inhibition of uvb-induced skin damage by exopolymers from aureobasidium pullulans ...

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Article Type: Original Article

Inhibition of UVB-induced Skin Damage by Exopolymers from Aureobasidium

pullulans SM-2001 in Hairless Mice

Kyung Hu Kim1a, Soo Jin Park1,2a, Young Joon Lee2,3, Ji Eun Lee1,2, Chang Hyun Song1,2,

Seong Hun Choi1,2, Sae Kwang Ku1,2* and Su Jin Kang2,3*

1Department of Histology and Anatomy, College of Korean Medicine, Daegu Haany

University, Gyeongsan, Repulic of Korea.

2The Medical Research Center for Globalization of Herbal Medicine, Daegu Haany

University, Gyeongsan, Repulic of Korea.

3Department of Preventive Medicine, College of Korean Medicine, Deagu Haany University,

Gyeongsan, Repulic of Korea.

aThese authors contributed equally to this work.

(Received 19 May 2014; Accepted 11 June 2014)

*Co-corresponding author


Sae Kwang Ku

Department of Histology and Anatomy, College of Korean Medicine, Daegu Haany

University, 1, Hannydaero, Gyeongsan, Gyeongsangbuk-Do, 712-715, Republic of Korea.

Tel: +82-53-819-1549

Fax: +82-53-819-1576

E-mail: [email protected]

Su Jin Kang

Department of Preventive Medicine, College of Korean Medicine, Daegu Haany University,

1, Hannydaero, Gyeongsan, Gyeongsangbuk-Do, 712-715, Republic of Korea.

Tel: +82-53-819-1296

Fax: +82-53-819-1576

E-mail: [email protected]

Abstract: Because antioxidants from natural sources may be an effective approach to the

treatment and prevention of UV radiation-induced skin damage, the effects of purified

exopolymers from Aureobasidium pullulans SM-2001 (“E-AP-SM2001”) were evaluated in

UVB-induced hairless mice. E-AP-SM2001 consists of 1.7% β-1,3/1,6-glucan, fibrous

polysaccharides and other organic materials, such as amino acids, and mono- and

di-unsaturated fatty acids (linoleic and linolenic acids), and shows anti-osteoporotic and

immunomodulatory effects, through anti-oxidant and anti-inflammatory mechanisms.

Hairless mice were treated topically with vehicle, E-AP-SM2001 stock and two- and four-

fold diluted solutions once per day for 15 weeks against UVB irradiation (three times per

week at 0.18 J/cm2). The following parameters were evaluated in skin samples:


myeloperoxidase (MPO) activity, cytokine levels (interleukin (IL)-1β and IL-10),

endogenous antioxidant content (glutathione, GSH), malondialdehyde (MDA) levels,

superoxide anion production; matrix metalloproteases (MMP-1, -9 and -13), GSH reductase

and Nox2 (gp91phox) mRNA levels, and immunoreactivity for nitrotyrosine (NT), 4-

hydroxynonenal (HNE), caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP).

Photoageing was induced by UVB irradiation through ROS-mediated inflammation, which

was related to the depletion of endogenous antioxidants, activation of MMPs and

keratinocyte apoptosis. Topical treatment with all three doses of E-AP-SM2001 and 5 nM

myricetin attenuated the UV-induced depletion of GSH, activation of MMPs, production of

IL-1β, the decrease in IL-10 and keratinocyte apoptosis. In the present study, E-AP-SM2001

showed potent inhibitory effects against UVB-induced skin photoageing. Thus, E-AP-

SM2001 may be useful as a functional ingredient in cosmetics, especially as a protective

agent against UVB-induced skin photoageing.

The skin is a vital organ, preserving water within the body, preventing infection and

protecting against ultraviolet (UV) radiation. Skin acts as a physical barrier but also regulates

the immune system and produces hormones and neurotransmitters [1, 2].

UVB radiation from sunlight is a major extrinsic factor in skin ageing [3]. When skin

is damaged due to repeated exposure to UV radiation, photoagwing occurs in skin tissue,

resulting in wrinkled, lax and coarse skin with uneven pigmentation and brown spots [4, 5].

UV radiation-induced skin damage is characterized by histological changes, including

damage to collagen fibers, excessive deposition of abnormal elastic fibers and increased

levels of glycosaminoglycans [4, 6]. These alterations were found in the dermal connective

tissues of photoaged skin in histological and ultrastructural studies [4, 7]. Chronic exposure


of the skin to UV radiation, particularly to UVB (290-320 nm), has been shown to induce

clinical and histological damage as a result of simultaneous skin destruction and repair [8].

UVB light is absorbed mainly in the epidermis, which consists primarily of keratinocytes [9].

Naturally aged skin is pale and smoothly wrinkled, while extrinsic ageing due to exposure to

sunlight causes photoaged skin to be coarsely wrinkled [10].

After UV radiation, reactive oxygen species (ROS) formation increases in skin and

ROS damage cellular lipids, proteins and DNA, leading to changes—and often to

destruction—of skin structures. As a consequence, regular skin function can be inhibited

[11]. Furthermore, UV exposure can trigger an imbalance between UV-related ROS and

endogenous antioxidant systems, such as glutathione (GSH) [12]. Neutrophils are also

stimulated, increasing the activity of myeloperoxidase (MPO), a ROS-generating enzyme

[13].

UV irradiation also induces the activity of matrix metalloproteases (MMPs),

regarded as the primary mediators of connective tissue damage in skin exposed to UV

irradiation and in premature ageing [14]. After UV exposure, a network of cytokines,

including interleukin (IL)-1β, responsible for the onset of cutaneous inflammation, is

activated and released. These molecules trigger vasodilatation, widening of interendothelial

junctions and separation of endothelial cells, increasing microvascular protein and fluid

leakage into the interstitium, resulting in oedema [15, 16].

A relationship between oxidative stress and inflammatory cytokines has been

reported. IL-1β can activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,

leading to an increase in superoxide anions. In turn, superoxide anions activate nuclear

factor-kappa B (NF-κB), stimulating cytokine release [17]. In contrast, phenolic antioxidants

inhibit the induction of inflammatory cytokines by inflammatory stimuli. The inhibition of


NF-κB by phenolic antioxidants correlates with their redox capabilities and suggests a redox-

sensitive protein factor as the target of this inhibition [18]. In this respect, antioxidants may

provide an effective approach to the treatment and prevention of oxidative-stress-mediated

alterations and diseases due to UV radiation [19].

The exopolymers from Aureobasidium pullulans SM2001 (E-AP-SM2001) may be

able to prevent these sun-exposure-related events. Purified E-AP-SM2001 consists of 1.7% β-

1,3/1,6-glucan, fibrous polysaccharides and other organic materials, such as amino acids and

mono- and di-unsaturated fatty acids (linoleic and linolenic acids) [20-23]. Recent studies

have shown that E-AP-SM2001 shows anti-osteoporotic [24], anti-inflammatory [25] and

immunomodulatory effects [26]. Additionally, E-AP-SM2001 has been suggested to exert

therapeutic effects in cisplatin-induced kidney damage [27] and in ligation-induced

experimental periodontitis through anti-oxidant and anti-inflammatory mechanisms [22].

