Orai1在糖尿病血管内皮损伤中的作用及机制研究

Impairment of endothelium-dependent vasorelexation is a characteristic change at the early stage of Diabetes, and it is also an important pathological factor to induce diabetic vascular injury. Although earlier studies have showed that disequilibrium of endothelial cell Ca2+ homeostasis is involved in endothelial dysfunction, which kind of calcium channel involved in this process remains elusive. Recently we have found that there was the store-operated calcium channel Orai1 expression in endothelial cells, high glucose could upregulate the expression of Orai1. Both the Orai1 blockers such as SK&F96365, 2-APB and transfection of the Orai1 siRNA could obviously reverse the downregulation of eNOS and reduction of NO production induced by high glucose. On the basis, in this study we intended to overexpress or knockdown of Orai1, to clarify the effect and of this channel on eNOS expression and the dysfunction of endothelium-dependent relaxation induced by high glucose, and to reveal the specific mechanism of this process. Moreover, we will further investigate the function role of Orai1 in regulating the endothelium-dependent vascular relaxation of aortas from db/db mice injected with Ad-Orai1 siRNA. Through this study, we can systematicly study the effect and mechanism of the store-operated calcium channel Orai1 in endothelial dysfunction in Diabetes, suggesting that Orai1 may be a novel and potential therapeutic target for diabetic vascular disease.

血管内皮依赖性舒张反应降低是糖尿病发病早期的标志性变化，是诱导糖尿病血管合并症的重要病理因素。尽管早期研究发现内皮细胞Ca2+稳态失调参与内皮损伤，但何种钙通道参与该过程，尚不清楚。我们最近的研究发现内皮细胞有orai1钙池操纵性钙通道表达，高糖处理可上调orai1蛋白水平。Orai1通道阻断剂SKF96365、2-APB以及orai1 siRNA转染可逆转高糖诱导的eNOS表达下调及NO生成减少。在此基础上，本项目拟通过过表达或下调orai1表达，阐明orai1钙通道在糖尿病诱导eNOS功能下调和内皮舒张反应障碍中的作用，并揭示其作用机理。并进一步通过在体实验明确下调orai1表达对糖尿病小鼠内皮依赖性血管舒张反应的影响。该研究工作将较系统认识orai1钙通道在糖尿病血管损伤中的作用及其机制，为评估orai1钙通道是否可作为防治糖尿病血管病变的新靶点提供理论依据。

糖尿病血管并发症是糖尿病临床常见的并发症，血管内皮功能不全是其早期病理变化。Ca2+作为生物体内一种重要的第二信使参与了几乎所有的生命过程，既往研究表明内皮细胞内Ca2+稳态失衡参与调控内皮损伤，但具体是何种钙通道目前并不十分明确。本研究通过细胞与整体水平实验观察钙池操纵性钙通道Orai1在糖尿病内皮损伤中的作用及其机制。首先我们研究发现在2型糖尿病db/db小鼠中，Orai1通道的表达增加，而db/db小鼠内皮依赖性血管舒张反应下降。在内皮细胞中高糖刺激可引起eNOS水平降低，而利用钙通道抑制剂及Orai1siRNA可以逆转该现象。以上结果提示Orai1可能参与调控糖尿病状态下的内皮功能损伤。而体内代谢紊乱同时也会诱发内质网应激反应，在糖尿病情况下机体内质网应激反应增加，参与体内一系列病理生理过程。已有研究证明内质网应激的发生与Ca2+失衡密切相关，我们的实验结果也表明在内皮细胞中，内质网应激诱导剂衣霉素（Tunicamycin）可促进内质网应激相关标志物Bip，CHOP表达增加，同时导致内皮细胞eNOS水平下降，另一方面衣霉素也上调Orai1通道的表达，而利用钙通道阻断剂SKF96365及Orai1siRNA能逆转上述蛋白的表达。进一步提示Orai1通道可能通过参与调控内质网应激反应进而参与糖尿病内皮功能损伤。通过该项研究可以为Orai1通道作为糖尿病血管并发症的潜在治疗靶点提供实验室依据。