Materials

Human HaCaT cells were purchased from Cell Lines Service (Eppelheim, Germany). Dulbecco’s modified Eagle’s medium (DMEM), 10% foetal calf serum (FCS), L-glutamine, antibiotics mixture (100 IU/ml penicillin and 100 μg/ml streptomycin) and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Biochrom AG (Germany). Human epidermal keratinocytes (HEK) cultured in EpiLife® medium supplemented with human keratinocyte growth supplement were purchased from Life Technologies (UK). A lyophilised sample of RJ that was standardized to include minimum 3.85% (E)-10-hydroxy-2-decenoic acid was obtained from Yamada Bee Company, Inc. (Japan). FlashBAC GOLD expression system and pOET vector were purchased from Oxford Expression Technologies (UK). BlueSript plasmid was purchased from GenScript (Hong Kong) and restriction enzymes XhoI and BamHI from New England Biolabs (UK). Spodoptera frugiperda Sf9 cells obtained from Invitrogen (Germany) were grown in Sf-900II serum-free medium from Gibco (USA). Anti-MMP-9 antibody was purchased from Merck (Germany). Rabbit polyclonal anti-bee defensin-1 (Def-1) was purchased from GenCust Europe (Luxembourg), and horseradish peroxidise-conjugated secondary antibodies were obtained from Promega (USA). The following inhibitors were used in the HaCaT scratch wound assay: PD98059 (extracellular signal-regulated kinase [ERK] inhibitor, 10 μM), SB203580 (p38 inhibitor, 20 μM), BAPTA-AM (cell-permeant calcium chelator, 30 μM) and rapamycin (mammalian target of rapamycin [mTOR] inhibitor, 100 nM). These agents were obtained from Calbiochem (USA). All other reagents including mitomycin C were purchased from Sigma-Aldrich (Germany) unless otherwise stated.

Cell culture

HaCaT keratinocytes were cultured in DMEM and sub-cultured every 4 days at 37 °C in 5% CO 2 . For all experiments, cells were grown to 70 to 80% confluence and incubated in serum-free DMEM 24 h prior to treatment with water RJ extract (WRJE) or recombinant Def-1 (rDef-1). Medium was then replaced with fresh serum-free DMEM, and cultures were treated with different concentrations of WRJE (0.25–1000 μg/ml) or rDef-1 (0.05–0.5 μg/ml) for 72 h.

HEK cells were sub-cultured according to the manufacturer’s instructions, and cultures were treated with WRJE or rDef-1 as mentioned above.

Cell viability

The cytotoxic effect of WRJE on HaCaT cells or HEK was measured by the Alamar Blue assay (Life Technologies, UK) according to the manufacturer’s protocol. Results were expressed as the percentage of cytotoxicity calculated according to the manufacturer’s equation.

Water royal jelly extract preparation

RJ was suspended in sterile deionised water at a concentration of 100 mg/ml. The supernatant of the WRJE was collected by centrifugation at 16,000 g for 30 min, divided into portions and stored at −80 °C until use. Total protein content in the WRJE was measured using the Quick Start Bradford protein assay (Bio-Rad, CA, USA) as described in the instruction manual.

Heat and proteinase K treatment

Heat and proteinase K treatment were performed by incubation of WRJE at 100 °C for 5 min and WRJE with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme.

WRJE fractionation

Heat-treated WRJE was fractionated by a reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate of 0.3 ml/min with elution using a 10 to 90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. HPLC fractions were freeze-dried under vacuum, re-dissolved in PBS, and assayed for MMP-9 induction. The fraction with maximal activity was used for identification of the MMP-9 inducer.

Defensin-1 cloning, expression and purification

The cDNA fragment optimised for codon usage in Spodoptera frugiperda (Sf9) cells coding the signal peptide and mature bee defensin-1 followed by a (His) 6 -tag with XhoI site on the N-terminal and BamHI site on the C-terminal was synthesised and cloned into a BlueSript plasmid (GenScript, Hong Kong). The purchased BlueScript-Def plasmid was digested with XhoI and BamHI. The cDNA fragment was purified and then ligated into similarly digested pOET2 vector (Oxford Expression Technologies Ltd., UK). The resulting recombinant plasmid was transformed into JM 109 Escherichia coli and verified by DNA sequence analysis (GATC Biotech, Germany). The correct plasmid encoded a translational peptide containing an N-terminal signal peptide, followed by the mature peptide sequence of bee Def-1 and a (His) 6 -tag (hereafter designated rDef-1).

