BV (You-Miel BV Ltd. Hwasoon, Jeon-nam, Republic of Korea) components were as follows: 45-50% melittin, 12% phospholipase A2, 4-5% 6-pentyl a-pyrone lipids, 4-5% carbohydrate, 2-3% amine, 2.5-3% mast cell degranulating peptide, 1% lysophospholipase A, 1.5-2% hyaluronidase, 1-1.5% histidine, 0.5% secarpin, 0.1% procamine, 0.1% tertiapin and 19-27% of others including protease inhibitor, glucosidase, acid phosphomonoesterase, norepinephrine, dopamine, invertase, unknown amino acids, with 99.5% purity.

According to instructions to users of BV (H&M pharmachem, Bucheon, Kyoung-ki, Republic of Korea), 8.2 g of BV was dissolved using 120 mL of 95% ethanol and shaking at room temperature, and insoluble substances were removed using Whatman filter paper. The ethanol was distilled off, at room temperature, so that the volume of the solution became about half the initial volume using a vacuum distiller. The remaining solution was transferred to a plate (with a thickness of about 5 mm), and the liquid component was removed using a vacuum freezer set at -20℃ for the first 3 hours, and then vacuum was applied overnight. The dry sample was collected with a spatula, ground in a mortar for 3 minutes, sealed, and stored at room temperature for later use.

Chungbuk National University-Institutional Animal Care and Use Committee approved the animal experimental protocol for this study (Approval no.: 00NU-2015-0976) and strict scientific and ethical care procedures were followed. Eight-week-old, male, HR-1 mice were randomly distributed into 5 treatment groups; (1) 3.5% phthalic anhydride (PA) solution [100 μL (20 μL/cm²)], was applied topically to the dorsal area of the ears, and the back, 3 times a week, for 4 weeks (n = 3); (2) 5% PA group, 5% phthalic anhydrous solution was administered topically to the back, 3 times a week, for 4 weeks (n = 3); (3) PA+BV 0.1 μg (n = 3); (4) PA+BV 0.1 μg liposomes (n = 3); and (5) PA+BV liposomes 0.05 μg (n = 3). All groups had PA administered, and 3 hours later, 100 μL of 0.1 μg of BV, 0.05 μg, and 0.1 μg of BV liposomes were administered. The mice in the control group were of the same age (n = 3).

During the 4 weeks of the experiment, the weight of each mouse was measured once a week on electronic scales (Mettler Toledo, Greifensee, Switzerland). Following euthanasia, the weight of lymph nodes extracted from mice was also measured. Ear thickness was measured using an instrument that specifically measures thickness (Digimatic Indicator, Mitsutoyo Co., Tokyo, Japan) to determine the extent of allergic dermatitis induced by the PA solution.

The skin from the ears and backs of the mice were f ixed in formalin (10%), embedded in paraffin wax, and 5 μm sections were prepared which were dyed with eosin and hematoxylin. Following dehydration and paraffin removal, the skin sections were stained with toluidine blue solution (0.25% Sigma Aldrich MO, USA). and examined under light microscopy. Using the Leica Application Suite, the thickness of the dermis and epidermis was measured, and the number of mast cells per unit area was counted (Leica Microsystem, Wetzlar, Germany).

For serum IgE concentration, the enzyme-linked immunosorbent assay kit (Gunma sibayagi Co., Ltd., Japan) instructions were followed. Antibodies were plated onto the Nunc C lower immunoplate (contained in the kit). The well was washed 3 times (pH 8.0, TWEEN 20 0.05%, 50 mM Tris, 0.14 M NaCl). Standards and serum samples were diluted with wash buffer and added to the well and cultured for 2 hours. To each well, 50 μL of biotin antiIgE conjugated antibodies were diluted 1,000 times, administered, and incubated for 2 hours to bind with captured IgE. After washing every well, horseradish peroxidase-conjugated detection antibodies were diluted 2,000 times and were added, and incubated for 1 hour. The enzyme reaction was initiated by adding tetramethylbenzidine substrate solution (100 mM sodium acetate buffer pH 6.0, 0.006% H₂O₂) and incubating on the plate at room temperature for 20 minutes in the dark. The reaction was ended by adding an acid solution (reaction stopper, 1M H₂SO₄) and the absorption of the yellow product was measured at 450 nm using a plate reader. The last concentration of IgE was quantified according to a standard curve.

Blood specimens were taken in the last period of the study. Levels of serum interleukin (IL)-1β, IL-6 and IL-4 were measured using enzyme-linked immunosorbent assay kits (Thermo Fisher Scientific Inc. Meridian Road, Rockford, IL USA) according to protocol.

The RiboEX RNA extraction kits (GeneAll Biotechnology, Seoul, Korea) were used to collect total RNA from the mice skins. The High-Capacity RNA-to-cDNA kits (Applied Biosystems, Foster City, CA, USA) was used to synthesize complementary deoxyribonucleic acid. Reverse transcription qualitative polymerase chain reaction was performed using specific primers within the StepOnePlus™ PCR System (Applied Biosystems, Foster City, CA, USA) as previously described [18], The fold change between groups was determined for all targets by the 2^ΔΔCt method. The mRNA levels were normalized to the 18S sequence as a housekeeping control.

