【摘要】 目的研究帕金森病(PD)细胞模型中,核孤儿受体Nur77激动剂真菌聚酮(Csn-B)对帕金森病神经细胞产生保护作用。方法将细胞分为空白组、对照组(Csn-B组)、模型组(MPP~+组)和实验组(MPP~++Csn-B组)。通过噻唑蓝(MTT)法检测不同浓度1-甲基-4-苯基吡啶离子(MPP~+)对人神经母细胞瘤细胞SH-SY5Y增殖影响;用Western blotting法检测微管相关蛋白1轻链3(LC3)-Ⅱ/Ⅰ和α-synuclein蛋白表达情况;用实时定量聚合酶链式反应(qPCR)检测LC3和α-synuclein mRNA表达情况;用免疫荧光观察LC3和α-synuclein蛋白在各组细胞内的表达情况。结果随着MPP~+浓度增高,其对SH-SY5Y细胞的抑制作用逐渐增强,抑制作用与浓度呈剂量依赖性。Western blotting结果显示,空白组、对照组、模型组和实验组LC3-Ⅱ/Ⅰ蛋白分别为0.21±0.02,0.23±0.03,0.32±0.03,0.69±0.05,模型组、实验组明显高于空白组(P<0.01),实验组明显高于模型组(P<0.01);空白组、对照组、模型组和实验组α-synuclein分别为0.30±0.05,0.51±0.06,2.18±0.12,0.73±0.08,模型组、实验组明显高于空白对照组(P<0.01),实验组明显低于模型组(P<0.01)。qPCR显示,空白组、对照组、模型组和实验组LC3 mRNA分别为3.94±0.18,4.04±0.13,6.01±0.21,7.43±0.35,模型组、实验组明显高于空白组(P<0.01),实验组明显高于模型组(P<0.01);空白组、对照组、模型组和实验组α-synuclein mRNA 3.09±0.19,3.47±0.24,7.18±0.30,4.60±0.27,模型组、实验组明显高于空白组(P<0.01),实验组α-synuclein明显低于模型组(P<0.01)。免疫荧光可见LC3蛋白主要定位在细胞质,模型组、实验组LC3表达量明显高于空白组,实验组LC3表达量高于模型组;α-synuclein蛋白在细胞质和细胞核中均匀分布,模型组、实验组表达量高于空白组,实验组表达量低于模型组。结论在帕金森病的细胞模型中,Nur77激动剂可以促进α-synuclein的降解,其机制可能与增加自噬水平有关。更多还原

【Abstract】 Objective To demonstrate the nuclear orphan receptor Nur77 agonist fungal polyketone( Cytosporone B,Csn-B) protects the neurons in Parkinson’ s disease cell model. Methods The SH-SY5 Y cells were divided into blank group,control group( Csn-B),model group( MPP~+) and experimental group( MPP~++ Csn-B). The relative viability of human neuroblastoma cells SH-SY5 Y induced by 1-methyl-4-pehny1-pyridine( MPP~+) was measured with MTT assay.The expression of microtubule associated protein 1 light chain3( LC3)-Ⅱ/Ⅰ and α-synuclein protein was detected by Western blotting. The expression of LC3 mRNA and α-synuclein mRNA were detected by qPCR. Immunofluorescence was used to observe the expression of LC3 and α-synuclein in each group. Results The MTT results showed that MPP+significantly inhibited proliferation of SH-SY5 Y cells in a dose dependent manner. Western blotting showed that,LC3-Ⅱand LC3-Ⅰgray scale in blank group,control group,model group and experimental group were 0. 21 ± 0. 02,0. 23 ± 0. 03,0. 32 ± 0. 03,0. 69 ± 0. 05,LC3-Ⅱ/Ⅰin model group and experimental group was significantly higher than that in blank group( P < 0. 01),and LC3-Ⅱ/Ⅰin experimental group was significantly higher than that in model group( P < 0. 01). The α-synuclein in blank group,control group, model group and experimental group were 0. 30 ± 0. 05, 0. 51 ± 0. 06, 2. 18 ± 0. 12,0. 73 ± 0. 08,the expression of α-synuclein in model group and experimental group were significantly higher than that in blank group( P < 0. 01). The expression of α-synuclein protein in experimental group was significantly lower than that in model group( P < 0. 01). The relative expression of LC3 mRNA in blank group,control group,model group and experimental group were 3. 94 ± 0. 18,4. 04 ± 0. 13,6. 01 ± 0. 21,7. 43 ± 0. 35,the LC3 mRNA in model group and experimental group were significantly higher than that in blank group( P < 0. 01),LC3 mRNA in experimental group was significantly higher than that in model group( P < 0. 01). The α-synuclein mRNA in blank group,control group,model group and experimental group were 3. 09 ± 0. 19,3. 47 ± 0. 24,7. 18 ± 0. 30,4. 60 ± 0. 27,the expression of α-synuclein mRNA in model group and experimental group were significantly higher than that in blank group( P < 0. 01). The expression of α-synuclein mRNA in experimental group was significantly lower than that in model group( P < 0. 01). Immunofluorescence showed that LC3 protein was mainly located in cytoplasm. The expression of LC3 in model group and experimental group were significantly higher than that in blank group,and the expression level of LC3 in experimental group was higher than that in model group. α-synuclein protein was distributed evenly in the cytoplasm and nucleus. The expression of α-synuclein in model group and experimental group was higher than that in blank group,experimental group was lower than that in model group. Conclusion In Parkinson’s disease cell model,Nur77 agonist can promote the degradation of α-synuclein,the mechanism may be related to increase autophagy level.更多还原