大鼠蛛网膜下腔出血后海马区磷酸化细胞外调节蛋白激酶1/2表达与细胞自噬的关系

The Relationship between Phosphorylated Extracellular Regulated Protein Kinase 1/2 Expression and Neurons Autophagy in Hippocampus after Subarachnoid Hemorrhage in Rat

目的探讨大鼠蛛网膜下腔出血(SAH)后海马区细胞外调节蛋白激酶1/2(ERK1/2)表达与神经细胞自噬的关系。方法采用枕大池二次注血法制作SAH大鼠模型,将120只雄性SD大鼠采用数字表法分成假手术组(Sham)、SAH组、U0126(ERK1/2抑制剂)低剂量组及U0126高剂量组。低剂量和高剂量U0126组分别于造模前30min侧脑室注射U0126 5和10g/L。水迷宫行为学测试大鼠的行为学变化;光镜观察海马区神经细胞形态结构;免疫组织化学法和免疫印迹法检测海马区磷酸化ERK1/2(p-ERK1/2)和自噬标志蛋白(Beclin-1及LC3-Ⅱ)的表达水平。结果与Sham组比较,SAH组大鼠在72h的逃避潜伏期时间明显延长、穿台次数明显减少(P<0.05),各时间点神经元存活比率均明显降低,且SAH组p-ERK1/2,Beclin-1及LC3-Ⅱ表达均增加,于24h达高峰;与SAH组比较,低、高剂量U0126组大鼠相应时间点的潜伏期明显延长、穿台次数明显减少(P<0.05),各时间点神经元存活比率均明显降低,且p-ERK1/2,Beclin-1和LC3-Ⅱ蛋白表达及存活神经细胞比率降低(P<0.05);与低剂量U0126组比较,高剂量U0126组的大鼠相应时间点的逃避潜伏期明显延长、穿台次数明显减少(P<0.05),p-ERK1/2及存活神经细胞比率降低(P<0.05),但2组间的Beclin-1和LC3-Ⅱ蛋白比较差别均无统计学意义(P>0.05)。结论大鼠SAH后海马区p-ERK1/2激活对神经细胞有保护作用,且这种保护作用与神经细胞自噬有关。

Objective To investigate the relationship between extracellular regulated protein kina- ses activation and neural cells autophagy in rats after subarachnoid hemorrhage. Methods 120 male SD rats were divided into sham operated group, SAH group, low dose ERK1/2 inhibitor U0126 group, and high dose ERK1/2 inhibitor U0126 group. The animal models were established by injecting the autolo- gous blood into cisterna magna twice. Two U0126 groups were injected U0126 （5 g/L or 10 g/L） in lat- eral ventricles 30 min before modeling. The morphology of hippocampal neurons was observed by light microscopy. The behavioral differences were detected using water maze behavior test. The expression levels of phosphorylated ERK1/2 （p-ERK1/2）and autophagy marker proteins （Beclin-1, LC3-Ⅱ ） in hippo- campus were detected by immunohistochemistry and Western-blot. Results Compared with those in Sham group, SAH group of rats in 72 h escape latency time significantly prolonged, wear number de- creased significantly （P〈 0.05）, neuron survival rate at each time point were significantly lower, whereas the expressions of p ERK1/2, Beclin-1, and LC3-Ⅱ increased in SAH group, and reached the peak at 24 h. Compared with those in SAH group, the latency time of both low and high dose of U0126 groups at the same time points was prolonged and the times for crossing the platform significantly reduced （P%0.05）, and at each time point neuron survival ratio was significantly decreased, and the expressions of p-ERK1/2, Beclin-1 and LC3-Ⅱ , and neuronal cell survival ratio decreased in two U0126 groups （P〈0.05）. Compared with those in low dose U0126 group, high dose group at the corresponding time points, the escape latency prolonged and the times for crossing the platform significantly decreased （P〈0.05）, and all the expressions of p-ERK1/2 and neuronal cell survival ratio decreased in high U0126 group （P〈0.05） ,while there was no statistically significant difference in Beclin-1 and LC3-Ⅱ between the two dose groups （P〉0.05）. Conclusions ERK1/2 activation had a protective effect on nerve cells after SAH, and this protective effect is partly related to enhancing neurons autophagy.