Citicoline对大鼠中脑原代细胞培养的神经保护作用

Neuroprotective effects of citicoline on 6-hydroxydopamine-treated mesencephalic dopaminergic neurons in primary culture

目的:探讨胞二磷胆碱(citicoline,CC)对6-OHDA诱导的帕金森体外细胞模型的保护作用及其机理。方法:孕15 d大鼠胚胎中脑原代培养,在培养第6、8及10天,实验组加不同浓度CC(2、1、0.1、0.01和0.001 mmol.L-1),并于第11天加50μmol.L-1的6-OHDA作用0.5 h,制作帕金森病细胞模型;6-OHDA组为原代培养细胞加50μmol.L-1的6-OHDA;对照组为原代培养细胞。培养11 d收集细胞。采用MTT法测细胞活力,通过流式细胞仪,用Fluo3/AM检测细胞内[Ca2+]i及用罗丹明123检测线粒体膜电位(Δψm)。结果:2、1和0.1 mmol.L-1CC组,细胞活力与对照组比较明显增高,并随着浓度的增加而增加,差异具有显著性(P<0.05)。1、0.1、0.01和0.001 mmol.L-1CC+6-OHDA组,细胞活力均高于6-OHDA组,差异有显著性(P<0.01)。1、0.1、0.01、0.001 mmol.L-1CC+6-OHDA组细胞内[Ca2+]i均明显下降,分别为(32.23±1.87)%、(17.09±7.45)%、(21.71±8.89)%及(29.18±4.71)%,与6-OHDA组(49.30±7.62)%相比,差异均有显著性(P<0.01);与对照组(42.40±0.81)%比较明显下降,差异均有显著性(P<0.01)。CC+6-OHDA各组与6-OHDA组比较均使Δψm升高,其中1 mmol.L-1CC+6-OHDA组Δψm高于对照组,差异有显著性(P<0.01)。结论:CC通过保护神经元细胞膜、增加细胞活力、降低细胞内[Ca2+]i以及提高Δψm,发挥其对神经元的保护作用。

Objective To study the neuroprotective effects of citicoline （CC） on the toxicity induced by 6-OHDA towards dopaminergic mesencephalic neurons in primary culture-Parkinson＇s disease （PD） model in vitro and its mechanism. Methods Mesencephalic neurons in culture were prepared from embryonic 15-day Wistar rats. Cultures were treated for 6, 8, 10 d with various concentrations of CC （2, 1, 0. 1, 0. 01 and 0. 001 mmol·L^-1）. At 11th day, the cultures were co-treated with the toxin 6-OHDA （50 μmol·L^-1） for 0.5 h, the cells were collected. Seven groups were categorized as follows. CC （2, 1, 0. 1, 0. 01 and 0. 001 mmol·L^-1） ＋ 6-OHDA, control and 6-OHDA group. The cell viability was evaluated with MTT assay. Intracellular free Ca^2＋＋ , [Ca^2＋] i, was labeled by using the fluorescent dye Fluo3-AM and detected by flow cytometer. By measuring the intracellular Rhodamine 123 fluorescence density with flow cytometer, mitochondrial membrane potential （MMP） was evaluated.Results Cultures were treated with 2, 1 and 0.1 mmol· L^-1 CC, the viability of cell was increased （P〈0. 05） . 1, 0. 1, 0.01 and 0. 001 mmol· L^-1 CC significantly attenuated 6-OHDA-induced neurotoxic effects. As compared with 6-OHDA, the viability of neurons increased, the mean [Ca^2＋ ] i was significantly lower in cells treated with CC （ 1, 0.1, 0.01 and 0. 001 mmol · L^-1） plus 6-OHDA （49.30±7.62）% than that in 6-OHDA group without CCtreatment （P〈0.01）. The densities of [Ca^2＋] i in CC （1, 0.1, 0.01 and 0.001 mmol· L^-1） plus 6-OHDA groups were （32.23± 1.87） %, （17.09 ± 7.45） %, （21.71 -± 8.89） %, （29.18 ± 4.71）%, respectively, and the density of mean [Ca^2＋ ] i in 6-OHDA group was （49.30 ± 7.62）^. MMP significantly increased （P〈0.01）. Conclusion CC has an important effect on dopaminergic cell survival in vitro in a validated model of PD. The neurotoxic effect of 6-OHDA can be reduced by CC and CC can increase the viability of neurons, decrease [Ca^2＋] i, maintain MMP at a relatively higher level.