养心安神法调控腺苷参与睡眠剥夺模型基底前脑能量代谢重构的机制研究

Sleep deprivation leads to the reconstruction of energy metabolism and increases the risk of obesity, insulin resistance, metabolic syndrome and other diseases. In previous work, it was found that the expression of AVP was enhanced by the heart-nourishing and mind-calming therapy, and AVP was the upstream activator of the AMPK signaling pathway. Chronic sleep deprivation induced basal forebrain astrocytes releasing adenosine, which activating A1 adenosine receptors. Heart-nourishing and mind-calming therapy can activate the AMPK/PPAR/PGC-1 signal pathway, which can regulate the astrocytes and neurons crosstalk, and participate in the regulation of sleep wake and energy metabolic remodeling via improve the expression of AVP. Accordingly, the project will observe the overall effect of sleep deprivation, biochemical indicator, metabolic pattern and metabolic profile; localization of AS, Glu, GABA through GFAP immunofluorescent double staining , study the interaction mechanism of AS and neuron through construction of A1ARsiRNA and A1AR overexpressed plasmid; AMPK/PPAR/PGC-1α signaling pathway changes based on bioinformatics screening of energy metabolism differentially expressed genes and key signaling pathways through Western blot, RT-PCR methods.It will provide new perspectives and ideas for prevention and treatment of sleep deprivation, and further enrich the scientific connotation of heart-nourishing and mind-calming therapy.

睡眠剥夺导致能量代谢重构，增加肥胖、胰岛素抵抗、代谢综合征等疾病风险。前期工作发现，养心安神法提高AVP表达，而AVP是AMPK信号通路的上游激活因子。慢性睡眠剥夺引起基底前脑星形胶质细胞释放腺苷，激活A1腺苷受体，养心安神法可通过提高AVP表达，激活AMPK/PPAR/PGC-1α信号通路，调控星形胶质细胞与神经元交流对话，参与调控睡眠觉醒和能量代谢重构。本项目在前期基础上，拟检测睡眠剥夺模型整体效应、代谢生化指标、代谢模式和代谢轮廓变化；GFAP免疫荧光双标法定位AS与Glu、GABA共表达，构建A1ARsiRNA和A1AR过表达质粒转染等研究AS与神经元交流对话机制；在生物信息学筛选SD中能量代谢差异表达基因和关键通路基础上，结合WB、RT-PCR检测AMPK/PPAR/PGC-1α信号通路变化，为睡眠剥夺防治提供新的视角和思路，进一步丰富和阐释养心安神法的科学内涵。

睡眠剥夺导致能量代谢重构，增加肥胖、胰岛素抵抗、代谢综合征等疾病风险。前期发现，养心安神法提高AVP表达，而AVP是AMPK信号通路的上游激活因子。提出科学假说：慢性睡眠剥夺引起基底前脑星形胶质细胞释放腺苷，激活A1腺苷受体，养心安神法可通过提高AVP表达，激活AMPK/PPAR/PGC-1α信号通路，调控星形胶质细胞与神经元交流对话，参与调控睡眠觉醒与能量代谢重构。主要研究内容：①研究睡眠剥夺模型能量代谢模式变化及养心安神法干预机制。②探究养心安神法对Ade及A1AR参与睡眠剥夺模型能量代谢过程中星形胶质细胞/Glu神经元交流对话机制。③筛选睡眠剥夺中关键能量代谢通路及关键蛋白相互关联关系，构建能量代谢在睡眠觉醒中的作用机制网络。④体内实验结合虚拟实验，研究养心安神法对睡眠剥夺AMPK/PPAR/PGC-1α能量代谢相关信号通路影响。研究结果显示：①多平台水环境法睡眠剥夺14d制备的失眠模型，模型大鼠总睡眠时间，慢波睡眠总时间，慢波睡眠第一第二阶段时间均显著缩短，学习记忆能力下降，内分泌激素分泌紊乱，糖脂代谢紊乱，大鼠海马神经元受损，养心安神法代表方剂天王补心丹干预后模型各指标改善较明显。②天王补心丹能清除睡眠剥夺大鼠海马中过量的谷氨酸，还能恢复谷氨酸转运体水平，天王补心丹含药血清可能会降低星形胶质细胞膜内钙离子浓度，减少细胞的凋亡，天王补心丹有效单体干预星型胶质细胞发现其对星形胶质细胞均能产生良好的促进增殖作用。③基因差异分析，测序得到321个差异表达基因。差异基因GO富集分析、差异基因KEGG富集分析、通路显著性分析等发现天王补心丹从多方面干预睡眠剥夺大鼠的能量代谢机制。④分子对接结合体内实验发现天王补心丹可通过特定位点与AMPK/PPAR/PGC-1α信号通路中的特定蛋白靶点结合模拟机体内能量代谢过程，发挥调控睡眠剥夺模型能量代谢的作用。养心安神法能对能量代谢和睡眠觉醒起到双重改善作用，进一步阐释了养心安神法在睡眠剥夺中的治则治法科学意义，丰富了中医“养心安神”理论内涵。