2.1 Chemicals and reagents
The tested benvitmod powder was donated by Beijing Wenfeng Tianjipharma Company. The dried extract powder was dissolved in dimethyl sulfoxide (DMSO) and diluted up to 100mM for storage at -20℃. Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) was used to dilute the chemicals to desired concentration. The final concentration of DMSO did not exceed 0.1% (v/v) and did not affect cell viability.
2.2 Subjects
A total of 6 AD patients (3 males and 3 females, range from 25 to 74 years old) were recruited in this study. All patients were at an active stage of the disease, before administration of systemic treatment. The informed consents were obtained from each patient. All AD patients satisfied the criteria of Hanifin&Rajka[15] and Chinese criteria for AD[16]. The study was approved by the ethics committee of Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College.
2.3 PBMC preparation
10 ml of venous blood was withdrawn from all patients and controls under total aseptic technique in blood collection tubes containing K2EDTA (dipotassium ethylenediamine tetra-acetic acid) for serum and PBMCs separation. PBMCs were separated from peripheral blood by Ficoll Hypaque density gradient centrifugation and resuspended in RPMI 1640 medium (Gibco, USA).
2.4 HaCaT cell culture
The HaCaT cell line was obtained from the Peking University People’s hospital (Chinese Type Culture Collection, Beijing, China). The human keratinocyte cell line and PBMCs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). In all experiments, cells were cultured under the atmosphere containing 5% CO2 at 37℃. Prior to further association studies, the growth medium was changed to serumfree medium. Cells in the control group were treated with DMEM alone. The HaCaT cells were pretreated with benvitimod (0.1µM, 1µM, 10µM, 100µM) for 1 h and subsequently treated with TNFα (10 ng/ml) and IFNγ (10 ng/ml) and incubated for 24 h at 37˚C. In another set of cultures, the cells were coincubated with 100nM StemRegenin1 (SR1, aryl hydrocarbon receptor antagonist, Solarbio, China) for 1 h at 37˚C in a humidifed 5% CO2 incubator.
2.5 Cell viability assay
Cell viability was assessed by the CCK-8 assay according to the manufacturer’s protocol. HaCaT cells and PBMCs were collected during the logarithmic growth phase and plated in triplicate in 96-well (2 × 104 cells/well, n = 4 per group). After 24 h of inoculation, the culture medium in benvitimod groups was replaced with DMEM containing different concentrations of benvitimod (0.1µM, 1µM, 10µM, 100µM, 1mM). A group of untreated control wells (cells treated with medium) and a group of blank control wells (without cells) were established at the same time. After another 24 h of incubation with benvitimod, 10µl of kit reagent was added to 100µl cell solution and incubated for a further 60 min at 37°C. The optical densities were measured at 450 nm using a quantitative automatic microplate reader. The cell viability rate (%) was calculated using the following formula: (OCexperimental group- ODblank control )/(ODcontrol group- ODblank control)×100%.
2.6 Measurement of chemokines and cytokines
PBMCs (2×106 cells/well) were cultured in 6-well plates. The cells were then washed and treated with benvitimod of different concentrations (0.1µM, 1µM, 10µM, 100µM) for 24 hours. A group of untreated control (cells treated with serum-free medium) and a group treated with SR1 (aryl hydrocarbon receptor antagonist, 100nM) were established. The supernatants were harvested and cytokine levels (IL-1β、IL-6、TNFα、IL-4 and IL-22) were analyzed by commercial ELISA kit (eBioscience, Germany) according to the manufacturer’s instructions.
HaCaT cells (1×106 cells/well) were cultured in 6-well plates. After reaching a confluent state, the cells were washed and treated with benvitimod of different concentrations(1µM, 10µM, 100µM) in 2ml medium that contained tumor necrosis factor-α(TNFα) and interferon-γ (IFNγ) (each 10 ng/mL; R&D Systems, USA) for 24h. A group of untreated control well (cells cultured in serum-free medium), a group of treated with only TI stimulants (TNFα and IFNγ, each 10 ng/mL) and a group of treated with 100nM SR1 were established at the same time. The supernatants were harvested and IL-4, IL-10, IL-22 and TARC levels were analyzed by commercial ELISA kit (eBioscience, Germany) according to the manufacturer’s instructions.
