B-induced hyperplasia and skin the inhibitory effect of GA treatment on the expression of intracel-
inflammation in BALB/c mice lular adhesion molecule (ICAM-1) in the mouse skin irradiated
Finally, using an in vivo model, we demonstrated the protective with UV-B. Exposure of mouse to UV-B irradiation resulted in a
effect of GA against UV-B-induced skin damage (Fig. 4a). Haema- distinct increase in the expression of ICAM-1, which was inhibited
toxylin and eosin staining showed detrimental effects of UV-B by the topical treatment of GA (Figure S6a, S6c).
irradiation as epidermal hyperplasia and leucocyte infiltration. As Discussion
shown in Fig. 4b, UV-B irradiation thrice a week for 16 weeks to Solar ultraviolet (UV)-B irradiation is the primary cause of
mice resulted in an increase in epidermal thickness along with environmentally acquired cutaneous pathologies that can culmi-
aggregates of chronic lymph mononuclear cells as compared to nate in severe morbidity and even mortality. The primary rea-
the control group that showed normal epidermis and dermal son for the loss of skin barrier integrity and tissue erosion,
appendages. Topical application of GA (100 lM) 1 h prior to associated with acute exposure to UV-B irradiation, is the gen-
UV-B irradiation reversed these effects (Figs 4b and S5c). Infiltra- eration of ROS (35,36). Elevated ROS disturbs equilibrium
tion of leucocytes was also markedly reduced by GA. Paralleling between pro- and antioxidants in the cell and activates various
our earlier observations in HaCaT cells, UV-B irradiation to the intracellular signalling pathways, leading to photodamage
mouse skin resulted in a distinct increase in phosphorylation of (37,38). Therefore, new strategies to ameliorate UV-B-induced
p38 and JNK and increased expression of COX-2 and p50 NF-jB damage deserve examination. One such strategy to reduce UV-

2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
444 Experimental Dermatology, 2016, 25, 440446
GA prevents UV-B -induced photodamage

