INTRODUCTION

Skin cancer is a significant health problem associated with

mortality and morbidity; therefore concerted efforts were made

aiming to develop novel strategies for the prevention of ultraviolet

radiation (UV)-mediated damages. Chemoprevention, defined as

“the use of agents capable of ameliorating the adverse effects of

UVB on the skin” by natural compounds, represents a new

concept in the attempt to control the carcinogenesis process UVB-

induced (1).

Calluna vulgaris L. Hull (known as common heather) is the

sole species in the genus Calluna in the Ericaceae family. It was

used in traditional folk medicine as an antiseptic, antibacterian

(2), cholagogue, diuretic (3), expectorant, antirheumatic and

antiinflammatory agent (4, 5).

Further, both in vitro (6) and in vivo studies (7-9) revealed

antioxidant and anti-inflammatory effects of Cv extract. Recently,

we demonstrated that pretreatment with Cv extract reduced lipid

peroxides and nitric oxide generation and inhibited the UVB-

induced apoptosis and inflammation in mice (8, 9) as well as the

formation of DNA photolesions in HaCaT keratinocytes (10).

These effects were assigned to the high content of polyphenols

identified in the extract composition (10).

It is known that phenolic compounds are plant secondary

metabolites with antioxidative, antiinflammatory and

photoprotective properties. In vitro and in vivo studies

demonstrated that kaempferol and quercetin, the most important

compounds identified in Cv extract, have important antioxidant,

anti-inflammatory and antiproliferative activities (11, 12). Lee et

al. (13) showed that kaempferol suppressed UVB-induced

cyclooxygenase-2 (COX-2) expression and Src kinase activity in

mouse skin epidermal JB6 P+ cells and attenuated the UVB-

induced phosphorylation of mitogen-activated protein kinase

(MAP kinase). Quercetin was found to suppress activation-UV

induced of phosphorylated extracellular signal regulated kinase

(ERK) and p38 MAP kinase in lipopolysaccharide-stimulated

macrophage, and to inhibit nuclear factor-κB (NF-κB)

activation, respectively proinflammatory cytokines and

metalloproteinases (MMPs) expression (14, 15).

Recent studies indicated that the MAPkinase signal

transduction pathways are involved in the cell function

regulation, including MMPs expression and cell growth (16).

Once activated, MAP kinases can translocate towards the nucleus

to phosphorylate and activate the activator protein 1 (AP-1) and

NF-κB factors. In the nucleus, NF-κB coordinates the

transcriptional activation of inflammatory, anti-apoptotic genes

and genes that positively regulate cell proliferation, metastasis

and angiogenesis (17). Taking into account the potential

therapeutic application of Cv extract on humans, this study

intended to evaluate the mechanisms involved in Calluna

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2012, 63, 4, 423-432

www.jpp.krakow.pl

G.A. FILIP1, I.D. POSTESCU2, C. TATOMIR2, A. MURESAN1, S. CLICHICI1

CALLUNA VULGARIS EXTRACT MODULATES NF-κB/ERK SIGNALING PATHWAY

AND MATRIX METALLOPROTEINASE EXPRESSION IN SKH-1 HAIRLESS MICE SKIN

EXPOSED TO ULTRAVIOLET B IRRADIATION

1Department of Physiology, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Department of Radiobiology and Tumor Biology, „Prof. I. Chiricuta” Oncologic Institute, Cluj-Napoca, Romania

Photochemoprevention with natural products represents a new concept in the attempt to reduce the occurrence of skin

cancer. However, the molecular mechanisms caused by ultraviolet light exposure remain still unclear. The aim of the study

was to assess the mechanisms involved in the action of a Calluna vulgaris (Cv) extract, administered in single or multiple

doses (10 consecutive days), on UVB-induced skin damage in SKH-1 hairless mice. The extract was topically applied 30

min before each UVB exposure in two doses (2.5 and 4 mg total polyphenolic content/40 µl/cm2). At 24 hours after the last

treatment, total mitogen-activated protein kinase (p44/42MAPkinase, ERK 1/2), nuclear factor-κB (phospho-NF-κB p65),

matrix metalloproteinases (MMP-2, MMP-9) and metalloproteinase inhibitor 1 (TIMP-1) levels were measured in skin

using enzyme-linked immunosorbent assay (ELISA). MMP-2 and -9 activities were additionally evaluated by zymography.

One topical application of Cv extract reduced the secretion (p<0.004) and inhibited MMP-9 activity UVB-mediated (54%

inhibition) via inhibition of NF-κB activation (68% inhibition). In multiple UVB exposures, both doses of Cv extract

induced the increase of ERK 1/2 level in correlation with activation of NF-κB and reduced the secretion (p<0.04) and

activation of MMP-9 (62% inhibition). Pretreatment with Cv diminished the MMP-2 protein secretion only in one dose

UVB-irradiated group (p<0.0001) and decreased TIMP-1 level (p<0.001). These results demonstrated the dual behavior of

Cv extract in skin protection against single versus multiple doses of UVB irradiation.

