基于脂肪细胞因子途径研究电针激活AMPK调控网络介导糖脂能量代谢的作用机制

Our previous study has proved that the meridian is a material energy information conversion and transmission channel，with active energy metabolism characteristic of high temperature, high oxygen partial pressure and abundant microcirculatory perfusion. Electroacupuncture (EA) enhances oxygen utilization and metabolism along the meridian. Adiponectin (APN) and leptin (LP) are obesity associated proteins which have attracted much attention in recent years. EA stimulation at specific acupoints along meridian line will cause a series of reactions associated with energy metabolism. Obesity is associated with a series of metabolic disorders in vivo. Taking this as the breakthrough point, our research intends to study the metabolic mechanism of meridian in virtue of obesity model in New Zealand rabbits, fed with high-sugar and high-fat diet. We will take EA at Tianshu, Zusanli and Fenglong acupoints, use enzyme linked immunosorbent assay (ELISA) to detect serum APN and LP levels before and after EA, real-time fluorescence quantitative PCR to detect the expression levels of mRNA of APN and LP in the adipose tissue, western blot to detect content of the two kinds of protein, and compare glucose and lipid values before and after EA. Then we analyze effect on glucose and lipid metabolism and target protein expression with EA, furthermore explore the related mechanisms, look for new experimental evidence to explore the material basis of meridian based on adipo-cytokines signal transduction pathway and energy metabolism, as well as provide scientific basis for the clinical acupuncture treatment of obesity.

经脉线上能量代谢旺盛，针刺可使沿经代谢增强。腺苷酸活化蛋白激酶（AMPK）作为细胞内的能量开关，其激活途径及下游级联反应参与机体多种代谢过程，与针刺的相关研究鲜见报道，其间联系值得深入发掘。脂联素(APN)和瘦素(LP)是脂肪组织特异表达的细胞因子，与AMPK共同参与调控能量稳态。肥胖者脾胃运化失常，作为代谢失衡疾病模型具有代表性。本实验选用临床减肥常用穴“足三里”和“内庭”，食饵法造模肥胖SD大鼠，检测电针干预后血清中糖脂、胰岛素、APN和LP水平；HE染色比较脂肪细胞大小； RT-PCR法和Western-blot法分别检测脂肪组织APN和LP的mRNA表达及蛋白含量；测定脂肪组织、肝脏、骨骼肌和下丘脑的AMPK活性亚基mRNA表达及蛋白磷酸化水平，外周与中枢系统结合，分析针刺效应对糖脂代谢和目标蛋白表达调节的影响及信号转导机制，从脂肪细胞因子途径和能量代谢角度探讨针灸作用的物质基础。

针刺效应与能量代谢密不可分，寻找二者之间的桥梁是发掘物质基础的关键所在，能量代谢依托体内的一系列生化反应来实现，生化反应又与蛋白的功能状态直接相关，寻找相关信号蛋白并研究其与针刺效应的相互作用可为研究能量代谢提供一个方向。本课题结合文献及预实验基础提出假说：“电针诱导脂肪细胞因子激活AMPK通路改善糖脂能量代谢。”选用临床减肥常用穴“足三里”和“内庭”，食饵法造模肥胖SD大鼠，生化自动仪及ELISA法检测电针干预后血清糖脂、胰岛素、APN和LP水平；HE染色比较脂肪细胞大小；RT-PCR法和Western-blot法分别检测脂肪组织APN和LP的mRNA表达及蛋白含量；测定脂肪组织、肝脏、骨骼肌和下丘脑的AMPK活性亚基mRNA表达及蛋白磷酸化水平，外周与中枢系统结合，分析针刺效应对糖脂代谢和目标蛋白表达调节的影响及信号转导机制，从脂肪细胞因子途径和能量代谢角度探讨针灸作用机制。主要结果为：喂食高脂饮食8周的大鼠出现肥胖、高脂血症和肝脂肪变性。单纯高脂饮食组体质量，血脂明显升高，肝组织出现明显脂肪浸润，差异有统计学意义（p<0.05）；经过30d电针疗程干预后，减肥降脂有效，并且电针降低了肥胖大鼠脂肪组织中脂肪细胞平均面积，改变脂肪组织及循环血清中的ADPN和LP含量，不同程度增加下丘脑、肌肉、脂肪组织和肝组织的AMPK蛋白表达，说明电针通过调节脂肪细胞因子的含量，激活AMPK通路、增加脂肪酸氧化作用而显著改善了高脂血症大鼠的高脂状态和蛋白代谢。该结果为阐释中医经络物质基础及相关能量代谢理论添砖加瓦，证实了电针的干预对肥胖以及可能的相关代谢综合征的有益作用，部分揭示了电针治疗肥胖症的体内分子机制，为临床应用提供了实验数据参考，具有重要的科学意义，也能为更好地使中医经络服务相关应用开发提供确切可行的途径。