S14G-humanin对β-淀粉样蛋白纤维化及其细胞毒性的影响

摘要

目的 探讨S14G-humanin(HNG)对β-淀粉样蛋白(Aβ)纤维化及其细胞毒性的影响.方法 于体外将Aβ1-42和不同浓度HNG共孵育,并设空白对照,应用硫黄素-T(Th-T)荧光法和透射电镜方法,观察HNG对Aβ1-42纤维化的作用;将Aβ1-42和不同浓度HNG共孵育后加入培养的大鼠肾上腺髓质嗜铬瘤细胞(PC12细胞),应用MTT法测定PC12细胞活性,观察不同浓度HNG对Aβ-42细胞毒性的拮抗作用.结果 (1)不同浓度HNG(100、200、400 μmol/L)+Aβ1-42(100μmol/L)组Th-T荧光强度(241.86±8.41,188.27±4.47,112.36±5.27)均较Aβl-42组(514.85±14.52)显著降低(P<0.01),且各组Th-T荧光强度随HNG浓度增加而降低,各组间比较有统计学差异(P<0.01).(2)透射电镜观察表明不同浓度HNG+Aβ1-42组纤维性Aβ均较Aβ1-42组显著减少.(3)不同浓度HNG(10、20、40 μmol/L)+Aβ1-42(10μmol/L)组PC12细胞活性C(67.83±3.57)%、(79.26±3.13)%、(89.67±3.25)%]均较Aβ1-42组[(57.52±5.41)%]显著增加(P<0.01),且各组细胞活性随HNG浓度增加而增加,各组间比较有统计学差异(P<0.01).结论 HNG在体外能够有效减少纤维性Aβ1-42形成并可抑制其细胞毒性作用,提示HNG有可能成为有效防治阿尔茨海默病的新药物.%Objective To investigate the effects of S14G-humanin (HNG) on the fibrillization and cytotoxicity of amyloid-beta protein (Aβ) in vitro. Methods After incubation of Aβ1-42 alone or with different concentrations of HNG (HNG + Aβ1-42), the fluorescence intensity of each sample was assessed by thioflavine-T (Th-T) fluorometric assay, and the fibrillar Aβ of each sample was examined by transmission electron microscopy. Then, the mediums of Aβ1-42 or HNG + Aβ1-42 incubations were added into cultured rat adrenal pheochromocytoma cells (PC12). The PC12 cell activity of each sample was examined by MTT method. The antagonistic effects of HNG with different concentrations on Aβ1-42 cytotoxicity were observed. Results The Th-T fluorescence intensity in various HNG (100, 200, 400 μmol/L, respectively) +Aβ1-42 groups was markedly lower than that in Aβ1-42 group (241.86±8. 41, 188. 27±4.47, 112.36±5.27 vs 514. 85±14.52,P＜0.01), with a dose-dependent manner (P＜0. 01). The fibrillar Aβ formation in each HNG (different concentrations) +Aβ1-42 group was significantly decreased than that in Aβ1-42 group. The cell activity measured by MTT method in each HNG (different concentrations) +Aβ1-42 group was markedly higher than that in Aβ1-42group [ (67.83±3.57)％, (79.26±3.13)％, (89.67±3.25)％ vs (57.52±5.41)％, P＜ 0.01], with a dose-dependent manner (P＜0. 01). Conclusions HNG may effectively reduce the fibrillization and neurotoxicity of Aβ1-42, suggesting that HNG may be a new therapeutic drug for AD.