糖尿病性心肌纤维化中SIRT1与MIF信号通路交互作用的分子机制研究

Myocardial fibrosis is the most important aspect in diabetic cardiac remodeling, but the mechanism underlying is not well elucidated. Our previous study showed that silent mating type information regulation 2 homolog-1 (Sirtuin1, SIRT1) protein was markedly decreased, and macrophage migration inhibitory factor（MIF）was markedly increased in the myocardium of 18-week-old diabetic db/db male mice with obvious myocardial fibrosis.A mutual negative regulation was found between SIRT1 and MIF in mouse cardiac fibroblasts.We hypothesize that MIF activates Smad3 to modulate ECM-related genes expression and negatively modulates SIRT1 protein expression via MIF-CD74-MAPK axis. Down-regulation of SIRT1 decreases the deacetylated forms of NF-kB, AP1 and Smad3, and reduces the level of reactive oxygen species(ROS) in cardiomyocytes, contributing to MIF expression, activation of MIF signaling pathway and ECM-related genes expression. The diabetic db/db male mice, MIF-knockout(MIF-KO) male mice and C57BL/6 male mice, and cardiac myofibroblasts derived from the adult MIF-KO male mice and adult C57BL/6 male mice will be used in the present project. We will study the roles of SIRT1 and MIF, cross-talk between SIRT1 and MIF signalling pathway in the process of diabetic myocardial fibrosis at the whole organism, cellular and molecular level,respectively. The effects of myocardium-specific over-expression of SIRT1 and suppression of MIF will be evaluated on the therapy effectiveness of diabetic myocardial fibrosis in diabetic db/db male mice. The present study is expected to provide scientific evidence and material for future therapy research on diabetic myocardial fibrosis.

心肌纤维化是糖尿病性心肌重构的重要方面， 但发生机制不明。我们证实发生纤维化的糖尿病db/db小鼠心肌中去乙酰化酶SIRT1和巨噬细胞移动抑制因子（MIF）分别显著降低和升高，SIRT1和MIF在心肌成纤维细胞中存在彼此负调控作用。推测MIF通过MIF-CD74-MAPK轴负调控SIRT1蛋白表达，活化Smad3并调控细胞外基质(ECM)相关基因表达；SIRT1降低使心肌消除活性氧簇(ROS)能力下降、去乙酰化NF-kB、AP1和Smad3减少，进而促进MIF表达、MIF信号通路激活和ECM相关基因表达。本课题将利用db/db小鼠和db/m小鼠，MIF基因敲除小鼠、C57BL/6小鼠及其心肌成纤维细胞，从整体-细胞-分子水平，研究SIRT1与MIF信号通路的交互作用，评价增强心肌SIRT1表达，抑制MIF表达对心肌纤维化的治疗作用，为进行糖尿病性心肌纤维化的治疗研究提供科学依据和资料。

心肌纤维化是糖尿病性心肌重构的重要方面， 但发生机制不明。巨噬移动抑制因子（MIF）是一种重要的前炎症因子，控制着炎症反应的起始点和反应程度，但MIF又具有明确的抗氧化活性。一般认为，炎症反应参与了糖尿病性心肌纤维化的过程，但MIF在发生纤维化的糖尿病心肌中的表达和作用机制不明。.利用MIF整体敲除（MIF-KO）及其野生型对照小鼠，建立链脲佐菌素(STZ)诱导的I型糖尿病动物模型，检测小鼠心肌中Sirt1，纤维化相关Cola1、Col3a1和α-SMA等目标基因的表达。分离培养小鼠心肌成纤维细胞，建立高葡萄糖/葡萄糖氧化酶（HG/Go）诱导的糖尿病性心肌纤维化的细胞模型。研究MIF对小鼠心肌成纤维细胞表型和纤维化相关基因表达调控的分子机制，并探讨MIF对小鼠心肌细胞中葡萄糖转运蛋白GLUT4表达的调控作用。.本项目从整体动物水平证实MIF敲除可加重糖尿病性心肌纤维化，但不影响SIRT1的表达。明确MIF通过上调Nrf2表达，增加抗氧化相关HO-1、SOD1、SOD2和TRX2表达，降低ROS水平，抑制Smad3信号通路活化。阐明MIF通过上调MLL和HCF1表达来增加Nrf2表达，进而使心肌成纤维细胞中抗氧化相关基因表达增加。发现MIF可特异上调具有抗纤维化作用的微小RNA-29b（miR-29b）表达来抑制纤维化相关基因表达。同时，证实MIF通过激活AMPK信号通路和MEF2A，-2C，-2D和Zac1上调心肌细胞中GLUT4表达，进而有利于抑制心肌纤维化的发生。本项目证实MIF具有抑制糖尿病性心肌纤维化的作用，并从整体-细胞-分子水平阐明了MIF通过增强Nrf2和抗氧化相关基因表达，降低ROS水平和Smad3信号通路激活，升高miR-29b和GLUT4表达来抑制心肌纤维化的分子机制。本项目为以MIF为靶点的糖尿病性心肌纤维化的治疗研究提供科学依据和资料，具有重要的科学意义和应用研究价值。