Colonies of natural honeybees (Apis mellifera L). used in this study were maintained at the National Academy of Agricultural Science, Wanju, Korea. BV was collected in collecting device (Chung Jin Biotech Co., Ltd., Ansan, Korea) in a sterile manner under strict laboratory conditions. In brief, the BV collector was placed on the hive, and the bees were given enough electric shocks which caused them to sting a glass plate from which dried BV was later scraped off. The collected BV was diluted in cold water and then centrifuged at 10,000 × g for 5 min at 4°C to discard residues from the supernatant. Purified BV was lyophilized by a freeze dryer and stored in a refrigerator for later use, as previoulsy described (32). BV used in the experiment was confirmed with size exclusion gel chromatography (AKTA Explorer; GE Healthcare Bio-Sciences, Piscataway, NJ, USA) by dissolving in 0.02 M phosphate-buffer with 0.25 M NaCl adjusted to pH 7.2 using a Superdex Peptide column (Amersham Biosciences, Piscataway, NJ, USA and GE Healthcare).

P. acnes (ATCC 6919; American Type Culture Collection, Rockville, MD, USA) were obtained from the Korean Culture Center of Microorganisms (Seoul, Korea) and were cultured at 37°C on Reinforced Clostridial Medium (BD Diagnostics, Sparks, MD, USA) under anaerobic conditions at 37°C until reaching OD₆₀₀ 1.0 (logarithmic growth phase). The log phase bacterial culture was centrifuged at 5,000 × g at 4°C for 15 min. Subsequently, the culture supernatant was removed, filtered (through a 0.22 μm pore size filter), and used immediately or stored at −20°C. The bacterial pellet was washed 3 times in 100 ml of phosphate-buffered saline (PBS) and finally suspended in 10 ml of PBS. The P. acnes suspension was incubated at 80°C for 30 min for heat-killing reaction. The heat-killed P. acnes suspension was stored at 4°C until use.

Eight-week-old ICR mice (n=30) were supplied from Samtako (Osan, Korea) and were individually housed in polycarbonate cages and maintained under constant temperature (22±2°C) and humidity conditions (55%). The mice were allowed free access to food and water, and were maintained in an environment with a 12:12-h light/dark cycle. All surgical and experimental procedures used in the current study were approved by the Institutional Review Board (IRB) committee at the Catholic University of Daegu Medical Center, Daegu, Korea and conformed to the US National Institutes of Health guidelines for the care and use of laboratory animals.

The mice were randomly divided into 6 different groups (5 mice/group) as follows: i) NC, normal control. ii) PA, living P. acnes [1.0×10⁷ colony-forming units (CFU)/20 μl in PBS] was intradermally injected into the left ears of the mice. The right ears received an equal amount (20 μl) of PBS. iii) PA/Vas, living P. acnes was intradermally injected into both the left and the right ears. After the injection, 0.05 g vaseline (Sigma, St. Louis, MO, USA) was applied to the surface of the skin of the right ear. For the next 3 groups, PA/BV1, PA/BV10 and PA/BV100, living P. acnes was intradermally injected into both the left and the right ears. After the injection, BV (1, 10 and 100 μg mixed with 0.05 g vaseline) was applied to the surface of the skin of the right ear in the mice in each group. At the end of each treatment period (24 h later), the animals were sacrificed by cervical dislocation and the ears were excised.

All tissue specimens were fixed in 10% formalin for at least 24 h at room temperature. Following fixation, perpendicular sections to the anterior-posterior axis of the wound were dehydrated in graded ethanol, cleared in xylene and embedded in paraffin. Thin sections (4-μm-thick) were mounted on glass slides, dewaxed, rehydrated in distilled water and stained with hematoxylin and eosin (H&E). As part of the histological evaluation, a pathologist blinded to the previous treatment examined all the sections under a light microscope (Nikon, Tokyo, Japan).

Paraffin-embedded tissue sections were deparaffinized with xylene, dehydrated in gradually decreasing concentrations of ethanol, and subsequently treated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. Tissue sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95°C. The final step was repeated using fresh 10 mM sodium citrate solution (pH 6.0). The sections were allowed to remain in the same solution while cooling for 20 min and rinsed in PBS. The sections were incubated with primary antibody (1:100) for 1 h at 37°C. Primary antibodies were the following: anti-TNF-α (Abcam, Cambridge, UK) and anti-IL-1β (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The signal was visualized using an EnVision System (Dako, Carpinteria, CA, USA) for 30 min at 37°C. 3,3′-Diaminobenzidine tetrahydrochloride (DAB) was used as the coloring reagent, while hematoxylin was used as the counterstain.

