间歇低氧对大鼠认知功能的影响及其分子机制的研究

Effects of intermittent hypoxia on the cognitive function of rats and the experimental study of molecular mechanisms

目的探讨间歇低氧对大鼠认知功能的影响及其分子机制。方法选用雄性Wistar大鼠64只,体质量(180±10)g,按随机数字表法分为正常对照组(UC组)和5%间歇低氧组(5%IH组),每组32只;每组按取材时间不同随机分为7、14、21、28 d四个亚组,每个亚组8只。UC组大鼠置于实验箱内持续注入空气,氧浓度为21%;5%IH组大鼠置于实验箱内,循环注入氮气及空气,使氧浓度在5%~21%之间变化,每120 s为1个周期。2组大鼠每日实验8 h,其余16 h常规饲养,分别持续进行7、14、21、28 d。各亚组实验后应用Morris水迷宫检测两组大鼠学习记忆功能。完成Morris水迷宫实验后处死并取出大鼠海马组织,制备切片标本:采用透射电镜观察大鼠海马CA1区神经细胞超微结构的改变;采用免疫组织化学法检测海马CA1区神经细胞凋亡蛋白酶激活因子-1(Apaf-1)、第二个线粒体衍生的caspase激活因子(Smac)蛋白的表达情况;采用Tunel法检测海马CA1区神经细胞凋亡情况,并计算神经细胞凋亡指数。采用Pearson检验,分析UC组不同时间点Apaf-1及Smac蛋白的表达与神经细胞凋亡指数的相关性。结果(1)Morris水迷宫实验:UC组大鼠间歇性低氧7、14、21、28 d逃避潜伏期分别为(20.83±3.25)、(22.17±2.79)、(20.50±4.51)、(21.17±4.17)s,跨越目标象限时间分别为(52.17±4.17)、(52.50±2.74)、(51.50±2.43)、(52.00±4.78)s,各时间点差异均无统计学意义(P值均>0.05);5%IH组间歇性低氧7、14、21、28 d逃避潜伏期分别为(33.17±2.14)、(44.33±3.45)、(52.17±3.87)、(64.33±2.73)s,跨越目标象限时间分别为(44.00±3.03)、(34.50±3.94)、(27.83±3.01)、(20.83±1.94)s,随间歇低氧时间的增加,逃避潜伏期延长,跨越目标象限时间明显缩短,差异均有统计学意义(F=106.335、61.772,P值均<0.01)。两组间比较:5%IH组不同时间点逃避潜伏期时间均大于UC组,跨越目标象限时间均小于UC组,差异均有统计学意义(P值均<0.01)。(2)透射电镜观察神经细胞超微结构:UC组大鼠海马CA1区神经元核较大,核膜结构完整,呈椭圆形或圆形,染色质分布均匀呈细颗粒状,细胞器丰富,形态结构正常;5%IH组从7 d开始出现神经元水肿,细胞核变形,核膜模糊,核仁逐渐消失,细胞器肿胀溶解等,随着时间的延长,改变程度越重。(3)Apaf-1、Smac蛋白的相对表达量:UC组各个时间点海马CAl区神经细胞Apaf-1、Smac蛋白的相对表达量差异均无统计学意义(P值均>0.05);而5%IH组各个时间点Apaf-1、Smac蛋白的相对表达量均随间歇低氧时间的延长而升高,差异均有统计学意义(F=25.328、42.923,P值均<0.01)。两组间比较:5%IH组各个时间点Apaf-1、Smac蛋白的相对表达量均明显高于UC组,差异均有统计学意义(P值均<0.01)。(4)神经细胞凋亡情况:UC组各个时间点海马CAl区神经细胞神经细胞凋亡少,AI差异无统计学意义(P>0.05);5%IH组随间歇低氧时间的延长而升高,AI差异有统计学意义(F=25.799,P<0.01)。两组间比较:各个时间点海马CAl区神经细胞凋亡明显高于UC组,AI差异均有统计学意义(P值均<0.05)。(5)Pearson检验显示:5%IH组各个时间点大鼠海马CA1区Apaf-1、Smac蛋白的表达与凋亡指数均呈正相关(rApaf-1=0.735、0.736、0.685、0.747,rSmac=0.735、0.734、0.679、0.751,P值均<0.05)。结论间歇低氧可导致大鼠认知功能障碍,这可能与其引起大鼠海马CA1区神经细胞超微结构的改变,诱导凋亡相关蛋白Apaf-1、Smac的表达,进而造成神经元凋亡有关。

