CysLT_(1)R对APP-HEK293细胞Aβ生成的调控作用及其机制

Effect of CysLT_(1)R on Aβproduction and the underlying mechanism in APP-HEK293 cells

目的探究CysLT_(1)R对APP-HEK293细胞中Aβ生成的影响并探寻其可能的作用机制。方法构建慢病毒载体LV-CysLT_(1)R-EGFP并转染APP-HEK293细胞,激动剂YM17690单独处理或拮抗剂普仑司特和YM17690共同处理细胞,采用ELISA检测Aβ;Western blot检测APP、BACE1、PS1、PS2;细胞免疫荧光和Western blot检测CysLT_(1)R和NF-κB p65在细胞的分布。转染clathrin、β-arrestin-2、Rab-5、siRNA;Co-IP检测CysLT_(1)R和NF-κB p65相互作用。结果激活CysLT_(1)R增加细胞分泌Aβ、上调BACE1的表达、但对APP、PS1、PS2表达无明显影响,升高细胞核中CysLT_(1)R和NF-κB p65水平;敲减clathrin和β-arrestin-2抑制CysLT_(1)R激活后的上述效应;受体激活后CysLT_(1)R与NF-κB p65在细胞核内相互作用。结论激活CysLT_(1)R促进Aβ的生成,其作用机制与该受体发生核转位有关。

Aim To investigate the effect of CysLT_(1)R on Aβproduction in APP-HEK293 cells and explore the underlying mechanism.Methods The lentivirus vector carrying CysLT_(1)R(LV-CysLT_(1)R-EGFP)was constructed and LV-CysLT_(1)R-EGFP was transfected into APP-HEK293 cells.The cells were treated with CysLT_(1)R agonist YM17690 alone or YM17690 in combination with CysLT_(1)R antagonist pranlukast.The production of Aβwas detected by ELISA,and the expression levels of APP,BACE1,PS1,PS2 were detected by Western blot.The distribution of CysLT_(1)R and NF-κB p65 in cells was detected by immunofluorescence staining and Western blot.The clathrin siRNA,β-arrestin-2 siRNA and Rab-5 siRNA were transfected into the cells.The interaction between CysLT_(1)R and NF-κB p65 was detected by Co-IP.Results The activation of CysLT_(1)R increased Aβproduction,up-regulated the expression level of BACE1,and increased levels of CysLT_(1)R and NF-κB p65 in cell nucleus,while no effects on APP,PS1 and PS2 expression.The knockdown of clathrin andβ-arrestin-2 inhibited the above effects induced by CysLT_(1)R activation.CysLT_(1)R and NF-κB p65 interacted in cell nucleus after the receptor activation.Conclusions The activation of CysLT_(1)R promotes the production of Aβ,and the underlying mechanism is related to the nuclear translocation of CysLT_(1)R.