K e y w o r d s : flavonoids, metalloproteinases, signal transduction, skin, ultraviolet radiation, metalloproteinase inhibitor 1

vulgaris effects in single or multiple doses of UVB-induced

damage to the skin of SKH-1 hairless mice. Specifically, we

assessed comparatively the modulatory effects of Cv extract on

UVB-mediated activation of ERK1/2 and NF-κB by using

ELISA tests. Also, we assessed the MMP-2 and -9 activities by

zymography and MMP-2, MMP-9 and TIMP-1 levels by ELISA.

MATERIALS AND METHODS

Reagents

Sodium dodecylsulfate (SDS), Triton X-100, 2,2-diphenyl-l-

picryl-hydrazyl (DPPH free radical), 2,2’-azino-bis(3-

ethylbenzothiazoline-6-sulfonate (ABTS), Bradford and Folin

Ciocalteu reagents were purchased from Sigma-Aldrich

Chemicals GmbH (Germany). ELISA tests for measuring total

p44/42MAPKinase (ERK1/2) and phospho-NF-κB p65 (ser536)

were purchased from Cell Signaling Technology, Inc. (Danvers,

MA, USA). ELISA tests for MMP-2, MMP-9 and TIMP-1 levels

were obtained from R&D Systems (Minneapolis, MN, USA).

Other chemicals were of reagent grade and purchased from

Sigma-Aldrich Chemicals GmbH (Germany).

Plant material

The aerial parts of Calluna vulgaris L. Hull (known as

Common Heather, fam. Ericaceae) were used. The aerial parts of

Calluna vulgaris L. Hull were collected in August 2010 from

Ciucea (altitude 650 m, 46.95 N 22.82 E), Cluj, Romania. The

plant was identified by Prof. Dr. Mircea Tamas and a voucher

specimen was deposited at the Herbarium of the Botany

Department (index no. 825), Faculty of Pharmacy, University of

Medicine and Pharmacy, Cluj-Napoca.

Sample preparation and total polyphenolic content

The Calluna vulgaris (Cv) fluid extract was obtained as

described (9). Total polyphenolic content (TPC) was assessed by

the Folin-Ciocalteu colorimetric reaction (18) and expressed as

equivalents (Eq) gallic acid (GA) per unit of volume. The

analysis was performed in triplicate.

High-performance liquid chromatography-mass spectrometry

analysis of polyphenols from Calluna vulgaris extract

The composition of the Cv extract in polyphenols was

determined by high-performance liquid chromatography

(HPLC) analysis as previously described (10), using an Agilent

1100 HPLC Series system (Agilent, USA) coupled with an

Agilent 1100 mass spectrometer (LC/MSD Ion Trap VL).

Determination in vitro of antioxidant capacity

2,2-diphenyl-l-picryl-hydrazyl (DPPH)·free radical assay

(19) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate

(ABTS) test were used to evaluate the antioxidant activity (8).

Animals and experimental protocol

Female SKH-1 hairless mice, 8 weeks old, from Charles

River (Germany) were acclimatized for one week in a 12-h

light/12-h dark cycle, 35% humidity, and free access to water

and normocaloric standard diet (VRF 1).

Two series of experiments were performed in order to

evaluate the effects of Cv extract on mice exposed to a unique

dose of UVB (experiment I) and respectively on mice treated

with multiple doses of UVB irradiation, 10 days consecutively

(experiment II).

In experiment I, 6 groups of 7 animals each, were treated as

follows: Group 1 – control; Group 2 – vehicle (acetone 40%);

Group 3 – UVB irradiation (240 mJ/cm2); Group 4 – vehicle +

UVB irradiation; Group 5 – Cv 2.5 mg TPC/cm2 + UVB

irradiation; Group 6 – Cv 4 mg TPC/cm2 + UVB irradiation.

For experiment II we used the following groups: Group 7 –

control; Group 8 – vehicle; Group 9 – UVB (multiple doses,

240 mJ/cm2/day, 10 days); Group 10 – vehicle + UVB; Group 11

– Cv 2.5 mg TPC/cm2 + UVB irradiation; Group 12 - Cv 4 mg

TPC/cm2 + UVB irradiation.

