高压氧预处理抑制神经元细胞外信号调节激酶活化及与死亡相关蛋白激酶1的相互作用研究

Inhibition effect of hyperbaric oxygen preconditioning on extracellular signal-regulated kinase activation and its interaction with death-associated protein kinase 1

目的 研究高压氧预处理对类缺血神经元细胞外信号调节激酶（ERK）和死亡相关蛋白激酶1（DAPK1）的影响. 方法（1）体外实验部分：将培养的小鼠神经元分为5组,即对照组、类缺血组、高压氧预处理组、空质粒组、ERK过表达组.对照组不做任何处理,类缺血组细胞经95％NO2＋5％ CO2混合气体处理30 min,高压氧预处理组细胞预先经98％O2＋2％CO2的混合气体处理2h后同类缺血组细胞处理,空载质粒组转染p3XFlag-CMV空质粒,ERK过表达质粒组细胞转染p3XFlag-CMV-ERK质粒.噻唑蓝（MTT）法检测不同时间点前3组细胞的生长情况,Western blotting法检测前3组细胞中磷酸化的细胞外信号调节激酶（p-ERK）的表达,以及对照组、空质粒组、ERK过表达组细胞中活化caspase-3蛋白及DAPK1的表达.同时采用免疫共沉淀的方法检测前3组细胞中ERK与DAPK1分子的相互作用.（2）体内实验部分：将BALB/c小鼠分为对照组、类缺血组和高压氧预处理组,每组15只.采用免疫共沉淀的方法检测小鼠海马区神经元中ERK与DAPK1分子复合体的表达情况. 结果 细胞分组后24h、48 h、72 h及96h,对照组、类缺血组、高压氧预处理组细胞存活率差异均有统计学意义（P＜0.05）,高压氧预处理可以显著抑制类缺血处理引起的细胞存活率的下降.同时3组细胞内p-ERK表达水平差异亦有统计学意义（P＜0.05）,高压氧预处理可以显著抑制类缺血处理引起的p-ERK的升高.此外,过表达ERK基因能促进细胞中活化caspase-3的表达,而高压氧预处理可以降低ERK与DAPK1的相互作用. 结论 高压氧预处理可通过抑制ERK的活化及其与DAPK1相互作用,阻断细胞凋亡的激活,从而诱导细胞缺血耐受.

Objective To investigate the effect of hyperbaric oxygen preconditioning （HBO） on activation of extracellular signal-regulated kinase （ERK） and death-associated protein kinase 1 （DAPK1）in ischemia-like treated neurons.Methods （1） Cultured mouse primary neurons were allocated into control,ischemia-like condition treated and HBO preconditioning groups.Neurons in ischemia-like condition treated group were treated with 95% NO2＋5% CO2 for 30 min,and cells in the HBO preconditioning group were pretreated with 98% O2＋2% CO2 for 2 h,followed by ischemia-like condition treatment.At the end ofthe treatment,the growth of neurons was measured by MTT assay and expression of phosphorylated extracellular signal-regulated kinase （p-ERK） was detected by Western blotting.Neurons were transfected with ERK over-expression vector and pretreated with HBO and ischemia-like condition,and then,the expressions of activated caspase-3 （cleavage caspase-3） and DAPK1 in neurons were analyzed by Western blotting.The interactions of ERK with DAPK1 were also detected by irnmunoprecipitation.（2） Forty-five BALB/c mice were randomized into control,ischemia treated and HBO preconditioning groups.After finishing the treatments,the interactions of ERK with DAPK1 in hippocampal neurons were detected by immunoprecipitation.Results As compared with control group,cells in ischemia-like condition group showed a reduction of cell survivals,while cells in HBO preconditioning group exhibited an inhibition effect on ischemia-like condition-induced cell survival reduction.The expressions of p-ERK were increased by ischemia-like condition,while decreased by HBO preconditioning,with significant difference （P〈0.05）.Moreover,over-expression of ERK promoted level of cleavage caspase-3 in neurons; however,DAPK1 expressions were not affected by ischemia-like condition or HBO preconditioning.The interactions of ERK with DAPK1 were attenuated by HBO preconditioning as compared with those by ischemia-like condition treatment.Conclusion HBO preconditioning inhibits ERK activation and disturbs the interaction between ERK and DAPK1,which blocks cell apoptosis and thus induces ischemic tolerance.