摘要
目的探讨ADDLs对细胞质p—ERK1/2的影响,揭示ADDLs影响突触可塑性导致认知功能障碍的分子机制。方法采用体外培养的大鼠海马神经元。免疫印迹方法观察ADDLs对p—ERK1/2的影响,细胞组分分离方法探索ADDLs影响ERK1/2在神经元各部位活化的规律;免疫共沉淀的方法检测ERK1/2与h3 Calponin的结合以及ADDLs对两者结合的影响。结果ADDLs以时间依赖的方式降低了神经元中p—ERK1/2水平;ADDLs对细胞质和细胞核部分ERK1/2的影响具有时序性,ADDLs作用1h显著降低细胞质中p—ERK1/2水平,作用6h细胞核中的p—ERK1/2才显著降低;免疫共沉淀结果显示ADDLs作用1h即可降低ERK1/2与h3Calponin的结合,而此时细胞核中P—CREB水平无显著变化。结论ADDLs作用早期首先通过阻断ERK1/2与h3Calponin的结合影响细胞质ERK1/2活化。
Objective To discuss the impact of ADDLs on the activation of cytosolic ERK1/2 and possible molecular mechanisms by which ADDLs affect synaptic plasticity, resulting in cognitive disorder. Methods The effects of ADDLs on phosphorylated ERK1/2 within primary hippocampal neurons were determined by immunoblotting. Subcellular fractions were isolated to further explore the effect of ADDLs on the levels of p - ERK1/2 within different parts of hippocampal neurons. The effect of ADDLs on ERK1/2 binding to h3 Calponin was detected by co - immunoprecipitation. Results ADDLs reduced the amounts of p - ERK1/2 in a dose - dependent manner, while different effects were observed on p -ERK1/2 localized within cytoplasmic and nuclear compartments as exposure times went. ADDLs could remarkably weaken the quantities of cytoplasmic p - ERK1/2 after 1 hour of treatment. In contrast, the levels of nuclear p - ERK1/2 were significantly declined after treatment for 6 hours. According to co - immunoprecipitation results, the binding of ERK1/2 to h3 Calponin was dissociated 1 hour after ADDL administration when no obvious changes were seen as to the quantities of p - CREB in the nucleus. Conclusion ADDLs can suppress the activation of cytosolic ERK1/2 via blockage of ERK1/2 binding to h3 Calponin in hippocampal neurons at an early stage.
出处
《徐州医学院学报》
CAS
2015年第4期211-215,共5页
Acta Academiae Medicinae Xuzhou
基金
国家自然科学基金(81273489,81471101)
江苏省高校自然科学研究重大项目(12KJA180008)
江苏省自然科学基金(BK2012582)