Hossain Shahdat, Hashimoto Michio, Katakura Masanori, Al Mamun Abdullah, Shido Osamu
Department of Environmental Physiology, Shimane University Faculty of Medicine, Izumo, 693-8501, Japan.
Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka, Bangladesh.
BMC Complement Altern Med. 2015 Apr 14;15:118. doi: 10.1186/s12906-015-0620-9.
Identifying agents that inhibit amyloid beta peptide (Aβ) aggregation is the ultimate goal for slowing Alzheimer's disease (AD) progression. This study investigated whether the glycoside asiaticoside inhibits Aβ1-42 fibrillation in vitro.
Fluorescence correlation spectroscopy (FCS), evaluating the Brownian diffusion times of moving particles in a small confocal volume at the single-molecule level, was used. If asiaticoside inhibits early Aβ1-42 fibrillation steps, more Aβs would remain free and rapidly diffuse in the confocal volume. In contrast, "weaker or no inhibition" permits a greater number of Aβs to polymerize into oligomers, leading to fibers and gives rise to slow diffusion times in the solution. Trace amounts of 5-carboxytetramethylrhodamine (TAMRA)-labeled Aβ1-42 in the presence of excess unlabeled Aβ1-42 (10 μM) was used as a fluorescent probe. Steady-state and kinetic-Thioflavin T (ThT) fluorospectroscopy, laser-scanning fluorescence microscopy (LSM), and transmission electron microscopy (TEM) were also used to monitor fibrillation. Binding of asiaticoside with Aβ1-42 at the atomic level was computationally examined using the Molegro Virtual Docker and PatchDock.
With 1 h of incubation time for aggregation, FCS data analysis revealed that the diffusion time of TAMRA-Aβ1-42 was 208 ± 4 μs, which decreased to 164 ± 8.0 μs in the presence of asiaticoside, clearly indicating that asiaticoside inhibited the early stages Aβ1-42 of fibrillation, leaving more free Aβs in the solution and permitting their rapid diffusion in the confocal volume. The inhibitory effects were also evidenced by reduced fiber formation as assessed by steady-state and kinetic ThT fluorospectroscopy, LSM, and TEM. Asiaticoside elongated the lag phase of Aβ1-42 fibrillation, indicating the formation of smaller amyloid species were impaired in the presence of asiaticoside. Molecular docking revealed that asiaticoside binds with amyloid intra- and inter-molecular amino acid residues, which are responsible for β-sheet formation and longitudinal extension of fibrils.
Finally, asiaticoside prevents amyloidogenesis that precedes neurodegeneration in patients with Alzheimer's disease.
鉴定抑制淀粉样β肽（Aβ）聚集的药物是减缓阿尔茨海默病（AD）进展的最终目标。本研究调查了糖苷积雪草苷在体外是否抑制Aβ1-42纤维化。
使用荧光相关光谱法（FCS），在单分子水平评估小共聚焦体积内移动颗粒的布朗扩散时间。如果积雪草苷抑制Aβ1-42纤维化的早期步骤，更多的Aβ将保持游离状态并在共聚焦体积中快速扩散。相反，“较弱抑制或无抑制”会使更多的Aβ聚合成寡聚体，进而形成纤维，导致溶液中的扩散时间变慢。在过量未标记的Aβ1-42（10μM）存在的情况下，使用痕量的5-羧基四甲基罗丹明（TAMRA）标记的Aβ1-42作为荧光探针。还使用稳态和动力学硫黄素T（ThT）荧光光谱法、激光扫描荧光显微镜（LSM）和透射电子显微镜（TEM）来监测纤维化。使用Molegro Virtual Docker和PatchDock在原子水平上对积雪草苷与Aβ1-42的结合进行了计算研究。
聚集孵育1小时后，FCS数据分析显示TAMRA-Aβ1-42的扩散时间为208±4μs，在积雪草苷存在的情况下降至164±8.0μs，这清楚地表明积雪草苷抑制了Aβ1-42纤维化的早期阶段，使溶液中留下更多游离的Aβ，并使其在共聚焦体积中快速扩散。稳态和动力学ThT荧光光谱法、LSM和TEM评估的纤维形成减少也证明了其抑制作用。积雪草苷延长了Aβ1-42纤维化的延迟期，表明在积雪草苷存在的情况下较小淀粉样物质的形成受到损害。分子对接显示积雪草苷与淀粉样蛋白分子内和分子间的氨基酸残基结合，这些残基负责β-折叠的形成和原纤维的纵向延伸。
最后，积雪草苷可预防阿尔茨海默病患者神经变性之前的淀粉样蛋白生成。