Before treatment, the animals were anaesthetized with an i. p.

injection of ketamine xylazine cocktail (90 mgkg–1 b.w. ketamine,

10 mgkg–1 b.w. xylazine). The extract was applied topically on

the skin in a dose of 2.5 (low dose) respectively 4 mg/cm2 (high

dose), in vehicle, 30 min before each UVB exposure. UVB

irradiation was performed with Waldmann UV 181 broadband

UV-B source as described previously (8, 9). The UVB emission

was monitored with a radiometer Variocontrol radiometer

(Waldmann GmbH, Germany). Animals were weighed and

observed during the experiment. 24 hours after the last treatment,

the skin was macroscopically examined, animals were weighed

and under anesthesia, fragments of dorsal skin were excised from

each mouse to be used for investigations. All experiments were

performed according to the approved animal-care protocols of the

Ethical Committee on Animal Welfare of the “Iuliu Hatieganu”

University of Medicine in accordance with the internationally

accepted principles for laboratory animal use and care (European

Community Guidelines, EEC Directive of 1986; 86/609/EEC).

Isolation of skin samples

After the removal of subcutaneous tissue, skin tissue

fragments were homogenized with a Polytron homogenizer

(Brinkman Kinematica, Switzerland) for 3 min, on ice, in

phosphate buffered saline (pH 7.4) added at a ratio of 1:4 (w/v).

The suspension was centrifuged for 5 min at 3000 g and 4°C in

order to prepare the cytosolic fraction. The proteins content in

homogenates was measured with the Bradford method (20).

Quantitative estimation of total p44/42MAPkinase (ERK 1/2)

and phospho-nuclear factor-κB p65 by ELISA

Total p44/42 MAPK (ERK1/2) protein and phospho-NF-KB

p65 (ser536) level were evaluated by path scan sandwich ELISA

tests according to the manufacturer’s protocol (Cell Signaling

Technology Inc.). Results are expressed in terms of OD units/mg

protein of different treatment samples.

Measurement of MMP-2, MMP-9 and TIMP-1 levels by ELISA

For the analysis of MMP-2, MMP-9 and TIMP-1 ELISA

assays were used according to the producer instructions (R&D

Systems). The two MMPs levels and their inhibitor were

expressed as pg/mg protein.

Determination of MMP-2 and MMP-9 activities by zymography

The activity of gelatinases was determined by zymography

(21). Skin samples were homogenized with an Ultra Turrax

homogenizer in a medium with 2% glycerin and 2% SDS in 62.5

mM Tris-HCl buffer. After centrifugation at 12,000 g, the protein

content in the supernatant was determined by a slight modification

of Lowry’s method. Five-microgram protein samples were

subjected to electrophoresis on 10% polyacrylamide gels

copolymerized with 1 g/mL gelatin at 100 V for 2 hours. After

424

electrophoresis, the gels were washed with 2.5% Triton X-100 for

1 hour, then incubated for 18 hrs, at 37°C, in 50 mM Tris-HCl

buffer, pH 7.4, containing 5 mM CaCl2, 200 mM NaCl and 0.02 %

BRIJ-35. The gels were then stained with 1% Coomassie brilliant

blue R-250, for 2 hours and discolored in 10 % acetic acid solution.

Activity was evaluated with an automated Vilber-Lourmet (France)

image analyzer using the Bio 1-D program. Proteolytic activity was

determined against a weight marker, confirmed with an enzymatic

marker and reported as arbitrary units/µg protein.

Statistical analysis

Experimental data were analyzed by one-way analysis of

variance (ANOVA) followed by the Tukey´s multiple

comparisons post-test using the GraphPad Prism 5.0 software

(GraphPad, San Diego, Ca., SUA). The four groups treated with

Cv extracts were compared with control group (untreated),

groups treated with vehicle and UVB irradiated. The data were

expressed as means ±standard deviation. The effects of Cv

extracts and UVB and their interaction were considered

statistically significant at p<0.05.

RESULTS

Composition of Calluna vulgaris extract and the antioxidant

capacity

Chemical analysis of the polyphenols content indicated a

value of TCP in Cv extract of 67.50±3.25 mg GAE/ml (in 3

repeated extractions). The antioxidant activity of Cv extract

evaluated by ABTS test was 38.92±0.01 eq. mM Trolox, in

agreement with the results obtained previously (8). The

antioxidant activity can be explained by the amount of phenolic

compounds found in Cv extract as shown in Fig. 1 (10). The

most representative compounds identified by HPLC-MS

analysis in non-hydrolyzed sample were: hyperoside (855.7

µg/ml), quercetin (388.32 µg/ml) quercitrin (273.88 µg/ml) and

kaempferol (99.912 µg/ml). The hydrolyzed sample revealed

elevated quantities of quercetol (1290.636 µg/ml) and

kaempferol (307.528 µg/ml).