Recombinant baculovirus was obtained using the approach of Posse et al.43. Briefly, a Sf9 cell monolayer was co-transfected with flashback baculovirus (Oxford Expression Technologies, UK) and recombinant pOET2 transfer vector (described above), using Lipofectin (Thermo Fisher Scientific, MA, USA) following the manufacturer’s instructions. Recombinant virus was amplified by infection of Sf9 cells in serum-free Sf-900 II SFM culture at a low multiplicity-of-infection (moi), and the amplified virus was used to infect Sf9 liquid cultures at moi = 2 for protein expression. Viral titre was assessed by plaque assay. Then, 72 h after infection, culture medium was cleared by centrifugation (2,000 g, 5 min). Supernatants were loaded onto a SP Sepharose FF column (GE Healthcare, UK) equilibrated with 0.05 M phosphate buffer (pH 6.6, buffer A) and eluted using 0.5 M NaCl in buffer A.

The rDef-1 containing eluate fractions were pooled, adjusted with Triton X-100 to a final concentration of 0.1%, loaded onto 4 ml Ni Sepharose Excel resin (GE Healthcare), and eluted using 0.5 M imidazole. The peptide-containing eluate fractions were pooled again and desalted using desalting PD-10 columns (GE Healthcare). The purity of prepared rDef-1 in distilled water was determined by 16.5% Tricine-SDS-PAGE. The gel was stained with Serva Blue (Serva, Germany), and the concentration of rDef-1 was measured using the Quick Start Bradford Protein Assay (Bio-Rad).

Gelatine zymography

Conditioned media of HaCaT and HEK cell cultures were subjected to gelatine zymography as previously described44. Briefly, non-reducing LDS sample buffer (Life Technologies, UK) was added to aliquots of culture medium supernatants at a ratio of 1:4. Twenty microlitre aliquots were separated on 8% SDS-PAGE gels containing 0.5 mg/ml gelatine under non-reducing conditions. The gels were washed in 2.5% Triton X-100 for 60 min at room temperature to remove the SDS and were subsequently incubated in a developer buffer [50 mM Tris (pH 7.8), 5 mM CaCl 2 and 0.2 M NaCl] for 24 h at 37 °C. Gels were stained with 0.5% Coomassie Brilliant Blue G-250, and the bands of proteolytic activity were quantified by densitometry (Quantity One, Bio-Rad, USA).

Western blot analysis

Western blot analysis was performed using the semi-dry blotting method45. Concentrated supernatants derived from HaCaT cells cultured as described above were prepared as follows. An initial volume of 0.5 ml of culture supernatant was collected from each well (control and treated cultures). A 10-fold concentration was obtained using ultracentrifugal filter devices (10,000 MWCO; Sartorius, Germany). Equal volumes (15 μl) of the concentrated supernatants were subjected to electrophoresis using 10% SDS-PAGE gels. Proteins were transferred onto nitrocellulose membranes and probed with the anti-MMP-9 antibody diluted at 1:400 in blocking buffer. Detection was performed using horseradish peroxidise-conjugated secondary antibodies. Visualisation of the immunoreactive bands was performed using the enhanced chemiluminescence kit (Kodak, USA). Quantification was performed by densitometry (Quantity One, Bio-Rad).

For detection of native Def-1 and its recombinant form (rDef-1), a rabbit polyclonal anti-bee Def-1 antibody was utilised according to Valachova et al. (2016). Briefly, fractions from Def-1 or rDef-1 purification were electrophoresed (15 μl) on a 16.5% Tricine-SDS-PAGE gel. Proteins were semi-dry blotted as mentioned above and probed with the rabbit polyclonal anti-honeybee Def-1-1 antibody diluted 1:2000 in blocking buffer. Detection was performed using horseradish peroxidise-conjugated secondary antibodies. Immunoreactive bands were detected using a solution containing dissolved SigmaFast 3,3-diaminobenzidine tablets (Sigma-Aldrich, UK).

In vitro scratch wounding

Scratch wound analysis was performed on confluent HaCaT monolayers as described by Ranzato et al.46 with modifications. The width of the wound space was measured at wounding and at the end of treatment, via an inverted microscope equipped with a camera (Leica, Microsystem, Milan, Italy) and NIH ImageJ software (Bethesda, MD, USA). Wound closure was determined as the difference between wound width at 0 and 24 h. Briefly, HaCaT cells were seeded at 1 × 104 cells/well in 96-well plates and grown to confluency in a complete DMEM medium. A linear wound was then generated in the monolayer with a sterile 200-μl plastic pipette tip. WRJE and rDef-1 were used at 100 to 1000 μg/ml and 0.05 to 0.5 μg/ml, respectively. Where indicated, the cells were treated with various inhibitors and 0.5 μg/ml rDef-1 for 24 h.

