抑制生物钟蛋白CRY相关炎症信号：交泰丸改善睡眠和胰岛素抵抗双重功效的共同分子机制？

Jiaotai pills, a Rhizoma Coptidis and Cinnamon-containing Chinese herbal formula, has been widely used to treat insomnia (heart and kidney disharmony) in China. Previous evidence has demonstrated the dual efficacy of Jiaotai pills at improving sleep and insulin resistance (IR),for which the precise mechanism remain unclear. Recent researches have shown that the development of IR is related to sleep loss. Given that elevated inflammatory mediators during sleep deprivation contribute to IR, we hypothesize that Jiaotai pills cocurrently improve sleep and IR through inhibiting circadian clock protein cryptochrome (CRY) associated inflammatory signaling. To test our hypothesis, we will establish animal models of chronic partial sleep deprivation in rats in which CRY gene expression is down-regulated. Experiments have been designed to examine sleep structure, the levels of blood glucose, the concentrations of inflammatory biomarkers and insulin sensitivity. For mechanistic study, we will examine CRY expression in hypothalamus,peripheral blood mononuclear cells and peripheral tissues, as well as subsequent changes in CRY downstream nuclear transcrition factor kappa-B(NF-κB) and the expression of molecules involved in insluin signaling pathway. Different experimental techniques including HPLC/HPLC-MS,ELISA, real time PCR, immunohistochemistry, Western blot will be used. Furthermore, we will optimize the dosage regimen of Jiaotai pills. The successful completion of the proposed research at integrative and molecular levels will illustrate the mechanisms of the dual actions of Jiaotai pills, based on which we will be able to develop a new biological rhythm-based treatment for diabetes. Additionally, the proposed research will provide scientific foundations for the efficacy of Jiaotai pills at "coordinating heart and kidney" and promote its wide use in clinical practice.

交泰丸由黄连、肉桂组成，主治心肾不交之不寐证。前期研究显示，交泰丸能改善睡眠，也能改善胰岛素抵抗（IR），但具体机制不明。新近发现，IR的发生与睡眠不足有关。本课题以"炎症是睡眠剥夺引发IR的病理生理学基础"为切入点，提出"抑制生物钟蛋白CRY相关炎症信号是交泰丸改善睡眠和IR双重效应的共同分子机制"的假说，建立大鼠慢性不完全性睡眠剥夺模型，在睡眠描记和检测糖代谢、炎症标志物和胰岛素敏感性的同时，采用ELISA、实时荧光定量PCR、免疫组化、Western blot、HPLC/HPLC-MS等方法，检测下丘脑、外周血单核细胞、外周组织CRY基因表达的变化，以及核转录因子NF-κB炎症信号和胰岛素信号转导通路分子的活性和表达，探讨交泰丸作为CRY激活剂的可能性，优化给药方案，促生一种基于生物节律的糖尿病治疗新方法，初步揭示交泰丸"交通心肾"的科学内涵，为其临床应用提供科学依据。

本项目在分析交泰丸配方颗粒主要有效成分的基础上，通过噪音干扰4周成功建立肥胖抵抗（OR）大鼠慢性不完全性睡眠剥夺模型，比较不同剂量交泰丸主要效应成分小檗碱、肉桂酸在OR大鼠体内的药代动力学参数，同时探索交泰丸对失眠大鼠睡眠结构、糖代谢、外周血、下丘脑、肝脏和脂肪组织炎症因子、生物钟基因CRY、cAMP/PKA/NF-κB和胰岛素PI-3K信号通路的影响。结果显示：①交泰丸可显著改善失眠大鼠的睡眠结构，表现为增加总睡眠时间、总慢波睡眠（SWStotal）、慢波睡眠1期（SWS1）时间；②降低大鼠体重、空腹血糖（FPG）、空腹胰岛素（FINS）、胰岛素抵抗指数（HOMA-IR）；③降低外周血hs-CPR、TNF-α、IL-6、IL-1β的浓度，降低下丘脑、肝脏和脂肪组织TNF-α、IL-6、IL-1βmRNA的表达；④降低外周血单核细胞（PBMC）NK-κBmRNA的表达，降低下丘脑、肝脏和脂肪组织NF-κB（p-P65/ P65）的活性；⑤降低下丘脑cAMP含量和PKA（p-VASP/VASP）的活性；⑥增加下丘脑、肝脏和脂肪组织胰岛素PI-3K/AKT信号通路各分子（PI-3Kp85、p-AKT/AKT、GLUT3、GLUT2、GLUT4）的表达；⑦增加PBMC的Cry1mRNA和Cry2mRNA表达，增加下丘脑、肝脏和脂肪组织Cry1和Cry2蛋白的表达。上述结果提示：对于慢性不完全性睡眠剥夺OR大鼠，交泰丸具有改善睡眠和胰岛素抵抗的双重功效，且这两种功效均与调节生物钟基因Cry相关炎症信号通路相关。