B-induced adverse effects is the use of botanicals that have the MAPK in UV-B-irradiated cells (Fig. 2a). These results were also
ability to scavenge or quench ROS (39). Glycyrrhizic acid, a confirmed in the mouse model of photodamage (Fig. 4a). UV-B
botanical, found in the roots and rhizomes of liquorice plant irradiation also activates NF-jB, a transcription factor that activates
(Glycyrrhiza glabra) has been used as traditional medicine for multiple genes involved in the expression of various proinflamma-
several chronic ailments (21). In this study, we found that tory mediators (51). The present study has shown that GA treat-
human skin keratinocytes (HaCaT) cells, irradiated with UV-B, ment inhibited the translocation of NF-jB from cytosol to the
resulted in an enhanced ROS formation. This ROS formation nucleus and reduced its protein content in UV-B-irradiated HaCaT
was markedly reduced by GA treatment (Fig. 1a). This study cells and BALB/c mouse skin (Figs 2b, S3d, S6a, S6c). Hence, it
highlights the protective effect of GA on HaCaT cells and seems that GA exerts its anti-inflammatory effect, at least in part, by
BALB/c mouse skin against UV-B irradiation-mediated photo- modulating MAPK, p38/JNK and NF-jB.
damage and elucidates a clear role for mitogen-activated protein UV-B irradiation also leads to the release of various MMPs that
kinases, nuclear factor kappa B and mitochondrial apoptotic are involved in the degradation of epidermal basement membrane
pathway in this damage. (52). Our results on HaCaT cells demonstrate that GA treatment
UV-B-induced DNA damage (via p53), death receptor could abrogate the activities of MMP-2 and MMP-9 (Figure S3e).
activation (via TNF-a, Fas, activating caspase 8) and ROS genera- Histopathological analysis of skin of BALB/c mouse exposed to
tion initiate a cascade of events leading to apoptotic sunburn cells UV-B irradiation suggests that chronic UV-B exposure alters epi-
in the epidermis of skin (18). Reactive oxygen species predomi- dermal morphology by increasing epidermal thickness and induces
nantly activate intrinsic or mitochondrial apoptotic pathway by an inflammatory response with aggregates of chronic lymph
inducing Bax translocation to mitochondria that trigger the open- mononuclear cells (Fig. 4b). Topical treatment of GA to BALB/c
ing of mitochondrial permeability transition pores (MPTP), caus- mice markedly decreased these signs of UV-B-induced inflamma-
ing uncoupling of the respiratory chain with reduction of Dm, tory response (Fig. 4b). Further, in the skin of UV-B irradiation-
ATP depletion, mitochondrial release of apoptotic proteins and exposed BALB/c mouse, GA treatment was shown to decrease the
activation of caspases (40,41). In the present study, we studied the expression of ICAM-1 (Figure S6a), an adhesion molecule
mechanistic basis for the antiapoptotic effects of GA treatment in involved in the interaction between keratinocytes and T cells dur-
UV-B-irradiated HaCaT keratinocytes. Annexin V/PI staining of ing UV-B-induced pro-inflammatory responses (53). Overall, these
UV-B-irradiated HaCaT cells indicated that GA treatment results suggest that GA exhibits potent anti-inflammatory activity
inhibited UV-B irradiation-induced apoptotic process (Figure S1e). in UV-B-irradiated HaCaT keratinocytes and BALB/c mouse
GA treatment could prevent UV-B-induced apoptosis by inhibit- exposed to chronic UV-B irradiation.
ing the translocation of Bax from cytosol to mitochondria while Our hypothesis that GA could inhibit oxidative stress-mediated
increasing Bcl-2 levels (Fig. 1b). The antiapoptotic effect of GA events was further confirmed by additional experiments, wherein
was further confirmed by studying downstream cascade of the levels of cellular antioxidants were chemically depleted. BSO is
apoptotic events. GA treatment significantly inhibited UV- known to deplete the cellular antioxidant, glutathione (GSH), and
B-induced alteration in mitochondrial membrane potential, DNA thereby to increase ROS levels in keratinocytes (54). In our study,
fragmentation and cleavage of PARP-1 and caspase-3 (Figures GA could abolish the exacerbated oxidative stress and its down-
S2b,a and 1b). stream cytotoxic effects induced by BSO in UV-B-exposed cells
Inflammation is an immediate response to UV-B-mediated (Figure S4a), thus suggesting that quenching ROS is an important
injury and photodamage, characterized by the development of cytoprotective mechanism mediated by GA.
erythema, elevated levels of proinflammatory mediators and an In conclusion, our results demonstrate that treatment using GA
increase in inflammatory infiltrates into the dermis (42). Ker- prevents UV-B-induced oxidative stress in HaCaT keratinocytes and
atinocytes are the main source of cytokines synthesis after UV expo- BALB/c mouse skin. GA ameliorated UV-B-induced photodamage
sure and predominantly produce IL-1a, IL-1b, IL-6, and TNF-a at least in part by inhibiting ROS-induced MAPKs and NF-jB sig-
(4345). Besides these mediators, COX-2 plays a vital role in nalling and via modulation of a Bcl-2 protein family. By inhibiting
inflammation as it is involved in the biosynthesis of PGE-2, a well- the pathways involved in UV-B-induced photodamage, GA has a
recognized mediator of active inflammation (46). An increase in the strong potential to be developed as a therapeutic or cosmetic pro-
production of PGE2 has been observed in skin erythema 24 h after duct against various cutaneous disorders caused by UV-B irradia-
UV-B exposure (47). Results of the present study indicate that GA tions.
treatment attenuated UV-B-induced release of pro-inflammatory Contributions
cytokines (IL-1a, -1b,-6 and TNF-a) and inhibited the expression of QA, PKJ, LAN and RAR performed the experiments. QA, RS and
COX-2 and subsequent PGE-2 synthesis (Figures S3a, 2a and S3b). SAT analysed the data. SCB performed and analysed the
To further characterize the mechanistic basis for the photoprotec- histopathology. SAT and PKJ designed the research study. QA, RS
tive effects of GA, we investigated its effect on the p38, JNK MAPK and SAT wrote the manuscript. SAT and RAV supervised the
and NF-jB subpathways. UV-B irradiation-mediated ROS produc- research and procured the funding.
tion is known to activate p38 and JNK MAPKs, but not ERK Acknowledgements
(48,49). We have previously shown that UV-B-treated fibroblasts This study was supported by Council of Scientific and Industrial Research
did not show any significant change in ERK phosphorylation, (CSIR), New Delhi, India, under its 12th Five Year Plan Project titled,
although GA reduced pERK significantly as compared to control Towards Understanding Skin Cell Homeostasis (TOUCH), Project No.
cells (50). In the present study, treatment of UV-B-irradiated BSC 0302. Financial assistance to QA and LAN by University Grants
HaCaT cells with GA decreased phosphorylation of p38 and JNK Commission (UGC), New Delhi, India; RAR by Council of Scientific and

2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2016, 25, 440446 445
Afnan et al.