As mentioned above, because E-AP-SM2001 has antioxidant effects [28-30], it may

protect against photoageing. Thus, we investigated inhibition by E-AP-SM2001 of the

photoageing effects induced by UVB irradiation. For this purpose, a mouse photoageing

model was used because intentionally damaging human skin by long-term exposure to UVB

light is ethically questionable [7, 10]. Myricetin was selected as a reference material because

it protects against UVB-induced skin photoageing [7, 31], an effect mediated via anti-oxidant

and anti-inflammatory pathways.


Methods and Materials

Animals and husbandry

In total, 100 6-week-old female HR-1 hairless mice (SLC, Shizuoka, Japan; body weight

range 16-24 g on receipt) were prepared. Six groups (intact vehicle control group,

UVB-exposed group, UVB-exposed + 5 nM myricetin (0.32 ng/cm2)-treated hairless mice,

UVB-treated and E-AP-SM2001 stock solution (equivalent to 3.40 mg/cm2 of

β-1,3/1,6-glucan)-treated hairless mice, UVB-treated and twofold-diluted E-AP-SM2001

solution (equivalent to 1.70 mg/cm2 of β-1,3/1,6-glucan)-treated hairless mice, UVB-treated

and fourfold-diluted E-AP-SM2001 solution (equivalent to 0.85 mg/cm2 of β-1,3/1,6-glucan)-

treated hairless mice) of eight mice each were selected, based on body weights at 8 days after

acclimatization (20.81±0.98 g, range, 19.0-23.0 g/head).

Animals were allocated four per polycarbonate cage in a temperature (20-25°C)- and

humidity (50-55%)-controlled room. The light/dark cycle was 12/12 hr and standard rodent

chow (Samyang, Seoul, Korea) and water were available ad libitum.

All laboratory animals were treated according to the national regulations for the

usage and welfare of laboratory animals. The protocol was approved by the Institutional

Animal Care and Use Committee of Daegu Haany University (Gyeongsan, Gyeongbuk,

Korea).

Preparation and application of test materials

A light brown viscous solution of E-AP-SM2001, consisting of filtered and purified

exopolymers from Aureobasidium pullulans SM2001, was supplied by Aribio (Seoul, Korea)

as the test material. Based on a previous report [20], the E-AP-SM2001 exopolymers


consist of β-1,3/1,6-glucan (17%), β-1,4-glucan (18%), α-(1,4)-(1,6)-glucan (8%), glucose

(37.7%), galactose (0.8%), mannose (1.5%), protein (3.1%) and ash (7.2%). Off-white

myricetin powder was obtained and used as a positive control. All test materials tested were

stored at 4°C in a refrigerator to protect from light and humidity until use. Aliquots (200 μL

each) of E-AP-SM2001 stock, two- and fourfold diluted solutions (diluted with distilled

water), were applied to a 1 × 1-cm constant patch on the left dorsal back skin near the gluteal

area, once per day for 15 weeks from the day of initial UVB exposure, as equivalent to 3.40,

1.70 and 0.85 mg/cm2 of β-1,3/1,6-glucan, respectively. Myricetin was dissolved in acetone

at 5 nM, and applied topically, 200 μL on a 1 × 1-cm dorsal back skin patch (0.32 ng/cm2).

On each UVB treatment day, each test material was applied at 1 hr after irradiation. In the

intact and UVB control mice, distilled water (200 μL) was applied instead of the test

material.

UVB irradiation

Skin photoageing was induced by UVB irradiation, three times per week at 0.18 J/cm2,

according to a previously established method [7] using a UV Crosslinker system (Hoefer

Scientific Instruments, San Francisco, CA, USA) with a peak emission at 312 nm.

Unexposed intact vehicle control mice were also ‘exposed’ to the non-emitting Crosslinker

system for the same duration as the UVB-exposed hairless mice.

Changes in body weight

Changes in body weight were measured once per week from 1 day before UVB radiation

throughout the 15 weeks of the experimental period using an automatic electronic balance


(Precisa Instruments, Dietikon, Switzerland). To reduce individual differences, the body

weight gains after 15 weeks of topical treatment were calculated as follows. From the

initiation of test material application to the end of 15 weeks (105 days) of test material

application, gain = body weight at sacrifice – body weight at start of topical application (1 hr

after first UVB irradiation).

Generation of replicas and image analysis

Replicas of mouse dorsal skin were obtained using the Repliflo Cartridge Kit (CuDerm

Corp., Dallas, TX, USA). A photograph of the dorsal skin around the test patch region was

taken before the animals were sacrificed. Impression replicas were set on a horizontal sample

stand, and wrinkle shadows were produced by illumination with a fixed-intensity light at a

40° angle using an optical light source. Black and white images were recorded with a CCD

camera and analysed using the Skin-Visiometer VL650 software (Courage & Khazaka,

Cologne, Germany). The parameters used in the assessment of skin wrinkles were the

average length and average depth of wrinkles, according to methods established previously

[9, 10], with some modifications (fig. 4).

Oedema evaluation

The effects of the treatments on UVB-induced skin oedema were measured as increases in

dorsal skin weight. After dorsal skin removal after 15 weeks of continuous topical treatment,

once per day, a constant area (6-mm diameter) was delimited with the aid of a punch; this

area was the weighed according to a method described previously [19]. The result was


calculated by comparing the weight of the skin between groups and expressed as g/6-mm

diameter of dorsal skin.

Measurement of MPO activity

UV-B-induced leukocyte migration to the skin was evaluated using the MPO kinetic-

colorimetric assay, as described previously [12, 19]. Skin samples were collected in 400 μL

of 50 mM K2HPO4 buffer (pH 6.0; Sigma-Aldrich, St. Louis, MO, USA) containing 0.5%

hexadecyltrimethylammonium bromide (Gibco, Carlsbad, CA, USA) and homogenized in an

ice bath (15 sec.). Then, the homogenates were centrifuged (1,000×g, 2 min., 4°C). The

supernatant was removed for assay. Briefly, 30 μL of sample were mixed with 200 μL of 0.05

M K2HPO4 buffer (pH 6.0), containing 0.167 mg/mL o-dianisidine dihydrochloride (Sigma-

Aldrich) and 0.05% hydrogen peroxide. The absorbance at 450 nm was determined

spectrophotometrically (Optizen Pop, Mecasys, Daejeon, Korea) after 5 min. The MPO

activity of samples was compared to a standard curve of neutrophils. Protein levels in the skin

homogenates were measured using the Lowry method [32]. The results are presented as MPO

activity (number of total neutrophils/mg protein).

Detection of IL-1β and IL-10 in skin tissue

Dorsal back skin tissue from the area around the test patches was collected at 105 days of

UVB irradiation. The tissue collected was homogenized and processed as described by

Safieh-Garabedian et al. [33] and Botelho et al. [34]. IL-10 and IL-1β concentrations were

determined by enzyme-linked immunosorbent assay as described previously [35]. Microtiter

plates were coated overnight at 4°C with antibody against rat IL-10 or IL-1β (10 μg/mL).


After blocking, the samples and standard at various dilutions were added in duplicate and

incubated at 4°C for 24 hr. The plates were then washed three times with buffer. After

washing, 100 μL of biotinylated sheep polyclonal anti-mouse IL-10 or anti-rat IL-1β (diluted

1/1000 with assay buffer 1% BSA; Abcam, Cambridge, UK) were added to the wells. After

further incubation at room temperature for 1 hr, the plates were washed and 100 μL of avidin-

HRP (Abcam), diluted 1:5000, were added. The colorimetric reagent o-phenylenediamine

(100 μL; Sigma-Aldrich) was added 15 min. later, and the plates were then incubated in the

dark at 37°C for 20 min. The enzyme reaction was stopped by addition of H2SO4 and

absorbance at 490 nm was measured using a microplate reader (Tecan; Männedorf,

Switzerland).

