BMAL1-SIRT1调控的内质网应激在糖尿病心肌再灌注易损性增加中的作用及分子机制

Excessive endoplasmic reticulum stress (ERS) is a major why hearts from diabetic subjects are more vulnerable to ischemia reperfusion (IR) injury. Our recent study showed that SIRT1 is a negative regulator of ERS, but the underlying mechanisms are still unclear. It has been known that there was an innate circadian rhythm to the regulation of ERS, and BMAL1 plays an important role as the key regulator of circadian rhythm. Recent study indicated that the interaction between BMAL1 and SIRT1 involved in the process of energy metabolism. Our preliminary study showed that myocardial IR significant down-regulated BMAL1 and SIRT1 expression, accompanied by increased post-ischemic infarct size and down-regulated phosphorylation of pro-survival protein Akt and GSK-3βin diabetic rats. Accordingly, we speculate that the regulation of ERS by BMAL1-SIRT1 plays a key role in increasing diabetic heart vulnerability to IR injury. In this proposal, we will establish in vivo and in vitro models of diabetes and IR by transfection with adeno-associated viral vector to investigate the roles of BMAL1-SIRT1 in mediating ERS, and to observe the effects on increased vulnerability of IR injury in diabetic hearts and its molecular mechanisms. This proposed studies may provide a new point of view and a potential therapeutic target site in combating ischemic heart disease in diabetes.

过度内质网应激（ERS）是糖尿病心肌缺血再灌注（IR）易损性增加的重要机制。我们前期研究发现，ERS受SIRT1的负性调控，但其机制尚未阐明。研究表明，ERS受生物钟节律的调控，且BMAL1作为生物钟核心调控基因发挥着重要作用；新近发现，BMAL1与SIRT1相互调控参与机体能量代谢。我们预实验表明，糖尿病心肌IR期间BMAL1和SIRT1蛋白表达下调，同时伴随着心肌梗死面积增加及促存活蛋白Akt、GSK-3β的磷酸化表达下调。据此，我们推测BMAL1-SIRT1调控的ERS是糖尿病心肌易损性增加的关键机制之一。本项目拟制备糖尿病和IR模型，以在体心脏与离体心肌细胞为研究对象，通过腺相关病毒转染等方法，探讨BMAL1-SIRT1调控的ERS在糖尿病心肌IR易损性增加中的作用及分子机制，为糖尿病心肌IR损伤的防治提供新靶点和新策略。

过度内质网应激（ERS）是糖尿病心肌缺血再灌注(I/R)易损性增加的重要机制，我们前期研究表明，ERS受生物钟节律的调控，且糖尿病心肌I/R期间生物钟核心调控基因BMAL1和SIRT1蛋白表达下调。本研究主要探讨BMAL1调控SIRT1表达，进而介导ERS节律紊乱可能是糖尿病心肌易损性增加的关键机制。研究结果表明：(1)非糖尿病大鼠心肌缺血再灌注损伤具有明显的昼夜节律性，即在ZT12-7PM时间点（黑夜相开始时）心肌损伤相较ZT0-7AM时间点（光相开始时）较重，而糖尿病大鼠在不同时间点心肌损伤无明显昼夜节律性；(2)非糖尿病大鼠心肌生物钟基因BMAL1及ERS相关蛋白GRP78 mRNA和蛋白表达具有昼夜节律性，而糖尿病大鼠在相同时间点mRNA和蛋白表达均下降；(3)相比非糖尿病大鼠，糖尿病大鼠心肌I/R后心肌损伤增加，且伴随着BMAL1蛋白表达的下降和内质网应激水平的显著增加；(4)HBAAV2/9-CMV-BMAL1转染糖尿病大鼠后，心肌BMAL1和SIRT1蛋白表达显著增加，I/R后心肌损伤显著减轻，内质网应激水平显著下降；(5)高糖高脂状态及缺氧复氧刺激下，心肌细胞BMAL1和SIRT1蛋白表达显著下降，内质网应激水平显著增加；(6)腺病毒过表达心肌细胞BMAL1表达后，细胞损伤及凋亡显著下降，内质网应激水平显著下降，表现为ERS相关蛋白GRP78、ERS凋亡蛋白（CHOP、Caspase12）表达下降，该保护作用可以被PI3K/Akt通路抑制剂和ERS激动剂所逆转。基于以上研究，探讨了BMAL1调控SIRT1表达，进而介导PI3K/Akt信号通路进一步调控ERS水平可能是糖尿病心肌易损性增加的关键机制，期待为糖尿病心肌I/R损伤的治疗提供新靶点和新策略。