类叶升麻苷对β-淀粉样蛋白聚集影响的体外研究

摘要

目的：研究类叶升麻苷（Acteoside，AS）在体外对β-淀粉样蛋白片段（Aβ1-42）聚集的影响。方法采用硫黄素 T（Th-T）结合实验研究各个浓度 AS 对单体态 Aβ1-42聚集的抑制作用以及对聚集态 Aβ1-42的解聚作用、呈现出的量效关系；通过对 AS 和 AS＋Th-T 进行300～600 nm 全波长吸收光谱扫描以及（160、320、640μmol／L）AS与（1．25、2．5、5、10、20、40、80、160和320μmol／L）Th-T 竞争结合实验，排除实验中可能存在的假阳性因素。结果硫黄素 T 结合实验表明，加入 AS 共同孵育24 h 后，单体态或聚集态 Aβ1-42的荧光强度均能减弱；全波长吸收光谱扫描显示在450 nm 和485 nm 处，AS、AS＋Th-T 均无特异性吸收峰，在450 nm 处给予刺激后，485 nm 附近也没有出现强的吸收；竞争结合实验表明，固定 AS 浓度，增加 Th-T 浓度，不会使荧光强度恢复至未加入 AS 组，则两者不存在相关结合位点；在 AS 影响 Aβ1-42聚集的量效关系实验中，AS 抑制 Aβ1-42聚集的 IC50为254．3μmol／L，对聚集态 Aβ1-42解聚的 IC50为282μmol／L。结论AS 可有效抑制单体态 Aβ1-42的聚集，对聚集态的 Aβ1-42也有明显解聚作用，提示 AS 对老年痴呆具有潜在的治疗作用。%Objective To investigate the effect of Acteoside on the aggregation of Aβ1-42 in vitro.Methods Thioflavin T(Th-T)fluorescence was used to detect the effect of various concentrations of AS on the ag-gregation of monomer Aβ1-42 and on the degradation of the aggregated Aβ1-42 ;the competitive binding ex-periment between Th-T and Acteoside and the absorption spectrum (from 300 nm to 600 um)were used to rule out the potential false positive factors.Results Thioflavin T experiment showed that AS could de-crease the fluorescence intensitywhen incubated with monomer Aβor aggregated Aβfor 24 h.Absorption spectra showed that AS,AS+Th-T had no specific absorption peak at 450 nm and 485 nm.There was no strong absorption near 485 nm after stimulation at 450 nm;Competitive binding assay showed that fixed AS concentration was shown to be able to increase the concentration of Th-T,but not to restore the inten-sity of fluorescence to Th-T without addition of Acteoside,indicating that there was no relevant binding sites between the two substances;.The IC50 was 254.3 μmol/L when Acteoside was observed to be able to inhibit the aggregation of Aβ1-42 ,whereas IC50 was 282 μmol/L when Acteoside was observed to be able to segregate the aggregation of Aβ1-42 .Conclusion Acteoside was shown to be effectively capable of inhibiting the aggregation of Aβ1-42 and of segregating the aggregation of Aβ1-42 ,suggesting that Acteoside could be potentially therapeutic in the therapy of Alzheimer′s disease.