ES preparation
The dried ES was purchased from Wonkwang PHARMACEUTICAL CORPORATION (Iksan, Korea). ES was washed twice with distilled water followed by drying, then extracted with 70% ethanol at room temperature for 3 days. The extract was concentrated with a vacuum evaporator and stored at 4 °C before experiments. The yield of ES extract was 6.14%. A voucher specimen (JUHES-1660) has been deposited at Department of Oriental Medicine Resources, Chonbuk National University (Iksan, Korea).
Preparation of P.acnes
P. acnes (KCTC 3315, Daejeon, Korea) was obtained from the Korean Collection for Type Culture (KCTC, Daejeon, Korea) and grown under anaerobic condition in 10 ml of GAM (Nissui Pharmaceutical, Japan) liquid medium at 37 °C for OD600 = 1.0 (logarithmic growth phase). A total cell count of 10 ml of P.acnes suspension was approximately 1.34 × 109 colony forming unit (CFU). P. acnes were harvested by centrifugation at 4000 rpm for 15 min at 4 °C to remove supernatant. Bacterial pellets were washed three times with 10 ml of PBS and finally suspended in 1 ml of PBS. The P. acnes suspension was incubated at 80 °C for 30 min for heat-killing reaction. To use cell stimulation heat-killed P. acnes suspension was stored at 4 °C until use. To use in vivo experiment living P. acnes suspension was stored at − 80 °C until use.
Antibacterial assay
ES was dissolved in dimethyl sulfoxide (DMSO) at different concentrations (100, 200 mg/ml). Each concentration of ES was then impregnated onto a paper disc (8 mm in diameter) and placed on the top of GAM agar plate containing 100 μl of bacterial solution containing P. acnes. These plates were incubated at 37 °C for 48 h under anaerobic condition. Tetracycline was employed as a positive control. Minimum inhibitory concentration (MIC) test was performed in sterile 96-well plates using broth dilution method. Briefly, bacteria were cultured to stationary phase for 48 h at 37 °C. The turbidity and cell numbers were measured 0.418 at 620 nm and 1.64 × 107 CFU, respectively. The cultivated bacteria was added into microplate at 0.5% of total volume (200 μl). ES extract was adjusted to concentrations through serial dilution in culture medium into 0 to 9 mg/ml. After incubating at 37 °C in an anaerobic jar for 48 h, the turbidity was obtained on a microplate ELISA reader as an indicator of bacterial growth.
To test minimum bactericidal concentration (MBC), 1 μl of various concentrations of the ES extract mixed with diluted solution of P. acnes for 48 h, 37 °C. And then MBC was performed by sub culturing the MIC dilutions on the sterile GAM agar broth. The lowest concentration of the extract in which bacteria failed to grow (99% no growth) was reported as MBC.
Lipase activity
P.acnes was grown in brain heart infusion (BHI) broth, and 100 μl amounts of cell suspensions (5.0 × 108 cells/mL) in BHI broth with final concentrations of 0.01, 0.1, 1, 10, 100 μg/mL ES were added to wells of 96well plates. The plates were anaerobically incubated at 37 °C for 24 h. Fifty microliter amounts of supernatants were centrifuged and the supernatants mixed with 50 μl of 10 mM 4-methyl umbelliferyl oleate (4-MUO) (Sigma Aldrich, St. Louis, USA) dissolved in 13 mM Tris-HCL, 0.15 M NaCl, and 1.3 mM CaCl2 (pH 8.0). The mixtures were incubated for 30 min at 25 °C under light illumination. Enzymatic reactions were terminated by adding 100 μl of 0.1 M sodium citrate (pH 4.2). The levels of 4-methylumbelliferone released by the lipase were measured using a fluorometric microplate reader (Fluoroskan Ascent™; Thermo Fisher Scientific, MA, USA); the excitation wavelength was 355 nm and the emission wavelength was 460 nm.
Cell viability assay
Human monocyte THP-1 cells were maintained in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, WELGENE, South Korea) and 1% penicillin (Gibco, USA) at 37 °C in an atmosphere with 5% CO2.
MTT assay was performed to measure cell viability. Briefly, THP-1 cells (3.0 × 104 cells/well) was incubated with various concentration of ES (0.1–10 μg/ml) for 24 h. MTT solution (500 μg/ml) was then added to each well and incubated at 37 °C for 8 h. Formazan crystal produced by living cell was dissolved in DMSO. The absorbance of each well was measured at wavelength of 540 nm on a microplate ELISA reader.
Enzyme-linked immunosorbent assay (ELISA)
THP-1 cells (3.0 × 105 cells/well) were pre-treated with indicated concentrations of ES (0.1–10 μg/ml) for 1 h followed by stimulation with heat-killed P. acnes for 18 h. The levels of IL-1β, IL-8, and TNF-α in culture media were measured with an ELISA kit (BD Pharmingen, San Diego, CA, USA). The absorbance of the ELISA plate was measured at wavelength of 405 nm using an automated microplate ELISA reader.
