运动对小鼠肌肉组织miR-200b表达的影响及其改善胰岛素抵抗的分子机制研究

Exercise could improve insulin resistance through activating AMPK signal pathway; however, the underlying mechanism remains unclear. It has been proved that a variety of miRNAs played important roles in the development of insulin resistance. Recently, miR-200b, a microRNA involved in the epithelial to mesenchymal transition (EMT), has been found that it could inhibit the expression of FOG2, which could bind to PI3K to form a complex and reduce the activity of insulin pathway. In the previous work, we have found that miR-200b was significantly reduced in the skeleton muscles in the high fat diet induced mice with insulin resistance, whereas the exercise could increase the expression of miR-200b in the HFD mice. Furthermore, it was indicated that miR-200b was regulated by AMPK through the cellular experiment. We hypothesized that AMPK could up-regulate the expression of miR-200b, subsequently repress the expression of FOG2, and improve insulin resistance during exercise. Moreover, we also found that the key molecules in the NF-kB pathway, IKKβ and IRAK4, significantly reduced in the skeleton muscles of miR-200b transgenic mice, which suggested that miR-200b might repress the activity of NF-kB and alleviate inflammation induced by high fat diet so that improve insulin resistance. In this proposal, the mouse model with insulin resistance will be established and intervened by long term exercise, to investigate whether insulin resistance would be improved through the mentioned pathways regulated by miR-200b and its relationship with AMPK.

运动可以激活AMPK信号通路而改善胰岛素抵抗（IR），但其机制未完全阐明。已发现多种miRNAs在IR形成中发挥重要作用。最近发现， miR-200可抑制FOG2表达，降低其与PI3K蛋白结合，从而缓解IR。我们在前期研究中发现，在高脂诱导的IR小鼠模型肌肉组织中，miR-200b表达显著下降，而运动却可以提高其表达水平，并且在细胞水平上发现，AMPK可以调控miR-200b的表达。因此，我们推测运动通过激活AMPK升高miR-200b的表达，进而抑制FOG2，而起到改善IR的作用。另外，我们在已经构建的miR-200b转基因小鼠肌肉组织中发现IKKβ和IRAK4表达显著降低，提示miR-200b也可能对抑制NF-kB信号通路活性，减轻炎症反应，而改善IR。本课题拟通过长期运动来干预IR小鼠模型，研究miR-200b是否可以通过上述机制在运动改善胰岛素抵抗中发挥作用，及其与AMPK的关系。

胰岛素抵抗（Insulin Resistance, IR） 是 2 型糖尿病的重要特征和病理生理学基础, 运动（Exercise）能够有效的改善胰岛素抵抗[4]，但是其分子机制仍需要进一步阐明。本课题通过高脂诱导胰岛素抵抗大鼠模型来研究运动对miR-200b 表达的影响和调控机制，并进一步阐明运动改善胰岛素抵抗的新的分子机制，了解运动调节胰岛素信号通路的表观遗传学机制。 .一、模型建立 本课题采用脂肪含量 45%高脂饲料（试验组）和脂肪含量 10%普食饲料（对照组）分别饲喂养大鼠8周，每周检测两组大鼠的体重，8周后糖耐量（GTT）及胰岛素耐量（ITT），将所得数据绘制出每周的糖耐量和胰岛素耐量曲线，比较高脂组及对照组曲线下面积（AUC），两组之间 AUC 有明显的统计学差异，且高脂组的糖耐量和胰岛素耐量出现异常，成功制造出高脂诱导胰岛素抵抗大鼠模型。.二 运动方案 运动组：动物跑台坡度0°,速度10m/min，运动强度 74%VO 2 max，持续时间 60min，运动频率 1 次/天，5 天/周。共12周。.安静对照组：将大鼠置于跑台的跑道上，但不开启跑台，跑道不转动。共12周。.三、指标检测.试验动物运动训练12周后处死，取后腿肌肉进行指标检测。.1、.QPCR检测rno-mir-200b-3p.2、.WB检测Ikkβ，p-Ikkβ，P65，p-P65，PI3K，p-PI3K，AKT，p-AKT，IRS，p-IRS，Fog2，GLUT4.3、双荧光素酶报告基因实验验证Rat-mir-200b-3p与FOG2(Zfpm2) 的相互关系。.四、实验结果.1、mir-200b-3p在高脂组中表达下调；高脂运动后，表达上调；.2、p-Ikkβ， p-65在高脂组中表达明显上调；高脂运动后，表达明显下调；表明这些信号通路在高脂组中是被激活的，而运动后则被抑制；.3、p-PI3K， p-AKT， p-IRS在高脂组中表达下调；高脂运动后，表达明显上调；表明这些信号通路在高脂组中是被抑制的，而运动后则被激活；.4、.Fog2在高脂组中表达明显上调；高脂运动后，表达明显下调；.5、GLUT4在高脂组中表达下调；高脂运动后，表达上调；.6、Rat-mir-200b-3p对靶基因FOG2(Zfpm2)是有调控作用的，而且主要对1号位点（TTTATTTTAC CAGCAGTATT