Nrf2-ARE通路在氧化应激致终末期肾病血管钙化中的作用及机制研究

Oxidative stress (OS) is an important mechanism of vascular calcification(VC) in end-stage renal disease (ESRD), how to control excessive ROS is the current difficulty. The transcription factor erythroid 22p45-related factor -2 (Nrf2) and its downstream target gene ARE is by far the most important endogenous antioxidant pathway. But if the weaked ESRD antioxidation ablity were related to the role of Nrf2-ARE and the strengthening of Nrf2- ARE pathway can improve VC is not clear.The project intends to investigate Nrf2 agonists and the adenovirus AdSM-MHC/SM22aNrf2siRNA on the regulation role of human aortic smooth muscle cells (VSMCs) oxidation state and inflammatory mediators expression and VC based on our founds of "decreased Nrf2 expression in ESRD radial artery have negative correlation with vascular calcification and ROS damage". We also detection of Nrf2, ARE, ROS and inflammatory mediators expression and the VSMCs osteoblast phenotype changes; then preparation of rat renal failure vascular calcification model to observe the effect of AdSM-MHC/SM22aNrf2siRNA or Nrf2 agonists on Nrf2, ROS and inflammatory mediators expression and VC changes in vivo. Our aim is to provide the role and mechanism of Nrf2- ARE pathway on the oxidative stress and vascular calcification.

氧化应激（OS）是终末期肾病（ESRD）血管钙化的重要机制，如何控制过高的OS是目前的难点。核因子NF-E2 相关因子(Nrf2) -ARE通路是迄今最重要的内源性抗氧化通路。但ESRD抗氧化作用薄弱是否与Nrf2-ARE作用受限有关以及强化Nrf2-ARE通路能否改善血管钙化尚不清楚，本项目拟在发现"ESRD桡动脉平滑肌细胞（VSMCs）Nrf2表达下降与血管钙化、OS损伤负相关"的基础上，分别以Nrf2激动剂和腺病毒AdSM-MHC/SM22 a Nrf2siRNA调控人VSMCs氧化状态，检测Nrf2及其下游基因、炎症介质和VSMCs成骨样细胞表型变化；其次体内观察AdSM-MHC/SM22 a Nrf2siRNA、Nrf2激动剂对大鼠肾衰竭血管钙化模型VSMCs上Nrf2及下游靶基因、炎症介质和血管钙化指标等的影响，为Nrf2-ARE通路在氧化应激致血管钙化中的作用及机制提供依据。

通过在体外建立高磷诱导的血管平滑肌钙化细胞模型，观察重组腺病毒载体Ad-SM-MHC/SM22a-Nrf2siRNA转染、Nrf-2激动剂和Nrf-2抑制剂调控Nrf2/ARE通路活性对钙化血管平滑肌细胞的氧化应激反应及其向成骨样细胞转分化的作用，在体外确认了Nrf-2抗氧化应激所致血管钙化和线粒体损伤以及其抑制钙化细胞成骨样转分化的能力。NFκB抑制剂和ROS抑制剂干预高磷诱导的血管钙化细胞模型实验再次验证了氧化应激致血管钙化、线粒体损伤和钙化细胞成骨样转分化的作用。最后体内激动Nrf-2观察其对大鼠血管钙化动物模型氧化应激反应及血管钙化的作用，进一步明确Nrf2-ARE通路在抗氧化应激所致血管钙化中的作用与机制，为选择新的药物靶点提供策略。