Fig. 3
From: Shuang-Huang-Lian prevents basophilic granulocyte activation to suppress Th2 immunity
a SHL decreased early IL-4 produced by BG-rich splenocytes. Splenic BGs, preliminarily purified by a MACS system, were seeded in a 96-well plate (1 × 106 cells/well) and treated with SHL (0.5–2%) and ST (100 μg/mL) or active papain (100 μg/mL) in the presence of IL-3 (1 μg/mL) for 24 h. IL-4 levels in the supernatants were analyzed by ELISA. ##P < 0.01 vs. NS; $$P < 0.01 vs. papain alone; **P < 0.01 vs. ST alone. b-c SHL inhibited BG CD200R surface expression. The whole blood from the ST-sensitized mice was collected and stained with anti-IgE FITC and anti-CD200R antibodies after an incubation with ST (5 μg/mL) for 2 h at 37 °C. BGs in the whole blood were identified by an anti-IgE FITC antibody. The surface expression of CD200R on the BGs was assayed by flow cytometry. Isotype control and unstained cells were used as the negative controls. b Representative dot plots of BG CD200R surface expression. c Effect of SHL on the mean fluorescence intensity (MFI) of CD200R in the BGs after stimulation with ST. d SHL decreased IL-4 release in the sensitized RBL-2H3 cells. The cells were sensitized with anti-ST IgE (25 μg/mL) at 37 °C overnight. The cells were pretreated with or without SHL at 37 °C for 30 min and were then stimulated with ST (20 ng/mL) for 6 h. IL-4 levels in the supernatants were assayed using a commercial ELISA kit. e SHL suppressed ST-induced NFAT activation in BGs. RBL-2H3 cells stably transfected with the pNFAT-luc plasmid were sensitized with anti-ST IgE (25 μg/mL) at 37 °C overnight. The cells were pretreated with or without SHL at 37 °C for 30 min and were then stimulated with ST (20 ng/mL) for 6 h. The cells were lysed and the luciferase activity was measured using the luciferase assay system. f SHL reduced Ca2+[c] levels in the RBL-2H3 cells. The RBL-2H3 cells were sensitized with anti-ST IgE (25 μg/mL) at 37 °C overnight. The cells were loaded with Fluo-3 AM (4 μM) at 30 °C for 30 min. The stained cells were treated with or without SHL for 30 min and were then exposed to ST (20 ng/mL). The fluorescent intensity (λex 485 nm and λem 538 nm) was recorded every 30 s. #P < 0.05 and ##P < 0.01 vs. the control; *P < 0.05, **P < 0.01 vs. ST alone