Citicoline对大鼠中脑原代细胞培养的神经保护作用

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50 μmol·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50 μmol·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^-1 CC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^-1 CC+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^-1 CC+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。

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

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 meanCa~(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 CC treatment(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.