Macroscopic changes

It is known from literature that UVB radiation induces

erythema, edema and hyperplasia of epidermis (22). Thus, we

first compared the visual aspect of the skin of non-irradiated

and irradiated animals. No visible macroscopic changes

(redness, swelling) in the skin of unirradiated mice and of

animals treated with vehicle were observed. The groups

exposed to a single dose of UVB (240 mJ/cm2), unprotected or

protected with vehicle and Cv extracts, had the same

macroscopic aspect as the unirradiated skin (Fig. 2A-E). In the

10 days experiment, multiple doses of UVB induced

hyperplasia of the skin, scaling and erythema (Fig. 2F, 2G).

Topical application of Cv extracts on 2 cm2 skin area protected

the skin, the epidermis was normal as color, thickness, without

scaling or pigmentation (Fig. 2H). No toxic effects of Cv extract

on treated animals (weight loss, morbidity and mortality) and

on the organs (liver, brain, spleen) were not observed.

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Fig. 1. High-performance liquid

chromatogram of the phenolic

compounds isolated from the extract

obtained from aerial parts of Calluna

vulgaris: (A) nonhydrolyzed sample:

p-coumaric acid (1), ferulic acid (2),

hyperoside (3), isoquercitrin (4),

rutoside (5), quercitrin (6), quercetol

(7), luteolin (8), kaempferol (9); (B).

hydrolized sample: p-coumaric acid

(1), ferulic acid (2), sinapic acid (3),

quercetol (4), luteolin (5), kaempferol

(6), apigenin (7).

Effects of Calluna vulgaris extract on UVB-induced activation

of p44/42 MAPkinase in SKH-1 hairless skin

The p44/42 MAPK (ERK1/2) signaling pathway can be

activated as response to a diverse range of extracellular stimuli

including mitogens, growth factors and cytokines (23) and is an

important target in cancer diagnosis and treatment. One single

dose of UVB did not change the protein level of total form of

enzyme (1.64±0.02 vs. 1.71±0.04 U OD/mg protein). Both doses

of Cv extract administered before UVB exposure maintained the

total ERK1/2 concentration close to control or irradiated groups

(1.62±0.06 respectively 1.62±0.08 U OD/mg protein; p>0.05)

(Fig. 3A).

In the 10 days experiment the total p44/p42 ERK 1/2 level,

evaluated after the last treatment, revealed in UVB irradiated

groups (0.16±0.04 vs. 0.33±0.15 U OD/mg protein; 50%

inhibition; p>0.05) lower but insignificantly amounts of protein

compared to control or vehicle treated groups (0.16±0.04 vs.

0.33±0.15 U OD/mg protein; 50% inhibition; p>0.05) (Fig. 3B).

Topical applications of Cv extracts increased significantly

phosphorylation of ERK (p<0.0001), suggesting that this

pathway was activated in skin by treatment with natural

products. The activation was not dose dependent, the highest

value of ERK 1/2 being found in group treated with low dose of

polyphenols (0.96±0.07 U OD/mg protein; 6.0 times; p<0.0001).

High dose of TPC (4 mg/cm2) maintained a high concentration of

ERK 1/2 in skin (0.73±0.21 U OD/mg protein).

Effects of Calluna vulgaris extracts on UVB-induced activation

of nuclear factor-κB in SKH-1 hairless skin

Our study showed low levels of phospho-NF-κB p65 protein

in control skin or in skin treated with vehicle, both in acute

(0.15±0.03 respectively 0.19±0.04 U OD/mg protein) and in the

10 days experiment (0.09±0.01 and 0.08±0.02 U OD/mg protein).

Single dose of UVB stimulated phosphorylation of NF-κB p65 at

Ser 536 in skin, as shown in Fig. 3C (0.37±0.03 U OD/mg protein;

p<0.0001). The same results were obtained when the skin was

pretreated with vehicle (0.32±0.05 U OD/mg protein; p<0.0001).

Topical application of Cv extracts before single UVB exposure

inhibited the NF-κB phosphorylation but not in a dose-dependent

manner. The greater inhibition was observed at low concentration

of polyphenols (0.22±0.06 U OD/mg protein; p<0.001; 68%

inhibition). In the 10 days experiment the results obtained were

different. Multiple doses of UVB maintained low levels of NF-κB

in skin, close to control (0.08±0.01 vs. 0.09±0.01 U OD/mg

protein) or vehicle treated groups (0.08±0.02 U OD/mg protein)

(Fig. 3D). Pretreatment with Cv extract increased phospho-NF-kB

p65 protein level but insignificantly compared to UVB group

(0.12±0.03 and 0.12±0.05 U OD/mg protein; p>0.05).

Effects of Calluna vulgaris extract on UVB-induced secretion

and activity of MMP-2 and MMP-9 in SKH-1 hairless skin

Exposure to UVB radiation is known to upregulate the

synthesis of matrix degrading enzymes. MMPs are a family of at

least 24 zinc-dependent endopeptidases involved in photoageing,

angiogenesis and metastasis (24). Among the family members,

MMP-2 and MMP-9 have mainly been characterized as

important factors involved in skin ageing and tumor invasion.