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

1 Brenneisen P, Sies H, Scharffetter-Kochanek K. 20 Afaq F, Katiyar S K. Mini Rev Med Chem 2011: 38 Lim T G, Jung S K, Kim J E et al. Exp Dermatol
Ann N Y Acad Sci 2002: 973: 3143. 11: 1200. 2013: 22: 428430.
2 Matsumura Y, Ananthaswamy H N. Toxicol Appl 21 Asl M N, Hosseinzadeh H. Phytother Res 2008: 39 Lee Y-R, Noh E-M, Han J-H et al. Eur J Pharma-
Pharmacol 2004: 195: 298308. 22: 709724. col 2012: 674: 8086.
3 Perluigi M, Di Domenico F, Blarzino C et al. Pro- 22 Haleagrahara N, Varkkey J, Chakravarthi S. Int J 40 Van Laethem A, Nys K, Van Kelst S et al. Free
teome Sci 2010: 8: 13. Mol Sci 2011: 12: 71007113. Radic Biol Med 2006: 41: 13611371.
4 Clydesdale G J, Dandie G W, Muller H K. Immu- 23 Kao T-C, Shyu M-H, Yen G-C. J Agric Food 41 Assefa Z, Garmyn M, Vantieghem A et al. FEBS
nol Cell Biol 2001: 79: 547568. Chem 2008: 57: 754761. Lett 2003: 540: 125132.
5 Warin A P. Br J Dermatol 1978: 98: 473477. 24 Kao T-C, Shyu M-H, Yen G-C. J Agric Food 42 Katiyar S K, Mukhtar H. J Leukoc Biol 2001: 69:
6 Tsoyi K, Park H B, Kim Y M et al. J Agric Food Chem 2010: 58: 86238629. 719726.
Chem 2008: 56: 89698974. 25 Van Rossum T, De Man R. Aliment Pharmacol 43 Takashima A, Bergstresser P R. Photochem Pho-
7 Hildesheim J, Awwad R T, Fornace A J. J Invest Ther 1998: 12: 199205. tobiol 1996: 63: 397400.
Dermatol 2004: 122: 497502. 26 Visavadiya N P, Soni B, Dalwadi N. Int J Food Sci 44 Kim Y, Lee S K, Bae S et al. Immunol Lett 2013:
8 Heck D E, Vetrano A M, Mariano T M et al. J Nutr 2009: 60: 135149. 149: 110118.
Biol Chem 2003: 278: 2243222436. 27 Li J-y, Cao H-y, Liu P et al. Biomed Res Int 2014: 45 Mizutani T, Masaki H. Exp Dermatol 2014: 23:
9 Piao M J, Zhang R, Lee N H et al. Photochem 2014: 115. 2326.
Photobiol 2012: 88: 381388. 28 Afnan Q, Adil M D, Nissar-Ul A et al. Phy- 46 Isoherranen K, Punnonen K, Jansen C et al. Br J
10 Afaq F, Ahmad N, Mukhtar H. Oncogene 2003: tomedicine 2012: 19: 658664. Dermatol 1999: 140: 10171022.
22: 92549264. 29 Cherng J-M, Tsai K-D, Yu Y-W et al. Radiat Res 47 Black A K, Greaves M, Hensby C et al. Br J Clin
11 Sharma S D, Meeran S M, Katiyar S K. Mol Can- 2011: 176: 177186. Pharmacol 1978: 6: 261266.
cer Ther 2007: 6: 9951005. 30 Farrukh M R, Nissar U A, Afnan Q et al. J Der- 48 Lopez-Camarillo C, Arechaga Ocampo E, Lo pez
12 Kim A L, Labasi J M, Zhu Y et al. J Invest Der- matol Sci 2014: 75: 2435. Casamichana M et al. Int J Mol Sci 2011: 13:
matol 2005: 124: 13181325. 31 Piao M J, Lee N H, Chae S et al. Biol Pharm Bull 142172.
13 Muthusamy V, Piva T J. Arch Dermatol Res 2012: 35: 873880. 49 Ding M, Lia J, Leonard S S et al. Mol Cell Bio-
2010: 302: 517. 32 Yu W, Sanders B G, Kline K. Cancer Res 2003: chem 2002: 234: 8190.
14 Zarubin T, Jiahuai H. Cell Res 2005: 15: 1118. 63: 24832491. 50 Farrukh M R, Nissar U-A, Kaiser P J et al.
15 Fernau N S, Fugmann D, Leyendecker M et al. J 33 Zhang X D, Zhang X Y, Gray C P et al. Cancer J Photochem Photobiol, B 2015: 148:
Biol Chem 2010: 285: 38963904. Res 2001: 61: 73397348. 351357.
16 Tanaka K, Hasegawa J, Asamitsu K et al. J Phar- 34 Oh J-H, Chung A-S, Steinbrenner H et al. Arch 51 Shin M H, Park R, Nojima H et al. PLoS One
macol Exp Ther 2005: 315: 624630. Biochem Biophys 2004: 423: 218226. 2013: 8: e61696.
17 Hur S, Lee Y S, Yoo H et al. J Dermatol Sci 35 Haratake A, Uchida Y, Schmuth M et al. J Invest 52 Zaid M A, Afaq F, Syed D N et al. Photochem
2010: 59: 163169. Dermatol 1997: 108: 769775. Photobiol 2007: 83: 882888.
18 Assefa Z, Van Laethem A, Garmyn M et al. Bio- 36 Afaq F, Adhami V M, Ahmad N et al. Oncogene 53 Imokawa G, Takagi Y, Higuchi K et al. J Invest
chim Biophys Acta 2005: 1755: 90106. 2003: 22: 10351044. Dermatol 1999: 112: 9196.
19 Van Laethem A, Van Kelst S, Lippens S et al. 37 Simmler C, Antheaume C, Lobstein A. PLoS One 54 Zhu M, Zhang Y, Cooper S et al. Mol Carcinog
FASEB J 2004: 18: 19461948. 2010: 5: e13713. 2004: 41: 179186.

2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
446 Experimental Dermatology, 2016, 25, 440446