Preparation of Bacillus/Trapa japonica fruit extract ferment filtrate (TJFs)
T. japonica (Texa and representative voucher specimen number: KP255650) was purchased from BS corporation (BS corporation, KOREA). It was grown in China. The hot water extraction of T. japonica was heated in a 37 °C by adding T. japonica and distilled water, then filtered out the vacuum and concentrated in the water bath. Then, the hot water extraction of T. japonica was fermented using two species of microorganisms: Bacillus methulotrophicus and Bacillus subtilis. T. japonica and medium were mixed with glucose, yeast extract and soytone. The final fermentation was performed by mixing microorganisms. It was fermented 72 h in a 37 °C fermentor (Fermentec, KOREA). After fermentation was completed, it was centrifuged and filtered with a 0.2 μm filter to completely remove the microorganisms. The fermented T. japonica extract was sequentially separated into Hexane, CH2Cl2, EtOAc, N-BuOH and water layers. Fractions were run in the order Hexane, CH2Cl2, EtOAc, and N-BuOH. Afterwards, the water layer was separated by using the Silica gel column (Waters, USA) was performed to divide into six fractions (fraction 1 ~ 6). Afterwards Sehpadex-LH20 column (Waters, USA) was performed to divide into six fractions. Compound 1–6 was then obtained via HPLC prep HPLC Xterra C18 ODS column (Waters, USA). fraction was refined using HPLC (Waters, USA). The mobile phase consisted of distilled water: ACN: MeOH solution (90:5:5, v/v) pumped at a rate of 1 ml/min. The combined filtrate was concentrated under vacuum, and completely dried by freeze drying. TJFs (consisting of 6 compounds) powder was dissolved in distilled water. GH Nam and SM Kang undertook the identification of TJFs in this study.
The amount of peptide in TJFs were measured using a lowry assay. It were Calculated by a standard curve using Bovine serum albumin (Sigma, USA). Lowry solution consists of a mixture of solution A and B (Solution A: 4 mg/mL NaOH and 20 mg/mL Na2CO3 in water Added 2 g of NaOH and 10 g of Na2CO3 to 400 mL water while stirring until completely dissolved, then adjust volume to 500 mL. Solution B: 10 mg/mL Potassium Sodium Tartrate and 5 mg/ mL CuSO4 in water). Add 100 mg Potassium Sodium Tartrate and 50 mg CuSO4 (cupric sulfate to 8 mL of water in a tube. Shake the mixture until solids are completely dissolved, adjust volume to 10 mL (50:1 mix of solutions A and B). Prepare samples by adding 2, 5, and 10 μL of sample into a glass tube and adjust total volume to 200 μL. To each tube added 1 mL Lowry’s Solution, vortex, wait for 15 mins. To each tube added 100 μL 1.0 N Folin’s Phenol reagent (Sigma, USA) while vortexing, wait for 30 mins. Absorbance is measured at 750 nm.
Wst-1 assay kit was purchased from Daeillab (Daeillab, Korea). LY294002 (PI3K/Akt inhibitor) and PD98059 (Erk inhibitor), L-Ascorbic acid were purchased from Sigma Aldrich (Sigma Aldrich, USA). BIO (GSK-3β inhibitor) was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Specific antibodies such as p-Akt, total-Akt, β-actin, Cyclin E, p-cdk2, p21 were obtained from Cell Signaling Technology (Beverly, USA). and p-GSK-3β, total-GSK-3β, p-Erk, total-Erk, Active-caspase-3 antibodies were purchased from Abcam (Cambridge, USA). Muse™ Muse™ Cell Cycle Kit (MCH100106) and Muse™ Cell Analyzer (PB4455ENEU) were purchased from Millipore (EMD Millipore Corporation, Germany). 3M™ Tegaderm (sterile barrier to external contaminants) was purchased from 3 M (3 M, USA).
Human hair Follicle dermal papilla cells were obtained from CEFO (CEFO, KOREA). Human hair Follicle dermal papilla cells were grown in DMEM medium (Hyclone, USA) containing 10% Fetal bovine serum (Hyclone, USA) and 1% antibiotics (100 mg/streptomycin, 100 U/ml penicillin) at 37 °C in a 5% CO2 atmosphere.
Cells were seeded at 3.8 × 105 cells/ml in a 12-well plate for 24 h and were incubated with TJFs (0.1 and 1%) for 24–48 h. Certain inhibitors (LY294002, BIO, PD98059) were treated for 24 h. Following incubation with the TJFs, the cells were incubated with a 100 μl/ml Wst-1 solution (Daeillab, Korea) for 60 min. Then, the optical densities of the solutions were quantified at a 450 nm wavelength by using a FLUOstar Omega (BMG labtech, Germany).
Cell migration assay
Seed the cells in a 6-well plate and culture until confluent. Then, using a 200 μl pipette tip (CORNING, USA) make a straight scratch, simulating a wound and were incubated with various concentrations of TJFs (0.1 and 1%) for 24 h. The wound healing area was photographed under a microscope (Carl Zeiss, USA). The photographs were taken at a magnification of × 200.