Therefore, we evaluated the effect of Cv extract on UVB-induced

MMPs protein secretion and activity in skin after UVB exposure.

A single dose of UVB increased 3.7 fold MMP-2 (24.07±7.26 vs.

6.28±3.10 ng/mg protein; p<0.0001) and 2.0 fold MMP-9 protein

levels (33.04±7.73 vs. 13.73±2.23 ng/mg protein) (Figs. 4A and

4B). Pretreatment of skin with Cv extracts given 30 minutes prior

426

Fig. 2. Macroscopic changes in UVB radiation-induced skin of SKH-1 hairless mice. Photograph showing no visible macroscopic

changes (redness, swelling) in the skin of mice from experiment I (A, B, C, D, E). In the 10 days of experiment, multiple doses of

UVB- induced hyperplasia of the skin, scaling and erythema (F, G). In groups protected with Cv extracts the epidermis was normal

as color, thickness, without scaling or pigmentation (H).

to single UVB exposure reduced the MMP-9 level (11.10±9.26

respectively 17.05±2.44) and significant differences were

obtained at low dose of TCP (p<0.004). ELISA data indicated

that Cv extracts decreased the secretion of MMP-2 in skin

(5.84±3.89 ng/mg protein; respectively 5.78±1.36; 41%

inhibition p<0.0001). Analysis of zymographic images revealed

the presence of MMP-9 (active form) and MMP-2 (active and

inactive forms) in all groups studied (Figs. 4C-E). The inactive

form of MMP-9 in skin was not detected. The activity of total

MMP-9 was significantly increased (3.0 fold) in UVB irradiated

group (523.8±2.05 U/µg protein) and in group pretreated with

vehicle (410.0±120.1 U/µg protein; 2.0 fold; p<0.005). Low dose

of Cv extract (2.5 mg TPC/cm2) significantly inhibited the total

MMP-9 activity (54% inhibition; 283.6±34.22 U/µg protein;

p<0.05). The activity of total MMP-2 evaluated by zymography

did not change significantly after UVB irradiation (227.3±72.79

vs. 177.1±18.51 U/µg protein) or after treatment with Cv extracts

(174.04.5±39.57 respectively 200.70±67.25 U/µg protein;

p>0.05).

In the 10 days experiment, multiple UVB exposures did not

increase the secretion of MMP-2 protein (6.85±1.16 U/µg

protein vs. 6.28±3.10 respectively 7.05±2.83 pg/mg protein in

control and vehicle treated groups; p>0.05) (Fig. 5A). Similar

values were obtained in groups pretreated with both doses of Cv

extract (6.22±1.79 and 4.74±1.08 pg/mg protein; p>0.05). Total

MMP-2 activity was maintained at the same level in UVB

irradiated groups and in groups treated with Cv extracts (p>0.05)

as in the control group (Figs. 5C and 5E).

MMP-9 protein level quantified by ELISA (pg/mg protein)

showed that the multiple UVB exposures insignificantly

increased MMP-9 protein synthesis in skin cells (17.79±7.11)

compared to control or vehicle treated groups (13.73±2.23

respectively 14.68±5.32 pg/mg protein) (Fig. 5B). MMP-9

protein secretion decreased 4.0 fold in group treated with 2.5

TPC/cm2 (4.33±0.14; p<0.04). High concentration in TPC (4

mg/cm2) significantly maintained the reduced MMP-9 level

(3.99±0.40 pg/mg protein) compared to UVB irradiated group

(78% inhibition; p<0.04). Also, the activity of total MMP-9

decreased significantly in the group treated with 2.5 mg

TPC/cm2 (158.5±44.68 U/µg protein; 62% inhibition; p<0.01)

compared to irradiated groups (381.3±120.3 respectively

417.4±6.78 U/µg protein) (Figs. 5D and 5E).

Effects of Calluna vulgaris extract on UVB-induced secretion

of TIMP-1 in SKH-1 hairless skin

Extracellular matrix metabolism is controlled by MMPs and

their tissue inhibitors, TIMPs. The balance between MMPs and

TIMPs plays an important role in the homeostasis of tissue.

Therefore, we studied the effect of Cv extract on UVB-mediated

altered TIMP-1 level. One single UVB irradiation (240 mJ/cm2) did

not change the TIMP-1 level in skin compared to non UVB exposed

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Fig. 3. Effects of Cv extract on UVB-induced activation of p44/42 MAPKinase (2A, 2B) and NF-κB (2C, 2D) in SKH-1 hairless skin.

