In sensitized group, the mast cells were much bigger, with more shrink on the cell membrane, bubbles in the cytoplasm and degranulation vehicles around the cells check details (Fig. 2A). Furthermore, ultrastructure analysis of mast cells by transmission electron microscope showed that the cell membrane was obscure, and degranulation vehicles was less evenly distributed in the cytoplasm of mast cells (Fig. 2A). The number of mast cells was significantly increased in OVA-treated RPLS (Fig. 2B). The

ratio of mast cell degranulation as indicated by vehicles (at least five) around the cells was also dramatically increased by ~3 fold (Fig. 2B). Mast cell degranulation was further confirmed by increased histamine levels in serum and RPLS (Fig. 2C). It has been suggested that an increase in intracellular Ca2+ through SOC channel is essential for mast cell degranulation

[13]. We therefore examined whether food allergen–induced mast cell activation is related to stimulation of Ca2+ mobilization. As shown in Fig. 3, the TG-evoked Ca2+ influx was dramatically enhanced in OVA-sensitized rat peritoneal mast cells, suggesting mast cell activation in the food-allergic model is related to upregulation of Ca2+ entry through SOCs. STIM1 and Orail are the two subunits of SOCs [23, 24]. Overexpression of STIM1 and Orail caused a significant increase in store-operated Ca2+ entry in RBL cells [16]. We thus examined the see more expression levels of both subunits. The results show that the mRNA (Fig. 3A,B) and protein levels Ketotifen (Fig. 3C,D) of both subunits were significantly increased in allergic animals as compared with controls (all P < 0.01). Furthermore, immunofluorescence study revealed that

the STIM1 subunits were translocated to the cell membrane, which is required for the activation of SOCs in OVA group, while it was evenly distributed in cytoplasm in control group (Fig. 4). Collectively, these data indicate that OVA-induced food allergy increased SOCs activity by enhancing transcription and expression of SOCs subunits, as well as increasing SOCs activity. Reactive oxygen species production in RPMCs isolated from control or allergic animals was examined by ELISA. The results demonstrated that ROS production in allergic mast cells was increased by 1.5-folds as compared with controls (Fig. 5A). Administration with ROS scavenger Ebselen (100 μm, 30 min) to OVA-challenged RPMCs reduced ROS production by ~30% (Fig. 5A). In parallel, clearance of intracellular ROS by Ebselen decreased histamine release by ~30% (Fig. 5B). Similarly, OVA challenge–induced Ca2+ increase through SOCs in activated mast cell was decreased by 30% by Ebselen treatment (Fig. 5C,D). The results indicate that mast cell activation is partially attributed to increased ROS production. Quantification of the protein levels of Orai1 and STIM1 demonstrated that Ebselen reduced both protein expressions by ~40% and ~30%, respectively (Fig.