针刺通过调控下丘脑SIRT1/FoxO1治疗胰岛素抵抗肥胖的机制研究

Insulin resistance (IR) is the key pathologic mechanism of obesity developing into type 2 diabetes(T2DM). In the hypothalamus, which controls normal body weight and glucose homeostasis, SIRT1 in the different nucleus deacetylates FoxO1 and the acetylation status of FoxO1 in hypothalamic neurons controls genes transcription of appetite-related neuropeptides, resulting in modulation on energy metabolism and insulin sensitivity. Hypothalamic SIRT1 is a new molecular target for the treatment of the rapidly-spreading metabolic disorders of obesity and T2DM. Our previous research demonstrated that acupuncture could control the body mass increase of high-fat-diet-induced IR rat model and reduce food intake, improve insulin sensitivity by stimulating SIRT1/PGC-1α in the skeletal muscle and mitochondrial metabolism. In this study we will intervene high-fat-diet-induced IR obese rat model by "The Combination of Biao-Ben Acupoints" acupuncture, with calorie restriction (CR), CR combined with acupuncture as control. Double immunofluorescence techniques, Western blotting, in situ hybridization, Dual-Luciferase Reporter Assay and RT-PCR will be applied to measure the gene transcription, mRNA and protein expression of SIRT1, deacetylation of FoxO1, and the expression of downstream appetite-related neuropeptides in the different nucleus of hypothalamus. Reactive oxygen species (ROS) changes will be tested as well. The aim of the study is to investigate the possible mechanism of acupuncture in modulating energy metabolism and IR through hypothalamic SIRT1 / FoxO1. The project will disclose the important mechanism of acupuncture in improving IR and energy metabolism centre of cntral nervous system, providing new proof for clinical application of acupuncture in the treatment of IR obesity and the related diseases.

胰岛素抵抗（IR）是肥胖发展成2型糖尿病的关键病理基础。能量代谢中枢下丘脑不同核团SIRT1去乙酰化FoxO1，控制食欲相关肽的基因表达，影响能量代谢和胰岛素敏感性，从而成为对抗肥胖和2型糖尿病的重要分子靶点。前期研究显示针刺能够通过激活骨骼肌SIRT1/PGC-1α、影响线粒体氧化代谢有效改善胰岛素敏感性，同时降低摄食量和体重。本项目拟采用"标本配穴"针刺干预胰岛素抵抗肥胖大鼠模型，运用荧光素酶报告基因技术、实时定量RT-PCR、免疫荧光双标等技术，观察针刺对下丘脑不同核团SIRT1激活FoxO1及其下游食欲相关肽基因转录、mRNA和蛋白表达、活性氧自由基的影响，并通过脑室微量给药特异性抑制SIRT1，观察针刺效应的变化，探讨针刺可能通过调控下丘脑SIRT1/ FoxO1影响能量代谢和胰岛素敏感性的机制。本项目的完成将为临床运用针灸腧穴配伍防治胰岛素抵抗肥胖及其相关性疾病提供新的科学依据

肥胖是引起胰岛素抵抗的主要因素，而胰岛素抵抗是肥胖导致2型糖尿病等代谢相关疾病的核心病理机制。课题组的前期研究结果表明：“标本配穴”针刺能够改善高脂饮食所致胰岛素抵抗大鼠的胰岛素敏感性，这种作用与针刺促进胰岛素靶器官——骨骼肌中的SIRT1、PGC-1α基因和蛋白表达有关。在此基础上，本课题提出“标本配穴”针刺改善胰岛素抵抗肥胖大鼠能量代谢的机制与下丘脑SIRT1/ FoxO1的调控参与有关，并且通过调控SIRT1/ FoxO1改善胰岛素抵抗肥胖大鼠下丘脑的氧化应激反应，发挥对能量代谢相关神经元的保护作用而调节体重的新假说。围绕这一假说，选用高脂饮食诱导的胰岛素抵抗肥胖大鼠为模型，观察到“标本配穴”针刺对胰岛素抵抗肥胖大鼠能量代谢中枢和胰岛素敏感性的整体调节作用，电针与SIRT1激动剂一样，具有降低餐后血糖的作用，且这一作用能够被SIRT1抑制剂部分阻断。电针激活SIRT1是降低餐后血糖的作用机制之一，电针还可能通过别的途径来调节血糖；电针能够促进胰岛素抵抗肥胖大鼠下丘脑中SIRT1的蛋白表达，电针介导的SIRT1激活能够调控转录因子FoxO1的乙酰化状态，SIRT1上调介导的FoxO1去乙酰化能够调控下丘脑食欲肽的基因NPY、POMC表达水平，抑制下丘脑氧化应激相关的酶类；电针及热量控制可以通过上调中枢下丘脑OB-Rb的表达，缓解瘦素抵抗，调节“脂肪-胰岛素”轴的负反馈机制，抑制促食欲肽AGRP的表达，从而防治胰岛素抵抗肥胖。本课题验证了针刺通过调控下丘脑SIRT1/ FoxO1影响能量代谢和胰岛素敏感性的机制。本项目的完成为临床运用针灸腧穴配伍防治胰岛素抵抗肥胖及其相关性疾病提供新的实验依据。