交泰丸调节生物钟蛋白CLOCK/BMAL1相关信号通路改善糖尿病抑郁障碍的机制研究

Diabetes complicated with depressive disorder is harmful with high morbidity, but the treatment is limited.Studies have shown that the CLOCK/BMAL1 signaling pathway not only regulates the expression of cryptochrome (CRYs) and deacetylase (SIRT1) to increase insulin sensitivity, but also regulates the expression of depression-related neurotransmitters. Jiaotai pill, used to treat insomnia, has a significant effect on diabetes and depressive disorder, but the mechanism is unknown.On the basis of the previous confirmation that jiaotai pill can up-regulate the circadian protein CRYs to improve sleep and glucose metabolism disorder, our research group propose the hypothesis that "Jiaotai pill regulates the circadian protein CLOCK/BMAL1 related signaling to improve diabetes with depressive disorder". To verify our hypothesis, we will establish animal models of depressive db/db mice and CLOCK/BMAL1 gene knock-down cells to test glucose metabolism, depression-related exercise experiments and depression-related neurotransmitters. Immunology, molecular biology and other methods will be used to detect the changes in the expression of CLOCK/BMAL1 gene and protein in hypothalamus, peripheral tissues and gene knock-down cells, as well as the changes in insulin signaling pathway, the expression of SIRT1 and CRYs, so as to elucidate the mechanism of the dual effects of Jiaotai pill in improving diabetes and depressive disorder and to provide scientific basis for its clinical application.

糖尿病并发抑郁障碍的发病率高，危害大，但治疗措施有限。研究表明，生物钟CLOCK/BMAL1信号通路，既可调节隐花色素（CRYs）和去乙酰化酶（SIRT1）的表达增加胰岛素敏感性，又可调节抑郁相关神经递质的表达。治疗不寐症的交泰丸，对糖尿病和抑郁障碍均有显著疗效，但机制不明。本课题组在前期证实交泰丸可上调生物钟蛋白CRYs改善睡眠和糖代谢紊乱的基础上，提出“交泰丸调节生物钟蛋白CLOCK/BMAL1相关信号通路改善糖尿病抑郁障碍”的假说，建立db/db糖尿病小鼠合并抑郁模型，构建CLOCK/BMAL1敲减细胞，检测糖代谢、抑郁行为学改变和抑郁相关神经递质的同时，采用免疫学、分子生物学等方法，检测下丘脑、外周组织和基因敲减细胞中CLOCK/BMAL1基因和蛋白的表达，以及胰岛素信号转导通路、SIRT1、CRYs表达的变化，阐明交泰丸改善糖尿病抑郁障碍双重功效的机制，为临床运用提供科学依据。

糖尿病合并抑郁障碍发病率高，危害大，但临床重视不足且治疗棘手。交泰丸是治疗失眠的经典方剂，本课题组前期研究已证实其良好的降糖效果，近年来已有学者发现其具有显著的抗抑郁作用，而交泰丸是否能发挥降糖和抗抑郁的双重功效及其作用机制尚不明确。本课题通过慢性束缚应激建立糖尿病合并抑郁障碍小鼠模型，给予不同剂量交泰丸干预，检测其对小鼠糖脂代谢、抑郁相关神经递质、炎症因子、凋亡相关蛋白及生物钟蛋白CLOCK/BMAL1相关信号通路的影响。我们发现交泰丸对于关键生物钟蛋白CLOCK和BMAL1的确有显著影响，但因其蛋白浓度过低，为了提升整个实验的可行性，我们进一步研究其下游的cAMP/PKA/CREB信号通路，来验证交泰丸发挥双重功效的可能作用机制。结果显示，交泰丸干预可以显著改善糖尿病合并抑郁小鼠的糖脂代谢紊乱，缓解抑郁样症状，抑制全身炎症状态，减少海马神经元损伤，而这可能与激活cAMP/PKA/CREB信号通路有关。此外，结合指纹图谱分析结果及课题组前期研究基础，选择小檗碱进行体外实验。我们发现，小檗碱可以改善葡萄糖联合皮质酮干预所导致的HT22细胞损伤，而使用PKA蛋白抑制剂后，小檗碱的治疗效果也被抑制，因此，小檗碱的这种治疗作用也与cAMP/PKA/CREB信号通路有关。上述结果提示：交泰丸及其单体成分可以改善糖尿病合并抑郁障碍，其作用机制可能与激活cAMP/PKA/CREB信号通路有关。这可以为交泰丸的临床推广应用提供科学依据。