2.7 Quantitative Real-Time-PCR (qRT-PCR)
HaCaT cells in 6-well plates were treated with TI (10ng/ml) and benvitimod of different concentrations (0.1µM, 1µM, 10µM) for 24 hours. A group of untreated control wells and a group of treated with 100nM SR1 were also established. Total RNA was isolated from the treated cells using RNA blood mini kit (Qiagen, Germany) according to the manufacturer’s instructions. The concentration and purity of RNA were measured at 260nm and 280nm using NanoDrop2000 Spectrophotometer (Thermo Scientific, USA). A260: A280 ratio greater than 1.8 and lower than 2.1 indicated highly pure RNA. The integrality of RNA was observed by agarose gel electrophoresis. RNA was then reverse transcribed with the Revert Aid First Strand cDNA Synthesis Kit (Reverse Transcription Kit, Tiangen, China). The reverse transcription mixture was incubated for 15 min at 42°C followed by 5 min at 95°C. The mRNA levels of AhR, CYP1A1, FLG, IVL and TSLP were tested by real-time quantitative polymerase chain reaction (RT-PCR). RT-PCR was performed using the SYBR Green Dye method which was carried out using cDNAs supplemented with SYBR Green supermix (Bio-rad, Hercules, CA, USA). The PCR amplification used gene-specific primers for β-actin (forward, 5’-TGGCACCCAGCACAATGAA-3’; reverse, 5’-CTAAGTCATAGTCCGCCTAGAAGCA-3’), AhR(forward, 5’-ATACCGAAGACCGAGCTGAAT-3’; reverse, 5’-CCAGCAGACACCTTAGACGACT-3’), CYP1A1(forward, 5’-GTCATCTGTGCCATTTGCTTTG-3’; reverse, 5’-CAACCACCTCCCCGAAATTATT-3’), FLG(forward, 5’- TGAAGCCTATGACACCACTGA-3’; reverse, 5’- TCCCCTACGCTTTCTTGTCCT-3’), FLG(forward, 5’- TGAAGCCTATGACACCACTGA-3’; reverse, 5’- TCCCCTACGCTTTCTTGTCCT-3’), IVL(forward, 5’- ACAAGGGAAGAGAGAGCCACTG-3’; reverse, 5’- TGTAGAGGGACAGAGTCAAGTTCA-3’), TARC(forward, 5’- ACTGCTCCAGGGATGCCATCGTTTT-3’; reverse, 5’- ACAAGGGGATGGGATCTCCCTCACTG-3’), TSLP(forward, 5’- CCCAGGCTATTCGGAAACTCAG-3’; reverse, 5’- CGCCACAATCCTTGTAATTGTG-3’). The PCR protocol consisted of a cycle at 95°C for 60 seconds followed by 40 cycles consisting of 15 s at 95°C, 15 s at 60°C and 15 s at 72°C. β-actin was used as an endogenous reference. The average Ct was calculated for the target genes and internal control (β-actin). ΔCt (Cttarget - Ctβ−actin) values were determined. The expression levels of target genes in AD patients was determined relative to controls as 2−ΔΔCt, where ΔΔCt = ΔCt (patient or control) – ΔCt (average for controls). The results were converted into relative expression in this article.
2.8 Immunofluorescence
HaCaT cells (1×106 cells/well) were cultured in 6-well plates. After washing with PBS, they were fixed with acetone for 10 min. After 24 h of inoculation, the culture medium in benvitimod groups was replaced with DMEM containing different concentrations of benvitimod (1µM, 10µM, 20µM). A group of untreated control wells (cells treated with medium) was established at the same time. After washing in PBS with 0.5 mg/ml Tween 20 (PBST), the fixed HaCaT cells were treated with 100 mg/mL bovine serum albumin (BSA) in PBST for 1 hour. Samples were then incubated with anti-AhR rabbit IgG (1:100 dilution; Abcam, Cambridge, MA, USA) overnight at 4 ℃. Next, samples were washed with PBST for three times and incubated with Alexa Fluor 546-conjugated anti-rabbit secondary antibody(Beyotime Biotechnology, China) for 1 hour at room temperature. Samples were then washed with PBST and mounted with SlowFade Gold Antifade Mountant (Beyotime Biotechnology, China) after nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, China) for 10 minutes. All samples were analyzed using an fluorescence microscope (Keyence, Osaka, Japan).
2.9 Western-blot
HaCat cells (1×106 cells/well) were collected by centrifugation, washed twice with phosphate buffered saline, and suspended in extraction lysis buffer (Solarbio, China) containing protease inhibitors (Solarbio, China). The protein concentration was determined using a protein assay reagent (Solarbio, China) according to the manufacturer’s instructions. Equal amounts of nuclear extract (30µg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was incubated with blocking solution (5% bovine serum albumin), followed by overnight incubation at 4°C with the appropriate primary antibody. The following primary antibodies and dilutions were used: mouse anti human GAPDH antibody (1:1000 dilution; Beyotime Biotechnology, China), rabbit anti-human FLG antibody (1:1000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-human IVL antibody (1:1000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-human TSLP antibody (1:1000 dilution; Abcam, Cambridge, MA, USA), anti-signal transducer and activator of transcription (STAT) 1, and anti-phospho-STAT1 antibody (1:1000 dilution; Cell Signaling Technology, Danvers, USA). The membranes were washed three times with Tris-buffered saline (TBS, 20 mM Tris, 0.2 M NaCl, pH 7.5) containing 0.05% Tween 20 (TBST), and then incubated with a 1:2000 dilution of a horseradish peroxidase-conjugated secondary antibody (Beyotime Biotechnology, China) for 1 hour at room temperature. The membranes were again washed three times with TBST, and then developed using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). Image capture was performed using ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA). To investigate multiple protein targets under the same treatment condition, the blot was stripped and reused. Equal loading of samples was confirmed by GAPDH levels in the whole-cell lysates. The integrated optical density for the protein bands was then analyzed using ImageJ software (version 1.46r; NIH, USA), and the values were normalized to GAPDH. The relative absorbance ratios of target protein to GAPDH were defined as the respective relative values of target proteins.
2.10 statistical Analysis
All the data in this study were obtained from at least four independent experiments and are presented here as the mean ± SEM, with n = 5. A two-tailed unpaired Student’s t test was used to analyze data between two groups. One-way analysis of variance was used to detect significant differences between the control and treatment groups. Dunnett’s test was used for multiple comparisons. Chi-square, Mann-Whitney U tests and Kruskal-Wallis test were used for non-normally distributed variables. These analyses were performed using GraphPad Prism (GraphPad Software 5.0, USA). Differences between groups were considered significant at P < 0.05.