吡虫啉对IgE和Toll样受体2介导的肥大细胞脱颗粒的影响

Lots of researchers suggested that pesticide environmental exposure hold strong associations with the increasing trend of type I hypersensitivity, but some studies suggest that imidacloprid, the best known neonicotinoid, may inhibit type I hypersensitivity. IgE-mediated degranulation of human mast cells is the key to the development of type I hypersensitivity, and studies have shown that TLR2-mediated activation of mast cells can promote type I hypersensitivity. In this proposal, the IgE-mediated and TLR2-mediated activation of human mast cell in vitro and passive systemic anaphylaxis in mice will be treated with imidacloprid only or α7 nAchR antagonists and imidacloprid in sequence, Then, the preformed granule-associated chemical mediators, such as histamine and β-hexosaminidase (β-hex), newly synthesized arachidonic acid metabolites, such as leukotrienes C4 (LTC4),proinflammatory cytokines including TNF-α and IL-6, Ca2+ influx in mast cells, and the phosphorylation antibodies in FcεRI signaling pathway such as p-Lyn, p-Syk, p-ERK, p-JNK, p-P38, p-PLC-γ and p-NF-κB will be determined. This work aims to illustrate the effects of imidacloprid on the IgE-mediated and TLR2-mediated activation of human mast cells and further explore the mechanisms by which imidacoprid affect IgE-mediated and TLR2-mediated mast cell degranulation. These results will provide fundamental knowledge for deeply assessing its effects on type I hypersensitivity, which is based on the IgE-mediated degranulation of mast cell, furtherly providing more theoretical guidance for assessing the effects of environmental factors, especially the use of pesticides on allergic diseases.

研究表明农药环境暴露能促进Ⅰ型超敏反应的发生，但有研究提示吡虫啉可能会抑制I型超敏反应。IgE介导的人源肥大细胞脱颗粒是I型超敏反应发生的关键，且研究表明TLR2介导的肥大细胞激活能促进Ⅰ型超敏反应的发生。本研究将建立IgE和TLR2介导的体内外（人源）肥大细胞活化模型，先用吡虫啉处理以上模型，进而用α7 nAchR拮抗剂MLA（或a7-nAChRs特异性siRNA）和吡虫啉前后处理以上模型，通过检测与过敏因子如β-氨基己糖苷酶和组胺、LTC4、IL-6和TNF-α以及FcεRI通路中钙离子内流、相关磷酸化抗体如p-P-PLC-γ 和 p-NF-κB表达等变化，最终揭示吡虫啉对IgE和TLR2共同介导的肥大细胞脱颗粒的影响及作用机制，为评价吡虫啉对以IgE介导的肥大细胞脱颗粒为基础的Ⅰ型超敏反应的影响提供理论依据，进而为剖析环境因素尤其是农药使用对过敏性疾病的影响提供更丰富的理论指导。

本项目以烟碱型乙酰胆碱受体nAChR 激动剂杀虫剂吡虫啉为研究对象，建立了IgE和TLR2介导的肥大细胞活化和小鼠PCA模型，尤其是牛奶β-乳球蛋白通过IgE介导的人源肥大细胞活化，用吡虫啉和α7 nAchR拮抗剂处理模型，检测与过敏因子和FcεRI通路相关信号分子等的变化，结果提示吡虫啉能明显抑制 IgE介导的肥大细胞活化时β-hex和组胺、LTC4、IL-6 和 TNF-α、肥大细胞钙离子内流、FcεRI信号通路中磷酸化抗体p-PLC-γ、p-NF-κB和小鼠PCA血管渗出，且α7-nAchR 拮抗剂能消除吡虫啉的抑制效应；此外，吡虫啉能明显抑制体外TLR2介导的肥大细胞活化时前炎因子TNF-α和IL-6的产生和小鼠PCA皮肤炎症中性粒细胞的浸润，但不抑制TLR2介导的RBL-2H3活化时组胺、β-hex和LTC4 的释放。这些结果表明吡虫啉能明显抑制IgE介导的肥大细胞脱颗粒，我们推测吡虫啉和其他新烟碱类杀虫剂与一个国家农村地区的较低的过敏性疾病发病率可能存在一定关联。鉴于吡虫啉与烟碱分子结构相似，我们推测α7-nAChRs 可能参与了吡虫啉对肥大细胞脱颗粒的抑制作用，提示肥大细胞上的α7-nAChRs可能是未来治疗过敏性疾病的潜在靶点。此外，鉴于TLR2在宿主免疫应答中的重要作用，我们推测吡虫啉可能通过抑制肥大细胞活化从而抑制宿主对致病原的免疫应答，导致宿主感染性疾病的发生。本项目研究结果为评价吡虫啉对以肥大细胞活化为基础的过敏性疾病和宿主防御免疫的影响提供了科学依据，进而为剖析环境因素尤其是食物农药残留对以过敏性疾病的影响提供了更丰富的理论指导。