瘦素对睡眠剥夺小鼠下丘脑GABA含量及其受体表达的影响

Effects of leptin on GABA content and receptor expression in hypothalamus of mice with sleep deprivation

目的观察瘦素对睡眠剥夺小鼠下丘脑γ-氨基丁酸(GABA)含量及其受体表达的影响,并探讨可能的机制。方法采用随机数字表法将雄性C57BL/6小鼠分为对照组、睡眠剥夺组、瘦素补充组,每组8只。对照组设置水环境不剥夺睡眠;睡眠剥夺组利用改良多平台水环境法建立小鼠睡眠剥夺模型;瘦素补充组在剥夺同时予以每天两次瘦素1.3 mg/kg腹腔给药。睡眠剥夺7 d后,观察小鼠一般情况,测量体重并采集下丘脑组织和血浆标本,ELISA法检测各组小鼠血浆瘦素水平,下丘脑GABA、谷氨酸(Glu)含量。Western blotting检测下丘脑组织中GABA关键合成酶谷氨酸脱羧酶67(GAD67)及GABAA受体α1亚型蛋白(GABAARα1)的表达水平。结果与对照组相比,睡眠剥夺组小鼠体重明显减轻[(22.03±0.42)g vs.(17.75±0.75)g,P<0.01],而瘦素补充组小鼠体重[(15.10±0.38)g]较对照组和睡眠剥夺组均明显减轻(P<0.01)。与对照组比较,睡眠剥夺组小鼠皮毛失去光滑,对光线和声音特别敏感,表现为轻微躁狂,攻击性强,血浆瘦素水平明显降低[(483.81±21.72)ng/ml vs.(359.14±16.69)ng/ml,P<0.01],下丘脑GABA水平明显降低[(152.37±8.14)ng/g vs.(111.31±2.96)ng/g,P<0.05]。瘦素补充组小鼠下丘脑GABA水平[(132.19±3.38)ng/g]明显高于睡眠剥夺组[(111.31±2.96)ng/g,P<0.05],但与对照组比较差异无统计学意义(P>0.05)。睡眠剥夺组、瘦素补充组小鼠下丘脑Glu水平[分别为(686.56±10.01)ng/g、(668.64±9.93)ng/g]较对照组[(577.11±16.36)ng/g]明显升高(P<0.05),而瘦素补充组与睡眠剥夺组比较差异无统计学意义(P>0.05)。睡眠剥夺组小鼠下丘脑GAD67、GABAARα1蛋白表达[分别为0.68±0.06、0.69±0.07]较对照组(分别为1.09±0.13、0.99±0.07)明显降低(P<0.05),而瘦素补充组(分别为1.39±0.19、1.33±0.14)较睡眠剥夺组及对照组均明显升高(P<0.05)。结论瘦素可上调睡眠剥夺小鼠下丘脑内GABA关键合成酶GAD67的表达,升高下丘脑GABA含量,提高下丘脑GABAARα1蛋白表达,这可能是瘦素影响睡眠的重要机制。

Objective To observe the effects of the adipocyte hormone leptin on GABA content and receptor expression in hypothalamus of mice with sleep deprivation,and explore the possible mechanisms.Methods Male C57BL/6 mice were randomly divided into three groups(8 each):control group,sleep deprivation(SD)group and leptin supplement(L-SD)group.Mice in control group were set up in a water environment without sleep deprivation,mice in SD group were set up in a"modified multi-platform water environment"to establish a sleep deprivation model,and mice in L-SD group were given leptin 1.3 mg/kg intraperitoneally twice daily in conjunction with sleep deprivation.Seven days after sleep deprivation,the general conditions of mice were observed,body weight was measured and hypothalamic tissues and plasma specimens were collected.ELISA was used to detect the plasma leptin levels,hypothalamicγ-aminobutyric acid(GABA)and glutamate(Glu)contents.Western blotting was performed to detect the expression levels of GABA key glutamate decarboxylase 67(GAD67)and GABAA receptorα1 subtype protein(GABAARα1).Results Compared with control group,the weight of mice in SD group significantly reduced[(22.03±0.42)g vs.(17.75±0.75)g,P<0.01],while the weight of mice in L-SD group[(15.10±0.38)g]significantly reduced compared with that in control group and SD group(P<0.01).The mice in SD group lost their smooth hair,were particularly sensitive to light and sound,and were slightly manic and aggressive;Compared with control group,the plasma leptin level in mice of SD group significantly reduced[(483.81±21.72)ng/ml vs.(359.14±16.69)ng/ml,P<0.01],and the hypothalamic GABA level significantly reduced[(152.37±8.14)ng/g vs.(111.31±2.96)ng/g,P<0.05].GABA levels of mice hypothalamus in L-SD group[(132.19±3.38)ng/g]were significantly higher than that in SD group[(111.31±2.96)ng/g,P<0.05],but no statistical difference was when compared with that in control group(P>0.05).The hypothalamic Glu levels were obviously higher in SD group[(686.56±10.01)ng/g]and L-SD group[(668.64+9.93)ng/g]than that in control group[(577.11±16.36)ng/g](P<0.05),but no statistical significance was found when compared between SD group and L-SD group(P>0.05).The expressive levels of GAD67 and GABAARα1 protein in the hypothalamus of mice in SD group[0.68±0.06,0.69±0.07]were significantly lower than that in control group(1.09±0.13,0.99±0.07)(P<0.05);While the expressive levels of GAD67 and GABAARα1 proteins in the hypothalamus of mice in L-SD group(1.39±0.19 and 1.33±0.14,respectively)were significantly higher than those in SD group and control group(P<0.05).Conclusion Leptin can up-regulate the expression of the key GABA synthase GAD67,increase the content of GABA and the expression of GABAARα1 protein in hypothalamus of sleep-deprived mice,which may be an important mechanism of leptin affecting sleep.