慢性睡眠剥夺致生物钟表达紊乱在发育期下丘脑食欲调控失衡机制中的作用

In recent years, the problem of sleep curtailment in children around the world has facing increasingly grave challenges. Sleep curtailment can affect the appetite regulation in hypothalamus of children and increase the risk of overeating and obesity. Epidemiological studies have shown that sleep deprivation can cause circadian clock disorders. Previous studies suggested that the expression of circadian clock had a regulatory effect on appetite regulating proteins in the hypothalamic arcuate nucleus, but the mechanism has been still unclear. Our study also found that chronic sleep deprivation could cause mRNA levels of CLOCK and BMAL1 in hypothalamus and protein contents of phosphorylated STAT3 and POMC decreased, which suggested that chronic sleep deprivation could make JAK2/STAT3 signal pathway compromised, changing expression of appetite peptides. We hypothesize that chronic sleep deprivation could cause the circadian clock disorder, then lead to imbalance of hypothalamus appetite regulation. In the present project, sleep deprivation in developmental rats was carried out by using rodent sleep deprivation devices, and adenovirus vectors containing different genes for the overexpression of circadian clocks were injected into the lateral ventricle by using brain locator. Our study was designed to investigate the role of the circadian clock disorder caused by sleep deprivation in the imbalance of hypothalamus appetite regulation. The effects of expression of different circadian clock gene on the JAK2/STAT3 signal pathway could be clarified by cell experiments, in order to provide experimental references for revealing the mechanism of imbalanced appetite regulation in children caused by sleep curtailment.

近年来世界各国儿童睡眠不足问题日益严峻。睡眠不足可影响儿童下丘脑食欲调控，增加饮食过量和患肥胖风险。流行病学研究显示睡眠剥夺可引起生物钟紊乱。前人研究结果提示，生物钟的表达对下丘脑弓状核中食欲调节蛋白具有一定调控作用，但其机制尚不清楚。课题组前期研究发现慢性睡眠剥夺可引起发育期大鼠下丘脑CLOCK和BMAL1的mRNA水平下降，pSTAT3和POMC蛋白含量下降，提示慢性睡眠剥夺可使JAK2/STAT3信号通路转导障碍。我们假设：慢性睡眠剥夺可引起生物钟表达紊乱进而导致下丘脑食欲调控失衡。本项目利用啮齿类睡眠剥夺设备对发育期大鼠进行慢性睡眠剥夺，通过侧脑室注射含过表达生物钟基因的腺病毒载体，探究睡眠剥夺致生物钟表达紊乱在下丘脑食欲调控失衡机制中的作用。通过细胞实验明确不同生物钟基因的表达对JAK2/STAT3信号通路的调控作用，为揭示睡眠时间减少所致儿童食欲调控失衡的机制提供实验参考数据。

本项目研究慢性睡眠剥夺所致发育期小鼠生物钟蛋白表达紊乱对中枢食欲失衡的影响，明确生物钟BMAL1的表达对食欲相关通路JAK2/STAT3的调控作用，揭示睡眠剥夺所致发育期小鼠中枢食欲调控失衡的机制，进一步探讨儿童肥胖发生的原因及为其防治靶点提供实验参考数据。通过动物实验发现：1.下丘脑SCN区BMAL1过表达改善了慢性睡眠剥夺引起的过量饮食行为。SDGFP组小鼠的体重增长明显高于CONGFP组和SDBMAL1组。与CONGFP组和SDBMAL1组相比，睡眠剥夺第二周开始，SDGFP组小鼠食物摄入量显著增加。2.下丘脑SCN区BMAL1过表达挽救了慢性睡眠剥夺引起的生物钟基因表达的下降。与CONGFP组相比，SDGFP组小鼠下丘脑中的BMAL1、CLOCK、CRY1、PER1和PER2蛋白水平显著降低。clock、bmal1、cry1、per1和per2 mRNA水平的变化与蛋白的变化一致。3.下丘脑SCN区BMAL1过表达改善了慢性睡眠剥夺诱导的JAK2/STAT3信号通路转导障碍。与CONGFP组相比，SDGFP组下丘脑中JAK2和STAT3磷酸化水平降低，促食欲肽AGRP和NPY表达上调，抑食欲POMC蛋白和α-MSH表达下调。POMC、AGRP和NPY mRNA水平的变化与蛋白的变化一致。p-STAT3的抑制因子SOCS3，在慢性睡眠剥夺过程中显著上调。SDBMAL1组逆转了JAK2/STAT3信号通路在睡眠剥夺条件下蛋白及mRNA水平的变化。结论：睡眠剥夺所致发育期小鼠中枢食欲调控失衡，引起饮食体重增加这可能与生物钟蛋白表达下调，JAK2/STAT3信号通路转导障有关；睡眠剥夺小鼠下丘脑SCN区生物钟基因BMAL1过表达后，慢性睡眠剥夺所致JAK2/STAT3信号通路转导障碍及食欲调节紊乱得到改善，说明BMAL1可通过直接调控JAK2/STAT3信号通路从而影响食欲调控。