GSH assay

Cutaneous GSH levels were determined using a fluorescence assay, as described previously

[14, 19]. Firstly, the skin (1:3 w/w dilution) was homogenized in 100 mM NaH2PO4 (pH 8.0;

Sigma-Aldrich) containing 5 mM EDTA (buffer 1). Then, homogenates were treated with

30% trichloroacetic acid (Sigma-Aldrich) and centrifuged twice (1,940×g, 6 min. and 485×g,

10 min.); the fluorescence of the resulting supernatant was measured using a fluorescence

spectrophotometer (RF-5301PC; Shimadzu Corp., Tokyo, Japan). Briefly, 100 μL of the

supernatant were mixed with 1 mL of buffer 1 and 100 μL of o-phthalaldehyde (1 mg/mL in

methanol; Sigma-Aldrich). The fluorescence was determined after 15 min. (λexc = 350 nm;

λem = 420 nm). A standard curve comprising 0.0-75.0 μmol GSH was prepared. Protein

levels in the skin homogenates were measured using the Lowry method [32]. Results are

presented as μmol GSH/mg protein.


Lipid peroxidation

Firstly, the protein content of the homogenate (10 mg/mL in 1.15% KCl) was measured using

the Lowry method [32]. Thiobarbituric acid reactive substance (TBARS) measurements were

used to evaluate lipid peroxidation, as described previously [36]. For this assay,

trichloroacetic acid (10%; Sigma-Aldrich) was added to the homogenate to precipitate

proteins. This mixture was then centrifuged (1,000×g, 3 min.). The protein-free sample was

extracted and thiobarbituric acid (0.67%) was added. The mixture was kept in a water bath at

100°C for 15 min. MDA, an intermediate product of lipoperoxidation, was determined from

the difference between the absorbances at 535 and 572 nm using a microplate

spectrophotometer (Tecan). The results are reported as nmol/mg protein.

Superoxide anion production

Superoxide anion production in tissue homogenates (10 mg/mL in 1.15% KCl) was

quantitated using the nitroblue tetrazolium (NBT) assay. Briefly, 50 μL of homogenate were

incubated with 100 μL of NBT (1 mg/mL; Sigma-Aldrich) in 96-well plates at 37°C for 1 hr.

The supernatant was then removed carefully and the reduced formazan was solubilized by

adding 120 μL of 2 M KOH and 140 μL of DMSO. NBT reduction was measured at 600 nm

using a microplate reader (Tecan). Data were normalized to the protein content.

Quantitative RT-PCR

Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA),

according to a method described previously [19]. The RNA concentrations and quality were

determined using a CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA). To


remove contaminating DNA, samples were treated with recombinant DNase I (Ambion,

Austin, TX, USA). RNA was reverse-transcribed using the High-Capacity cDNA Reverse

Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the

manufacturer’s protocol. The β-actin mRNA level was used as a control for tissue integrity in

all samples. Primer sequences for mice genes were: matrix metalloprotease-1 (MMP-1), 5’-

AAG GTT AGC TTA CTG TCA CAC GCT T-3’ and 5’-CGA CTC TAG AAA CAC AAG

AGC AAG A-3’; matrix metalloprotease-9 (MMP-9), 5’-CCC GGA CCA AGG ATA CAG-

3’ and 5’-GGC TTT CTC TCG GTA CTG-3’; matrix metalloprotease-13 (MMP-13), 5’-CAT

CCA TCC CGT GAC CTT AT-3’ and 5’-CAT CCA TCC CGT GAC CTT AT-3’;

glutathione (GSH) reductase, 5’-TGC GTG AAT GTT GGA TGT GTA CCC-3’ and 5’-TGC

GTG AAT GTT GGA TGT GTA CCC-3’; gp91phox subunit of the phagocyte NADPH

oxidase (Nox2), 5’-AGC TAT GAG GTG GTG ATG TTA GTG G-3’ and 5’-AGC TAT

GAG GTG GTG ATG TTA GTG G-3’; β-actin, 5’-AGC TGC GTT TTA CAC CCT TT-3’

and 5’-AAG CCA TGC CAA TGT TGT CT-3’.

Histopathology

Samples from dorsal back skins around the test patch were separated and fixed in 10%

neutral buffered formalin, then embedded in paraffin wax, sectioned (3-4 μm) and stained

with hematoxylin and eosin (H&E) for general histopathology or Masson’s trichrome (MT)

for collagen fibers, according to our established methods [37]. The histopathological profiles

of each sample were observed under a light microscope (Nikon, Tokyo, Japan).

To quantify the changes in more detail, the number of microfolds formed on the

surface of epithelium (folds/mm of epithelium), mean epithelial thicknesses (μm/epithelium),

and mean numbers of inflammatory cells infiltrated into the dermis (cells/mm2 dermis) were


determined in a general histomorphometrical analysis using image analysis software

(iSolution FL, ver. 9.1, IMT i-solution Inc., Quebec, Canada) under H&E staining and in

collagen fiber-occupied regions of the dermis (%/mm2 of dermis) under MT staining. The

histopathologist was blinded to the group distributions when conducting this analysis.

Immunohistochemistry

After dewaxing of the prepared skin histological paraffin wax sections, citrate buffer antigen

(epitope) retrieval pre-treatment was conducted. Briefly, a water bath was pre-heated with a

staining dish containing 10 mM citrate buffer (pH 6.0) to 95-100°C. The slides were

immersed in the staining dish and the lid was placed loosely. After a 20-min. incubation, the

staining dish was placed at room temperature for 20 min. to cool. Sections were

immunostained using avidin-biotin complex (ABC) methods for caspase-3, PARP, NT, 4-

HNE, iNOS and MMP-9 (table 3) according to a previous report [38]. Briefly, endogenous

peroxidase activity was blocked by incubation in methanol and 0.3% H2O2 for 30 min., and

non-specific binding was blocked with normal horse serum blocking solution (Vector Labs.,

Burlingame, CA, USA, diluted 1:100) for 1 hr in a humidity chamber. Primary antibodies

used were: anti-cleaved caspase-3 (Asp175) polyclonal antibody (Cell Signaling Technology

Inc., Danvers, MA, USA), anti-cleaved PARP (Asp214) specific antibody (Cell Signaling

Technology), anti-4-hydroxynonenal polyclonal antibody (Abcam, Cambridge, UK), anti-

nitrotyrosine polyclonal antibody (Millipore Corporation, Billerica, CA, USA), and mouse

anti-MMP9 antibody (Abcam). They were incubated overnight at 4°C in a humidity chamber

and then incubated with biotinylated universal secondary antibody (Vector Lab., Burlingame,

CA, USA, Dilution 1:50) and ABC reagents (Vectastain Elite ABC Kit, Vector Labs; diluted

1:50) for 1 hr at room temperature in a humidity chamber. Finally, sections were reacted with


a peroxidase substrate kit (Vector Labs.) for 3 min. at room temperature. All sections were

rinsed in 0.01 M PBS three times between steps.