Cv extract was applied topically on the skin in a dose of 2.5 respectively 4 mg TPC/cm2, in vehicle, 30 min before each UVB exposure

(240 mJ/cm2). The mice were exposed to a single dose of UVB (2A, 2C: acute experiment) and to multiple doses of UVB (2B, 2D: 10

consecutive days). Skin pretreatment with both doses of Cv extract increased significantly the phosphorylation of ERK 1/2 24 hours

after the last treatment, in the 10 days experiment. The low dose of Cv extract reduced the level of the phosphorylated form of NF-κB

p65 subunit in acute experiment. Values are means ±standard deviation. Statistical analysis was done by a one-way ANOVA, with

Tukey´s multiple comparisons posttest (**p<0.001; ***p<0.0001 compared to UVB group).

group (603.2±115.2 vs. 611.1±62.4 pg/mg protein; p>0.05) (Fig.

6A). Cv extracts applied before one UVB exposure insignificantly

reduced the secretion of protein in skin (498.0±129.4 respectively

409.3±77.35 pg/mg protein; p>0.05). Multiple doses of UVB

maintained the same levels of TIMP-1 in skin as non UVB

irradiated skin (658.1±80.98 vs. 611.1±62.44 pg/mg protein) (Fig.

6B). Pretreatment with 2.5 mg TPC/cm2 Cv extract resulted in

significant inhibition of TIMP-1 level (311.2±82.11 pg/mg protein;

p<0.001) compared to UVB alone (48% inhibition). Also, 4.0 mg

TPC/cm2 significantly decreased the TIMP-1 generation (2.6 fold;

253.4±45.81 pg/mg protein; p<0.0001).

DISCUSSIONS

The present study examined some parameters assumed to be

involved in of putative photochemopreventive effects of a

natural extract obtained from Calluna vulgaris against the

photodamaging action of a single dose and multiple doses of

UVB. To define the efficacy of Cv extract at the molecular level

we assessed the secretion and activation of MAPK/ERK 1/2,

NF-κB and MMPs in SKH-1 hairless mice skin following UVB

irradiation and the modulatory effect of Cv extract on these

events. Perde et al. reported the isolation of quercetin,

isoquercitrin, quercitrin, hyperozid and kaempferol from the

plant of Romanian origin (10). It seems that the accumulation of

high concentration of phenolic compounds depends on the

collection area and period, as proved by phytochemical studies

on Calluna vulgaris (25, 26). The protective effect of Cv extract

was primarily confirmed by the macroscopic aspect of the skin,

the epidermis of mice being normal in color, thickness, without

scaling or pigmentation after UVB exposure. The macroscopic

changes in UVB radiation-induced skin of SKH-1 hairless mice

are suggestively presented in Fig. 2 as supplementary material.

It is well known that MAPKs, which belong to a family of

serine/threonine protein kinases, have the role to mediate signal

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Fig. 4. Effects of Cv extract on a single dose UVB-induced secretion and activity of MMP-2 and MMP-9 in SKH-1 hairless skin: (A)

MMP-2 level, (B) MMP-9 level, (C) MMP-2 and MMP-9 (D) activities in skin 24 hours after the treatment. The animals were treated

as under Fig. 3. Topical application of Cv extract diminished the MMP-2 and MMP-9 protein secretion and inhibited the MMP-9

activity. Data are means ±standard deviation. Statistical analysis was done by a one-way ANOVA, with Tukey´s multiple comparisons

posttest (*p<0.01; **p<0.001; ***p<0.0001 compared to UVB group); (E) Zymographic patterns of MMP-2 and MMP-9 activities in

UVB and Cv treated groups in SKH-1 hairless skin.

transduction from the cell surface to nucleus. Among the three

distinct MAPK pathways identified in mammalian cell, ERK1/2

is activated through UVB-induced oxidative stress (27), growth

factors and protein kinase C. It has been also documented that

NF-κB is a downstream target of the MAPK signal transduction

pathway and that its activation is important in inflammation and

cellular proliferation (27). Further, UV-induced MAPkinase

signaling is also involved in the activation of MMPs, enzymes

involved in the degradation of skin tissues matrix. Reactive

oxygen (ROS) and nitrogen (RNS) species and mediated

activation of MAPK and NF-κB signaling pathways in UVB

exposure has been shown to be inhibited by using plant origin

antioxidants (28, 29). Therefore, we considered the activation of

these pathways as being molecular targets for potential

photochemopreventive agents.

In our study, exposure to a single dose of UVB with or

without treatment with Cv extract, did not change the protein

level of total ERK1/2 after 24 hours. These findings are in

agreement with those of Vicentini et al. (30) which demonstrated

that quercetin, an important component of Cv extract, did not

have effect on activation-UV induced of the three MAP kinases,

ERK, JNK, or p38, in human keratinocytes. It seems that a single

dose of UVB induces the activation of ERK 1/2 with a maximum

after 12-24 hours, without any change in total ERK1/2 protein

level in first hours (31). Our results could be explained either by

a transient effect of UVB, in the first hours after irradiation, or

by activation of enzyme without increasing of total protein level.

