梓醇活性成分对MPP^+诱导的BV2细胞损伤的神经保护作用

Neuroprotective effects of Catalpol on BV2 cells injured by MPP^+

目的观察大脑多巴胺神经营养因子(CDNF)在梓醇保护多巴胺胶质细胞中的作用。方法①将小鼠小胶质BV2细胞随机分为对照组、梓醇组、1-甲基-4-苯基吡啶离子(MPP+)模型组和MPP+模型+梓醇组,采用RT-PCR技术检测处理0、6、24、48和72 h后BV2细胞CDNF mRNA表达的变化。②采用终浓度分别为0.1、1和10μmol/L的梓醇处理BV2细胞,24 h后加入MPP+,48 h后Western blotting检测CDNF蛋白表达;以正常细胞为对照组,设未加入梓醇预处理的MPP+模型组。③设对照组、MPP+模型组、梓醇组、抗CDNF抗体组和梓醇+抗CDNF抗体组;除对照组外,其余各组均经MPP+处理;采用[3H]-多巴胺放射分析法检测CDNF抗体阻断后梓醇对多巴胺摄取的影响。结果①在MPP+加入后的0～72 h,梓醇组和MPP+模型组的CDNF mRNA表达与对照组比较均无显著变化;与MPP+模型组比较,在MPP+加入后48 h,MPP+模型+梓醇组CDNF mRNA表达显著升高(P<0.01)。②MPP+加入后48 h,MPP+模型组CDNF蛋白表达量(0.679±0.013)低于对照组(1.009±0.015),差异有统计学意义(P<0.001);以10μmol/L梓醇预处理的BV2细胞CDNF蛋白表达量(0.812±0.011)显著高于MPP+模型组(P<0.01);0.1、1μmol/L梓醇预处理的BV2细胞CDNF蛋白表达量与MPP+模型组比较无明显变化。③MPP+模型组多巴胺摄取力(63.5±2.5)低于对照组(99.9±0.8),差异有统计学意义(P<0.01);梓醇组多巴胺摄取力(87.2±2.4)显著高于MPP+模型组(P<0.01);梓醇+抗CDNF抗体组多巴胺摄取力(73.6±2.7)显著低于梓醇组(P<0.01),而高于抗CDNF抗体组(P<0.05)。结论梓醇对MPP+诱导的BV2细胞损伤的神经保护作用与上调CDNF基因和蛋白表达有关。

Objective To explore the role of cerebral dopamine neurotrophic factor（CDNF） in the protection effect of Catalpol on the injured dopaminergic glial cells.Methods ① Mouse glial BV2 cells were randomly divided into control group,Catalpol group,1-methyl-4-phenylpyridinium（MPP＋） model group and MPP＋ model＋Catalpol group.The expression of CDNF mRNA in BV2 cells 0,6,24 and 48 h after treatment was detected by RT-PCR.② BV2 cells were treated with 0.1,1 and 10 μmol/L Catalpol respectively,MPP＋ was added 24 h later,and the expression of CDNF protein was determined by Western blotting 48 h later.Normal cells were served as control group,and those without pretreatment with Catalpol were established as MPP＋ model group.③ Control group,MPP＋ model group,Catalpol group,anti-CDNF antibody group and Catalpol＋ anti-CDNF antibody group were established,all groups were treated with MPP＋ except control group,and the effect of Catalpol on dopamine uptake after CDNF antibody blockade was examined by -dopamine radiometry.Results ① Compared with control group,the expression of CDNF mRNA in Catalpol group and MPP＋ model group was not significantly changed 0 to 72 h after administration of MPP＋.However,the expression of CDNF mRNA in MPP＋ model＋ Catalpol group was significantly higher than that in MPP＋ model group 48 h after administration of MPP＋（P0.01）.② The expression of CDNF protein in MPP＋ model group was significantly lower than that in control group 48 h after administration of MPP＋（0.679±0.013 vs 1.009±0.015,P0.001）.The expression of CDNF protein in BV2 cells after pretreatment with 10 μmol/L Catalpol（0.812±0.011） was significantly higher than that in MPP＋ model group（P0.01）,while the expression of CDNF protein in BV2 cells after pretreatment with 0.1 and 1 μmol/L Catalpol was not significantly changed compared with MPP＋ model group.③ The dopamine uptake in MPP＋ model group was significantly lower than that in control group（63.5±2.5 vs 99.9±0.8,P0.01）.The dopamine uptake in Catalpol group（87.2±2.4） was significantly higher than that in MPP＋ model group（P0.01）.The dopamine uptake in Catalpol＋anti-CDNF antibody group（73.6±2.7） was significantly lower than that in Catalpol group（P0.01）,while was higher than that in anti-CDNF antibody group（P0.05）.Conclusion The neuroprotection effect of Catalpol against MPP＋ toxicity may be associated with the upregulation of expression of CDNF mRNA and protein in BV2 cells.