About 100 mg of skin or 1 × 10⁶ cells were homogenized with lysis buffer [pH 8.0, 50 mM Tris, 150 mM NaCl, 0.2% sodium dodecylsulfate, 10 μL/mL aprotinin, 0.02% sodium azide, 1 mM phenylmethylsulfonyl fluoride, 1% igapel 630 (Sigma Chem. Co. St. Louis, MO, USA), 0.5 mM EDTA, 10 mM NaF, 0.5% sodium deoxycholate, 0.1 mM EGTA]. After centrifuging at 23,000 g for 1 hour, the same amount of protein (20 μg) was separated on a sodium dodecyl sulfate/10%-polyacrylamide gel, and it was transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The nitrocellulose membrane was fixed at room temperature for 2 hours with 5% (w/v) fat free dried milk in Tris buffered saline solution (10 mM Tris, pH 8.0, 150 mM NaCl) with 0.05% TWEEN 20. The membrane was incubated for 4 hours at room temperature using specific antibodies. Mouse monoclonal antibodies directly targeting p50 (1:500, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), p-p38, p38, p-ERK, ERK, p65 (1:1,000, Cell signaling Technology, Beverly, MA, USA) were used. Rabbit polyclonal antibodies directly against IκB, IκB-α (1:500, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) were also used. The anti-rabbit immunoglobulin G-horseradish peroxidase (Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) were equivalently conjugated and used for the western blots’ and incubated. For the detection of immunoreactive proteins, the enhanced chemiluminescent western blotting detection system was used.

In this study, all experiments were repeated 3 times and had similar results. Every statistical analysis was worked by GraphPad Prism 5 software (Version 5.03, GraphPad software Inc., CA, USA). The difference between groups was analyzed with one-way ANOVA followed by Tukey’s multiple comparison test. All values were shown as the mean ± standard deviation, and statistical significance was p < 0.05.

The change in body weight was measured and no significant difference was observed between the groups of mice (Fig. 1). Changes in morphology of the skin from the backs of the mice, compared with the control group, included erythema, edema, and erosion in the PA treatment groups. These morphological changes were improved with 0.1 μg/mL of BV treatment, which were dramatically improved by BV in liposomes (0.05 and 0.1 μg/mL; Fig. 1).

To investigate the inhibitory effect of BV liposomes, the weight of each auricular lymph node, and serum IgE concentrations were evaluated. PA application caused an increase in the weight of the lymph node compared with the control group. After application of the BV liposomes, the lymph node weight reduced significantly in the 0.05 μg/mL, and 0.1 μg/mL BV liposome treated groups, as well as in the 0.1 μg/mL BV treated group (Fig. 2). Repeated local administration of PA solution caused a significant increase in serum IgE concentration in the blood of the mice as the allergic response developed. However, serum IgE concentration was significantly lower in the 0.05 μg/mL, and 0.1 μg/mL BV liposome treated groups, as well as in the 0.1 μg/mL BV treated group (Fig. 2).

Histology was performed on the skins from the backs of the mice and was analyzed to observe potential changes in the epidermis caused by the BV liposomes. The epidermis thickness of the PA group was greater than control group (Fig. 3). The epidermis became thinner in the BV treated group (0.1 μg/mL), and where BV in liposomes were used (0.05 μg/mL, and 0.1 μg/mL BV liposome group) this action was synergistic. Moreover, there was a significant dose-dependent decrease in epidermal thickness in the 0.05 μg/mL, and 0.1 μg/mL BV liposome treated groups (p < 0.05).

In addition, using a microscope to count the change in the number of infiltrated mast cells, the number was significantly higher in the PA treatment group compared with the control group (Fig. 3). However, the number of mast cells after treatment was observed as less in the 0.05 μg/mL, and 0.1 μg/mL BV liposome treated groups, as well as in the 0.1 μg/mL of BV treated group. Mast cell infiltration was concomitantly, synergistically, and dose-dependently inhibited in groups treated with BV in liposomes (0.05 μg/mL, and 0.1 μg/mL; Fig. 3).

The mouse serum levels of IL-1β, IL-4 and IL-6 were measured to observe how BV liposome administration was involved in potential changes when inflammatory cytokines are released from dermatitis induced by PA in the control, PA treatment, PA+BV, and PA+BV liposome treatment groups. After PA solution was applied topically and repeatedly, a significant increase of IL-1β, IL-4 and IL-6 levels was induced in serum (p < 0.05). However, those were significantly and dose-dependently decreased in the 0.05 μg/mL, and 0.1 μg/mL BV liposome treated groups, as well as in the 0.1 μg/mL of BV treated group compared with 5% PA treated group (p < 0.05; Fig. 4).

To examine the production of chemokines and pro-inflammatory cytokines in the skin of the backs of the mice, real-time PCR was performed. The levels of pro-inflammatory cytokines such as IL-1β, IL-4, and IL-6, and of chemokines such as TSLP, and CCL22 increased due to the repeated application of PA solution topically. However, those were significantly and dose-dependently decreased in the 0.05 μg/mL, and 0.1 μg/mL BV liposome treated groups, as well as in the 0.1 μg/mL BV treated group by comparison with 5% PA treated group (p < 0.05; Fig. 5).

To observe the potential action of BV liposomes involved in MAP kinase pathway on PA-induced inflammation in mice, the MAP kinase activation (phosphorylation of p38 and ERK), induced by PA treatment, was measured in cells of the skins from the backs of the mice. The analysis of western blot from individual 3 experiments appeared that BV liposomes (0.05 and 0.01 μg/mL) as well as BV (0.1 μg/mL) strongly dose-dependently blocked PA (5%)-induced activation of p-ERK and p-p38 in cells of the skin from the backs of the mice (Fig. 6).

To examine the potential involvement of signal molecules in the NF-κB signal pathway in the suppressive effect of BV liposomes on PA-induced inflammatory dermatitis in mice, the activation of p65, IκB, p50 and p-IκB induced by PA treatment was evaluated in cells of skins from the backs of the mice. The analysis of western blots from 3 different experiments showed that BV liposomes (0.05 and 0.01 μg/mL) as well as BV (0.1 μg/mL) strongly and dosedependently blocked PA (5%)-induced activation of NF-κB in cells from the skins of the backs of the mice (Fig. 7).