慢性间歇性低氧对幼鼠脑区AMPK通路的影响

Effect of chronic intermittent hypoxia on AMPK pathway in young rats

目的：探讨慢性间歇性低氧对幼鼠脑区腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）通路的影响。方法：第一部分：将3～4周龄的SD幼鼠随机分为4组（n=8）：2周空气模拟对照（2AC）组、2周慢性间歇低氧（2IH）组、4周空气模拟对照（4AC）组和4周慢性间歇低氧（4IH）组。第二部分：将3～4周龄的SD幼鼠随机分为2组（n=8）：4周慢性间歇性低氧组（4IH）和4周慢性间歇性低氧用药组（4IHI）。造模结束后行八臂迷宫测试,TUNEL法检测细胞凋亡,RT-qPCR法了解腺苷A2a受体的mRNA表达,Western blot法测定AMPK及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）的磷酸化水平。结果：与对照组相比,2IH组和4IH组的参考记忆错误（RME）、工作记忆错误（WME）和总错误（TE）次数明显增加,差异有统计学显著性（P〈0.01）;4IH与2IH组比较,各项错误次数亦明显增加（P〈0.01）。4IHI组比4IH组RME、WME和TE次数减少,差异有统计学显著性（P〈0.01）。与对照组相比,2IH组和4IH组海马和前额皮层区的神经元凋亡增多,以4IH组凋亡明显（P〈0.05）;4IHI组凋亡较4IH组减少（P〈0.05）。2IH组和4IH组海马和前额皮层区腺苷受体A2a的mRNA和AMPK磷酸化蛋白的水平升高,mTOR磷酸化蛋白的水平下降,4IH组较2IH组改变明显（P〈0.05）。4IHI组较4IH组海马和前额皮层区的AMPK磷酸化蛋白水平下降,mTOR磷酸化蛋白水平升高（P〈0.05）。结论：慢性间歇性低氧诱导神经元凋亡,从而引起幼鼠学习记忆障碍,呈时间依赖性。慢性间歇性低氧上调腺苷A2a受体,激活海马和前额皮层区AMPK,抑制mTOR的活性,诱导神经元凋亡,进而影响幼鼠学习记忆能力。

AIM： To investigate the effect of chronic intermittent hypoxia on AMP-activated protein kinase（ AMPK） pathway in the brain of young rats. METHODS： Part one： SD mice（ 3 ~ 4 weeks old） were randomly divided into 4 groups（ n = 8） ： simulated air control group for 2 weeks（ 2AC）,chronic intermittent hypoxia group for 2 weeks（ 2IH）,simulated air control group for 4 weeks（ 4AC） and chronic intermittent hypoxia group for 4 weeks（ 4IH）. Part two： SD mice（ 3 ~ 4 weeks old） were randomly divided into 2 groups（ n = 8） ： chronic intermittent hypoxia group for 4weeks（ 4IH） and chronic intermittent hypoxia group treated with AMPK inhibitor for 4 weeks（ 4IHI）. After modeling,the eight-arm maze test was performed. TUNEL method was used to detect the neuronal apoptosis in the hippocampal and prefrontal cortical tissues. The mRNA expression of adenosine A2 a receptor was examined by RT-qPCR,and the protein levels of phosphorylated AMPK（ p-AMPK） and mammalian target of rapamycin（ p-mTOR） were determined by Western blot.RESULTS： Compared with control group,the numbers of reference memory error（ RME）,working memory error（ WME）and total error（ TE） in 2IH group and 4IH group significantly increased（ P〈 0. 01）. Compared with 2IH group,the numbers of errors in 4IH group also increased significantly（ P〈 0. 01）. Compared with 4IH group,the values in 4IHI group significantly decreased. Compared with control group,the neuronal apoptosis of hippocampus and prefrontal cortex in 2IH group and 4IH group increased,and that in 4IH group was more evident（ P〈 0. 05）. In 4IHI group,the neuronal apoptosis decreased. The mRNA expression of adenosine A2 a receptor in the hippocampal and cortical tissues in 2IH group and4 IH group was higher than that in control group. The protein level of p-AMPK was higher,and p-mTOR was lower in 2IH group and 4IH group,and those in 4IH group were more evident（ P〈 0. 05）. Compared with 4IH group,the protein level of p-AMPK was lower,and p-mTOR was higher in 4IHI group. CONCLUSION： Chronic intermittent hypoxia induces neuronal apoptosis,resulting in impairment of learning and memory in a time-dependent manner by upregulating adenosine A2 a receptor,activating AMPK activity,and inhibiting mTOR phosphorylation in rats.