Notch1受体调控GSK-3beta抑制糖尿病心肌病的机制研究

Cardiac hypertrophy is one of distinct characters of diabetic cardiomyopathy, but the mechanism is still unclear. Reactive oxidative species (ROS) activate downstream signaling pathway to promote cardiac hypertrophy by inhibiting GSK-3beta. Notch1 receptor modulates the heart homeostasis, but what is the effect of Notch1 receptor on cardiac hypertrophy induced by diabetes and what is the mechanism underlying still unknown. In addition, there are few reports about the effect of Notch1 receptor on diabetic cardiomyopathy at home and abroad. Our previous research had reported that decreasing the level of ROS was able to alleviate the cardiac cell injury. In our pre-experiment, we observed that the expressions of beta-myosin heavy chain and atrial natriuretic peptide were up-regulated, and cardiac cells became hypertrophy after treated with high glucose for 48 hours; when Notch1 receptor was inhibited, the cardiac hypertrophy was obviously deteriorate. So we hypothesize that Notch1 receptor is activated, acts on its target genes, then modulates the activity of GSK-3beta in order to inhibit the cardiac hypertrophy induced by diabetes. In this project, we will duplicate the diabetic cardiomyopathy animal model by Notch1 knock-out and wide type mice and diabetic cardiomyopathy cell model induced by high glucose, and then we will prove our hypothesis in vivo and in vitro by siRNA, co-immunoprecipitation, transmission electron microscope and immunofluorescence ect. Our research will provide a new theory and drug target for the prevention and cure of diabetic cardiomyopathy.

心肌肥大是糖尿病心肌病的显著特征，但发病机制不详。活性氧物质（ROS）激活下游信号通路，拮抗GSK-3beta的活性，促进心肌肥大的发生。Notch1受体参与心脏稳态的调节，但它在糖尿病诱发的心肌肥大中的作用及其作用机制尚不清楚，且国内外鲜有报道。本课题组已证实，降低ROS水平可以减轻心肌细胞损伤程度。预实验结果显示：高糖培养心肌细胞48小时后，beta-肌球蛋白重链和心钠肽的mRNA表达上调，心肌细胞明显肥大；抑制Notch1受体后，心肌细胞肥大程度显著加剧。因此，我们提出“Notch1受体活化并激活其下游靶基因，通过调控GSK-3beta的活性抑制糖尿病诱发的心肌肥大”的假说。本课题拟制备Notch1敲除鼠和野生鼠的糖尿病心肌病动物模型和高糖细胞模型，运用siRNA、免疫共沉淀、透射电镜和免疫荧光等技术，从整体和细胞层次证明我们的假说。本研究将为糖尿病心肌病的防治提供新理论和新靶点。

心肌肥大是糖尿病心肌病的显著特征，但发病机制不详。Notch1受体参与心脏稳态的调节，但它在糖尿病诱发的心肌肥大中的作用及其作用机制尚不清楚，且国内外鲜有报道。本项目制备Notch1敲低和野生型的糖尿病心肌病小鼠模型和高糖细胞模型，运用透射电镜、激光共聚焦、分子生物学技术、小动物超声、激光散斑成像等技术，从整体和细胞层次进行研究。本课题在糖尿病动物模型和细胞模型中的主要研究成果如下：1）敲低Notch1受体引起小鼠心脏代偿性轻度扩张；敲低Notch1受体的糖尿病小鼠表现出严重扩张性心肌病，其心功能显著下降，心脏对脑部皮质的供血显著降低；证实Notch1受体在维护正常心功能方面具有保护作用。2）证实Notch1受体有利于维持心肌纤维正常形态和超微结构。3）敲低Notch1受体的糖尿病小鼠GSK-3alpha, GSK-3beta 和p-GSK-3beta蛋白的表达显著降低，Notch1受体胞内段与GSK-3beta的相互作用受到干扰，其机制主要与Wnt/beta-catenin、Akt/GSK-3beta和PKCbeta2信号通路相关。4）敲低Notch1受体导致心肌细胞自噬水平显著被抑制，证实Notch1受体通过AMPK信号通路调节自噬。5）细胞模型证实抑制Notch1受体会加剧高糖诱导的原代心肌细胞的肥大；激活Notch1受体可以一定程度的缓解高糖诱导的原代心肌细胞的肥大，其机制与降低活性氧物质生成有关。本课题已发表相关SCI论文5篇；会议论文1篇；申请专利1项。本研究揭示了Notch1受体发挥心肌保护作用的机制，为糖尿病心肌病的防治提供新理论和新靶点。