蓝莓花青素抑制内皮细胞氧化应激损伤的作用机理研究

Currently, atherosclerosis has become one of the major causes of death in Western countries and the incidence is also increasing year by year in China. Our previous studies found that blueberry anthocyanins could significantly inhibit oxidative stress-induced injury in human vascular endothelial cell, having a potential role in the prevention and treatment of atherosclerosis, but the molecular mechanism have not yet been clear. Recent studies reported that ROS/p38MAPK/NF-κB signal pathway was closely related with oxidative stress-induced injury in endothelial cell. The project is designed to build TNF-α-induced endothelial cell injury model using cultured vascular endothelial cells in vitro. The molecular mechanism of blueberry anthocyanins against oxidative stress-induced endothelial injury will be studied by the intervention of the indentified blueberry anthocyanins in the cell model. The confocal laser scanning, real-time PCR, Western blot analysis and adding the specific inhibitor will be used to elucidate the molecular mechanism of blueberry anthocyanins against oxidative stress-induced endothelial injury, from the aspect of ROS/p38MAPK/NF-κB signaling pathways and expression of cell apoptosis related gene. On this basis, the role of the molecular structure of blueberries anthocyanins in inhibiting oxidative stress induced injury in endothelial cell will also be studied to reveal the structure-activity relationship between blueberry anthocyanins and the inhibitory effects of oxidative stress induced injury in endothelial cell. The proposed project will provide a theoretical basis for the development of functional activity in blueberry with vital scientific significance.

动脉粥样硬化已成为西方发达国家引起死亡的主要病因之一，在我国的发病率也逐年上升。我们的前期研究发现，蓝莓花青素能够显著抑制氧化应激引起的血管内皮细胞损伤，对动脉粥样硬化具有潜在的防治作用，但其分子机制尚不明确。近年来的研究发现，ROS/p38MAPK/NF-κB信号通路与内皮细胞氧化损伤密切相关。本项目以分子结构明确的蓝莓花青素为研究对象，构建TNF-α诱导的内皮细胞氧化应激损伤模型，通过蓝莓花青素干预细胞模型，运用激光共聚焦、实时PCR、Western blot以及加入特异性抑制剂干预等实验方法，从ROS/p38MAPK/NF-κB信号通路、细胞凋亡相关基因表达等方面，阐明蓝莓花青素抑制内皮细胞氧化应激损伤的分子机制。在此基础上，研究蓝莓花青素分子结构特征在抑制内皮细胞氧化应激损伤中的作用，由此揭示其抑制内皮细胞氧化应激损伤的构效关系，为蓝莓功能活性挖掘提供理论依据，具有重要科学意义。

本项目以蓝莓花青素为研究对象，利用体外原代培养的血管内皮细胞，构建TNF-α诱导的内皮细胞氧化应激损伤模型，通过蓝莓花青素干预细胞模型，运用激光共聚焦、实时PCR、Western blot等实验方法，从信号通路、细胞凋亡相关基因表达等方面，阐明蓝莓花青素抑制内皮细胞氧化应激损伤的分子机制，研究了蓝莓花青素分子结构特征在抑制内皮细胞氧化应激损伤中的作用。结果表明，蓝莓花青预处理TNF-α诱导损伤细胞时，显著降低TNF-α对细胞增殖的抑制作用。蓝莓花青素可提高细胞POD、SOD活性，且随浓度增加而升高。当蓝莓花青素预处理浓度从50μg/ml增加到100μg/ml时，细胞早期凋亡从12.96%降到6.22%，晚期凋亡从12.16%降到3.45%，与TNF-α处理比，极显著降低（P＜0.01）。这表明蓝莓花青素通过提高细胞抗氧化酶活性、维持细胞内氧化还原平衡和抑制细胞凋亡，对导致的内皮细胞损伤起到保护作用。.TNF-α可诱导内皮细胞ROS生成，下调抑制凋亡基因Bcl-2的mRNA的表达，上调促凋亡基因p53、Bax、Bcl-xl的mRNA的表达，增加凋亡相关蛋白p38MAPK、p53和NF-κB的蛋白表达水平。蓝莓花青素显著降低内皮细胞中ROS生成（P＜0.05），随着蓝莓花青素浓度的增加，且呈现浓度依赖关系。蓝莓花青素极显著提高细胞内抑制凋亡基因Bcl-2、Bcl-xl的mRNA 相对表达含量（P＜0.01），且随浓度的增加而升高。不同浓度蓝莓花青素均可下调促凋亡基因Bax、p53的mRNA的表达，降低凋亡相关蛋白p38MAPK、p53和NF-κB的蛋白表达水平，提示蓝莓花青素能够显著抑制ROS/p38MAPK/NF-κB信号通路，从而起到抗氧化应激损伤的效果。.比较研究了4种花青素矢车菊素-3-O-葡萄糖苷、矢车菊素-3-O-半乳糖苷、飞燕草素-3-O-葡萄糖苷、锦葵色素-3-O-半乳糖苷在TNF-α诱导的内皮细胞氧化应激的作用，研究其对内皮细胞存活率的影响。在50-150μg/ml浓度范围内，4种花青素提高细胞存活率与其浓度呈显著正相关，且高于对照组，差异极显著（P＜0.01）。相同浓度下条件下，锦葵色素-3-O-半乳糖苷处理组细胞存活率高于飞燕草素-3-O-葡萄糖苷、矢车菊素-3-O-葡萄糖苷、矢车菊素-3-O-半乳糖苷。构效关系研究结果表明，花色苷含有的总-OH