黄绿青霉素致血管内皮损伤机制及硒元素的抗损伤作用研究

The mortality rates of cardiovascular disease are not alike in different areas with the characteristics of distinct spatial distribution in the world, and this characteristics contributes to the analysis of the pathogenesis of the disease. The mortality rate of cardiovascular disease is higher in the zonal distribution area from Northeast of China to Southwest than any other regions of the country. There are two obvious environmental problems in the area. Firstly, the pollution of citreoviridin (CIT) in food is serious. Secondly, the intake of selenium (Se) in food is low. The recent research indicates that the mycotoxin of CIT is produced by some fungi such as Penicillium Citreoviride. We find that CIT induced cell apoptosis and cell dysfunction when cultured with vascular endothelial cells. Vascular endothelial cells constitute endothelium in the surface of vessel lumen, and play important roles in regulating physiological function of vessel. The dysfunction of vascular endothelium is the leading cause of atherosclerosis and results in cardiovascular disease. Therefore it is supposed that CIT induces cardiovascular disease via impairment of vascular endothelial cells. In addition, as Se has protective effect on vascular endothelial cells preventing from impairment, the deficiency of Se may relate to cardiovascular disease. But the pathway of cardiovascular disease induced by CIT pollution has not been mentioned, the effects of CIT and Se on endothelial cells and the mechanism of action in endothelial cells impairment is still poorly understood..In the project, the mechanism of CIT-induced apoptosis vascular endothelial cells will be investigated by the culture method of human umbilical vein endothelial cells. The effect of CIT in process of atherosclerosis will be tested in animal experiments, and the biological markers of vascular endothelium injured by CIT will be detected..The regulatory effect of nuclear factor-kappa B in endothelial cells impairment induced by CIT will be analyzed. The mediate effects of Se on CIT-induced impairment will be studied in paralleled experiments by supplying with sodium selenite (Na2SeO3).

调查显示,在我国从东北-西南的带状分布区内，部分地区心血管病高发。该地区有两个突出环境问题：1.黄绿青霉素（CIT）的粮食污染较为严重；2.饮食中硒元素（Se）不足。最近研究发现CIT可引起内皮细胞凋亡和功能异常。血管内皮覆盖于血管腔内，为血液流动提供光滑的内表面，并具有重要的血管调节功能，内皮损伤是动脉粥样硬化等血管疾病的起点，另外Se有减轻内皮损伤的作用。以上研究提示CIT可能通过对内皮的损伤引发血管疾病，但有关CIT对内皮损伤和Se对该损伤的保护作用的研究尚不深入、对损伤的机制并不清楚，且该损伤能否导致动脉粥样硬化还缺乏证据支持。本项目旨在通过人脐静脉血管内皮细胞原代培养和动物实验两个途径，探索CIT引发内皮凋亡的机制，阐明核因子-κB在CIT引发内皮功能损伤过程中的调控作用，筛选内皮损伤标志物，发现CIT导致动脉粥样硬化的证据，研究Se对CIT所导致的内皮损伤的保护作用。

真菌毒素(mycotoxin)是由真菌产生的毒性代谢产物。联合国粮食及农业组织（FAO）将真菌毒素列为最危险的谷物天然污染物。在诸多真菌毒素中黄绿青霉素（citreoviridin，CIT）是与心血管损伤关系最为密切的一种， CIT可引发血管内皮损伤但其发生机制和致病结局尚未深入研究。由于血管内皮损伤和血管炎症性病变是动脉粥样硬化的始动因素，我们提出以下假设： CIT毒素可通过某种机制引发血管内皮细胞凋亡和调节功能异常，进而促进动脉粥样硬化的发病，并通过本研究加以阐释和验证。硒元素与动脉硬化及冠心病发病有关，WHO将硒列入与冠心病有关的微量元素之一，硒元素的摄入偏低有可能加重CIT引发的内皮损伤。.课题组围绕黄绿青霉素（CIT）对血管内皮损伤机理、对动脉粥样硬化发病的影响和硒元素的抗损伤作用三个方面研究开展研究，按照研究计划，运用动物实验、体外细胞培养、多重光谱技术、分子模拟蛋白质结合实验、动脉斑块特异染色等技术，开展研究，得出创新性结论如下：（1）CIT抑制了血管内皮细胞cyclinD1 表达和上调p53和p21表达，从而使细胞分裂周期滞留在G0/G1期，降低细胞的增殖活性；（2）CIT干扰凋亡调节蛋白Bax、Bcl-2的表达，上调活化caspase-9，形成级联反应进一步活化caspase-3，引发内皮细胞凋亡；（3）CIT通过增强TNF-α诱导的内皮细胞转录因子NF-κB的核内易位，上调相关基因转录，导致ICAM-1, VCAM-1, E选择素等粘附分子及趋化因子MCP-1的表达增高，进而促进内皮细胞的粘附作用；（4）CIT加重载脂蛋白E基因敲除小鼠（ ApoE-/-）动脉粥样硬化斑块的形成，起到促进动脉粥样硬化发病的作用；（5）CIT与血清白蛋白、人免疫球蛋白和溶菌酶的结合过程发生了能量转移和二级结构的变化，CIT与血清白蛋白的作用力主要是疏水力和氢键，与人免疫球蛋白和溶菌酶的作用力主要是疏水力和静电作用，并兼有氢键；（6）补硒抑制了内皮细胞的凋亡，促进了内皮细胞的增殖，减轻了内皮细胞的损伤；（7）补硒降低了小鼠脂质过氧化程度，保护了血管内皮功能，通过补硒降低了低硒小鼠动脉粥样硬化的病变程度。依托本项目发表论文8篇（其中SCI收录6篇）。