Paraffin-embedded ear tissue sections were deparaffinized with xylene and dehydrated in gradually decreasing concentrations of ethanol. The tissue sections were subsequently placed in blocking serum (5% bovine serum albumin in PBS) at room temperature for 1 h. Primary antibody (1:100 dilution) was added followed by overnight incubation at 4°C, and incubation with secondary antibody (1:200 dilution) was performed at room temperature for 4 h. Antibodies used in the experiments were the following: CD14, TLR2 (Santa Cruz Biotechnology, Inc.) and anti-mouse, anti-rabbit-biotinylated secondary antibodies conjugated with FITC or Texas Red (Invitrogen, Carlsbad, CA, USA). The slides were mounted using VECTASHIELD^® Mounting Medium (VECTOR Laboratories, Inc., Burlingame, CA, USA). Specimens were examined and photographed using a fluorescence microscope (Nikon).

The tissues were lysed and homogenized in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 100 mM PMSF, 1 M DTT, 10 mg/ml leupeptin and aprotinin; all from Sigma). Following incubation on ice for 30 min, the samples were centrifuged at 8,000 × g at 4°C for 30 min, and the supernatant was collected. The total protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA). Total protein (10–50 μg) was separated on 8–12% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore Corp., Billerica, MA, USA) using a standard SDS-PAGE gel electrophoresis procedure. The membranes were blocked in 5% skim milk in TBS-T (10 mM Tris, 150 mM NaCl and 0.1% Tween-20) for 2 h at room temperature and probed with primary antibody for 4 h. A horseradish peroxidase (HRPO)-conjugated secondary antibody (anti-mouse, anti-rabbit and anti-goat) was used for detection. Signals were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). The primary antibodies used in this study were the following: anti-TNF-α from Abcam, and anti-IL-1β and anti-glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) from Santa Cruz Biotechnology. All primary antibodies were diluted at 1:1,000. Signal intensity was quantified using an image analyzer (LAS-3000; Fujifilm, Tokyo, Japan).

Total RNA was extracted from ear tissue with TRIzol reagent (Gibco, Grand Island, NY, USA) according to the manufacturer’s recommendations. The purity and quantity of the RNA preparation were measured at optical densities of 260 and 280 nm. First-strand cDNA was synthesized using oligo(dT) primers and M-MLV reverse-transcriptase (Promega Corp., Madison, WI, USA). An aliquot of cDNA was used for PCR using primer sets specific to mouse TNF-α, IL-1β and GAPDH. Primer sequences were as follows: TNF-α forward, 5′-AGT GGT GCC AGC CGA TGG GTT GT-3′ and reverse, 5′-GCT GAG TTG GTC CCC CTT CTC CAG-3′; IL-1β forward, 5′-CAT GAG CAC CTT CTT TTC CT-3′ and reverse, 5′-TGT ACC AGT TGG GGA ACT CT-3′; GAPDH forward, 5′-GTG GAC ATT GTT GCC ATC AAC G-3′ and reverse, 5′-GAG GGA GTT GTC ATA TTT CTC G-3′. PCR products were visualized by 1.5% agarose gel electrophoresis with ethidium bromide staining.

Tissues were trypsinized and incubated for 15 min in a hypo-osmotic buffer (10 mM HEPES, pH 7.9, 10 mM KCI, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT) for 20 min, and vortexed and centrifuged at 5,000 × g for 15 min at 4°C. The nuclear supernatant was collected as the nuclear lysate fraction. Each buffer also contained 0.2 mM phenylmethanesulfonyl fluoride (PMSF) and 10 μg/ml protease inhibitors.

A DIG Gel Shift kit (Roche, Mannheim, Germany) was used for EMSA. The nuclear factor-κB (NF-κB) and activator protein (AP)-1 oligonucleotide probe (NF-κB, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, AP-l, 5′-CGC TTG ATG ATG CAG CCG GAA-3′; only sense strands are shown) was end-labeled with DIG-ddPUT. For binding reactions, 9 μg nuclear extract protein was mixed with binding buffer (0.5 μg poly dI-dC, 0.1 μg poly L-lysine and 0.8 μg labeled oligonucleotide, final volume of 20 μl) and was incubated at 37°C for 30 min. The nuclear protein-DNA complex was separated on a 6% non-denaturing polyacrylamide gel in TBE buffer at 80 V for 1.5–2 h at 4°C. The samples were transferred to Hybond-XL membranes (Amersham Biosciences) by electroblotting for 30 min. The membranes were cross-linked for 10 min and washed with solution containing 100 mM maleic acid, 150 mM NaCl and 0.3% Tween-20, pH 7.5 for 10 min, were then moved to blocking solution (DIG Gel Shift kit) for 40 min. The membranes were incubated with anti-digoxigenin-AP Fab fragments (1:10,000) for 30 min, and the nuclear protein-DNA bands were developed with detection solution including 100 mM Tris-HCl, 100 mM NaCl, pH 9.5 and 100 μg/ml disodium 3-{4-methoxyspiro [1,2-dioxetane-3,2′(5′-chloro)tricyclo(3.3.1.1³,⁷)decan]-4-yl} phenyl phosphate. NF-κB and AP-1 bands from the films were quantitated using an image analyzer (LAS-3000; Fujifilm).