Moon et al. (32) confirmed that ERK and JNK kinases were

activated within 30 min of UVB irradiation followed by a

decline of enzymes to a baseline level.

The treatment with Cv extracts before each UVB exposure,

10 consecutive days, resulted in an increase of total ERK 1/2

level, especially at low dose (2.5 mg TPC/cm2). The activation

of MAP kinase by Cv extract is indeed surprising as well as

intriguing. Generally, the chemopreventive effect of polyphenols

is due on the one hand to action free radical scavengers and on

429

Fig. 5. Effects of Cv extract on UVB-induced secretion and activity of MMP-2 and MMP-9 in SKH-1 hairless skin, in the 10 days

experiment: (A) MMP-2 level, (B) MMP-9 level, (C) MMP-2 and MMP-9 (D) activities in skin 24 hrs after the last treatment. The

animals were treated as under Fig. 3. Pretreatment with Cv extract inhibited the protein secretion and activity of MMP-9. Data are

means ±standard deviation. Statistical analysis was done by a one-way ANOVA, with Tukey´s multiple comparisons posttest (*p<0.01

compared to UVB group); (E) Zymographic patterns of MMP-2 and MMP-9 activities in UVB and Cv treated groups in SKH-1

hairless skin.

the other hand to the induction of phase II detoxifying enzymes

via antioxidant response element (ARE). Recent studies showed

the involvement of MAP kinase in the regulation of phase II

enzymes gene expression and in induction of apoptosis (33).

However, the active components from Cv extract which would

activate the MAP kinase pathway remain unclear. Other

investigators have found both prooxidant and antioxidant effects

of quercetin depending on concentration used (34) and

experimental design (35). There is evidence that quercetin, used

in different doses, increased 1.3- to 2.0-fold the phospho-

ERK1/2 levels in human epidermoid carcinoma A431 cells (36).

These results suggested that multiple doses of quercetin could

induce phosphorylation of ERK 1/2. The effect was probably

due to quercetin degradation products which became unstable in

any aqueous-based topical formulation. They may have

prooxidant effects and initiate the apoptosis and also potentate

the UVB-induced c-fos gene expression and increase p38 and

cyclic AMP response element binding (CREB) phosphorylation

(37). Addition of ascorbic acid to quercetin stabilizes it and

inhibits formation of reactive intermediate species (ortho-

semiquinone radical and ortho-quinone) (38) and consequently

reduces its proapoptotic effect on UVB-irradiated human

keratinocytes (35).

An activating effect on MAPkinase expression was observed

in treatments with other natural compounds. Thus, Chen et al.

(33) have shown that (-)-epigallocatechin gallate (EGCG)

induced the MAP kinase activation, with maximum effect at 2

hours after treatment. Other in vitro studies revealed that

silibinin amplified the phosphorylation of ERK 1/2 induced by

15 and 30 mJ/cm2 of UVB, without noticeable changes in total

ERK 1/2 protein levels (39).

It is well known that ROS play a key role in enhancing

inflammation through the activation of stress kinases and redox

sensitive transcription factors, such as NF-κB. In keratinocytes,

NF-κB activates p21 which in turn is involved in growth arrest

(40). Our results indicated that one exposure of skin to UVB

activated NF-κB p65 compared to non UVB-exposed.

Pretreatment with 2.5 mg TPC/cm2 inhibited the single dose

UVB-induced NF-κB p65 activation, while in the 10 days

experiment Cv extract increased the phospho-NF-κB p65 protein

level. The results obtained in acute experiment are consistent

with those published by Vicentini et al. (30). The authors showed

that quercetin decreased one single UV irradiation-induced NF-

κB DNA-binding by 80%. In the 10 days experiment the data

obtained suggested a possible role of ERK 1/2 in NF-κB

activation in Cv treated groups. It was shown that NF-κB DNA

binding activity was one of the essential events for the protection

of cells under various kinds of stress (39). Probably by an up-

regulation of phospho-NF-κB p65, Cv extract had protective

effect against UVB-induced damage. More detailed studies,

however, are needed to support this idea. Moreover, some

authors demonstrated that natural extracts acted differently: in

lower doses or if the damage was moderate, they protected the

skin cells, but in higher doses or if the UVB damage was severe,

they induced apoptotic cells death. This could be assumed as

protective mechanism of natural compounds in prolonged

exposure to ultraviolet radiation.

UVB radiation has been reported to cause induction of

different MMPs, enzymes able to cleave components of cell-cell

and cell-matrix junctions within the epithelium to promote re-

epithelialization. MMPs activities are modulated on several

levels including transcription, pro-enzyme activation or by

inhibitors, endogenous (tissue inhibitors of metalloproteinases -

TIMPs) (14) or exogenous (doxycycline) (41). Gelatinases

belong to MMPs group and include two members, MMP-2

(gelatinase A, 72kDa) and MMP-9 (gelatinase B, 92kDa).

