基于SGLT-1靶标和NLRP3信号通路探讨血糖波动诱导糖尿病心肌细胞焦亡的机制

Diabetes Cardiomyopathy(DCM) is one of most popular diabetes complications, while its mechanism is still not very clear.Pyroptosis is a new way of programming cell death which recently was found in DCM. Sodium -glucose cotransporter 1(SGLT-1) is a very important glucose transporting protein which is significantly upregulated in diabetes cardiomyocyte.However, few research is about what function SGLT-1 play on the injury of cardiomyocyte. Our researching group is the first one who found the best glucose lowering rate existing. Pyroptosis and SGLT-1 upregulation of Cardiomyocyte of mice suffered from type 2 diabetes mellitus (T2DM) were also proved to be improved by Blood glucose fluctuation in our preliminary experiment. Meanwhile, pyroptosis related factors were also found arising in clinical patients administrated with SGLT inhibitor. Therefore, we assume that fasting blood glucose fluctuation would upregulate SGLT-1 of cardiomyocyte, which in turn lead to natrium overload, hence NLRP3 pyroptosis pathway is activated, and as a result, myocardial injury may occure. We will test the effection of blood glucose fluctuation on pyroptosis and SGLT-1 of cardiomyocyte in mice, and investigate whether the cardiomyocyte pyroptosis is induced by SGLT-1 upregulation, natrium overload and NLRP3 activation. The results are expected to contribute to the clarification of the pyroptosis mechanism of DCM and provide theoretical evidence for prevention and treatment of DCM.

糖尿病心肌病（DCM）是常见心血管并发症，但其机制并不透彻。细胞焦亡是近年新发现的程序性细胞死亡方式，最近研究发现DCM时出现细胞焦亡。钠糖转运蛋白1（SGLT-1）是重要的葡萄糖转运蛋白，糖尿病时心肌SGLT-1明显上调，但其参与的心肌损伤作用研究甚少。本课题组前期研究首次发现2型糖尿病（T2DM）患者存在最佳降糖速率，预实验验证了血糖波动能够促进T2DM小鼠心肌细胞焦亡发生及SGLT-1上调，临床观察发现SGLT类抑制剂可能抑制细胞焦亡相关因子产生。据此我们假设：快速血糖波动导致心肌细胞SGLT1上调，进而导致钠超载，NLRP3细胞焦亡通路激活，促进心肌损伤发生。我们将在T2DM小鼠体内验证血糖波动对心肌细胞SGLT-1及焦亡的影响。同时通过基因沉默等方法验证心肌细胞SGLT-1上调后，通过激活NLRP3通路促进心肌细胞焦亡，阐明DCM的细胞焦亡机制，为DCM的临床防治提供实验基础。

糖尿病心血管并发症因其是糖尿病致残和死亡的首要原因而备受关注。其中糖尿病性心肌病（DCM）独立于心血管疾病，是糖尿病患者致死的主要原因之一。因此阐明DCM的病理机制，寻找可能的生物学标志物及新的治疗靶点，对指导DCM的临床防治有重要价值。SGLT1在小肠、肾、肝、肺、心肌细胞中表达，心肌 SGLT1 的表达水平可能反映心力衰竭的严重程度。糖尿病时心肌SGLT-1明显上调，但其参与的心肌损伤作用研究甚少。血糖波动（GV）与DCM密切相关。在糖尿病心血管并发症的发病机制中，GV比持续高血糖更有害。然而，血糖波动对心脏功能的损害机制仍不清楚。我们构建了血糖波动的糖尿病小鼠模型，模型构建后第6周利用SGLT1沉默腺相关病毒对小鼠进行尾静脉注射，病毒干预后测量小鼠体重及血糖，超声心动仪检测小鼠心功能相关指标，观察小鼠心肌病理结构及心肌组织超微结构。通过应用免疫组织化学染色，western blot，流式细胞术，ELISA等技术检测心脏组织学结构及凋亡、焦亡和氧化应激的变化。并进一步在体外细胞模型中证实了血糖波动诱导心肌细胞SGLT-1上调后，通过激活NLRP3 通路促进心肌细胞焦亡。本课题主要发现如下：（1）：SGLT1的敲低可通过调节氧化应激和对抗线粒体功能障碍来改善由葡萄糖水平波动引起的心肌细胞凋亡（2）：快速血糖波动导致心肌细胞SGLT1上调，NLRP3细胞焦亡通路激活，促进心肌损伤发生。SGLT1沉默可部分阻断糖尿病小鼠血糖变异性引起的心脏损伤以及所诱发的炎症反应和心肌细胞焦亡；（3）：血糖波动加重心肌纤维化，SGLT1可能通过直接调节心脏巨噬细胞和成纤维细胞的活化，参与血糖波动诱导的心脏纤维化；（4）在GVDM小鼠左心室组织中，血糖波动驱动M1巨噬细胞极化，抑制SGLT1部分逆转了M1巨噬细胞极化并促进巨噬细胞向M2表型发展。我们的研究揭示了GV通过SGLT1诱导心肌细胞的焦亡和凋亡，阐明了糖尿病心肌损伤的机制，为DCM的临床防治提供实验基础。