Hesperidin was obtained from Wako (Wako Pure Chemicals, Osaka, Japan). UVB irradiation was carried out using a UVM-225D Mineralight UV Display Lamp (UVP, Phoenix, AZ, USA). Replicas of mouse dorsal skin were obtained using a Repliflo Cartridge Kit (CuDerm Corp., Dallas, TX, USA). Antibodies against ERK, phospho ERK, MEK, phospho MEK, and anti MMP-9 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.).
Experimental animals and oral administration
Male hairless mice (Hos/HR-1, 6-week-old) were purchased from Japan SLC, Inc. (Sizuoka, Japan). Mice were housed in a climate-controlled room at 24 °C and 50% humidity under a 12 h light/dark cycle. They were acclimatized for 1 week prior to the study, and provided free access to food and water. They were divided into 3 groups randomly (n = 7 per group): control, UVB-treated vehicle, and UVB-treated hesperidin (100 mg/kg) groups. Hesperidin treated group was orally administered 0.1 mL of water containing 100 mg/kg body weight per day hesperidin in the study period (5 days a week for 12 weeks). Animals in the vehicle group were orally administered drinking water, whereas the unexposed control group animals did not receive any treatment. All experimental protocols were approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee (12–045).
UVB irradiation in mice
This experiment investigated the effect of orally administered hesperidin on the dorsal skin of UV-irradiated mice. Mice were irradiated 3 times at 48 h intervals per week for 12 weeks. The amount of irradiation was progressively increased from 60 mJ/cm2 per exposure at week 1 to 90 mJ/cm2 at week 7.
Evaluation of wrinkle formation and TEWL
Replicas of mouse dorsal skin were obtained using Repliflo Cartridge Kit (CuDerm Corp., U.S.A.). A dorsal skin sample was taken after the animals were sacrificed. Animals were sacrificed by lethal inhalation of carbon dioxide. The impression replicas were set on a horizontal sample stand, and wrinkle shadows were produced by illumination with a fixed-intensity light at 35° angle using an optical light source. Black and white images were acquired with a CCD camera and analyzed by Skin-Visiometer VL 650 software (Courage & Khazhka, Cologne, Germany). The parameters used in the assessment of skin wrinkles were the average length and average depth of wrinkles. For the evaluation of skin hydration, TEWL, as a marker of epidermal skin barrier function, was measured with Tewameter® TM300 mounted on a Multi Probe Adapter (CK Electronics GmbH, Germany).
Doral skin was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5-μm thickness. Skin sections were stained with hematoxylin and eosin (H&E). The thickness of the epidermis was measured under light microscopy with an eyepiece micrometer (Olympus, Japan).
After taken of dorsal skin, all dorsal skin was snap frozen in nitrogen and stored at −80 °C for analysis. The skin tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 10-μm thickness using a microtome. After deparaffinization and rehydration, the sections were incubated with 5% BSA (in PBS) at room temperature for 1 h to block non-specific binding. Sections were then incubated with following primary antibodies: anti-TNF-α (1:200; Abcam, Cambridge, MA) and anti-IL-8 (1:200; Immuno-biological laboratoriesCo, Fuhioka, Japan) in a humidified chamber overnight at 4 °C. After washing, the tissue sections were incubated with appropriate fluorescence conjugated Alexa 488 (1:2000; ThermoFisher Scientific, Waltham, CA) secondary antibodies for 1 h and counter-stained with DAPI (ThermoFisher scientific) for 15 min. Subsequently, the sections were washed, mounted using Vecta shield mounting media (Vector Laboratories, Burlingame, CA), and imaged using a fluorescence microscope (Leica, Wetzlar, Germany).
Preparation of skin lysates
At the end of the experiments, mice were sacrificed by cervical dislocation, and the dorsal skin was excised. The fat was removed, and the skin was immediately pulverized with liquid nitrogen using a mortar and pestle. The pulverized skin was homogenized on ice with Precellys®24 (Bertin, USA) tissue homogenizer. Subsequently, proteins were extracted with 20% SDS solution containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM iodoacetamide, 1 mM leupetin, 0.1 mM sodium orthovanadate, and 5 mM sodium fluoride. The obtained lysates were centrifuged at 13,000 rpm for 30 min, and the protein content in the supernatant was determined using a Bio-Rad protein assay kit (Bio-Rad).
To assess the gelatinolytic activities of MMP-9, equal amounts of the protein extract were mixed with non-reducing sample buffer, incubated for 10 min at room temperature, and subjected to gelatin zymography (0.1% gelatin) under non-reducing conditions. After electrophoresis, the gel was washed with 2.5% Triton X-100 for 1 h to remove SDS, and then incubated for 24 h at 37 °C in developing buffer (1 M Tris-HCl, pH 7.5, 10 mM CaCl2). Subsequently, the gels were stained with Coomassie brilliant blue followed by destaining (25% ethanol, 8% acetic acid). Gelatinolytic activity could be observed as horizontal white bands on a blue background. Relative band densities were analyzed using Image J 1.44 software (NIH, Bethesda, MD, U.S.A.)
RNA extraction and quantitative real-time polymerase chain reaction
Total RNA was extracted from UVB-irradiated mouse skin tissue using the TRIzol reagent and the protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TaqMan assays (Applied Biosystems, Foster City, CA, USA) specific for MMP-9 on a QuantStudio™ 6 Flex Real-Time PCR system (Applied Biosystems). Each sample was assayed in triplicate, and relative mRNA expression levels were calculated using the ΔΔCt method and normalized to the β-actin mRNA level in each sample.
The lysates prepared from 100 μg of the fat-removed dorsal skin of mice, were centrifuged at 13,000 rpm for 30 min, and aliquots of the supernatant containing 100 μg of proteins were subjected to 10% SDS-PAGE. After electrophoresis, the proteins were blotted onto nitrocellulose membrane. Subsequently, the membranes were blocked by incubation with TBS-T (0.1% Tween 20) containing 5% BSA. The membranes were then incubated with primary antibodies (anti-phospho-ERK, anti-phospho–MEK, anti-MMP-9, or anti-ERK), and washed with TBS-T. Protein bands were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech) after hybridization with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG or goat anti-mouse IgG Ab diluted 1:1000). The relative amounts of proteins were detected using an enhanced chemiluminescence Western Blotting Detection Kit (Amersham, Little Chalfont, Buckinghamshire, UK).
All measurements are presented as means ± S.D. values of triplicate. Differences were a p value of <0.05 was considered significant.