蛋白激酶D调控内质网应激参与糖尿病心肌病心肌细胞凋亡的作用及机制研究

In human and animal models of diabetes, a heart muscle specific disease in the absence of other vascular pathology has been described, termed diabetic cardiomyopathy (DCM). The pathogenesis of diabetic cardiomyopathy is a chronic and complex process, which is still unclear. Cardiocyte apoptosis as an early cellular event has been considered to play a critical role in the development of diabetic cardiomyopathy. Because myocytes rarely proliferate in adult cardiac muscles, the loss of cardiomyocytes would eventually lead to compromised cardiac function. The persistent ER(endopasmic reticulum) stress can trigger the activation of c-JUN NH2-terminal kinase (JNK) or transcriptional induction of C/EBP homologous protein (CHOP) and/or caspase-12 dependent pathways to promote the initiation of the apoptotic process. Recently, numerous studies have demonstrated that the apoptosis pathway induced by ER stress was involved in the pathogenesis of diabetic heart failure. The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases. Increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system. Recent studies suggest that PKD is an important regulator of different intracellular signaling pathways, including cell apoptosis. The persistent activation of PKD in the heart of DCM rat was observed in our preliminary research. Based on the existing studies and our preliminary experimental results, we hypothesize that PKD plays an important role in cardiocyte apoptosis of DCM, and this effect is mediated by ER stress. The present study is designed to explore the effects and underlying mechanisms of PKD activation in DCM. In our study, type 2 DM rats with cardiomyopathy in vivo and rat primary cardiomyocytes in vitro will be used for discussing the relationship between PKD and ERS during the cardiomyocyte apoptosis. Meanwhile, the role of atorvastatin which may inhibit the activation of PKD and reduce the cardiomyocyte apoptosis will be investigated in vivo. This project will provide a new theoretical support in understanding the pathological mechanism of diabetic cardiomyopathy，which may be helpful to provide a novel target for drug discovery and therapeutic intervention of diabetic cardiomyopathy.

心肌细胞凋亡是糖尿病心肌病（DCM）的主要病理改变，也是糖尿病心功能障碍的重要病理生理机制。内质网应激（ERS）是介导DCM心肌细胞凋亡的重要途径。蛋白激酶D（PKD）家族在心血管系统中发挥了重要的调节作用。PKD的激活参与细胞凋亡的调控过程。我们的前期研究显示糖尿病心肌组织中PKD活化表达明显增加。本课题综合国内外研究现状和预实验结果，提出PKD调控ERS相关信号通路诱导心肌细胞凋亡是糖尿病心肌病重要分子机制的假说。拟以2型糖尿病心肌病大鼠模型为基础，探讨PKD对DCM心肌细胞凋亡及心功能的影响，并初步探讨阿托伐他汀的对糖尿病的心肌保护作用是否通过PKD介导。同时，通过腺病毒转染技术，分别在体内及体外实验中诱导PKD基因沉默，进一步探讨ERS相关信号通路在PKD致DCM心肌细胞凋亡中的作用。本课题的实施将为阐明糖尿病心肌病的发生机制提供新的理论依据，为其防治提供新的靶点。

蛋白激酶D（PKD）在心血管系统中发挥重要作用。然而，PKD在糖尿病心肌病（DCM）发病机制中的潜在作用仍不清楚。厄贝沙坦可保护糖尿病引起的心脏损伤，其机制尚不明确。我们旨在探讨厄贝沙坦的心脏保护作用是否通过PKD和内质网（ER）应激介导。通过建立2型糖尿病大鼠模型，并对各组大鼠进行代谢检测，超声心动图和组织病理学检测，我们发现厄贝沙坦可剂量依赖性的改善2型糖尿病大鼠的左室重构及功能障碍，厄贝沙坦对DCM的心脏保护作用与其抑制心肌组织PKD和内质网应激的激活有关。在体外实验，采用H9c2心肌细胞研究PKD-ERS在HG诱导的细胞凋亡中的作用机制，结果显示PKD基因沉默可通过下调IRE1α/CHOP信号通路抑制内质网应激，从而降低高糖诱导的心肌细胞凋亡。. 此外，有文献表明溴区包含蛋白7（BRD7）可通过调控XBP-1s核转位参与内质网应激信号通路。然而，BRD7对高血糖诱导的心肌细胞凋亡的分子机制仍不清楚。为验证该问题，在体内，我们建立了1型糖尿病大鼠模型病采用慢病毒转染抑制BRD7的表达。研究结果示糖尿病大鼠心肌细胞中BRD7的表达明显上调，BRD7基因沉默对糖尿病引起的心脏损伤具有保护作用。在体外，采用H9c2心肌细胞研究BRD7在HG诱导的细胞凋亡中的作用机制。我们发现高糖通过激活ERK1/2信号通路诱导BRD7表达，同时BRD7通过调控内质网应激信号通路介导高糖诱导的心肌细胞凋亡。. 科学意义：这些研究结果将促进对PKD及BRD7生物学功能的认识，有助于进一步揭示内质网应激糖尿病心肌病心肌细胞凋亡中的作用，从而为更好地防治糖尿病心肌病提供新的理论依据和策略。