3,6ʹ-二芥子酰基蔗糖神经保护作用研究

Studies on Neuroprotective Effects of 3,6'-Disinapoyl Sucrose

目的:研究远志中寡糖酯类成分3,6ʹ-二芥子酰基蔗糖(DISS)对神经的保护作用。方法:将不同浓度的DISS与APP-SH-SY5Y细胞共同孵育24 h,四甲基偶氮唑盐(MTT)法检测各组细胞的存活率,免疫荧光法检测细胞内活性氧(ROS)水平,生化法检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,ELISA法检测细胞内和培养液中β-淀粉样蛋白(Aβ)含量,蛋白质免疫印迹法(Western blot)检测细胞内淀粉样前体蛋白(APP)、微管相关蛋白轻链3-Ⅰ(LC3-Ⅰ)和LC3-Ⅱ的表达。结果:DISS可以提高APP-SH-SY5Y细胞的存活率(P<0.05),减少细胞内ROS的生成(P<0.01),提高SOD和GSH-Px活力(P<0.05),降低细胞内和培养液中的Aβ含量(P<0.01,P<0.05),明显升高细胞内LC3Ⅱ蛋白表达水平,升高LC3Ⅱ/LC3Ⅰ水平(P<0.05),对APP表达无明显影响。结论:DISS可能通过降低细胞内氧化应激,减少细胞Aβ表达及调节自噬发挥神经保护作用。

Objective:To study the neuroprotective effects of 3,6'-disinapoyl sucrose(DISS),an oligosaccharide ester component of the traditional Chinese medicine Polygala tenuifolia Willd.Methods:Different concentrations of DISS were co-incubated with APP-SH-SY5Y cells for 24 h.The survival rate was detected by methyl thiazole tetrazolium(MTT)assay,The intracellular reactive oxygen species(ROS)levels were detected by immunofluorescence.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were detected by biochemical methods.The intracellular and culture medium contents ofβ-amyloid protein(Aβ)level were detected by enzyme-linked immunosorbent assay(ELISA).The expression of amyloid precursor protein(APP),microtubule-associated protein lightchain 3-Ⅰ(LC3-Ⅰ)and LC3-Ⅱin cells were detected by western blotting.Results:DISS could increase the viability of APP-SH-SY5Y cells(P<0.05),decrease the production of intracellular ROS(P<0.01),and increase the activities of SOD and GSH-px in APP-SHSY5Y cells(P<0.05).Meanwhile,DISS also decreased the Aβcontent in APP-SH-SY5Y cells(P<0.01)and cell culture medium(P<0.05),significantly increased the expression level of intracellular LC3Ⅱprotein and increased the ratio of LC3Ⅱ/LC3Ⅰ(P<0.05).However,DISS had no significant effect on APP expression.Conclusion:DISS may exert neuroprotective effects by reducing intracellular oxidative stress,decreasing cellular Aβexpression and regulating autophagy.