Hossain Shahdat, Hashimoto Michio, Katakura Masanori, Miwa Koji, Shimada Toshio, Shido Osamu
Department of Environmental Physiology, Shimane University Faculty of Medicine, Izumo, Japan.
J Neurochem. 2009 Oct;111(2):568-79. doi: 10.1111/j.1471-4159.2009.06336.x. Epub 2009 Aug 17.
The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n-3), one of the essential brain nutrients, on in vitro fibrillation of amyloid beta (Abeta(1-42)), Abeta(1-42)-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Abeta(1-42)-fibrill formation with a concomitant reduction in the levels of soluble Abeta(1-42) oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5%), Tris-Tricine gradient(4-20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Abeta(1-42) oligomers of 15-20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Abeta(1-42) backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern-Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Abeta(1-42)-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Abeta(1-42) toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.
二十二碳六烯酸（DHA；C22:6，n-3）是一种必需的脑营养物质，本研究利用硫黄素T荧光光谱法、激光共聚焦显微荧光法和透射电子显微镜，研究了其对β淀粉样蛋白（Aβ(1-42)）体外纤维化、Aβ(1-4)寡聚体及其对SH-SY5Y细胞毒性作用的机制。结果清楚地表明，DHA抑制Aβ(1-42)纤维形成，并同时降低可溶性Aβ(1-42)寡聚体水平。用DHA处理预形成的寡聚体后，其聚合成纤维的过程受到抑制，这表明DHA不仅阻碍其形成，还抑制其向纤维的转化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（12.5%）、Tris-三羟甲基氨基甲烷梯度（4-20%）凝胶电泳和蛋白质免疫印迹分析表明，DHA抑制至少两种15-20 kDa的Aβ(1-42)寡聚体，表明它在纤维化过程中阻碍这些途径上的三聚体/四聚体中间体。DHA还降低了二酪氨酸水平和酪氨酸固有荧光强度，表明DHA中断了Aβ(1-42)主链中酪氨酸的微环境。此外，如斯特恩-沃尔默常数降低所示，DHA保护酪氨酸免受丙烯酰胺碰撞猝灭。3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐还原效率和免疫组织化学检查表明，DHA抑制Aβ(1-42)诱导的SH-SY5Y细胞毒性。综上所述，这些数据表明，通过抑制Aβ(1-42)有毒三聚体/四聚体寡聚体，DHA可能会限制淀粉样变性神经退行性疾病——阿尔茨海默病。
