褐藻多糖硫酸酯对MPP+损伤的MN9D细胞氧化应激及凋亡的保护作用(1)
[摘要] 目的 探讨褐藻多糖硫酸酯(FUC)对帕金森病(PD)细胞模型的保护作用及机制。 方法 采用100 μmol/L MPP+损伤的MN9D细胞制作PD模型，用MTS检测FUC预处理后PD细胞模型的细胞存活率。荧光检测法测定细胞内活性氧(ROS)，荧光素酶法检测细胞内Caspase-3的活性变化，Western blot检测凋亡相关蛋白Bax及Bcl-2的变化。 结果 FUC 100 μmol/L作用24 h明显增加了MPP+损伤的MN9D细胞存活率，差异有高度统计学意义(P < 0.01);FUC预保护明显降低了MPP+损伤3 h的MN9D细胞内ROS活性及6 h时Caspase-3的表达，差异有高度统计学意义(P < 0.01);FUC、司来吉兰预保护后MN9D细胞凋亡相关蛋白Bax表达明显下降，而Bcl-2表达明显增加，差异有高度统计学意义(P < 0.01)。 结论 褐藻多糖硫酸酯可能通过抗氧化、降低Caspase-3表达、抑制细胞凋亡来保护PD细胞模型。
[关键词] 褐藻多糖硫酸酯;帕金森病;MN9D细胞;氧化应激;细胞凋亡
[中图分类号] R742.5 [文献标识码] A [文章编号] 1673-7210(2019)03(a)-0020-05
[Abstract] Objective To explore the protective effect of fucoidan (FUC) on the cell model of Parkinson′s disease (PD) and its mechanism. Methods PD model was established by MN9D cells which were damaged by 100 μmol/L MPP+. The cell viability of PD cell model was detected by MTS after FUC pretreatment. The reactive oxygen species (ROS) was measured by fluorescence detection， the activity of Caspase-3 in cells was measured by luciferase， the expression changes of apoptosis-related proteins Bax and Bcl-2 were measured by Western blot. Results After application of 12 h， FUC 100 μmol/L significantly increased the cell viability of MN9D cells damaged by MPP+， the difference was highly statistically significant (P < 0.01). The FUC pretreatment reduced the activity of intracellular ROS of MN9D cell damaged by MPP+ in 3 h and the expression of Caspase-3 in 6 h， there were highly statistically significant differences (P < 0.01). After FUC and Selegiline pretreatment， the expression of MN9D cell apoptosis-related protein Bax was decreased， and the expression of Bcl-2 was increased， the differences were highly statistically significant (P < 0.01). Conclusion Fucoidan may protect the PD cell models through anti-oxidation， reducing the expression of Caspase-3 and inhibiting cell apoptosis.
[Key words] Fucoidan; Parkinson′s disease; MN9D cell; Oxidation stress; Cell apoptosis
帕金森病(PD)是一种常见于中老年的中枢神经系统变性疾病，目前其确切发病机制尚不清楚。研究认为氧化应激与细胞凋亡在PD的发病中起重要作用[1]。褐藻多糖硫酸酯(FUC)是从褐藻中提取的具有抗氧化、抗凝及抗老化作用的多糖。研究表明，FUC具有对阿尔茨海默病(AD)模型的神经保护作用[3]，我们推测FUC对于PD亦可能具有一定作用。本研究通过MPP+损伤小鼠中脑多巴胺能细胞系(MN9D)细胞制作PD细胞模型，观察FUC对细胞模型氧化应激及凋亡的影响，以探讨其对PD模型的保护作用。
1 材料与方法
1.1 材料