Objective To investigate the effects of intermittent hypoxia on the cognitive function of rats and the molecular mechanisms.Methods Sixty-four male Wistar rats(weight 180±10g)were selected and divided into normal control group(UC group)and 5%intermittent hypoxia group(5%IH group)by random number table method.Thirty-two rats in each group were randomly divided into four time subgroups(7 d,14 d,21 d and 28 d)according to different sampling time,with 8 rats in each subgroup.In the experiment chamber,the rats in the UC group were continuously injected with air with oxygen concentration of 21%,and the rats in the IH group were continuously injected with nitrogen and air while the oxygen concentration varied from 5%and 21%.Each cycle cost 120 s and the experiments conducted for 8 hours every day.Because of the four time subgroups of the two groups,the experiment time was different(7 d,14 d,21 d and 28 d).After the first experiment,the Morris water maze was performed to detect the learning and memory function of the rats in each group on day 7,day 14,day 21 and day 28.After the Morris water maze,hippocampal tissues of rats were taken out and made into section preparation.The transmission electron microscopy was adopted to observe the ultrastructural changes in the hippocampus CA1 pyramidal cells.Immunohistochemistry was used to detect the expression of apoptotic protease-activating factor(Apaf-1)and second mitochondria-derived activator of caspase(Smac)proteins in hippocampal CA1 region.The apoptosis of hippocampal CA1 neurons was detected by TUNEL method and the apoptosis index of neurons was calculated.Pearson test was used to analyze the correlation between Apaf-1 and Smac protein expression and apoptosis index of nerve cells at different time points.Results(1)Morris water maze:the escape latencies of intermittent hypoxia of rats in UC group respectively were(20.83±3.25)s,(22.17±2.79)s,(20.50±4.51)s and(21.17±4.17)s on day 7,day 14,day 21 and day 28;the cross target quadrant time respectively were(52.17±4.17)s,(52.50±2.74)s,(51.50±2.43)s and(52.00±4.78)s,and the difference was not statistically significant(all P values>0.05).The escape latencies of intermittent hypoxia of rats in the 5%IH group respectively were(33.17±2.14)s,(44.33±3.45)s,(52.17±3.87)s and(64.33±2.73)s on day 7,day 14,day 21 and day 28;the cross target quadrant time respectively was(44.00±3.03)s,(34.50±3.94)s,(27.83±3.01)s and(20.83±1.94)s;with the increase of intermittent hypoxia time,the escape latent period was prolonged,and the time to cross the target quadrant was significantly shortened.The differences were statistically significant(F=106.335,61.772,all P values<0.05).Comparing the two groups:the escape latent of 5%IH group were longer than that of UC group at each time point,and the cross target quadrant time of 5%IH group were shorter than that of UC group at each time point;the differences were statistically significant(all P values<0.01).(2)The transmission electron microscopy was adopted to observe the ultrastructural changes of nerve cells.The nucleus of neurons in hippocampal CA1 area of the rats in UC group was large;the nuclear membrane structure was complete,of which the shape was oval or round;the chromatin distribution was uniform in the shape of fine particles;the cell organelles were abundant,and the morphological structure was normal.In the 5%IH group,neurons started to appear edema,nuclear deformation,blurring of nuclear membrane,gradual disappearance of nucleoli,swelling and dissolution of organelles from Group 7d.With the prolongation of time,the level of change was more serious.(3)The relative expressions of Apaf-1 and Smac proteins:there was no statistically significant difference in the relative expression levels of Apaf-1 and Smac proteins in nerve cells in the CAl region of the hippocampus at each time point in the UC group(all P values were>0.05).The relative expressions of Apaf-1 and Smac proteins in the 5%IH group at all time points increased with the extension of intermittent hypoxia time,and the differences were statistically significant(F=25.328,42.923,P<0.01).Comparison between the two groups showed the relative expressions of Apaf-1 and Smac proteins of the 5%IH group were significantly higher than that of UC group at each time point and the differences were statistically significant(all P values<0.05).(4)The apoptosis of nerve cells:there were less apoptosis of nerve cells in the hippocampus CAl region of the UC group at each time point and AI differences had no statistical significance(P>0.05).Apoptosis of nerve cells in 5%IH group increased with the extension of intermittent hypoxia time and the AI differences were statistically significant(F=25.799,P<0.01).Comparison between the two groups showed the apoptosis index of nerve cells in the hippocampus CAl region of the 5%IH group were obviously higher than that in the UC group,and AI differences were statistically significant(all P values<0.05).(5)Pearson tests revealed:the expressions of Apaf-1,Smac protein in the hippocampal CA1 region of the rats in the 5%IH group at all time points were positively correlated with the apoptosis index(rApaf-1=0.735,0.736,0.685 and 0.747,rSmac=0.735,0.734,0.679 and 0.751,all P values﹤0.05).Conclusions Intermittent hypoxia can lead to cognitive dysfunction in rats,which may be related to the ultrastructural changes of nerve cells in the hippocampal CA1 region of rats and the induction of the expressions of apoptotic proteins Apaf-1 and Smac,resulting to the apoptosis of neurons.