基于蛋白质酪氨酸硝化的铁过载促进胰岛素抵抗的分子机制及黄芩苷的干预作用研究

Excessive iron may promote the progress of type 2 diabetes, however, the mechanism is still unclear. It has been reported that insulin receptor (IR) and insulin rceptor substrate (IRS) were nitrated in type 2 diabetes and subsequently the tyrosine phosphorylation was interfered, leading to insulin resistance. In our previous study, we found that iron overload significantly promoted protein tyrosine nitration in animal tissues. Since one of the pathognomonic feature of type 2 diabetes is insulin resistance, we hypothesis that one of the mechanism of iron promoting type 2 diabetes, may due to iron catalyzing the nitration of tyrosine residues of insulin signaling proteins. In this project, by using immuno precipitation, western blotting, immunofluorescence, HPLC-MS/MS and other biochemical analysis, we will study the effects of iron overload on site selection of tyrosine nitration of insulin signaling proteins under different nitric oxide producing conditions, and the changes of insulin signaling protein function in mouse liver, muscle and adipose tissues and cells, as well as cell free system. These results will elucidate the mechanism of excessive iron on promoting type 2 diabetes. Meanwhile, this project will also study the inhibitory effect of baicalin, which is adjunctive therapy medicine for diabetes and posseses both iron chelation and antioxidant activity, on protein tyrosine nitration, these results will provide rationality for developping new adjunctive therapy medicine for type 2 diabetes.

铁过载可促进2型糖尿病的发生发展，但其机理尚不清楚。文献报道，胰岛素受体或受体底物酪氨酸发生硝化，会影响其磷酸化，导致胰岛素抵抗；我们发现，铁过载可显著促进组织蛋白质酪氨酸的硝化水平。由于2型糖尿病的特征之一是胰岛素抵抗，因此，我们推测铁催化胰岛素信号蛋白酪氨酸硝化，引起胰岛素抵抗，是铁过载促进2型糖尿病的主要原因之一。本课题拟在整体动物、细胞和无细胞体系上，通过调控一氧化氮生成，采用免疫沉淀、免疫印迹、HPLC-MS/MS及其他生化技术，研究铁过载对胰岛素敏感组织和细胞中胰岛素信号蛋白的硝化情况，及硝化对蛋白功能的影响。从蛋白质酪氨酸硝化的角度，阐明铁过载促进2型糖尿病发生发展的分子机理。此外，通过这个新靶点，探讨兼有铁络合和抗氧化活性及辅助治疗糖尿病的黄芩苷抑制2型糖尿病的分子机理。研究结果可为防治2型糖尿病及研发相关药物提供科学依据和新的作用靶点，具有重要的科学意义和潜在的应用前景。

本项目通过整体动物、细胞水平和体外化学水平的多层次研究，从蛋白质酪氨酸硝化的角度，研究铁过载引起胰岛素抵抗的分子机制。研究发现，铁，特别是卟啉铁，易于和多肽和蛋白结合，并在过氧化氢和亚硝酸盐存在下，能够有效促使胰岛素、胰岛素受体底物、胰岛淀粉样多肽等与2型糖尿病密切相关的蛋白发生酪氨酸硝化。胰岛素氧化和硝化后会导致胰岛素功能缺失，胰岛素受体底物硝化后抑制胰岛素受体底物的磷酸化，从而引起胰岛素抵抗。胰岛淀粉样多肽的硝化，则会引起胰岛淀粉样多肽聚集体存留在寡聚体阶段，导致细胞毒性更强，损伤胰岛细胞。这些因素都与2型糖尿病的发生密切相关。黄芩苷可以一方面可以络合铁，另一方面能够有效清除活性氧和活性氮，抑制酪氨酸硝化，还可以作为胰岛淀粉样多肽聚集的抑制剂，从多种分子机制上减轻胰岛素抵抗，抑制2型糖尿病的发生发展，对于2型糖尿病具有潜在的辅助疗效。黄芩苷具有这些功能的重要原因之一与其分子中含有邻二酚结构有关。此外，在本项目的研究过程中，发现了作为体内过氧亚硝酸盐抑制剂的铁卟啉水溶性衍生物，也具有催化蛋白质酪氨酸硝化的功能，并系统研究了铁卟啉水溶性衍生物催化酪氨酸硝化与其衍生基团之间的关系，提出了合理的分子设计手段，为开发蛋白质酪氨酸硝化抑制剂指明了方向。