Substrates of both enzymes are: collagen denatured by MMP-1

UVB-induced (42), vitronectin, fibronectin and gelatin (43).

MAPkinases also regulate the expression of MMP-9. Moreover,

some MMP genes contain NF-κB binding sites and are shown to

be involved in their active synthesis (44).

In our study, one single UVB exposure increased

significantly the secretion of MMP-2 and MMP-9 in skin cells.

Also, UVB increased MMP-9 activity, both in acute and in the

10 days experiment. It was known that the recruitment of

inflammatory cells, including neutrophils, macrophages and

mast cells in skin exposed to UVB, could lead to release of

MMP-9 protein. It was also demonstrated, that UVB irradiation

stimulated activation of MMP-9 in keratinocytes in a dose-

dependent manner via hydroxyl radical and lipid peroxides

generation (45). In our study, pretreatment with Cv extract

inhibited induction and activation of MMP-9. This effect was

430

Fig. 6. Effects of Cv extract on UVB-induced secretion of TIMP-1 in SKH-1 hairless skin in a single dose of UVB (A) and 10 days

experiment (B). The animals were treated with Cv extract in a dose of 2.5 and respectively 4 mg TPC/cm2, in vehicle, 30 min before

each UVB exposure (240 mJ/cm2). Both doses of Cv extract resulted in significant inhibition of TIMP-1 secretion compared to UVB

in the 10 days experiment. Data are means± standard deviation. Statistical analysis was done by a one-way ANOVA, with Tukey´s

multiple comparisons posttest (*p<0.01; **p<0.001 compared to UVB group).

noticed at low concentration of polyphenols and was explained

by antiinflammatory and antioxidant effects of Cv extract, as

demonstrated in our previous in vivo studies (8). These results

are in agreement with those of Fisher et al. which reported that

the inflammatory cytokines induced MMP-9 expression through

NF-κB in keratinocytes exposed to UVB (46).

Analysis of the gelatinase MMP-2 in skin after one single

UVB irradiation showed increased levels of MMP-2 secretion,

this effect being inhibited by Cv extracts. The MMP-2 activity in

skin was not significantly changed in these groups, neither in

experiment I, nor in experiment II. A possible explanation is the

absence of critical elements in the promoter region of this gene,

critical elements present in other MMP promoters in response to

UVB exposure (47).

TIMP-1 is important for extracellular matrix metabolism

because it can inhibit the activity of all MMPs, except membrane-

type MMP. Generally, UV irradiation induces a significant

increase in TIMP-1 level (47). However, UV induction of MMPs

exceeds that of TIMP-1, resulting in an imbalance in MMP and

TIMP levels (48). Therefore, we evaluated the TIMP-1 secretion

in skin in correlation with the secretion and activity of MMPs. In

our experimental model, UVB irradiation did not influence the

level of TIMP-1 in skin. Pretreatment with Cv extracts reduced

the secretion of protein, especially after multiple doses of UVB

irradiation. These results suggested that the inhibitory effect of

Cv extract on MMPs induction was due mainly to inactivation of

MMPs activators (ROS and cytokines) and not to the stimulation

of inhibitors secretion.

In summary, Cv extract attenuated UVB-induced

photodamage by modulating the activation of MMPs,

MAPkinases and NF-κB. The results demonstrated the dual

efficacy of Cv extract in skin protection against different doses

of UVB irradiation and suggested the role of Cv as a UVB

damage sensor. In lower doses or if the damage was moderate,

Cv protected the skin cells, but when the UVB damage was

severe or in multiple UVB exposures, activated ERK 1/2 kinase

and NF-κB, and possibly induced apoptotic cells death. The

effect might be important in human skin protection that is

exposed to sunlight in their daily life. Further studies must be

carried out on other biomarkers and signaling pathways in order

to elucidate the molecular mechanisms involved in Cv effect in

chronic UV irradiation.

Acknowledgements: This work was supported by the

Ministry of Education, Research and Youth by PN II Program

(42-104/2008). We would like to express our gratitude to Dr.

Mircea Tamas for providing the Cv extract, and to Dr. Doina

Daicoviciu and Nicoleta Decea for their technical contribution.

We also thank Mr. Remus Moldovan for animal handling.

Conflict of interests: None declared.

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R e c e i v e d : April 18, 2012

A c c e p t e d : August 20, 2012

Author’s address: Dr. Adriana Filip, Department of

Physiology, 1-3 Clinicilor Street, “Iuliu Hatieganu” University

of Medicine and Pharmacy, Cluj-Napoca, Romania.

E-mail: adrianafilip33@yahoo.com

432