佛手多糖对1-甲基-4-苯基-吡啶离子诱导人神经母细胞瘤(SH-SY5Y)细胞损伤的保护作用研究

Protective effect of bergamot polysaccharide on SH-SY5Y cells injured by 1-methyl-4-phenyl-pyridine ion

该文探究佛手多糖对1-甲基-4-苯基-吡啶离子(1-methyl-4-phenyl-pyridine ion,MPP+)诱导人神经母细胞瘤(SH-SY5Y)细胞损伤的保护作用及其机制。佛手多糖经大孔吸附树脂AB-8进行纯化。体外培养SH-SY5Y细胞,构建帕金森病(Parkinson′s disease,PD)细胞模型,实验分为对照组、MPP+模型组、佛手多糖组。采用噻唑蓝(methye thiazdye telrazlium,MTT)法检测细胞存活率,Hoechst33258染色法观察细胞形态,2′,7′-二氯荧光黄双乙酸盐荧光探针检测细胞活性氧(reactive oxygen species,ROS)水平,JC-1荧光探针法检测线粒体膜电位,蛋白免疫印迹(Western blot)检测磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)、蛋白激酶B(protein kinase B,Akt)、磷酸化细胞外调节蛋白激酶(phosphorylated extracellular regulated protein kinases1/2,p-ERK1/2)和细胞色素c(cytochrome c,Cyt-c)蛋白表达水平。结果表明,佛手多糖的得率4.86%,纯度为44.46%,经过AB-8纯化后,纯度提高到60.81%;与对照组相比,模型组细胞的存活率显著降低,Hoechst33258染色下可见细胞破碎,细胞核皱缩,细胞内ROS显著增加,线粒体膜电位显著降低。与模型组相比,佛手多糖组的细胞存活率显著增加,细胞形态明显得到改善,ROS水平下降,线粒体膜电位升高。Western blot结果显示,佛手多糖能抑制MPP+引起的p-Akt和p-ERK1/2的降低,以及Cyt-c的上升。综上,佛手多糖对MPP+诱导SH-SY5Y细胞损伤具有保护作用,其机制可能是通过调节线粒体ROS的产生和Cyt-c的释放,进而维持线粒体稳态,激活Akt信号通路和ERK信号通路,抑制细胞的凋亡,从而起到保护作用。研究结果可为缓解帕金森病的发生发展提供理论依据,同时也能更好地开发和利用佛手资源。

This study aimed to explore the protective effect and mechanism of bergamot polysaccharide on damaged SH-SY5Y cells induced by 1-methyl-4-phenyl-pyridine ion(MPP+).The bergamot polysaccharide was extracted and purified with AB-8 macroporous resin.SH-SY5Y cells were cultured in vitro to construct a Parkinson′s disease(PD)cell model.The experiment was divided into a control group,the MPP+model group,and the bergamot polysaccharide group.The cell viability was detected with the methye thiazdye telrazlium(MTT)method,and the cell morphology was observed by the Hoechst33258 staining method.The reactive oxygen species(ROS)level was detected by the DCFH-DA fluorescence probe,and mitochondrial membrane potential was measured by the JC-1 fluorescence probe method.The protein expression levels of phosphorylated protein kinase B(p-Akt),protein kinase B(Akt),phosphorylated extracellular regulated protein kinases1/2(p-ERK1/2),and cytochrome c(Cyt-c)were detected by Western blot.Results showed that the yield of bergamot polysaccharide was 4.86%and the purity was 44.46%.After purification by AB-8,the purity of the polysaccharide was increased to 60.81%.Compared with the control group,the cell viability of the MPP+-treated group was significantly reduced,the cells were broken and the nucleus was shrunk after Hoechst33258 staining.Meanwhile,the intracellular ROS was significantly increased,and the mitochondrial membrane potential was significantly decreased.Compared with the model group,the cell viability of the bergamot polysaccharide group was significantly increased,the cell morphology was significantly improved,the enhancement of intracellular ROS was inhibited,and the mitochondrial membrane potential was increased.The results of the Western blot showed that bergamot polysaccharide could inhibit the decrease of p-Akt and p-ERK1/2 as well as the increase of Cyt-c caused by MPP+.Based on these data,bergamot polysaccharide had a protective effect on SH-SY5Y cells against the injury induced by MPP+,which might be correlated with antioxidant activity as well as ERK and Akt activation pathway.The research results could provide a theoretical basis for alleviating the occurrence and development of Parkinson's disease,and be also beneficial for the development and utilization of bergamot resources.