Data are presented as the means ± SE. The Student’s t-test was used to assess the significance of differences between independent experiments. A value of p<0.05 was considered to indicate a statistically significant difference.

To investigate the therapeutic effects of BV against P. acnes-induced inflammatory skin disease, P. acnes was intradermally injected into both the left and the right ears of ICR mice. After the injection, various concentrations of BV (1, 10 and 100 μg) mixed with 0.05 g of vaseline were applied to the surface of the skin of the right ear. As shown in Fig. 1A, significant ear swelling, redness and erythema were observed 24 h after the P. acnes injection. Histological observation revealed that the P. acnes injection induced a considerable increase in the number of infiltrated inflammatory cells. By contrast, the BV-treated ears showed noticeably reduced ear thickness, swelling, erythema and inflammatory reactions. In particular, treatment with 1 μg of BV resulted in a 3-fold reduction of ear thickness compared to the ears injected only with living P. acnes (Fig. 1B). Based on these results, 1 μg of BV was used in the subsequent experiments.

It has previously been demonstrated that P. acnes stimulates the production of pro-inflammatory cytokines, such as TNF-α and IL-1β (6). Therefore, we investigated whether TNF-α and IL-1β are involved in the inhibitory effects of BV on P. acnes-induced inflammatory skin disease. Immunohistochemical staining for TNF-α and IL-1β revealed that the expression levels of these cytokines were barely detectable in the normal skin tissue from the NC group. As shown in Fig. 2A, the PA and PA/Vas groups showed a marked increase in the number of TNF-α and IL-1β-positive cells compared to the NC group. However, treatment with BV significantly inhibited the number of TNF-α- and IL-1β-positive cells compared to the PA and PA/Vas groups. Consistent with immunohistochemical staining, the results from western blot analysis revealed that treatment with BV suppressed the expression levels of these cytokines in the inflamed tissue (Fig. 2B). Moreover, the results from RT-PCR also indicated that treatment with BV markedly inhibited the expression of TNF-α and IL-1β in the PA/BV1 group (Fig. 2C). These observations suggested that BV effectively inhibited the inflammatory events in the animal model of P. acnes-induced inflammatory skin disease.

To determine whether BV effectively blocks TLR2 and the macrophage marker of CD14, we performed double immunoflurorescence staining. As shown in Fig. 3, the PA and PA/Vas groups showed significantly higher TLR2 and CD14 expression levels than the NC group. In addition, merged images show that the PA group predominantly expressed CD14 in the dermis and TLR2 in the epidermis, both of which are localized at different parts of the tissue. These results demonstrated that P. acnes induced the infiltration of macrophages and the activation of TLR2 in the inflamed skin tissue. By contrast, treatment with BV significantly inhibited TLR2 and CD14 expression in the P. acnes-injected tissue. Therefore, BV effectively blocks the expression of TLR2 and CD14 in P. acnes-induced inflammatory skin disease.

Pro-inflammatory cytokines are regulated at the transcriptional levels by several transcriptional factors, including AP-1 and NF-κB (22). Therefore, we examined the DNA-binding activity of NF-κB and AP-1 in inflamed skin tissue treated with or without BV by EMSA. As shown in Fig. 4A and B, the expression of NF-κB and AP-1 was increased in the PA and PA/Vas groups, but those levels were significantly inhibited in the PA/BV1 group. These results indicate that BV inhibits the expression of inflammatory cytokines, such as TNF-α and IL-1β by suppressing the DNA-binding activity of NF-κB and AP-1 in the inflamed skin tissue. Therefore, these results suggest that BV is effective in inhibiting NF-κB and AP-1 activation in inflamed skin tissue, which provides the molecular basis for the decreased expression of chemokines/cytokines in the inflamed skin treated with BV following P. acnes injection.