辛温通阳中药介导PKA/CREB通路活化PGC-1α调控非酒精性脂肪肝细胞糖脂代谢的研究

Based on research advances of the PGC-1 alpha as the center target of hepatic insulin-cAMP axis for regulating glucose and lipid metabolism, combined with our previous clinical research to activating yang herbal of acrid-warm flavor fistular onion stalk and fistular onion stalk extract on NAFLD patients, at the same time in the previous study, we found that fistular onion stalk extract could treat NAFLD in the modle of rats. We found that fatty degeneration of liver cell was associated with the high expression of PGC-1 alpha, which was the target of propelling the cell energy metabolism. This reasearch project aims to take experiments in vivo and in vitro experimental methods, with fat emulsion by making the rat models of NAFLD, fat emulsion induced fatty degeneration of liver cells for culture in vitro. We would observe fistular onion stalk extract on liver cell glycolipid metabolism level, detect signal pathway of PKA/CREB, PGC-1 alpha and downstream glycolipid metabolism speed enzyme gene CPT-1, PEPCK and G-6-Pase gene expression, write the change of ROS levels, respectively.At the same time,we would take the PKA inhibitors and cAMP agonists as controls, confirm fistular onion stalk extract to up-regulate PGC-1 alpha expression depending on the PKA / CREB signaling pathway activity. The research has very important scientific significance in the molecular mechanism of NAFLD and will make the great progress at the intervention approach of activating yang of acrid-warm flavor .

基于PGC-1α作为肝细胞胰岛素-cAMP轴的中心靶点调节糖脂代谢的研究进展，结合我们既往以辛温通阳中药葱白及葱白提取物抗NAFLD的疗效基础，同时在前期研究中，我们发现辛温通阳中药葱白提取物治疗NAFLD大鼠后，肝细胞脂肪变性的减轻与PGC-1α的高表达，从而与活化细胞能量代谢有关。本项目拟采取体内实验和体外实验的方法，以脂肪乳灌胃法建立NAFLD大鼠模型，用脂肪乳剂培养诱导体外肝细胞脂肪变性，观察辛温通阳中药葱白提取物对肝细胞糖脂代谢水平的影响，检测信号通路PKA/CREB、PGC-1α及下游糖脂代谢限速酶基因CPT-1、PEPCK和G-6-Pase基因的表达，动态观察ROS水平，分别以PKA抑制剂和cAMP激动剂作为对照，证实辛温通阳中药葱白提取物上调PGC-1α的表达依赖于PKA/CREB信号通路的活性。本研究对揭示NAFLD进展的分子机制及药物干预途径等都具有十分重要的科学意义。

基于PGC-1α作为肝细胞胰岛素-cAMP轴的中心靶点调节糖脂代谢的研究进展，结合我们既往以辛温通阳中药葱白及葱白提取物抗NAFLD的疗效基础，同时在前期研究中，我们发现辛温通阳中药葱白提取物治疗NAFLD大鼠后，肝细胞脂肪变性的减轻与PGC-1α的高表达，从而与活化细胞能量及线粒体代谢有关。.本课题运用体内实验和体外实验相结合的方法，以高脂饲料喂养法建立NAFLD大鼠模型，用脂肪乳剂培养诱导体外肝细胞脂肪变性，均以PKA抑制剂H89和cAMP激动剂Forskolin为对照，观察辛温通阳中药葱白提取物干预前后NAFLD大鼠血糖、血脂及肝脏形态及病理性改变，检测转录因子cAMP效应元件结合蛋白CREB、转录辅激活因子PGC-1α、脂肪酸β氧化限速酶CPT-1、肝糖异生限速酶PEPCK和G-6-Pase基因的表达水平，动态观察ROS及脂质过氧化物水平。其结果揭示了辛温通阳中药葱白提取物通过PKA/CREB通路上调关键因子PGC-1α表达，从转录水平激活一系列下游基因表达，诱导脂肪酸氧化及肝糖异生，减轻肝脏甘油三酯的堆积及胰岛素抵抗，从源头阻断非酒精性脂肪肝病的发生发展的分子机制。