Quercetin inhibits Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R related Ca2+ fluctuations

Abstract

An allergic reaction is a potentially fatal hypersensitivity response caused by mast cell activation, particularly histamine and lipid mediators. Histamine release caused by reaction to drugs is considered a pseudo-allergic reaction. Quercetin is known for its anti-allergic immune effect. However, at present, its anti-pseudo-allergic effect and its mechanism are less investigated. Therefore, the purpose of this study was to evaluate the anti-pseudo-allergic effect of Quercetin in vivo and to explore the mechanism in vitro. The anti-pseudo-allergic activity of Quercetin was evaluated in vivo using a mouse model, while Quercetin mechanism of action was examined in vitro using HEK293 cells expressing Mrgprx2, a mast cell specific receptor, and LAD2 mast cell line. Our in vivo results showed that Quercetin could attenuate Evans blue leakage in the paws and hind paw thickness in C57BL/6 mice in a dose-dependent manner, and could significantly inhibit serum histamine and chemokines release. In addition, it suppressed calcium mobilization and attenuated the release of histamine and MCP-1 in peritoneal mast cells in a dose-dependent manner. Furthermore, it inhibited the vasodilation due to histamine, the release of eosinophils, and the percentage of degranulated mast cells, indicating that Quercetin antagonized mast cell mediators in vivo, histamine-induced vasodilation and eosinophil release.In vitro results showed that Quercetin reduced pseudo-allergic induced calcium influx, suppressed degranulation and chemokines release in a similar way as dexamethasone (100 μM) (mast cell stabilizer) in LAD2 mast cell line. In addition, Quercetin inhibited Mrgprx2-induced both calcium influx and pseudo-allergic reaction in HEK293 cells expressing Mrgprx2. C48/80, a histamine promoter, and Substance P (a neuropeptide) EC50 was higher when combined with Quercetin compared to the EC50 of these compounds alone, suggesting that Quercetin could inhibit Mrgprx2-induced pseudo-allergic reaction. Furthermore, Quercetin decreased PLCγ-IP3R signaling pathway activation induced by C48/80 in LAD2 mast cell line. In Mrgprx2 knockdown LAD2 cells, the effect of Quercetin (200 μM) reduced C48/80 induced calcium flux and the release of β‑hexosaminidase, histamine, MCP-1 and IL-8 compared with non-atopic control (NC) transfected LAD2 human mast cells, suggesting that Quercetin anti-pseudo-allergic effect was related to Mrgprx2. The docking results showed that Quercetin had a good binding affinity with Mrgprx2 similar to the one of Substance P and C48/80. Therefore, Quercetin inhibited Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations.Our results validated Quercetin as an effective small molecule inhibiting Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations, thus highlighting a potential candidate to suppress Mrgprx2 induced pseudo-allergic related diseases.