Cells or fibres comprising > 30% of the immunoreactivity for each antiserum

compared with intact dermal keratinocytes or dermal tissue were regarded as positive, and the

mean numbers of caspase-3, PARP, NT and 4-HNE-immunoreactive epithelial cells (%,

cells/100 epithelial cells) were counted using an established automated image analysis

process [39, 40], with some modifications. Additionally, the percentages occupied by MMP-9

immunoreactive fibres in the dermis were calculated (%/mm2 dermis). The histopathologist

was blinded to the group distribution when performing the analyses.

Statistical analyses

Multiple-comparison tests of the various dose groups were conducted. Variance homogeneity

was examined using the Levene test. If no significant deviation from variance homogeneity

was detected, the data were analysed by a one-way ANOVA followed by a least-significant-

differences multi-comparison (LSD) test to determine whether pairs of groups were

significantly different. In cases in which significant deviations from variance homogeneity

were detected by the Levene test, the non-parametric Kruskal-Wallis H comparison test was

conducted. When a significant difference was identified by the Kruskal-Wallis H test, the

Mann-Whitney U-test was used to determine whether specific pairs of groups were

significantly different. Statistical analyses were conducted using the SPSS software (ver.

14.0K for Windows; IBM SPSS Inc., Armonk, NY, USA).


Results

Body weight

Compared with the intact vehicle control, body weights were unchanged in all UVB-exposed

hairless groups. Thus, UVB did not influence body weight gain during 15 weeks of exposure

with or without test material application. Also, neither the three doses of E-AP-SM2001 nor 5

nM myricetin influenced body weight gains, compared with the intact and UVB control

groups, throughout the experiment (fig. 1). The gains in body weight were changed by -

10.74% in the UVB control group compared with those of the intact vehicle control group

over 15 weeks. Gains in body weight were changed by 4.94, -7.05, -6.02 and 7.26% in the 5

nM myricetin, E-AP-SM2001 stock and two- and fourfold dilution groups, respectively,

compared with the UVB control group.

Wrinkle measurements and analyses of skin replicas

The mean length and average depth of skin wrinkles were increased significantly in the skin

replicas of the UVB control group compared with the intact control group. Conversely,

compared with the intact vehicle control, the mean lengths and average depths of skin

wrinkles of skin replicas in the UVB control group were 80.43 and 93.18%, and were

changed by -33.73, -36.56, -29.72 and -19.10% in the 5 nM myricetin, E-AP-SM2001 stock,

and two- and fourfold dilution groups, respectively. The average depths of skin wrinkles were

changed by 33.59, -42.43, -24.40 and -21.77%, respectively, compared with the UVB control

group (figs. 2, 3).


UVB-induced skin oedema

Compared with the unexposed intact vehicle control group, 6-mm diameter skin weights and

the oedema scores were increased significantly in the UVB-exposed control group.

Conversely, significant decreases in skin weights were detected in mice to which 5 nM

myricetin and all three doses of E-AP-SM2001 were applied. Notably, E-AP-SM2001

showed marked dose-dependent decreases in skin oedema scores (fig. 4). The 6-mm-diameter

skin weights were changed by 183.96% in the UVB control compared with the intact vehicle

control group. The 6-mm-diameter skin weights were changed by -42.69, -47.84, -31.23 and -

21.59% in the 5 nM myricetin, E-AP-SM2001 stock, and two- and fourfold dilution groups,

respectively, compared with the UVB control group.

Skin MPO activity

Significant increases in skin MPO activities were detected in the UVB control group,

compared with intact control hairless group. Compared with the intact control group, the skin

MPO levels in the UVB control group were changed by 585.92%. In contrast, the skin MPO

levels were changed by -66.16, -73.32, -44.57 and -29.34% in the 5 nM myricetin, E-AP-

SM2001 stock, and two- and fourfold dilution groups, respectively, compared with the UVB

control group (fig. 5).

Skin IL-1β levels

Compared with the intact control hairless group, skin IL-1β levels were increased in the

UVB-exposed control group. Conversely, significant and dose-dependent decreases in skin

IL-1β levels were detected in the groups treated with the three doses of E-AP-SM2001 and


5 nM myricetin, compared with the UVB control group (fig. 6). Compared with the intact

control group, the skin IL-1β levels were changed by 139.70% in the UVB control group.

Skin IL-1β levels were changed by -44.34, -50.14, -31.79 and -19.31% in the 5 nM myricetin,

E-AP-SM2001 stock, and two- and fourfold dilution groups, respectively, compared with the

UVB control group.

Skin IL-10 levels

Compared with the unexposed intact control group, skin IL-10 levels were decreased

significantly in the UVB control group. However, significant and dose-dependent increases in

skin IL-10 levels were detected in the groups top which were applied the three doses of E-

AP-SM2001 and 5 nM myricetin, compared with the UVB control group (fig. 7). Compared

with the intact control group, the skin IL-10 levels were changed by -20.02% in the UVB

control group. They were changed by 62.66, 79.26, 39.88 and 24.60% in the 5 nM myricetin,


UVB control group.

Skin GSH content

Skin GSH contents were decreased significantly in the UVB-exposed control group

compared with those of the intact control hairless group, while significant increases in skin

GSH contents were detected in all E-AP-SM2001 and 5 nM myricetin groups compared with

the UVB control group (table 1). The skin GSH contents in UVB control groups were

changed by -67.92% compared with the intact control group. Skin GSH contents were


changed by 122.64, 171.92, 99.71 and 39.54% in the 5 nM myricetin, E-AP-SM2001 stock,

and two- and fourfold dilution groups, respectively, compared with the UVB control group.

Skin lipid peroxidation: MDA levels

Compared with the unexposed intact vehicle control group, skin MDA levels were increased

markedly in the UVB-exposed control group. In contrast, the E-AP-SM2001 stock, two- and

fourfold diluted solution and 5 nM myricetin groups showed decreases in skin MDA levels,

compared with the UVB control group (table 1). The skin MDA levels in the UVB control

group were changed by 307.94%, compared with the intact control group. Skin MDA levels

were changed by -58.83, -63.97, -43.97 and -30.27% in the 5 nM myricetin, E-AP-SM2001

stock, and two- and fourfold dilution groups, respectively, compared with the UVB control

group.

Skin superoxide anion production

Superoxide anion production in skin tissues of UVB-exposed mice were increased

significantly compared with those in intact control hairless mice. Compared with the intact

control group, skin superoxide anion production changed by 159.80% in the UVB control

group. In contrast, in the 5 nM myricetin and E-AP-SM2001 stock, and two- and fourfold

dilution groups, superoxide anion production was decreased by -47.42, -59.37, -36.48 and -

27.92%, respectively, compared with the UVB control group (table 1).


Skin MMP mRNA levels

Compared with the unexposed intact vehicle control group, the skin MMP-1, -9 and -13

mRNA levels (relative ratio to the control) were increased in the UVB-exposed control

group. Conversely, dose-dependent and significant decreases in skin MMP mRNA levels

were noted in the groups treated with the three doses of E-AP-SM2001 and 5 nM myricetin,

compared with the UVB control group (table 2).

Skin GSH reductase mRNA levels

Compared with the intact control group, the skin GSH reductase transcript levels were

decreased significantly in the UVB-exposed groups. The GSH reductase mRNA levels in the

skin of the UVB control group were changed by -18.50% compared with the intact control

group. In contrast, the skin GSH reductase transcript levels were changed by 60.83, 78.93,

47.63 and 22.55% in the myricetin 5 nM, E-AP-SM2001 stock, and two- and fourfold diluted

groups, respectively, compared with the UVB control group (table 2).

