电针通过mTORC1/S6K1自噬途径改善胰岛素抵抗大鼠认知功能损害的机制研究

Insulin resistance of CNS is the key pathogenesis of mild cognitive impairment. Autophagy is involved in insulin signal transduction, and is a target for improving the CNS insulin resistance. In our previous studies, electroacupuncture/EA have been shown to relieve CNS insulin resistance, and inhibit pathological changes of cognitive impairment. But the mechanism is still unclear. Therefore, it is proposed that EA can promote cognitive function by relieving CNS insulin resistance through mTORC1/S6K1 autophagy pathway. In this study, EA intervention in high-fat-diet-induced IR model rats with cognitive impairment, co-immunoprecipitation, mRFP-eGFP-LC3, real-time PCR, western-blot will be applied. Combined with mTOR inhibitor and activator, to analyze the correlation betweenhypothalamic autophagy activation and CNS insulin signal transduction. mTOR activator is applied to intervene the activity of mTORC1/S6K1pathway, so as to explore the mechanism of EA on improving the cognitive function by relieve CNS insulin resistance through the mTORC1/S6K1 autophagy pathway. In order to confirm that hypothalamic autophagy activation is the key to the effect of EA on regulating CNS insulin signal transduction, and improving CNS insulin resistance is the target of promoting cognitive function. This project will provide experimental basis for the clinical application of EA in the prevention and treatment of IR-related cognitive impairment and other diseases.

中枢胰岛素抵抗是认知功能损害的重要发病机制。自噬参与胰岛素信号传导，是改善中枢胰岛素抵抗的有效途径。前期研究发现电针对中枢胰岛素抵抗认知功能损害具有良性调节作用，但作用机制尚不明确。由此提出电针通过mTORC1/S6K1自噬途径促进中枢胰岛素信号传导，缓解中枢胰岛素抵抗，改善胰岛素抵抗认知功能损害的假说。本项目以电针干预胰岛素抵抗认知功能损害模型大鼠，采用免疫共沉淀、双荧光活细胞成像、real-time PCR, western-blot等检测技术，以mTOR抑制剂与激动剂为对照，分析下丘脑自噬与中枢胰岛素信号传导的相关性；以mTOR激动剂干预mTORC1/S6K1自噬途径，探究电针通过自噬途径改善中枢胰岛素抵抗，促进认知功能的作用机制，以期证实下丘脑自噬激活是电针改善中枢胰岛素信号传导的关键，改善中枢胰岛素抵抗是促进认知功能的靶点，并为临床运用电针防治胰岛素抵抗认知功能损害提供实验依据。

课题组在前期研究中发现，电针能够提高胰岛素抵抗模型大鼠中枢胰岛素敏感性，改善胰岛素抵抗状态，促进学习记忆功能。本项目从mTORC1/S6K1途径入手，探讨电针缓解中枢胰岛素抵抗，改善认知功能损害的作用机制。研究采用高脂饲料诱导胰岛素抵抗模型，选用足三里、丰隆、关元、中脘和百会为电针干预穴位，以mTORC抑制剂和假电针作为阳性对照，观察电针对胰岛素抵抗大鼠胰岛素敏感性、糖脂代谢、自噬水平的影响。.结果表明：①高脂饲料诱导的胰岛素抵抗大鼠体重、血糖、血脂升高，胰岛素敏感性显著降低，逃避反应延迟、学习记忆能力下降；下丘脑Tau、p-Tau、Aβ1-42与S100B蛋白表达明显升高，说明胰岛素抵抗大鼠认知功能减退。下丘脑胰岛素信号传导相关分子IRS1、Akt及其磷酸化水平明显降低，GSK3β及其磷酸化水平明显升高，说明出现中枢胰岛素抵抗。下丘脑Atg1、Atg13、WIPI2、LC3-β与Beclin1水平升高；自噬小体的双层膜囊泡结构不完整，致密物增加；Atg1在细胞核和细胞质中表达增加，说明下丘脑自噬活性升高。②电针能有效控制胰岛素抵抗大鼠的体重增长，降低外周血糖脂水平，提高全身胰岛素敏感性；③电针能升高下丘脑IRS1、Akt及其磷酸化水平表达，降低GSK3β及其磷酸化水平，说明电针能够缓解中枢胰岛素抵抗。④电针能降低下丘脑Tau和Aβ1-42表达，促进胰岛素抵抗大鼠学习记忆能力；⑤电针治疗后，下丘脑Atg1，Atg13表达减少，LC3B-Ⅱ表达减少，提示电针能调控胰岛素抵抗大鼠下丘脑自噬水平；⑥mTOR抑制剂与电针存在类似的作用，能够缓解大鼠中枢胰岛素抵抗，减少Tau和Aβ1-42的表达，mTOR激动剂抑制电针效应。.本项目电针治疗可以改善胰岛素敏感性，改善外周糖脂代谢和中枢自噬活性，并通过mTORC1/S6K1途径调控中枢胰岛素信号传导，缓解中枢胰岛素抵抗，改善胰岛素抵抗认知功能损害。为临床运用电针治疗胰岛素抵抗及其相关性疾病提供了实验证据。