海藻糖对帕金森病氧化损伤保护作用及机制研究

Protective effect of trehalose on oxidative damage in PD and its mechanism

目的研究海藻糖对1-甲基-4-苯基吡啶离子(MPP+)诱导的shsy5y细胞损伤的保护作用及机制。方法利用MPP+诱导损伤shsy5y建立帕金森病模型,分为对照组、损伤组、实验1组(海藻糖10mmol/L)、实验2组(海藻糖50mmol/L)和实验3组(海藻糖100mmol/L)。CCK-8法检测细胞存活率,利用二乙酰二氯氢化荧光素探针检测shsy5y细胞活性氧水平,hoechst 33342染色观察海藻糖对MPP+诱导的细胞凋亡保护作用,Western blot检测Nrf2、血红素氧化酶1(heme oxygenase-1,HO-1)抗氧化损伤蛋白表达。结果与对照组比较,损伤组shsy5y细胞存活率明显降低(P<0.01),Nrf2和HO-1表达明显降低(0.676±0.020 vs 1.000±0.067,P<0.05;0.546±0.049vs 1.000±0.048,P<0.01)。损伤组MPP+可以显著诱导shsy5y细胞发生凋亡,核碎裂,不同浓度实验组可以逆转这种诱导凋亡。实验1组Nrf2表达较损伤组明显升高,实验2组和实验3组Nrf2和HO-1表达较损伤组明显升高,差异有统计学意义(P<0.01)。损伤组shsy5y细胞内绿色荧光较对照组明显升高,不同浓度实验组随海藻糖浓度升高,shsy5y细胞内绿色荧光明显减少。结论海藻糖可以逆转MPP+作用的shsy5y细胞损伤,这一作用路径可能是通过促进Nrf2/HO-1信号通路激活,减少活性氧损伤,从而起到保护作用。

Objective To study the protective effect of trehalose on shsy5 y cell injury induced by MPP+and its mechanism.Methods A PD model was established by inducing shsy5 y cell injury with MPP+.The shsy5 y cells were divided into control group,injury group,experimental group A(trehalose 10 mmol/L),experimental group B(trehalose 50 mmol/L)and experimental group C(trehalose 100 mmol/L).The viability of shsy5 y cells was assayed with CCK-8 method.The reactive oxygen level in shsy5 y cells was measured with a diacetyl dihydrogen fluorescein probe.The protective effect of trehalose on apoptosis of shsy5 y cells induced by MPP+was observed with hoechst 33342 staining.The Nrf2 and HO-1 proteins were detected by Western blot.Results The survival rate of shsy5 y cells and the expression levels of Nrf2 and HO-1 were significantly lower in injury group than in control group(0.676±0.020 vs1.000±0.067,P<0.05;0.546±0.049 vs 1.000±0.048,P<0.01).MPP+could significantly induce the apoptosis and nuclear fragmentation of shsy5 y cells in injury group and reverse the apoptosis of shsy5 y cells in experimental groups A-C.The expression levels of Nrf2 were significantly higher in experimental group A than in injury group and those of Nrf2 and HO-1 were significantly higher in experimental groups A and B than in injury group(P<0.01).The green fluorescence in shsy5 y cells was significantly higher in injury group than in control group.The trehalose level was significantly higher while the green fluorescence in shsy5 y cells was significantly lower in experimental groups A-C than in control group.Conclusion Trehalose can reverse the MPP+-induced injury of shp5 y cells by activating the Nrf2/HO-1 signaling pathway and reducing the reactive oxygen damage.