Skin Nox2 mRNA levels

Compared with the unexposed intact vehicle control group, the skin Nox2 transcript levels

were increased significantly in the UVB-exposed control group. Conversely, decreases in the

levels of Nox2 were detected with all three doses of E-AP-SM2001 and 5 nM myricetin,

compared with the UVB control group (table 2). Compared with the intact control group, the

skin Nox2 transcript levels were changed by 61.86% in the UVB control group. Skin nox2

transcript levels were changed by 26.43, -33.95, -21.78 and -18.84% in the 5 nM myricetin,



UVB control group.

General histopathological changes in dorsal back skin tissues

UVB-exposed control mice showed a marked increase in mean epithelial thickness due to

hyperplasia/hypertrophy of epidermal keratinocytes. Furthermore, noticeable increases in the

numbers of inflammatory cells infiltrating the dermis, abnormal collagen deposition and

formation of microfolds on the surface of epithelial linings were observed. Compared with

the intact control, the mean numbers of epithelial surface microfolds were changed by

548.31% in the UVB control group. They were changed by -50.09, -64.30, -49.36

and -40.21% in the 5 nM myricetin, E-AP-SM2001 stock, and two- and fourfold dilution

groups, respectively, compared with the UVB control group. Additionally, the mean

epithelial thicknesses were changed by 134.83% in the UVB control group compared with the

intact control group, and by -40.57, -46.64, -39.44 and -17.46% in the 5 nM myricetin, E-AP-

SM2001 stock, and two- and fourfold dilution groups respectively, compared with the UVB

control group.

Furthermore, the UVB control group showed an increase of 2633.33% in the mean numbers

of infiltrated dermal inflammatory cells, compared to -25.84, -66.60, -52.16 and -40.10% in

the 5 nM myricetin, E-AP-SM2001 stock, and two- and fourfold dilution groups,

respectively, compared with the UVB control group. The percentages of collagen-

fibre-occupied dermal regions were changed by 76.05% in the UVB control group compared

with the intact control, and by -28.60, -36.88, -28.04 and -14.79% in the 5 nM myricetin, E-

AP-SM2001 stock and two- and fourfold dilution groups, respectively, compared with the

UVB control group (table 3, fig. 8).


Immunohistochemical changes in dorsal back skin tissues

To assess changes in numbers of epithelial NT-, 4-HNE-, caspase-3- and PARP-

immunoreactive cells, and dermal MMP-9 immunoreactivity, we performed a

histomorphometrical analysis. Compared with the intact control group, the number of

epidermal NT-immunoreactive cells was changed by 646.15% in the UVB control group. The

number of epidermal NT-immunoreactive cells was changed by -43.15, -77.32, -62.59

and -41.83% in the 5 nM myricetin, E-AP-SM2001 stock, and two- and fourfold dilution

groups, respectively, compared with the UVB control group.

The numbers of epidermal 4-HNE-positive cells were changed by 503.06% in the UVB

control group compared with the intact control groups. They were changed by -46.53, -76.31,

-59.73 and -40.27% in 5 nM myricetin, E-AP-SM2001 stock, and two- and fourfold dilution

groups, respectively, compared with the UVB control group.

The number of epidermal caspase-3-positive cells in the UVB control group was changed by

390.98% compared with the intact control group. The number of epidermal caspase-3-

positive cells was changed by -64.47, -74.58, -63.09 and -44.41% in the 5 nM myricetin, E-

AP-SM2001 stock, and two- and fourfold dilution groups, respectively, compared with the

UVB control group.

Compared with the intact control group, the number of epidermal PARP-immunolabelled

cells was changed by 437.72% in the UVB control group, and by -52.53, -72.59, -66.07 and -

45.68% in the 5 nM myricetin, E-AP-SM2001 stock, and two- and fourfold dilution groups,

respectively, compared with the UVB control group.

Dermal MMP-9 immunoreactivity in the UVB control group was changed by 163.07%

compared with the intact control group. Dermal MMP-9 immunoreactivity was changed by -


37.54, -55.99, -40.69 and -35.33% in the 5 nM myricetin, E-AP-SM2001 stock, and two- and

fourfold dilution groups, respectively, compared with the UVB control group (table 4, fig. 9).

Discussion

Exposure to UVB radiation is a primary cause of skin photoageing, characterized by skin

wrinkles, roughness, laxity, irregular pigmentation, telangiectasia, atrophy and neoplasia

[41]. The major aspects of the pathogenesis of skin photoageing induced by UVB include

skin wrinkle formation, epidermal thickening [7, 9, 10], degradation of matrix

macromolecules by MMPs [7], ROS-mediated inflammation [19] and keratinocyte apoptosis

[42, 43]. Because these processes in UVB-induced skin photoageing could be inhibited by

antioxidants of natural origin [3, 9, 10, 19], efforts have been made to identify antioxidants

that could protect against or prevent photoageing due to UVB exposure.

Compared with the UVB control, photoageing-related wrinkle formation and

hyperplasia/hypertrophy of epithelial keratinocytes were inhibited significantly in the

E-AP-SM2001 (stock, two- and fourfold-diluted solutions)- and myricetin (5 nM)-treated

UVB-irradiated mice. Thus, E-AP-SM2001 has photoprotective effects against wrinkle

formation due to UVB exposure.

Sunlight plus living in an oxygen-rich atmosphere increases involuntary and

detrimental damage to the skin, such as wrinkling, scaling, dryness, mottled pigment

abnormalities (hyper- or hypopigmentation) and neoplastic changes [19]. UV irradiation

activates the inflammatory reactions that are responsible for skin cancer and premature skin

ageing. Acute exposure to UV triggers migration of inflammatory cells, particularly

neutrophils, the “first defence” cells [44]. Additionally, chronic photoageing induces marked


hyperplasia of epidermal cells with hyperkeratosis, resulting in wrinkle formation [45]. Thus,

treatments that smooth wrinkle formation may provide an effective therapeutic approach to

counteracting photoageing.

Our results showed that E-AP-SM2001 inhibits skin oedema in UVB-irradiated

mice in a dose-dependent manner. Furthermore, E-AP-SM2001 alleviated xylene- and

formalin-induced ear and paw oedema [25]. These findings suggest that E-AP-SM2001 may

ameliorate UVB-induced erythema in human skin. Because of the similarity between oedema

in mice and erythema in human beings, the sensitivity to UV irradiation in mice is assessed

by measuring by skin oedema; this can be used as a predictor of a change in sensitivity in

human beings [19, 46]. UVB-induced inflammation and its mediators can cause oedema in

hairless mice and erythema in human beings [19]. A large body of evidence suggests that

inflammatory mediators, including cyclooxygenase-derived metabolites of arachidonic acid,

increase vascular permeability and blood flow [47], leading to oedema in mice.

Compared with the UVB-irradiated mice, UVB-irradiated mice with topically

applied E-AP-SM2001 showed inhibition of MMP-1, -9 and 13 activities and related

abnormal dermal collagen deposition. These observations indicated that E-AP-SM2001

inhibited UVB-induced skin sclerosis by regulating the activities of MMP-1, -9 and -13. The

MMPs are a family of structurally related endopeptidases. Because MMPs can degrade

various extracellular matrix components, they play important roles in tissue remodelling

during developmental morphogenesis and angiogenesis, tissue repair, arthritis, skin ageing

and tumour invasion [48]. MMPs can be classified into several subgroups; e.g., collagenases,

gelatinases, stromelysins, membrane-type MMPs and other MMPs, according to their

structures and substrate specificities [49].