Scratch wounding with HaCaT cells was also conducted by pre-treating these cells with 10 μg/ml MMC for 2 h in order to assess the contribution of cell proliferation in the WRJE- and rDef-1-induced in vitro wound closure.

In vitro cell migration assay

A cell migration assay was performed according to Ranzato et al.26 in transwell plates (8 μm pore size, ThinCert™ 24 Well Cell Culture Inserts for Multiwell Plates, Germany). Briefly, a total of 1 × 105 cells per well were seeded in the upper compartment of filters. The lower chamber was filled with 500 μl complete DMEM medium with WRJE and rDef-1 at concentrations 100 to1000 μg/ml and 0.05 to 0.5 μg/ml, respectively. After 24 h incubation at 37 °C, the filters were removed, stained with 0.5% crystal violet (145 mM NaCl, 0.5% formal saline and 50% ethanol) for 10 min and washed thrice with water. The upper side of the filters was scraped using a cotton swab to remove cells that had attached but not migrated. Following PBS washing of the filters, the dye was eluted from the cells with 33% acetic acid and measured at 540 nm in a plate reader (Infinite 200 Pro, Tecan).

In vivo wounding healing (excision model)

Adult male 10 to 13-week old Wistar albino rats (Velaz, Czech Republic) (n = 20) weighing 180–250 g were used. The experimental animals were housed separately and fed standard pelleted food with no restricted access to water and food during the course of experiment. The animals were anaesthetised with ketamine (Narkamon, Bioveta, Czech Republic) and xylazine (Cylariem, Germany) anaesthesia with inhalation introduction using isoflurane (Aerrane, Baxter, UK). After each painful intervention, the animals were supplied with tramadolum i.m. (Tramal, Grunenthal, Germany). Before the surgery, both flanks were shaved and disinfected with 70% alcohol. Four full thickness round excision wounds (1.5 cm in diameter) were induced (two on each side of the animal’s back). One wound was a untreated wound. The second wound was treated with vehicle only, namely carboxymethyl cellulose (CMC) gel. The third wound was treated with 5% RJ ointment in CMC. The fourth wound was treated with 0.1 mg/ml rDef-1 ointment in CMC. Wounds were dressed with Tegaderm sterile dressing (3 M Healthcare, MN, USA), which was changed every other day until wound closure. Digital photographs were taken at the time of surgery, and every other day until closure, which was defined as the time at which the wound was completely re-epithelialised and filled with new tissue. The diameter of the open wound area was measured. The wound closure area of each animal was assessed by tracing the wound diameter at days 0, 1, 3, 5, 7, 9 and 13 after wounding surgery and the wound closure rate was expressed as the percentage of open wound area.

Histological analyses

Full profile biopsies, including central wound zone and adjacent unwounded skin, were obtained on days 1, 3, 7, 11 and 15 (four animals per time interval) and fixed in 4% paraformaldehyde. Serial paraffin sections (4 to 5 µm thick) were cut and stained with haematoxylin/eosin (HE) for blinded general morphological description and an assessment of wound healing parameters and pathology, such as the presence of necrosis, inflammatory reaction, granulation, angiogenesis, epithelisation and general granulation tissue morphology. Microscopic images were captured (microscope Opton AxioPhot, Germany) with the Zeiss Axio Vision imaging software.

Study approval

All methods were performed in accordance with the national guidelines and regulations. The preparation of genetically modified Escherichia coli and recombinant peptide followed the guidelines of the Ministry of Environment of the Slovak Republic. All animal work was approved by the State Veterinary and Food Administration of the Slovak Republic (Project License 3743/14-221) following local ethical approval and was conducted in accordance with the Animal Care guidelines of the Slovak Medical University.

Statistical analysis

Data were collected from three and at least four independent in vitro and in vivo experiments, respectively. The results are presented as the means ± standard errors (SEM). All data were statistically analysed using t-tests or one-way ANOVAs for comparisons of two groups or groups greater than three, respectively. P-values less than 0.05 were considered to be significant. Analyses were performed using GraphPad Prism (GraphPad Software Inc., CA, USA).