士的宁对多巴胺能神经元的保护作用

Protective Effect of Strychnine on Dopaminergic Neurons

摘要:

目的: 选择2种神经毒物1-甲基-4-苯基吡啶离子(MPP⁺)和谷氨酸在体外诱导多巴胺能神经元退行性损伤,并检测士的宁对此细胞的药理毒理作用,及对其退行性损伤的协同作用或拮抗作用。 方法: 采用孕14 d小鼠,取出胎鼠中脑,分离出多巴胺能神经元,调整细胞密度7.5×10⁵/mL,于5%CO₂,37 ℃,100%湿度条件培养,于体外培养的第10天分别加入士的宁0.1, 1, 5, 10 μmol·L^-1,MPP⁺ 10 μmol·L^-1,谷氨酸500 μmol·L^-1作用48 h,第12天染色。选取细胞数,神经元树突长度和数量作为观察指标,并做出分析。 结果: 士的宁对未损伤的多巴胺能神经元在48 h培养过程中无毒性影响。在10 μmol·L^-1士的宁作用下,多巴胺能神经元数量明显高于对照组(P<0.05);在5~10 μmol·L^-1士的宁作用下,树突数量与对照组相比也有明显提高(P<0.05)。士的宁(10 μmol·L^-1)和MPP⁺共同培养48 h后,细胞计数明显高于MPP⁺单独培养组(P<0.05);这种保护作用更为明显的表现在细胞形态学变化方面,在10 μmol·L^-1的士的宁剂量下,神经树突长度明显高于MPP⁺单独培养组(P<0.05);神经树突的数量在5 μmol·L^-1士的宁作用下与MPP⁺单独培养组相比有明显增加(P<0.05)。在对谷氨酸(500 μmol·L^-1)诱导的神经元损伤模型中,与谷氨酸组相比,10 μmol·L^-1的士的宁对所有的观察指标的数值都有递增影响 (P<0.05)。 结论: 首次证实士的宁具有可在体外刺激中脑分离出的多巴胺能神经元的生长,并且对此神经元退行性损伤有保护作用。

Abstract:

Objective:In this study two substances 1-methyl-4-phenyl pyridineion(MPP⁺) and glutamate(Glu) were used to induce neurotoxicity and degenerative change in dopaminergic neurons in primary cell culture. By additional applying strychnine in such cellular toxic models we tested the pharmacological and toxicological influence of strychnine on neurodegenerative processes. Method: the pregnant OF1/SPF mice were choose, the midbrains were collected, the primary cells of dopaminergic neurons were cultured at 5%CO₂,37 ℃,100% humidity conditions in incubator. On the day in vitro(DIV) 10, strychnine at concentration of 0.1, 1, 5, 10 μmol·L^-1, MPP⁺ 10 μmol·L^-1, Glu 500 μmol·L^-1 was added for 48 h and performed staining on DIV 12.numbers of dopaminergic neurons, numbers of processes and length of processese were measured and analyzed. Result: The results demonstrated that strychnine at all range of concentrations (1-10 μmol·L^-1) had no significant toxic effect on normal dopaminergic neuron cells in 48 hours without MPP⁺ or glutamate. However, a significant stimulating effect on neuron occured, namely, in which under treatment of strychnine at dose 10 μmol·L^-1 cell number was enhanced compared with control (P<0.05); the value of length of neuronal processes also increased by 10 μmol·L^-1 strychnine (P<0.05); as well as number of neuronal processes elevated in the range of 5-10 μmol·L^-1 (P<0.05). When neurons were treated by MPP⁺ (10 μmol·L^-1), strychnine played anti-neurodegenerative effect. Under concentration 10 μmol·L^-1 of strychnine, the number of neurons was higher than that in MPP⁺ treatment (P<0.05); the value of length of neuronal processes was increased by strychnine at 10 μmmol·L^-1 (P<0.05); the number of neuron processes under 5 μmol·L^-1strychnine was also higher than that in MPP⁺ treatment group (P<0.05). In Glu(500 μmol·L^-1) induced neurotoxicity, strychnine showed significant neuroprotective action, which indicated by recovery of morphology or the number of dopaminergic neurons 10 μmol·L^-1 (P<0.05). Conclusion: The present results demonstrate that strychnine stimulated in vitro the growth of dopaminergic neurons from substantia negra par compacta, and played the neuronal protective effect against neurodegenerative damage in vitro.