Immunofluorescence (IF) staining
Cells were seeded at 1 × 105 cells/ml in a 12-well plate with cover glasses and incubated for 24 h. Following treatment with TJFs (1%), The cells were stained with 0.7 μM Hoechst 33342 for 10 min and fixed with 10% Neutral buffered formalin for 20 min. Then the cells were washed with PBS twice, Cells were permeabilized with 100% MeOH and blocking in 10% normal goat serum. Cells were then incubated overnight at 4 °C with active-caspase 3 primary antibody. On the second day, cells were washed with PBS and incubated with a secondary antibody for 1 h. The coverslips were mounted for fluorescence microscope observation. Subsequently, the cells were observed using a confocal microscope (Olympus, Japan).
Determination of cell cycle
Cells were seeded at 9.5 × 105 cells/ml in a 6-well plate. After 24 h incubation, cells were treated with various concentrations of TJFs (0.1 and 1%) for 24 h. Following incubation with TJFs, the cells were resuspended with PBS. And slowly add 0.2 ml of 70% ethanol. After incubate for at least 3 h at − 20 °C, the fixed cells were analyzed in Muse™ Cell Analyzer (Merck Millipore Co.).
Cells were seeded at 1 × 105 cells/ml in a 24-well plate for 24 h transfer serum-free media and were incubated with various concentrations of TJFs (0.1 and 1%) for 24 h. Following incubation with the TJFs, incubated with SRD5A1 covered 96-well microplate at 37 °C for 60 min. Transfer 100 μl of 1X Biotinylated SRD5A1 Detector Antibody into one 96-well plate and incubate at 37 °C for 60 min. Then, add 100 μl 1X Avidin-HRP Conjugate, seal the 96-well plate (e.g. with a foil) and stand for 60 min at 37 °C. Add 90 μl of TMB Substrate into each well and incubate at 37 °C for 15–30 min. Add 50 μl of Stop Solution into each well in same order as for substrate. Tap plate gently to mix. Measure the absorbance at 450 nm with a FLUOstar Omega (BMG labtech, Germany).
Cells were seeded at 1 × 106 cells/ml in a 6-well plate. After a 24 h incubation, cells were treated with various concentrations of TJFs (0.1 and 1%) for 24 h. Certain inhibitors (LY294002, BIO, PD98059) were pre-treated for 60–120 min prior to treatment with TJFs (1%). After a 24 h, cells were rinsed twice with PBS, scraped with a lysis buffer [50 mM Tris-HCl (pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate] with Halt™ Protease and Phosphatase inhibitor cocktail (ThermoFisher, USA) and subjected to the western blot analysis. Protein quantification was performed using a Bradford assay and 50 μg of protein were loaded per lane. Primary antibodies reacted overnight at 4 °C and secondary antibodies reacted for 120 min at 4 °C.
In ovo chicken Chorioallantoic membrane (CAM) assay
Sterilize the Fertile eggs with 70% ethanol and put them in 37 °C, 45 ± 5% Humidity incubator. After 72 h incubation, drill a hole in the air sac of the Fertile egg and check the vessel. Then, put a disk containing TJFs (0.1 and 1%) and Vitamin C (0.1%) with sterilized silicon ring in the Chicken Chorioallantoic Membrane. block it with a tegarderm (sterile barrier to external contaminants) and put it back into 37 °C, 45 ± 5% Humidity incubator. After 72 h, Verify the angiogenensis effects of the TJFs through a microscope (Carl Zeiss, USA).
Organotypic 3D cell culture model
6-well plates were pre-coated with type I collagen and incubated for 10 min in room temperature. 1 × 106 cells/ml Human hair Follicle dermal papilla cells were seeded on 0.3-μm pore size cell culture insert plate and incubation with Matrigel and type I collagen mixture in 45 min. After cell mixture was detached from the insert plate, incubated for 3 days in the complete medium. After incubation, the 3D cell formation medium was placed in the bottom well and cultured for 2 weeks while changing the medium with TJFs (1%) every 2 days.
The organotypic 3D cell cultures were fixed in 10% Neutral buffered formalin for 24–48 h, embedded in paraffin and sectioned into 5 μM thick slices. Consecutive thin cryosections (5 μM) of optimum cutting temperature compound (Sakura Finetek, USA) embedded sections were fixed in acetone at 4 °C for 10 min. Following washing in PBS, sections were treated with 3% H2O2 for 10 min to block endogenous peroxidase activity, and the sections were inhibited with 10% normal goat serum. Then, the sections were blocked and washed in PBS and incubated with a primary antibody overnight at 4 °C. On the second day, the sections were washed with PBS and incubated with a secondary antibody for 2 h, the sections were observed using a confocal microscope (Olympus, Japan).
All the experiments were repeated at least three times and analyzed using t-tests (SPSS 20.0, USA). p<0.05 was considered to indicate a statistically significant difference.