MN9D细胞(购自中国医学科学院，本院中心实验室保存);DMEM/F12培养基(美国Gibco BRL公司产品，11320-033);新生牛血清(NCS，奥地利PAA公司);胎牛血清(Gibco公司);MPP+ idione(美国Sigma公司);FUC(中国生物檢验中心，纯度99.8%，Z20030 053)。阳性对照药物司来吉兰(SEL，芬兰奥立安公司，H20040400)[4]。MTS细胞活力测定试剂盒(G1112)、Caspase-3活性荧光素酶检测试剂盒(PROMAGE公司，G8090);细胞内活性氧(ROS)荧光检测试剂盒(Cell Biolabs公司，K936);小鼠单克隆Bax(B8249)和Bcl-2(SAB4500003)抗体、GAPDH(G9545)购自Sigma公司。其他试剂均为分析纯(购自Sigma公司)。, 百拇医药(梁志刚 孙旭文 刘竹丽)
[关键词] 褐藻多糖硫酸酯;帕金森病;MN9D细胞;氧化应激;细胞凋亡
[中图分类号] R742.5 [文献标识码] A [文章编号] 1673-7210(2019)03(a)-0020-05
[Abstract] Objective To explore the protective effect of fucoidan (FUC) on the cell model of Parkinson′s disease (PD) and its mechanism. Methods PD model was established by MN9D cells which were damaged by 100 μmol/L MPP+. The cell viability of PD cell model was detected by MTS after FUC pretreatment. The reactive oxygen species (ROS) was measured by fluorescence detection， the activity of Caspase-3 in cells was measured by luciferase， the expression changes of apoptosis-related proteins Bax and Bcl-2 were measured by Western blot. Results After application of 12 h， FUC 100 μmol/L significantly increased the cell viability of MN9D cells damaged by MPP+， the difference was highly statistically significant (P < 0.01). The FUC pretreatment reduced the activity of intracellular ROS of MN9D cell damaged by MPP+ in 3 h and the expression of Caspase-3 in 6 h， there were highly statistically significant differences (P < 0.01). After FUC and Selegiline pretreatment， the expression of MN9D cell apoptosis-related protein Bax was decreased， and the expression of Bcl-2 was increased， the differences were highly statistically significant (P < 0.01). Conclusion Fucoidan may protect the PD cell models through anti-oxidation， reducing the expression of Caspase-3 and inhibiting cell apoptosis.
[Key words] Fucoidan; Parkinson′s disease; MN9D cell; Oxidation stress; Cell apoptosis
帕金森病(PD)是一种常见于中老年的中枢神经系统变性疾病，目前其确切发病机制尚不清楚。研究认为氧化应激与细胞凋亡在PD的发病中起重要作用[1]。褐藻多糖硫酸酯(FUC)是从褐藻中提取的具有抗氧化、抗凝及抗老化作用的多糖。研究表明，FUC具有对阿尔茨海默病(AD)模型的神经保护作用[3]，我们推测FUC对于PD亦可能具有一定作用。本研究通过MPP+损伤小鼠中脑多巴胺能细胞系(MN9D)细胞制作PD细胞模型，观察FUC对细胞模型氧化应激及凋亡的影响，以探讨其对PD模型的保护作用。
1 材料与方法
1.1 材料
MN9D细胞(购自中国医学科学院，本院中心实验室保存);DMEM/F12培养基(美国Gibco BRL公司产品，11320-033);新生牛血清(NCS，奥地利PAA公司);胎牛血清(Gibco公司);MPP+ idione(美国Sigma公司);FUC(中国生物檢验中心，纯度99.8%，Z20030 053)。阳性对照药物司来吉兰(SEL，芬兰奥立安公司，H20040400)[4]。MTS细胞活力测定试剂盒(G1112)、Caspase-3活性荧光素酶检测试剂盒(PROMAGE公司，G8090);细胞内活性氧(ROS)荧光检测试剂盒(Cell Biolabs公司，K936);小鼠单克隆Bax(B8249)和Bcl-2(SAB4500003)抗体、GAPDH(G9545)购自Sigma公司。其他试剂均为分析纯(购自Sigma公司)。, 百拇医药(梁志刚 孙旭文 刘竹丽)