Preparations of BLAB tea extract
The recipe of BLAB was adapted from the published book by Schleicher and Saleh [25]. Black cumin seeds were provided by the Institute of Traditional Chinese Medicine and Ethnic Medicine of Xinjiang Uygur autonomous region. Licorice, anise seeds, and black tea(Yunnan black tea) were purchased from Anhui Jiyou Chinese Medicine Decoction Pieces Co. LTD and were powdered using a pulverizer and sieved through a 100 mesh screen. Subsequently, the samples were mixed in a ratio of 3:1:1:1 to prepare BLAB samples. The BLAB powder was dissolved in distilled water with different concentrations of black cumin (100, 200, and 400 mg/mL) at a temperature of 90 ± 5 ℃ for 30 min. The mixture was centrifuged at 3000 rpm for 10 min and then the supernatant was collected by centrifuging at 3000 rpm for 10 min. The prepared aqueous extract of BLAB was stored at 4 °C for further analysis. Fresh BLAB extracts with different concentrations of black cumin (100, 200, and 400 mg/mL), were prepared for the treatment of AR model in mice. Dexamethasone (Dex, 2.5 mg/kg) was used as the positive control group.
Experimental animals
Seventy-two specific pathogen-free five-week-old BALB/c male mice were purchased from Shanghai Jinhui Experimental Animal Co. Ltd (Shanghai, China). The mice were housed in a laminar air-flow cabinet under standard laboratory conditions at a temperature 23 ± 3 °C and relative humidity of 55 ± 10% with a 12 h dark/light cycle. The mice were allowed to acclimatize for 1 week before the start of the experiments. All procedures and experiments were approved in accordance with the guidelines of the Institutional Animal Care and Use Committee of Zhejiang province (SYXK(浙)2015–0008).
Establishment of allergic rhinitis model and treatment
Male mice were randomly divided into six groups (n = 12 per group), as follows: control, OVA, BLAB100, BLAB200, BLAB400, and Dex groups. The OVA-induced AR mouse model and treatment were established according to Piao et al.[7, 8] (Fig. 1). Briefly, OVA-induced AR mice were sensitized by intraperitoneal injection with 200 μL phosphate-buffered saline (PBS), containing 100 μg OVA (Grade V, Sigma, St. Louis, MO, USA) emulsified in 1 mg aluminum hydroxide (Thermo Scientific, Rockford, MD, USA) on days 0, 4, 7, 10, 14, 18, and 21. From day 22, the mice were orally administrated with 200 μL of BLAB solution or Dex (2.5 mg/kg) once daily, 1 h before the intranasal challenge of OVA. Mice in the control and OVA group were given 200 μL of PBS solution. One week after the last sensitization, on days 28–42, mice received an intranasal challenge with 2 mg/mL OVA, 10 μL into each nostril every day. Mice were sacrificed 24 h after the last OVA challenge for further analysis.
Measurement of allergic symptoms
After each nostril was challenged with 10 μL OVA (2 mg/ml), the mice were placed into observation cages for evaluation of nasal symptoms. The frequencies of nasal rubbing and sneezing were measured for 10 min, immediately after the last OVA intranasal challenge [8, 9].
Nasal lavage fluid and blood sample collection and cell count
Twenty-four hours after the last OVA challenge, blood was obtained by removing the eyeball from anesthetized mice. Immediately, the blood samples were centrifuged at 1000 × g for 10 min at 4 ℃ to obtain the serum, and stored at -80 ℃ for further analysis. Next, nasal lavage fluid (NALF) was collected according to the method described by Bui et al. [26]. Briefly, a catheter was inserted in the direction of the upper airway via the partially resected trachea into the nasopharynx, and 1 mL cold PBS was used to perfuse the nasal cavity gently. The collected NALF was centrifuged at 1000 × g for 10 min at 4 ℃. The supernatant was stored at − 80 ℃ for further analysis. The cell pellets in NALF supernatant were resuspended in the same volume of cold PBS and the cell numbers were counted with a hemocytometer. NALF (150 μL) was centrifuged onto clean glass slides using a cytospin device (GMI, Inc, Ramsey, Minnesota, USA) to measure the number of differential inflammatory cells (1000 rpm, 10 min, 4 ℃). The cells were stained using Diff-Quik kit (Beijing Solarbio Science & Technology Co. Ltd, Beijing, China), according to the manufacturer’s instructions.
Histological examination
Referring to the method of Malmhäll et al. [5, 8], after the collection of NALF, the heads of mice were removed and fixed in 10% formalin solution for 3 days, followed by decalcification in EDTA for 5 days at 25 ± 3 ℃. Before embedding in paraffin wax, the samples were dehydrated with a series of ethyl alcohol and xylene. Nasal tissues were sectioned into 5 μm thickness and stained with hematoxylin and eosin (H&E) (Sigma-Aldich, St. Louis, MO, USA) for the examination of general morphology, periodic acid-Schiff (PAS) (Beijing Solarbio Science & Technology Co. Ltd, Beijing, China) for goblet cell hyperplasia and Giemsa (Shanghai Yuanye Biotechnology Co. Ltd, Shanghai, China) for eosinophil and mast cell infiltration. The number of goblet cells, eosinophils, and mast cells were counted, and epithelial damage was analyzed in randomly selected fields under 400 × magnification.
Measurements of Treg cells, cytokines, and histamine
Flow cytometry and the Human Regulatory T cell Staining Kit (eBioscience, USA) for Treg cell were used according to the manufacturer’s protocols [7]. The serum levels of total IgE, anti-OVA specific IgE, IgG1, and IgG2a were measured using ELISA kits (Invitrogen, San Diego, CA, USA) and serum levels of histamine were quantified using the histamine assay kit (R&D Systems Inc, USA) [8], following the manufacturers’ instructions. The levels of cytokines (IL-4, IL-5, IL-13, IFN-γ, IL-10, and IL-12) in nasal mucosa and serum [10] were quantified using cytokine quantification kits (Invitrogen, San Diego, CA, USA), according to the manufacturer’s protocols.
Analysis of chemical composition of BLAB tea
With reference to the method reported by Hajhashemi [15] and Shiozaki [23], Gas Chromatograph Mass Spectrometer (GC–MS) measurements were performed using a Shimadzu instrument equipped with GC: Shimadzu 2010Plus, MS: MS detector QP2020, ionization for MS: electron impact ionization, mass analyzer: single quadrupole, software: GC–MS solution, library: NIST 14 s, column: SH-Rxi-5il MS, dimensions: 30 m × 0.25 mm × 0.25 µm film thickness. The program conditions were as follows: the oven start temperature was 50 ℃ (1 min), the subsequent gradient was 25 ℃/min to 150 ℃ and then at a rate of 10 ℃/min to 300 ℃, with a final hold at 300 ℃ for 15 min and a total run time of 35 min. Carrier gas was helium, the flow rate was 1.2 mL/min, no split flow. The injection volume was 1 µL and the scan mass range was 50 m/z-500 m/z. The mass spectra in the electron ionization mode were recorded at 70 eV. The spectrum of the unknown compound was compared with the spectrum of the known compounds in the NIST 14 s library.
Statistical analysis
Each experiment was repeated thrice. The date was expressed as means ± standard error of means, and the statistical significance of comparisons among groups was performed using one-way ANOVA, followed by Student’s test. Statistical significance was considered at the 95% confidence level (P < 0.05) and 99% confidence level (P < 0.01).