RNA isolation and real- time RT PCR
Total cellular RNA was isolated from human monocyte THP-1 cells using easy-BLUE reagent Kit (iNtRON Biotechnology, Seoul, South Korea). Total RNA was used as template for first-strand cDNA synthesis using a Power cDNA Synthesis Kit (iNtRON Biotechnology, Seoul, South Korea) according to the manufacturer’s instructions. The transcription levels of genes were determined with a StepOnePlus Real-time PCR System (Applied Biosystems, Foster City, CA, USA). The relative gene expression was calculated using the comparative CT method with StepOne Software v2.1 (Applied Biosystems, Foster City, CA, USA). The expression of β-actin mRNA was used as an endogenous control. We used TNF-Forward primer 5’-TTACGCCTTTGAAGTTAGCAG-3′ and TNF-Reverse primer 5’-CGTCCAAATACATCGCAAC-3′ for TNF-α, 5′- TCTTTGAAGAAGAGCCCGTCCTC- 3′ /5’-GGATCCACACTCTCCAGCTGCA- 3′ for IL-1β, and 5′- GAATACTCTATTGCCGATGGT-3′/5’-CGATGGGTTTGCGTTTG-3′ primers for β-Actin as an internal control.
Western blot analysis
Stimulated cells were rinsed with ice-cold PBS and lysed using lysis buffer (iNtRon Biotech, Seoul, South Korea) for 1 h. Total cell lysates were centrifuged at 12,000×g at 4 °C for 10 min to obtain supernatants. After bicinchoninic acid (BCA, Sigma) protein quantification assay, the supernatant was mixed with 2× sample buffer, boiled at 95 °C for 5 min, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane (Roche Diagnostics, IL, US). These membranes were blocked with 5% skim milk in PBS-Tween-20 (PBST) for 1 h at room temperature followed by overnight incubation with anti-phospho-JNK, anti-p38, and anti-ERK antibodies at 4 °C. After washing three times with PBST, these membranes were incubated with secondary antibodies for 1 h at room temperature followed by three times of washes with PBST. The protein-antibody complexes were visualized with ECL Western blotting Luminol Reagent (Santa Cruz Biotech, CA, USA). Images were recorded with an LAS-4000 image reader (Fujifilm Life Sciences, Tokyo, Japan).
Experimental animal model
All experimental protocols (CBNU2016–085) were approved by the Committee on the Care of Laboratory Animal Resources, Chonbuk National University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Male BALB/c mice (6 weeks old) were obtained from SAMTAKO (Osan, South Korea). They were individually housed in polycarbonate cages and maintained under constant temperature (25–27 °C) with a 12 h light-dark cycle. They were provided free access to standard diet and tap water. These animals were allowed to acclimate to these conditions for at least 7 days before the experiment.
These mice were randomly divided into 4 different groups (4 mice/group) as follows: B: non-treatment, PA: Live P. acnes (1.34 × 109 CFU/ 20 μl PBS) was intradermally injected into the left ear. The right ear was received an equal amount of PBS. PA/ES 1 mg and PA/ES 10 mg with live P. acnes were intradermal injected into both the left and right ears. At 24 h after the injection, ES (1 or 10 mg/ml in PBS) was applied to the surface of the right ear skin of each group. At the end of each treatment period, these animals were sacrificed by cervical dislocation and their ears were measured using a micro-caliper (Mitutoyo, Kanagawa, Japan).
Histological analysis
Ear section sample was fixed with 10% formaldehyde, embedded in paraffin wax, routinely processed and sectioned into 4-μm-thick slices. These ear sections were stained with hematoxylin and eosin (H&E) followed by examination with a light microscope to determine the presence of edema and inflammatory cell accumulation.
HPLC-MS
The extract of ES was dissolved in MeOH into 0.1 mg/ml. Gallic acid (Sigma aldrich chemie GmbH, Germany), protocatechuic acid (Hwi analytik GmbH, Germany), quercetin (Tokyo Chemical Industry, Tokyo, Japan) and kaempferol (Santa Cruz Biotechnology Inc., USA) were dissolved in MeOH for analysis, either. HPLC was performed on an Agilent 1100 system (Agilent Technologies, Waldbronn, Germany) with a photodiode array detector DAD (G1315D) and Agilent 1100 series quard pump (G1311A), and an Agilent 6410 Triple Quadrupole LC/MS mass spectrometer (Agilent Technologies, Waldbronn, Germany) coupled with an ESI (electrospray ionization) interface and an ion trap mass analyzer. The ESI (electrospray ionization) source was operated in negative ionization modes. Analysis of included compounds were performed under the following conditions: column, TSK-gel ODS-80Ts (Tosoh Co., Tokyo, Japan 4.6 mm X 150 mm); mobile phase, 0.1% formic acid (solvent system A) and CH3CN (solvent system B) in a gradient mode (B from 20 to 80% in 30 min); sample injection, 5 μl; flow rate, 0.5 ml/min; temperature, 30 °C, UV wavelength, 254 nm and 350 nm. High-purity nitrogen was used as dry gas at a flow rate at 10 L/min, gas temperature at 300 °C; fragmentor voltage 150 V. Nitrogen was used as nebulizer at 30 psi and capillary voltage, ±4000 V.
Statistical analysis
All results are presented as mean ± S.E.M. Results were analyzed using Graph Pad Prism version 5.0 program (Graph Pad Software, Inc., La Jolla, CA, USA). One-way analysis of variance with Tukey hoc post test was used to determine the differences. Statistical significance was considered when P value was less than 0.05.
