白藜芦醇通过Wnt/b-catenin信号干预VSMC成骨样转化及CKD血管钙化的分子机制

Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality. Accumulated evidences suggested that vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation (VOT) is a crucial mechanism in promoting vascular calcification in CKD patients. In addition, Wnt/b-catenin signaling has been shown to play an essential role in the regulation of VOT in a high-phosphate environment which is a common phenomenon in CKD patients. Recently, the potential effect of resveratrol on interfering with VOT and vascular calcification has emerged while Wnt/b-catenin signaling has been presented to be a probable target of resveratrol in several other types of cells. Therefore, the hypothesis that through manipulating Wnt/b-catenin signaling, resveratrol plays a critical role in inhibiting the process of VOT and vascular calcification in CKD is put forword..This research project will be set up as follows. First of all, in vitro, ex vivo and in vivo models of chronic renal failure, a severe stage of CKD, followed by vascular calcification will be established, respectively. Then the effects of resveratrol on each key step during the process of VOT will be studied one by one and the pathological changes in the aorta tissue by resveratrol will be analysed in detail. Furthermore, the capacity of resveratrol in regulating the activity of Wnt/b-catenin signaling in VSMCs will be assayed. Finally, by manipulating the activity status of Wnt/b-catenin signaling either by ectopic expression of specific genes of the signaling pathway or by applying specific recombination proteins of it in vitro, the influence of resveratrol on VOT and vascular calcification will be detacted..The results presented in the studies of this project will demonstrate that through targeting Wnt/b-catenin signaling, which in turn impeding VOT, resveratrol possesses a potent effect on retarding vascular calcification in CKD.

血管平滑肌细胞(vascular smooth muscle cell, VSMC)成骨样转化是慢性肾脏病(chronic kidney disease, CKD)发生血管钙化的中心环节，而Wnt/b-catenin信号在VSMC成骨样转化和钙化程序中发挥重要的调控作用。白藜芦醇对血管钙化具有潜在的干预效应，而Wnt/b-catenin信号有可能是其关键的作用靶点。由此提出科学假说：白藜芦醇可通过调控Wnt/b-catenin信号活化状态，干预CKD环境中VSMC成骨样转化与血管钙化。本项目拟从体外、离体和体内三个层面，通过研究白藜芦醇对CKD环境中VSMC成骨样转化和钙化程序的影响，揭示白藜芦醇对CKD血管钙化的防治效应；通过研究白藜芦醇对VSMC内Wnt/b-catenin信号活化状态的影响，阐明其作用的分子机制，为其临床应用及探索防治CKD血管钙化的新措施提供有益思路。

高磷诱导的血管平滑肌细胞（VSMC）成骨样转化是慢性肾脏病（CKD）动脉中层钙化（AMC）的核心。前期研究表明，Wnt/β-catenin信号在高磷诱导VSMC成骨样转化和钙化中发挥了重要作用。白藜芦醇是一种天然多酚类化合物，具有抗炎、抗氧化和抗老化等活性。文献报道，白藜芦醇可以减轻高磷诱导的血管钙化，另有癌症领域研究证实，白藜芦醇能够抑制Wnt/β-catenin信号。然而，白藜芦醇能否通过调控Wnt/β-catenin信号干预高磷诱导的VSMC成骨样转化进而抑制CKD中的AMC，国内外尚无报道。本项目以雄性SD大鼠饲以腺嘌呤和高磷的饮食诱导CKD-AMC动物模型；离体培养小鼠主动脉环，建立高磷诱导VSMC成骨样转化和AMC的离体模型；β-甘油磷酸刺激培养的大鼠VSMC细胞株建立高磷诱导VSMC成骨样转化和钙化的体外模型，分别在上述钙化模型中设立白藜芦醇干预组。采用茜素红、硝酸银染色和钙盐测定等观察钙化的程度，采用qRT-PCR和Western blot等观察VSMC成骨样转化过程中血管平滑肌标志蛋白SM22α、成骨细胞转录因子Runx2及骨相关蛋白骨钙蛋白（OC）和骨桥蛋白（OPN）的表达变化，采用qRT-PCR和Western blot等观察高磷诱导VSMC成骨样转化过程Wnt//β-catenin信号成分促Wnt分泌蛋白PORCN和Wls的表达变化，以及对Wnt受体LRP6磷酸化的影响，采用质核分离和Western blot等观察β-catenin的表达和分布。研究显示，白藜芦醇明显减轻腺嘌呤加高磷饮食诱导的大鼠CKD-AMC程度，减轻高磷诱导的小鼠离体主动脉环AMC的程度及培养的大鼠VSMC钙化的程度。在离体和体外模型中，白藜芦醇抑制高磷所诱导的SM22α表达的减少以及Runx2、OC和OPN表达的增加。在培养的VSMC中，白藜芦醇明显抑制高磷诱导的PORCN和Wls的表达、p-LRP6上调、β-catenin入核以及两种形式活化β-catenin的增加，但其不能明显抑制Wnt3a诱导的Runx2的表达。本研究结果证实了白藜芦醇对高磷诱导VSMC成骨样转化和CKD-AMC的干预效应，揭示了白藜芦醇对高磷诱导的VSMC内Wnt/β-catenin信号活化的影响，进而部分阐明了白藜芦醇抑制高磷诱导VSMC成骨样转化和CKD-AMC的分子机制。