SIRT1对糖尿病冠状动脉平滑肌细胞Orai1通道的调控作用

The abnormal proliferation, migration and phenotypic change of vascular smooth muscle cells (VSMCs) are the key link of Diabetes to induce and accelerate coronary atherosclerosis process. but the molecular mechanism of VSMCs dysfunction is unclear. Past many researches suggest that it is related to intracellular calcium handling dysfunction. Recently Orai1 was identified to molecular basis of Ca2+ released-activated Ca2+ channel. It is an important channel of Ca2+ influx in VSMC. Our primary study suggests that orai1 channel expression levels up-regulate in relation to diabetic coronary artery smooth muscle cells dyfunction. Deacetylase SIRT1 can down-regulate orai1 expression levels and inhibit proliferation in VSMCs. This project bases on the above-mentioned study. The relationship of orai1 channel function change and coronary artery dysfunction was further investigated in 2 type diabetic model. Effect of Orai1 channel function change on intracellular calcium concentration, proliferation, migration was identified in VSMCs in diabetic cell model, and investigated the transcription regulation mechanism of deacetylase SIRT1 on Orai1. In vitro and in vivo, Orai1 channel function regulated by SIRT1 was validated to protect diabetic coronary artery dysfunction. This study will seek to the key molecule to improve Orai1 channel function form the upper stream. The new pathway was provided for the prevention and treatment of diabetic coronary artery disease.

血管平滑肌细胞(VSMCs)增殖迁移异常和表型改变是糖尿病诱发和加速冠状动脉粥样硬化形成的关键环节，但其分子机制不明。既往研究提示与胞内钙调控紊乱有关，近年确定了Orai1是钙释放激活钙通道的分子基础，是VSMCs外钙内流的重要通道。前期研究提示Orai1表达上调与糖尿病冠状动脉平滑肌功能异常密切相关，而去乙酰化酶SIRT1可以下调糖尿病VSMCs的Orai1表达，抑制VSMCs增殖。本课题基于上述研究基础，拟采用2型糖尿病动物模型，深入研究Orai1和SIRT1表达变化与冠状动脉功能受损的相关性。在细胞模型上，探讨SIRT1对Orai1表达变化的转录调控机制以及对糖尿病冠状动脉平滑肌细胞功能异常的影响。在器官和整体水平上，验证能否通过SIRT1调控Orai1表达来保护糖尿病冠状动脉功能受损。本研究从"上游"寻找能改善Orai1通道的关键分子，将为多靶点防治糖尿病冠心病提供新的理论依据。

本研究主要探讨SIRT1调控Orai1功能在糖尿病冠状动脉功能受损中的作用，主要发现：（1）正常冠状动脉平滑肌收缩调节机制存在明显的种属差异；（2）糖尿病时其功能明显异常，U46619和5-HT诱发离体糖尿病冠状动脉平滑肌收缩反应明显增加，与Orai1通道信号上调和去乙酰化酶SIRT1表达下调明显相关；（3）利用离体冠状动脉培养方法，建立器官水平的糖尿病损伤模型，进一步证实Orai1通道信号介导冠状动脉对5-羟色胺收缩异常的机制；（4）在AGE处理的细胞损伤模型上，Orai1通道表达上调，平滑肌细胞合成表型相关蛋白增加，收缩表型相关蛋白减少，Orai1通道介导的钙内流明显增加；去乙酰化酶SIRT1激动剂白藜芦醇可以明显逆转AGE诱导的细胞损伤过程，NF-κB特异性抑制剂吡咯烷二硫代氨基甲酸酯（PDTC）可以阻断白藜芦醇的效应；（5）在糖尿病冠状动脉整体动物模型和离体器官水平上，验证SIRT1激动剂白藜芦醇可以逆转冠状动脉平滑肌Orai1通道和血管功能变化。结果提示Orai1通道功能异常参与糖尿病冠状动脉功能异常，SIRT1激动剂通过下调Orai1通道功能对糖尿病冠状动脉功能受损有明显的保护作用。SIRT1是改善Orai1通道的关键分子，是防治糖尿病冠心病的重要靶点。