MMP expression is usually low in unstimulated skin cells or normal skin tissues,

but the expression of some MMPs is induced by various extracellular stimuli, such as UV or

infrared radiation, growth factors, cytokines and tumour promoters [48, 49]. These lines of

evidence suggest that MMPs are directly involved in skin photoageing, and that inhibition of

the activities of MMPs, either directly by a specific inhibitor or indirectly by reducing their

expression, may provide an effective therapeutic method of counteracting photoageing.

From our results, topical application of E-AP-SM2001 inhibited the UVB-induced

increase in 4-HNE and NT significantly, concomitantly with a decrease in GSH. These

findings suggest that E-AP-SM2001 exerted strong antioxidant activity, inhibiting lipid

peroxidation, and maintaining the glutathione system and defences against oxidative stress

despite UVB exposure. Various toxic substances produced by lipid peroxidation can destroy

surrounding tissues [50]. 4-HNE, an α,β-unsaturated hydroxyalkenal, is produced by lipid

peroxidation in cells and is used as a tissue lipid peroxidation marker. NT is a product of

tyrosine nitration, mediated by reactive nitrogen species, such as peroxynitrite anions and

nitrogen dioxide. It has been detected in many pathological conditions and is regarded as a

marker of iNOS-dependent, reactive-nitrogen-species-induced nitrative stress [51, 52].

4-HNE and NT are considered possible causal agents of numerous conditions, such as

chronic inflammation, neurodegenerative diseases, adult respiratory distress syndrome,

atherogenesis, diabetes and some types of cancer [53, 54].

GSH is a representative endogenous antioxidant that prevents tissue damage by

maintaining ROS at low levels. Its sulfhydryl group is highly polarizable and allows the

removal of radicals directly by hydrogen transfer, making it an optimum nucleophile for

reactions with electrophilic compounds [19]. Additionally, it acts as a co-factor for

glutathione peroxidase and glutathione reductase, which reduce hydrogen peroxide and lipid

hydroperoxides [55]. In this context, UVB irradiation induced the depletion of GSH and can


disrupt the balance between ROS production and antioxidant systems, leading to skin

damage.

Our data showed that E-AP-SM2001 decreased UVB-increased superoxide anion

and MPO activity but also dermis inflammatory cell infiltration. Furthermore, E-AP-SM2001

inhibited expression of NADPH oxidase sub-unit gp91phox, Nox2. These effects of E-AP-

SM2001 are likely responsible for the inhibition of neutrophil recruitment and activity [47,

56]. UV irradiation induces the migration of inflammatory cells; neutrophils are then

recruited rapidly from the peripheral blood to inflammatory sites [19]. UVB radiation leads to

generation of ROS, such as singlet oxygen (1O2), H2O2, the superoxide anion (O2.-) and nitric

oxide [13, 47]. The superoxide anion plays a pivotal role in neutrophil recruitment to

inflammatory foci. Although inflammatory cells play central roles in the removal of causes of

inflammation, activated polymorphonuclear leukocytes (PMNs) are also a potential source of

oxygen metabolites [57], which are associated with recruitment of neutrophils into injured

tissues [47]. Release of one of the activating cytotoxic enzymes from PMNs [58], MPO, is

increased markedly by UVB irradiation [12, 19]. Thus, a decrease in MPO activity is

implicated in the reduction of neutrophil influx into skin tissue [34]. Nox2 is generally

referred to as the gp91phox subunit of the phagocyte NADPH oxidase and is expressed

predominantly in white blood cells of myeloid lineage [59]. Nox enzymes are a major source

of endogenous ROS generation in response to inflammatory mediators, such as cytokines,

growth factors and hypoxic conditions, all of which are elevated in response to epithelial

damage [60]. In contrast, antioxidants inhibit Nox2 activity [61].

In this study, E-AP-SM2001 dose-dependently decreased UVB-increased IL-1β

levels following UVB irradiation, suggesting that inhibition of ROS-mediated IL-1β

production might mediate the protective effect of E-AP-SM2001 against UVB. Furthermore,

UVB induced decreases in IL-10 levels, while E-AP-SM2001 increased the production of IL-


10, as reported by Campanini et al. [19]. It is well documented that NF-κB plays a pivotal

role in ROS-induced inflammation. NF-κB binding to the promoters of the genes encoding

TNF-α and ILs (particularly IL-6 and IL-1) leads to induction of inflammation by controlling

their transcription [62]. UVB irradiation exposure in human skin cells triggers the production

of cytokines, such as TNF-α, IL-1α, IL-1β and IL-6 [19]. IL-1 is necessary for the initiation

of some forms of immune response [63], and is released by various cell types such as

macrophages, dendritic cells, lymphocytes, endothelial cells, fibroblasts and keratinocytes.

IL-1β stimulates neutrophils and other cells, increasing expression of adhesion molecules,

such as intercellular adhesion molecules and L-selectins.

UVB irradiation enhances the expression of NOD1 and NOD2 receptors, which

induce the production of pro-IL-1β [64]. UV irradiation also induces ROS-dependent

activation of inflammasomes, leading to caspase-1-dependent cleavage of pro-IL-1β to active

IL-1β [65].

In contrast, the anti-inflammatory cytokine IL-10 inhibits NF-κB and balances the

activating and inhibitory inflammatory signalling by reducing the transcription and

production of pro-inflammatory cytokines [62]. Indeed, several flavonoids can reduce the

production of IL-1β [66] and induce the production of IL-10, as an immunomodulatory effect

[67].

UVB-irradiated mice treated with E-AP-SM2001 showed decreased epidermal

caspase-3 and PARP immunoreactivities compared with UVB-irradiated mice. Exposure to

UV light, leading to mass apoptosis, could compromise the natural barrier function of the

skin and accelerate photoageing. This suggests that the inhibition of epidermal keratinocyte

apoptosis may represent a therapeutic method for blocking of photoageing [42, 43].


Apoptosis is a highly regulated process that involves the activation of a series of

cellular events, leading ultimately to cell death. Caspase-3 and PARP are key pro-apoptotic

factors [68], and the increase in the numbers of caspase-3- and PARP-immunoreactive

keratinocytes in skin epithelium indicates the occurrence of apoptosis and related damage

[43].

Our observations suggest that photoageing-related changes (ROS-mediated

inflammation, depletion of endogenous antioxidants, activation of MMPs and apoptosis)

were significantly and dose-dependently inhibited by topical application of E-AP-SM2001.

Additionally, E-AP-SM2001 showed more potent inhibitory effects than 5 nM myricetin

against UVB-induced skin photoageing. These results demonstrate that E-AP-SM2001 shows

protective effects against UVB-induced photoageing.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

This work was supported by a National Research Foundation of Korea (NRF) grant, funded

by the Korean government (MSIP; No. 2011-0030124).


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Table 1. Changes in skin antioxidant defence systems after 15 weeks of continuous topical

application of E-AP-SM2001 or myricetin in UVB-exposed mice.

