摘要
目的探究半胱氨酰白三烯受体1(CysLT_(1)R)对体外培养的APPswe/PS1ΔE9双转基因小鼠(APP/PSI小鼠)皮质原代神经元β淀粉样蛋白(Aβ)生成的影响及机制。方法①将携带CysLT_(1)R的慢病毒-绿色荧光蛋白载体(LV-CysLT_(1)R-EGFP)或空载体(LV-EGFP)转染至APP/PS1小鼠皮质原代神经元,使神经元过表达CysLT_(1)R(CysLT_(1)R-OE),采用Western印迹法检测神经元CysLT_(1)R蛋白表达水平。②分别以CysLT_(1)R激动剂YM17690(1μmol·L^(-1))单用或与CysLT_(1)R拮抗剂普仑司特(Pran 1μmol·L^(-1))联用处理CysLT_(1)R-OE神经元12 h;采用ELISA检测细胞培养基内Aβ_(1-40)和Aβ_(1-42)水平;Western印迹法检测神经元中淀粉样前体蛋白(APP)、β淀粉样前体蛋白切割酶1(BACE1)、早老素(PS)1和PS2表达水平。以YM17690(1μmol·L^(-1))单用或与Pran(1μmol·L^(-1))联用处理CysLT_(1)R-OE神经元2 h后,采用Western印迹法检测CysLT_(1)R和NF-κB P65亚基在细胞内的分布。③采用渗透差法在CysLT_(1)R-OE神经元中导入核定位序列多肽(NLS-pep,10 g·L^(-1))竞争性抑制CysLT_(1)R核转位或阴性对照多肽(NON-pep,10 g·L^(-1)),随即以YM17690(1μmol·L^(-1))处理CysLT_(1)R-OE神经元12 h,采用Western印迹法检测CysLT_(1)R激活后细胞培养基内Aβ水平、胞质中APP和BACE1表达水平;以YM17690(1μmol·L^(-1))处理导入多肽后的CysLT_(1)R-OE神经元2 h,采用Western印迹法检测CysLT_(1)R和NF-κB P65亚基在细胞内的分布。结果①CysLT_(1)R-OE神经元CysLT_(1)R蛋白表达水平较对照组显著增加(P<0.01)。②与CysLT_(1)R-OE组相比,CysLT_(1)R-OE+YM17690组神经元分泌Aβ_(1-40)和Aβ_(1-42)显著增加(P<0.05),胞质中BACE1及细胞核中CysLT_(1)R和NF-κB P65亚基表达水平显著增加(P<0.05),而神经元中APP,PS1和PS2表达水平无明显改变。YM17690和Pran联用(CysLT_(1)R-OE+YM17690+Pran组)可消除上述改变。③与CysLT_(1)R-OE+YM17690组相比,CysLT_(1)R-OE+NLS-pep+YM17690组CysLT_(1)R和NF-κB P65亚基在细胞核内�
OBJECTIVE To investigate the effect of cysteinyl leukotriene receptor 1(CysLT_(1)R)on amyloidβ-protein(Aβ)production in APPswe/PS1ΔE9 double transgenic(APP/PS1)mouse primary cortical neurons and the underlying mechanism.METHODS①The lentivirus-EGFP(LV-EGFP)vector and lentivirus-CysLT_(1)R-EGFP(LV-CysLT_(1)R-EGFP)vector were constructed and transfected into primary cortical neurons from APP/PS1 mice.The neurons over expressing CysLT_(1)R(CysLT_(1)R-OE)after trans⁃fection with LV-CysLT_(1)R-EGFP were referred to as CysLT_(1)R-OE neurons,and the expression of CysLT_(1)R protein was detected by Western blotting.②After the CysLT_(1)R-OE neurons were treated with 1μmol·L^(-1) of CysLT_(1)R agonist YM17690 alone or in combination with 1μmol·L^(-1) of CysLT_(1)R antagonist pranlukast(Pran)for 12 h,the secretary levels of Aβ_(1-40) and Aβ_(1-42) were detected by ELISA and the expres⁃sions of amyloid precursor protein(APP),β-site of amyloid precursor protein cleaving enzyme 1(BACE1),presenilin-1(PS1)and PS2 were determined by Western blotting.After the CysLT_(1)R-OE neu⁃rons were treated with YM176901μmol·L^(-1) alone or in combination with Pran 1μmol·L^(-1) for 2 h,the subcellular distribution of CysLT_(1)R and NF-κB P65 subunit was determined by Western blotting.③To competitively inhibit the nuclear translocation of CysLT_(1)R,10 g·L^(-1) of nuclear localization sequence peptide(NLS-pep)or 10 g·L^(-1) of none-nuclear localization sequence peptide(NON-pep)was delivered into CysLT_(1)R-OE neurons that were immediately treated with YM176901μmol·L^(-1) for 12 h.The levels of Aβ_(1-40) and Aβ_(1-42) in culture medium and the expressions of APP and BACE1 in cytoplasm were detected by Western blotting.After the delivery of NLS-pep or NON-pep,the CysLT_(1)R-OE neurons were treated with YM176901μmol·L^(-1) for 2 h,and the distribution of CysLT_(1)R and NF-κB P65 subunit was observed by Western blotting.RESULTS①The expression of CysLT_(1)R in the CysLT_(1)R-OE group was signifi⁃cantly increas
作者
龙燕
柯璇
洪浩
LONG Yan;KE Xuan;HONG Hao(Department of Pharmacology,School of Pharmacy,China Pharmaceutical University,Nanjing 211198,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2022年第2期81-89,共9页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(81573413)。
