睡眠剥夺对小鼠蓝斑神经元抗氧化水平及自噬活动的影响

Effects of sleep deprivation on antioxidant activity and autophagy in locus coeruleus neurons in mice

目的观察睡眠剥夺(sleep deprivation,SD)对蓝斑(locus coeruleus,LC)神经元抗氧化(antioxidant)水平和自噬(autophagy)活动的影响。方法成年(8~10周)雄性C57BL/6小鼠54只(23~26 g),采用轻柔刺激法建立全睡眠剥夺6 h(SD 6 h)(n=8只/组)和12 h(SD 12 h)(n=8只/组)模型,改良多平台法建立REM睡眠剥夺(REM sleep deprivation,RSD)(n=11只/组)模型。Western blot检测超氧化物歧化酶2(superoxide dismutase,SOD2)、过氧化氢酶(catalase)、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和P62(也称SQSTM1)的表达水平。免疫荧光染色观察溶酶体膜相关蛋白-1(lysosomalassociated membrane protein 1,LAMP-1)的表达。结果 SD 6 h后,蓝斑神经元SOD2(P<0.05)和catalase(P<0.05)表达水平显著上调。SD 12 h后,蓝斑神经元SOD2和catalase表达水平无明显变化。但是RSD后,蓝斑神经元SOD2(P<0.05)和catalase(P<0.05)表达水平显著下调。外侧下丘脑作为对照脑区,睡眠剥夺对其神经元SOD2和catalase表达水平均无明显影响。SD 6 h和SD 12 h对蓝斑神经元LC3-Ⅱ和P62表达水平均无明显变化。但是RSD显著降低蓝斑神经元LC3-Ⅱ表达水平(P<0.05),而P62表达水平升高(P<0.05),LAMP-1的表达也相应减弱。SD对外侧下丘脑神经元LC3-Ⅱ和P62表达水平均无显著影响。结论短时全睡眠剥夺引起蓝斑神经元抗氧化反应一过性升高而自噬活动无明显影响;长时程REM睡眠剥夺使蓝斑神经元的抗氧化水平和自噬活动均减弱。

Objective To observe the effects of sleep deprivation on the antioxidant activity and autophagy in the neurons in the locus coeruleus of mice. Methods Fifty-four adult male C57BL/6 mice （8-12 weeks old, weighing 23-26 g） were randomized into total sleep deprivation （induced by gentle stimulation） for 6 h group （n=8）, total sleep deprivation for 12 h group （n=8）, and rapid eye movement sleep deprivation （RSD; induced using a modified multiple platform method） group （n=11）, and each group was assigned a normal control group with the same number of mice. Western blotting was used to detect the expression levels of superoxide dismutase 2 （SOD2）, catalase, microtubuleassociated protein 1 light chain 3 Ⅱ （LC3-Ⅱ） and P62 （also known as SQSTM1）,and immunofluorescence staining was used to detect the expression of lysosomal-associated membrane protein 1 （LAMP-1） in the locus coeruleus and the lateral hypothalamus of the mice. Results The protein levels of SOD2 and catalase were significantly increased in the neurons of the locus coeruleus in the mice after sleep deprivation for 6 h （P〈0.05）, but showed no further increment after sleep deprivation for 12 h. RSD significantly decreased the protein levels of SOD2 and catalase in the locus coeruleus neurons （P〈0.05）. In the lateral hypothalamic neurons, sleep deprivation resulted in no significant changes in the expression levels of catalase or SOD2. Sleep deprivation for 6 and 12 h did not significantly affect the expression of LC3-Ⅱ and P62 in the locus coeruleus, whereas RSD caused a significant decrease in LC3-Ⅱ expression （P〈0.05）, increased P62 level （P〈0.05） and reduced LAMP-1 level in the locus coeruleus neurons （P〈0.05）. Sleep deprivation did not affect the expression levels of LC3Ⅱ or P62 in the lateral hypothalamic neurons. Conclusion Short-term total sleep deprivation causes a transient increase in the antioxidant activity whereas has no significant effect on autophagy in the locus coeruleus neurons. Long-term RSD significantly reduces both the antioxidant activity and autophagy in the locus coeruleus neurons of mice.