The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032, People's Republic of China.
Inflamm Res. 2012 Nov;61(11):1257-63. doi: 10.1007/s00011-012-0523-x. Epub 2012 Jul 21.
To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation.
NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting.
shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.
NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.
确定神经激肽-1 受体（NK1R）在 FcεRΙ 聚集后是否在 RBL-2H3 肥大细胞的激活中起作用。
使用针对 NK1R 的短发夹 RNA（shRNA）抑制 RBL-2H3 细胞中的 NK1R 表达，并通过 Western blot 确定。为了激活，NK1R 敲低和对照 RBL-2H3 细胞均通过二硝基苯酚（DNP）特异性 IgE 敏化，并与抗原 DNP-牛血清白蛋白（BSA）刺激。在 RBL-2H3 细胞激活后，通过 ELISA 和共聚焦显微镜测定分别监测单核细胞趋化蛋白（MCP-1）的产生和细胞内钙流。为了研究信号机制，通过 Western blot 评估 RBL-2H3 细胞激活后丝裂原活化蛋白激酶（MAPKs）的磷酸化。
shRNA-NK1R 介导了对 RBL-2H3 细胞中 NK1R 表达的有效抑制。与对照 RBL-2H3 细胞相比，NK1R 敲低的 RBL-2H3 细胞中的 MCP-1 蛋白产量减少了 55％以上。此外，由于 NK1R 表达的抑制，DNP-BSA 刺激（通过 FcεRΙ）后钙动员和 MAPK（Erk1/2、JNK 和 p38）的磷酸化水平均降低。
NK1R 是 FcεRΙ 结合后 RBL-2H3 细胞激活所必需的，并参与调节 MAPK 信号通路。