Group Glutathione

(μmol/mg protein)

Malondialdehyde

(nmol/mg protein)

Superoxide anion production (NBT reduction/OD600 nm)

Controls

Intact 1.36±0.38 0.39±0.16 0.38±0.09

UVB 0.44±0.09d 1.61±0.35a 0.99±0.19a

Reference

5 nM myricetin 0.97±0.13ef 0.66±0.16bc 0.52±0.15bc

E-AP-SM2001

Stock 1.19±0.20f 0.58±0.12bc 0.40±0.10c

Twofold dilution 0.87±0.14df 0.90±0.20ac 0.63±0.12ac

Fourfold dilution 0.61±0.12df 1.12±0.14ac 0.72±0.15ac

Values are expressed as means ± SD of eight hairless mice.

UVB = ultraviolet B.

NBT = nitroblue tetrazolium.

E-AP-SM2001 = exopolymers from Aureobasidium pullulans SM2001.

a p < 0.01 and b p < 0.05, versus intact control by LSD test.

c p < 0.01, versus UVB control by LSD test.

d p < 0.01 and e p < 0.05, versus intact control by MW test.

f p < 0.01, versus UVB control by MW test.


Table 2. Changes in skin mRNA levels after 15 weeks of continuous topical application of

E-AP-SM2001 or myricetin to UVB-exposed mice.

Group MMP-1 MMP-9 MMP-13 GSH

reductase Nox2

Control

Intact 1.01±0.06 1.07±0.09 1.05±0.09 1.03±0.10 1.00±0.07

UVB 1.96±0.24e 1.85±0.20a 2.22±0.26a 0.84±0.09b 1.61±0.14a

Reference

5 nM myricetin 1.31±0.19ef 1.26±0.08ac 1.36±0.07a

c 1.36±0.13a

c 1.19±0.10a

c

E-AP-SM2001

Stock 1.12±0.06ef 1.18±0.09c 1.20±0.09c 1.51±0.24a

c 1.07±0.04c

Twofold dilution

1.36±0.14ef 1.35±0.13ac 1.57±0.17a

c 1.24±0.18a

c 1.26±0.13a

c

Fourfold dilution

1.63±0.24eg 1.50±0.09ac 1.88±0.20a

c 1.03±0.11d

1.31±0.13a

c

Values are expressed as means ± SD of eight hairless mice, relative to β-actin mRNA.


MMP = matrix metalloprotease.

GSH = glutathione.

Nox2 = gp91phox subunit of the phagocyte NADPH oxidase.


a p < 0.01 and b p < 0.05, versus intact control by LSD test.

c p < 0.01 and d p < 0.05, versus UVB control by LSD test.

e p < 0.01, versus intact control by MW test.

f p < 0.01 and g p < 0.05, versus UVB control by MW test.


Table 3. General histomorphometrical analysis of skin samples after 15 weeks of continuous

topical application of E-AP-SM2001 or myricetin to UVB-exposed mice.

Group

Number of microfolds (folds/ mm epidermis)

Mean epithelial thickness

(μm/epidermis)

Mean inflammatory

cells (cells/mm2 dermis)

Collagen fiber-occupied regions (%/mm2 dermis)

Controls

Intact 11.13±3.44 20.20±2.63 9.75±2.82 46.25±6.48

UVB 72.13±8.10a 47.42±4.36a 266.50±47.44c 81.43±7.28a

Reference

Myricetin 5 nM 36.00±10.27ab 28.18±3.27ab 197.63±44.48ce 58.14±6.92ab

E-AP-SM2001

Stock 25.75±7.59ab 25.31±2.97ab 89.00±22.25cd 51.40±5.45b

Twofold dilution

36.50±6.44ab 28.72±3.54ab 127.50±18.38cd 58.59±4.13ab

Fourfold dilution

43.13±10.32ab 39.14±3.67ab 159.63±22.09cd 69.38±6.20ab




a p < 0.01, versus intact control by LSD test.

b p < 0.01, versus UVB control by LSD test.

c p < 0.01, versus intact control by MW test.

d p < 0.01 and e p < 0.05, versus UVB control by MW test.


Table 4. Immuno-histomorphometrical analysis of skin samples after 15 weeks of continuous

topical application of E-AP-SM2001 or myricetin to UVB-exposed mice.

Group

Epidermal immunoreactive cells (cells/100 epithelial cells) Dermis MMP-9 immunoreactivit

y (%) Nitrotyrosine 4-

Hydroxynonenal Caspase-3 PARP

Controls

Intact 11.38±4.07 12.25±2.66 16.63±3.16 14.25±2.49 30.13±8.15

UVB 84.88±10.66a 73.88±10.91c 81.63±11.82c 76.63±12.20c 79.25±11.25a

Reference

Myricetin 5 nM 48.25±7.65ab 39.50±7.11cd 29.00±3.46cd 36.38±8.73cd 49.50±7.21ab

BP

Stock 19.25±3.85b 17.50±2.45cd 20.75±5.39cd 21.00±5.78cd 34.88±10.74b

Twofold dilution

31.75±6.36ab 29.75±6.78cd 30.13±4.91cd 26.00±3.21cd 47.00±11.69ab

Fourfold dilution

49.38±11.35a

b 44.13±5.79cd 45.38±12.33cd

41.63±12.05c

d 51.25±5.50ab



PARP = cleaved poly(ADP-ribose) polymerase.

MMP = matrix metalloprotease.


a p < 0.01, versus intact control by LSD test.

b p < 0.01, versus UVB control by LSD test.

c p < 0.01, versus intact control by MW test.

d p < 0.01, versus UVB control by MW test.


FIGURE 1. Body Weight Changes during 15 Weeks of Continuous Topical Application of E-AP-SM2001 or Myricetin in UVB-exposed Mice

No significant changes in body weights were detected in all UVB-exposed hairless mice as compared to unexposed intact vehicle control. In addition, myricetin and all three different dosages of E-AP-SM2001s also did not influence body weights as compared to those of intact and UVB control mice, throughout all experimental periods.

Values are expressed mean ± S.D. of eight hairless mice, g

UVB = ultraviolet B

E-AP-SM2001

Before means 1 day before UVB/test material application

The day of 0 means start day of UVB irradiation and test patch topical application

All animals were overnight fasted before sacrifice (arrow)

Before 0 7 14 21 28 35 42 56 63 77 91 98 104 105

Bod

y w

eigh

ts (

g)

18

20

22

24

26

28

30

32

34 Intact vehicle control miceUVB vehicle control miceMyricetin 5nM applied miceBP stock solution applied miceBP 2-fold diluted solution applied miceBP 4-fold diluted solution applied mice

Days of test material apply


FIGURE 2. Changes of Mean Length of Wrinkles in Skin Replicas after 15 Weeks of Continuous Topical Application of E-AP-SM2001 or Myricetin in UVB-exposed Mice

Note that significant increases in mean length of skin wrinkles were detected in the skin replicas, taken from UVB control as compared to intact control. However, these increases in wrinkle formations were significantly decreased by topical application of all three different dosages of E-AP-SM2001s, dose-dependently. In addition, myricetin 5nM also showed significant decreases in average length of skin wrinkles as compared to UVB control mice.

