Reagents
AS-IV was purchased from Nanjing Zelang Med-Tech Co., Ltd (QZ-0811102;Nanjing, China; purity ≥ 99%). Dexamethasone was purchased from TianYao Pharmaceutical Co., Ltd (41303101; Hubei, China). Montelukast and Loratadine were from MSD Pharmaceutical Co., Ltd (K004567; Hangzhou, China) and XianLin BaoYa Pharmaceutical Co., Ltd (12CRXF1018; Shanghai, China), respectively.
Experimental AD relapsing model and medication
Male BALB/c mice of 6-8 weeks old were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (SCXK-2012-0002, Shanghai, China). Mice were maintained at Nanjing University of Chinese Medicine under specific pathogen-free conditions. All procedures and animals were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine and strictly performed according to the Guide for the Care and Use of Laboratory Animals. The FITC-induced AD-Re mouse model was established as reported previously [13]. Briefly, mice abdomens were shaved with an area of approximately 3×3cm2 on day 0, topically sensitized with 80 μl of 1.5% FITC (Sigma-Aldrich, St. Louis, MO, USA) solution on days 1 and 2, and the right ear was treated with 20 μl of 0.6% FITC solution on day 6 (elicitation). The initial allergic inflammation was established on day 7 (24 h after elicitation). From day 7 to day 14, mice were given no treatment, allowing inflammation to subsided naturally, which confirmed by the ear swelling measurement with a thickness gauge (7301; Mitutoyo, Kawasaki, Japan). Mice were then preventively treated with AS-IV (12.5, 25, 50 mg/kg/day, i.g.), Lor (1.3 mg/kg/day, i.g.), Mon (1.3 mg/kg/day, i.g.), or Dex (0.67 mg/kg/day, i.p.) for 10 days in remission phase of AD-Re. Medications were terminated at indicated time points before re-challenge with 0.6% FITC on the right ear (to induce AD relapse). Ear swelling was calculated as the thickness difference between the left and right ears. Mice were then sacrificed and samples taken. Thymus index was calculated as the ratio of the weight of thymus glands to the body weight of mice.
Determination of cytokines and IgE production
Production of IL-4, IL-5, IL-13, and IFN-γ in mice ear tissue homogenates and IgE in serum were measured by ELISAs (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions.
Histology
Ear tissue were fixed in 10% formalin immediately after mice were euthanized. Paraffin-embedded sections (4 μm) of the ear tissue were stained with hematoxylin and eosin (H&E) for histology analysis.
Pharmacokinetic study of AS-IV in vivo
To evaluate the pharmacokinetics of AS-IV in remission of AD-Re model, male BALB/c mice of 6-8 weeks old were divided into two groups (Control and AD-Re). Control: mice were treated with AS-IV (25mg/kg/day, i.g.) for 10 days; AD-Re: mice were treated with AS-IV (25mg/kg/day, i.g.) from day 14 to day 23 for 10 days in remission phase of AD-Re. Blood samples were collected into heparinised tubes via the oculi chorioideae vein at 0.167, 0.333, 0.667, 1, 2, 4, 8, 12 and 24 h after the final administration (4 animals/group were collected for each time point). The blood samples were centrifuged at 4000 rpm for 15 min, and the plasma samples obtained were stored at -20 oC until the analysis.
HPLC-MS determination of AS-IV
The determination of AS-IV was performed on the Waters ZQ 2000 LC/MS Waters 2695 HPLC System with 2996 PDA Detector (Waters, USA). The chromatographic analysis of AS-IV was performed on a Waters Chrom-matrix GP-C18 ODS column (4.6×250 mm, 5μ) at 30 oC. The mobile phase was methanol and water (75:25, v:v) at a flow rate of 0.2 mL/min. The positive ion electrospray ionization mode was used for mass spectrometry. The precursor ion and product ion are m/z 807.2→627.2+ for AS-IV and m/z 493.8→368.9+ for glibenclamide, respectively. The collision energy for AS-IV and glibenclamide was 53 and 14 eV, respectively. The MS conditions were optimized as following: ion spray voltage, 4600v; nebulizer gas pressure (N2), 15 psi; drying gas flow (N2), 10 L/min; Desolvation Temp, 360 oC.
Western blots
Ear tissue lysates were subjected to SDS-PAGE and Western blots analysis with the use of anti-TLR4 (1:1000 dilution; Santa Cruz Biotechnology), anti-TLR8 (1:1000 dilution; MultiSciences), anti-MyD88 (1:1000 dilution; Cell Signaling Technology), anti-TIRAP (1:1000 dilution; GeneTex), and anti-GAPDH (1:1000 dilution; Cell Signaling Technology) antibodies followed by horseradish peroxidase-electrochemiluminescence (HRP-ECL) detection (Millipore).
Quantitative real-time PCR
Gene expression of TLR2, TLR3, TLR4, TLR8, TLR9 and NF-κB in ear tissue were detected by quantitative real-time PCR as described previously [15]. The oligonucleotide sequences of primers (GenScript Biotech Corp, Nanjing, China) applied were 5’-ATCAGTCCCAAAGTCTAAAGTCG-3’ (S) and 5’-ATGCCAGCTTCTTCATCGGT-3’ (AS) for TLR2, 5’-AACGGTTCCTTCTCCTATCTCC-3’ (S) and 5’-CACTTTGCTTAGTAAATGCTCGC-3’ (AS) for TLR3, 5’-CTGTCTTACTACACCGCTATTTGG-3’ (S) and 5’-ATGGGAAGAAAACTACCCTTTACAG-3’ for TLR9, 5’-TCTCTATGACCTGGACGACTCTT-3’ (S) and 5’-GCTCATACGGTTTCCCATTTAGT-3’ (AS) for NF-κB; 5’-TGGTTTACACGTCCATCGGT-3’ (S) and 5’-ATCAATGGTCACATCACATAGTCC-3’ (AS) for TLR4; 5’-CTCCAGTATTTTCCTCACCTTCA-3’ (S) and 5’-GATTATGGCTCAGTAGCAGTGTCTC-3’ (AS) for TLR8; 5’-GGTTGTCTCCTGCGACTTCA-3’ (S) and 5’-TGGTCCAGGGTTTCTTACTCC-3’ (AS) for GAPDH.
Statistical analysis
Data are expressed as means ± standard deviations (SD). One-way ANOVA analysis was used for multiple groups comparisons and the unpaired two-tailed Student’s t-test was used for comparison between two groups, using GraphPad Prism 7 (GraphPad Software, CA, USA). A statistical value of p<0.05 was considered significant.