Briefly, after partial tracheal resection under deep anaesthesia, a 22-gauge catheter was inserted into the choana towards the heads of a portion of mice. Each nasal cavity was gently irrigated by l ml of sterile saline. Nasal lavage fluid (NLF) was collected and centrifuged, and the supernatant was stored at −20 °C for cytokines analysis using enzyme-linked immunosorbent assay (ELISA). Cytokine levels of IL-5, selleck inhibitor IL-10,
IL-17, TGF-β1, IFN-γ and endogenous LF in NLF were measured by ELISA according to the manufacturers’ instructions (Boster Biotech, Wuhan, China). The detection sensitivity of the ELISA kits was <2 pg/ml for all cytokines. Five mice per group were chosen for histopathology. Animals were decapitated
and the heads were decalcified, embedded in paraffin and sectioned as previously described . Histological changes in the nasal mucosa of all groups were examined using haematoxylin-eosin (HE) staining for eosinophils and periodic acid-schiff stain (PAS) for goblet cells. The cytoplasm of eosinophils in the nasal lamina propria (LP) stains red by HE, while the cytoplasm of goblet cells from the epithelium stains purple by PAS. Eosinophils in the LP were counted in four different fields, and eosinophil frequencies were expressed as cells/mm2. Goblet cells were expressed as cells/mm of epithelium. Th1, Th2, Th17 and Treg cell transcription factor and cytokine mRNA expression levels were determined each group (n = 5 per group). Nasal mucosa from samples was obtained using toothed microscopic tweezers under a stereo microscope (ZAS301; Beijing, China) and immediately frozen at −70 °C. Total buy RG7420 RNA was extracted by Trizol (Invitrogen, Carlsbad, CA, USA), and 0.5 μg total RNA was used for the reverse transcription reaction using
a Rever Tra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions and as previously described . The qPCR of T-bet (NM_019507.2), GATA-3 (NM_008091.3), ROR-C (NM_011281.2), FOXP3 (NM_001199348.1), IFN-γ (NM_008337.3), IL-5 (NM_010558.1), IL-17 (NM_010552.3), IL-10 (NM_010548.2), TGF-β1 (NM_011577.1), TNF-α (NM_013693.2) and LF (NM_008522.3) was performed with an ABI 7500 real-time PCR system (Applied Biosystems, Farnesyltransferase Foster City, CA, USA) using the SYBR qPCR mix (TOYOBO) according to the manufacturer’s protocol. Briefly, 1.0 μl cDNA was added to 10 μl SYBR qPCR mix, 7 μl RNase-free water and 1 μl of each primer (10 μm). The PCR conditions consisted of an initial denaturation at 95 °C for 50 s, followed by amplification for 40 cycles of 15 s at 95 °C, 15 s at 56–60 °C (varying between primer sets) and 50 s at 72 °C. An analysis of relative gene expression was calculated using the 2−ΔΔCT method on the ABI 7500 Sequence Detection System Software (Applied Biosystems). Gene expression was normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH, NM_008084.2).