从肠道菌群失调研究慢性失眠致认知障碍和糖代谢紊乱的共同机制及交泰丸的干预作用

Chronic insomnia not only impairs learning and memory ability but also decreases insulin sensitivity, thereby increasing the risk of developing dementia and diabetes mellitus. However, long-term use of sedative hypnotics, antidepressants and anxiolytics, which are commonly used to improve sleep, may aggravate cognitive decline and insulin resistance (IR). Jiao-Tai-Wan (JTW) is a famous prescription which can effectively treat insomnia caused by imbalance between heart and kidney. Our previous study had successfully proved that JTW showed beneficial effects on sleep, cognitive dysfunction and IR in the rats with chronic insomnia. Furthermore, these effects were related to maintaining intestinal barrier function and anti-inflammation. Recent studies have shown that sleep loss may cause gut microbial dysbiosis,leading to low-grade inflammation. Given that the weight ratio of Rhizome Coptidis and Cortex Cinnamomi contained is unique(10:1) and Rhizome Coptidis is often used to treat gut infection, we hypothesize that these novel effects of JTW in the treatment of insomnia are targeting the regulation of gut microbes and inflammation. To test our hypothesis, we will establish a rat model of chronic insomnia. Experiments have been designed to examine the differences in gut microbes and their metabolites. Meanwhile, LPS/TLR4/NF-κB signaling pathways in bacteria-gut-brain axis and bacteria-gut-peripheral tissue axis will be measured. Central amyloid metabolism, tau protein phosphorylation and peripheral insulin signaling will be also determined. In addition, the changes of these beneficial effects of JTW will be explored when depleting gut microbes by antibiotics treatment. Experimental techniques as metagenome sequencing will be used. We believe that the successful completion of the proposed research will illustrate the dual mechanism of JTW at treating cognitive decline and glucose metabolism disturbance in chronic insomnia,thereby providing scientific basis for clinical application.

慢性失眠既损害认知功能又降低胰岛素敏感性，增加罹患痴呆和糖尿病的风险。长期服用助眠类西药可改善睡眠，但却加重认知障碍和胰岛素抵抗（IR）。交泰丸是治疗心肾不交之不寐症的著名方剂，课题组前期证实其可改善失眠大鼠睡眠、认知和IR，机制与维持肠屏障功能和抗炎有关。基于交泰丸中高黄连、低肉桂（10:1）的特殊配伍比例且黄连常用于治疗肠道感染，而失眠可导致肠道菌群紊乱，进而引发低度炎症，我们提出“交泰丸调节肠道菌群、抑制炎症反应，从而改善慢性失眠认知障碍和糖代谢紊乱”的假说，建立大鼠慢性失眠动物模型，运用宏基因组学等技术，研究交泰丸对肠道菌群、“菌-肠-脑”和“菌-肠-外周组织”轴LPS/TLR4/NF-κB炎症信号通路、中枢Aβ和tau蛋白代谢、外周组织胰岛素信号通路的影响，同时探索抗生素处理去除肠道菌群后交泰丸功效的变化，阐明交泰丸改善慢性失眠认知障碍和糖代谢紊乱的共同机制，为临床应用提供依据。

本项目通过建立大鼠慢性失眠模型，给予不同剂量交泰丸干预，检测治疗前后大鼠睡眠结构、肠道菌群和肠道损伤、炎症信号通路、糖代谢和胰岛素信号转导通路、认知功能、血浆代谢物的影响，从“菌-肠-脑”和对“菌-肠-外周组织”轴探讨交泰丸改善慢性失眠相关认知障碍和糖代谢紊乱的机制，结果显示：①交泰丸干预可显著增加慢性失眠大鼠的睡眠时长，表现为增加非快速动眼睡眠时长、降低大鼠清醒期的delta波段能量和快速动眼睡眠的theta波段能量，且干预前后大鼠体重、肝肾功能和血脂无明显变化；②交泰丸干预显著改变慢性失眠大鼠的肠道菌群结构，表现为疣微菌门明显增加，肠道阿克曼菌丰度升高；③交泰丸可降低失眠大鼠肠道通透性，降低肠组织TLR4、MyD88、IKKβ、NF-κBp65的磷酸化水平，降低外周血LPS、LBP、IL-6及TNF-α浓度；④交泰丸干预未能慢性失眠大鼠肠组织ROS累积，但减少γH2Ax、TIA和Cleaved-caspase-3的表达，缓解肠道结构损伤，且能增加AMPK磷酸化水平和Sirt3、PGC1α、GSR、SOD1、Bcl2的表达；⑤交泰丸改善失眠大鼠糖耐量，降低附睾白色脂肪重量，增加脂肪组织AKT磷酸化水平；⑥交泰丸改善失眠大鼠认知功能，水迷宫实验显示逃避潜伏期显著缩短、目标象限游泳时间、目标象限游泳距离、平台穿过次数明显增加，降低脑脊液、海马、前额叶、颞叶皮质等脑区的Aβ42水平，降低脑组织IL-1β、IL-6、TNF-α等炎症因子水平，改善神经细胞凋亡；⑦交泰丸干预可影响慢性失眠大鼠的血浆中多种代谢产物，HPPA和NALT，这两种在交泰丸干预大鼠中增加的物质，可增加HT22、C2C12、Caco2细胞内生物钟蛋白Bmal1的震荡幅度；⑧交泰丸有效成分之一小檗碱可增加Caco2细胞内超氧阴离子水平，但没有明显增加细胞内H2O2水平，小檗碱干预可增加Caco2细胞AMPK磷酸化和Sirt3、GSR、Bcl2表达。上述结果提示：交泰丸可调节肠道菌群，增加阿克曼菌丰度，抑制肠源性内毒素血症、改善肠道和中枢炎症，从而改善慢性失眠相关认知障碍和糖代谢紊乱，为交泰丸的临床推广应用提供科学依据。