Values are expressed mean ± S.D. of eight hairless mice

UVB = ultraviolet B

a p<0.01 and b p<0.05 as compared to intact control by LSD test

Intact UVB Myricetin 5nM 0 2 4

Mea

n le

ngth

of

skin

wri

nkle

s (m

m)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Controls BP applied as diluted solution

a

bc ac ac

ac


c p<0.01 as compared to UVB control by LSD test

FIGURE 3. Changes of Average Depth of Wrinkles in Skin Replicas after 15 Weeks of Continuous Topical Application of E-AP-SM2001 or Myricetin in UVB-exposed Mice

Note that significant increases in average depth of skin wrinkles were detected in the skin replicas, taken from UVB control as compared to intact control. However, these increases in wrinkle formations were significantly decreased by topical application of all three different dosages of E-AP-SM2001s, dose-dependently. In addition, myricetin 5nM also showed significant decreases in mean depth of skin wrinkles as compared to UVB control mice.


UVB = ultraviolet B

a p<0.01 and b p<0.05 as compared to intact control by LSD test

c p<0.01 as compared to UVB control by LSD test


Ave

rage

dep

th o

f sk

in w

rink

les

(mm

)

0

20

40

60

80

100

120


a

bc

ac

ac ac


FIGURE 4. Changes of Skin Oedema Scores – 6-mm Diameter Skin Weights after 15 Weeks of Continuous Topical Application of E-AP-SM2001 or Myricetin in UVB-exposed Mice

Note that significant increases in 6-mm diameter of skin weights, the oedema scores, were detected in UVB-exposed vehicle control mice as compared to unexposed intact vehicle control hairless mice. However, significant decreases in skin weights were detected in myricetin 5nM and all three different dosages of E-AP-SM2001s applied to hairless mice, respectively. Especially, E-AP-SM2001s showed obvious dose-dependent decreases in skin oedema scores.



Skin

wei

ghts

(g/6

mm

-dia

met

er d

orsa

l ski

n)

0.00

0.02

0.04

0.06

0.08

0.10


a

ab ab

ab ab


UVB = ultraviolet B

a p<0.01 as compared to intact control by LSD test

b p<0.01 as compared to UVB control by LSD test

FIGURE 5. Changes of Skin MPO Activities after 15 Weeks of Continuous Topical Application of E-AP-SM2001 or Myricetin in UVB-exposed Mice

Note that significant increases in skin MPO activities were detected in UVB control mice as compared to intact control hairless mice. However, significant decreases in skin MPO activities were detected in all test material topically applied hairless mice, including myricetin 5nM as compared to UVB control, respectively. Especially, E-AP-SM2001s showed clear dose-dependent inhibition of skin MPO activities.


Skin

MP

O a

ctiv

ity

(Neu

trop

hils

x10

5 /mg

of p

rote

in)

0

2

4

6

8

10

12

14


a

ac bc

ac

ac



UVB = ultraviolet B

MPO = myeloperoxidase

a p<0.01 and b p<0.05 as compared to intact control by MW test

c p<0.01 as compared to UVB control by MW test

FIGURE 6. Changes of Skin IL-1β Levels after 15 Weeks of Continuous Topical Application of E-AP-SM2001 or Myricetin in UVB-exposed Mice


Skin

IL

-1b

leve

l (pg

/100

mg

of p

rote

in)

0

10

20

30

40

50

60

70


a

ab b

ab

ab


Note that significant increases in skin IL-1β levels were detected in UVB-exposed control mice as compared to intact control hairless mice. However, significant decreases in skin IL-1β levels were detected in all three different dosages of E-AP-SM2001s applied hairless mice as compared to UVB control, dose-dependently. In addition, myricetin 5nM also showed significant decreases in skin IL-1β levels as compared to UVB-exposed control hairless mice.


UVB = ultraviolet B

IL = interleukin

a p<0.01 as compared to intact control by LSD test

b p<0.01 as compared to UVB control by LSD test


Skin

IL

-10

cont

ents

(pg

/100

mg

of p

rote

in)

0

100

200

300

400


b

bc ac

c d


FIGURE 7. Changes of Skin IL-10 Levels after 15 Weeks of Continuous Topical Application of E-AP-SM2001 or Myricetin in UVB-exposed Mice

Note that Skin IL-10 levels in UVB control mice were significantly decreased when compared with unexposed intact control hairless mice. However, significant and dose-dependent increases in skin IL-10 levels were detected in all three different dosages of E-AP-SM2001s applied mice as compared to UVB control hairless mice, respectively. Myricetin also showed significant increases in skin IL-10 levels as compared to UVB-exposed control at 5nM concentration.


UVB = ultraviolet B

IL = interleukin

a p<0.01 and b p<0.05 as compared to intact control by MW test

c p<0.01 and d p<0.05 as compared to UVB control by MW test


FIGURE 8. Representative Histological Images of Dorsal Back Skin Tissues, Taken from Unexposed Intact or UVB-Exposed Hairless Mice

Note that marked increases in mean epithelial thicknesses due to hyperplasia/hypertrophy of epidermal keratinocytes were detected on the dorsal back skin tissues in UVB-exposed control mice with noticeable increases in the numbers of inflammatory cells infiltrated into dermis, abnormal


collagen depositions and formation of microfolds on the surface of epithelial lining, respectively. However, noticeable decreases in UVB-induced histopathological dermis sclerosis and inflammatory signs were observed in all E-AP-SM2001 stock, 2- and 4-fold diluted solution topically applied hairless mice, again dose-dependently, as compared to UVB control mice, respectively. Myricetin also showed meaningful decreases in epithelial surface microfolds, mean epithelial thicknesses, numbers of dermal infiltrated inflammatory cells and the percentages of collagen fibre occupied dermal regions as compared to UVB control at 5nM concentration.

A = Intact vehicle control mice

B = UVB vehicle control mice

C = Myricetin 5nM applied mice

D = E-AP-SM2001 stock solution applied mice

E = E-AP-SM2001 2-fold solution applied mice

F = E-AP-SM2001 4-fold solution applied mice

UVB = ultraviolet B

MT = Masson’s trichrome

Arrows indicate microfolds formed

Scale bars = 100µm.


FIGURE 9. Representative Immunohistochemical Images of Dorsal Back Skin Tissues, Taken from Unexposed Intact or UVB-Exposed Hairless Mice


Noticeable increases in immunolabelled cells for oxidative stress (NT and 4-HNE) and apoptosis (caspase-3 and PARP) markers among epidermal keratinocytes were detected on the dorsal back skin tissues in UVB-exposed control mice with marked increases in the dermal MMP-9 immunoreactivities, respectively. However, dramatic decreases in oxidative stress and apoptosis markers, and also dermal MMP-9-immunoreactivities were observed in myricetin 5nM, E-AP-SM2001 stock, 2- and 4-fold diluted solution applied hairless mice as compared to UVB control mice, respectively. Especially, E-AP-SM2001s also showed clear dose-dependent decreases in epithelial NT-, 4-HNE-, caspase-3- and PARP-positive cells and dermal MMP-9-immunoreactivities cells.

A = Intact vehicle control mice

B = UVB vehicle control mice

C = Myricetin 5nM applied mice

D = E-AP-SM2001 stock solution applied mice

E = E-AP-SM2001 2-fold solution applied mice

F = E-AP-SM2001 4-fold solution applied mice

UVB = ultraviolet B

4-HNE = 4-Hydroxynonenal

PARP = cleaved poly(ADP-ribose) polymerase

MMP = matrix metalloproteases

All ABC immunostain

Scale bars = 50µm.

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inhibition of uvb-induced skin damage by exopolymers from aureobasidium pullulans ...

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