Cell culture and reagents
BMMCs isolated from male BALB/c mice (n = 1) were cultured for up to 10 weeks in RPMI-1640 media containing 2 mM L-glutamine, 0.1 mM nonessential amino acids, antibiotics, and 10 % fetal bovine serum (FBS), with 20 % pokeweed mitogen-stimulated spleen condition medium (PWM-SCM) as a source of interleukin-3 (IL-3). After 3 weeks, greater than 98 % of the cells were verified as BMMCs according to a previously described procedure [6].
Male BALB/c mice (5 weeks old) were purchased from Samtako BioKorea (Osan, Korea). Mice were observed every day for one week during quarantine and acclimation. All animals were maintained under standard conditions of temperature (22.5 ± 0.5 °C), humidity (42.6 ± 1.7 %), 12 h lighting (8:00 AM–8:00 PM, 290 lx), ventilation (10 –15 times per hour), and diet (Teklad Global Diets, Harlan Laboratories Inc., USA). This study was conducted according to the guidelines listed in the Pharmaceutical Affairs Act of Korea Food and Drug Association (KFDA) and approved by the Animal Care and Use Committee of the Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea; reference number #14–035) and performed according to the guidelines of the Animal Care and Use Committee at KIOM.
The HaCaT cell line was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified 5 % CO2 incubator.
The ingredients of the complete cell culture medium, FBS, and antibiotics were purchased from Lonza (Basel, Switzerland). Recombinant human TNF-α, IFN-γ, and enzyme-linked immunosorbent assay (ELISA) kits to measure RANTES and TARC were purchased from BioLegend (San Diego, CA, USA). Cell counting kits (CCKs) for the cell cytotoxicity assay were purchased from Dojindo Molecular Technologies (Kumamoto, Japan). β-Hexosaminidase (p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside; PNP-GluNAc) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary and secondary antibodies used for Western blotting were purchased from Cell Signaling Technology (Boston, MA, USA). All other chemicals were of reagent grade. All experiments were performed at least in triplicate.
Preparation of WSR
Sanguisorbae Radix was obtained from Yeongcheon Oriental Herbal Market (Yeongcheon, Korea). All voucher specimens were deposited into the herbal bank of the KM-application center, Korea Institute of Oriental Medicine (KIOM; Daejeon, Korea) after verification by Professor Ki-Hwan Bae of the college of Pharmacy, Chungnam National University (Daejeon, Korea). To prepare the WSR, dried SR pieces (50.0 g) were placed in 1,000 mL distilled water and then extracted by 3 h of heating at 115 °C (Gyeongseo Extractor Cosmos-600, Inchon, Korea). Following extraction, the solution was filtered using standard testing sieves (150 μm) (Retsch, Haan, Germany) and freeze-dried. The freeze-dried extract powder was dissolved in dimethyl sulfoxide (DMSO) and centrifuged at 14,000 rpm for 10 min. The resulting supernatant was filtered (0.2 μm pore size) and then stored at 4 °C prior to use. The acquisition was 5.3642 g and the yield 10.73 %. The powder of WSR was dissolved in 10 % DMSO solution for all experiments. All experiments were performed at least in triplicate, and contain a control group as a vehicle control group containing 0.1 % DMSO.
Cell cytotoxicity assay
Cell cytotoxicity was analyzed using a CCK. Cells were seeded onto 96-well plates (2 × 105 cells/well). After 24 h, WSR was added at concentrations of 10, 50, 100, and 200 μg/mL, and the plates were incubated for 24 h at 37 °C in a 5 % CO2 incubator. CCK solutions were added to each well and the cells incubated for 1 h. Optical density was measured at 570 nm using an ELISA plate reader (Infinite M200, Tecan, Männedorf, Switzerland).
β-HEX release assay
β-Hexosaminidase (β-HEX) was quantified by spectrophotometric analysis of the hydrolysis of PNP-GluNAc. For cell stimulation, BMMCs (5 × 105 cells/mL) were sensitized overnight with 100 ng/mL anti-dinitrophenyl (DNP) and then stimulated for 15 min with 25 ng/mL DNP-human serum albumin (HSA). To investigate the effects of WSR, varying concentrations were added 2 h prior to the addition of DNP-HSA. The supernatants were harvested according to a previously described procedure [7].
Measurement of chemokine production
HaCaT cells (1 × 106 cells/well) were seeded onto 6-well plates. After 18 h, the cells were treated with WSR at concentrations of 1, 10 and 50 μg/mL for 2 h at 37 °C in an atmosphere of 5 % CO2. After treatment, TNF-α/IFN-γ (each 10 ng/mL) was added to each well and incubated for a further 24 h. The culture medium was then harvested and the levels of chemokines in the supernatants detected using ELISA kits according to the manufacturer's instructions.
RNA isolation and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from HaCaT cells using the RNA-Spin total RNA extraction kit (iNtRoN, Daejeon, Korea) according to the manufacturer’s instructions. Reverse transcription was carried out in a 20 μl reaction with 1 μg of total RNA transformed into cDNA using AccuPower CycleScript RT premix (Bioneer). For measurements of TARC, RANTES, MDC, IL-8 and β-actin mRNA, the PCR conditions were as follows: 12 cycles of primer annealing at 25 °C for 30 s, cDNA synthesis at 45 °C for 4 min, melting of the secondary structure and cDNA synthesis at 55 °C for 30 s, and heat inactivation at 95 °C for 5 min. The PCR-amplified primers used in this study are described in previously described [5]. Gene expression was quantified by real-time PCR using the AccuPower 2× Greenstar qPCR Master (Bioneer) according to the following protocol: pre-denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. Amplifications were carried out using QuantStudio 6 (LifeScience, ABI, USA), The fold change in the expression of the target gene relative to the control was normalized to β-actin using the 2-ΔΔCt method.
Western blotting
Protein expression was evaluated by Western blotting according to standard procedures. The cells were pre-treated with WSR and stimulated with TNF-α/IFN-γ during incubation for the indicated periods at 37 °C. Cells were then harvested and resuspended in radio-immunoprecipitation assay lysis buffer (Millipore, Bedford, MA, USA) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). After a further round of centrifugation, cell debris was discarded and the protein concentration in the supernatant was determined using Bradford’s reagent. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a nitrocellulose membrane (Millipore, MA, USA) followed by blocking with 3 % bovine serum albumin (BSA) in Tris-buffered saline containing 0.1 % Tween 20. The membrane was incubated first with the primary antibodies at 4 °C overnight followed with horseradish-peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Specific proteins were detected using Clarity™ West ECL Substrate (Bio-Rad Laboratories, CA, USA).
Statistical analysis
Data were analyzed using GraphPad Prism software (ver. 5.0 GraphPad Software, San Diego, CA, USA). Results are expressed as the mean ± standard error of the mean (SEM) and were evaluated using Student's t-test or analysis of variance (ANOVA). A p value less than 0.05 was considered statistically significant.
