同型半胱氨酸和褪黑素对离体帕金森病模型大鼠多巴胺能神经元的影响

Effect of homocysteine and melatonin on dopaminergic neuron in rat model of Parkinson disease in vitro

目的:观察同型半胱氨酸对离体帕金森病模型的影响及褪黑素的保护作用。方法:实验于2003-09/2004-02在华中科技大学同济医学院附属协和医院神经内科实验室完成。参考文献制备离体帕金森病大鼠模型,将2月龄健康SD雌性大鼠12只分成4组,6-羟多巴胺对照组及6-羟多巴胺+褪黑素、6-羟多巴胺+同型半胱氨酸对照组及6-羟多巴胺+同型半胱氨酸组+褪黑素组,每组3只。褪黑素组和对照组分别腹腔注射褪黑素(10mg/kg)或等量生理盐水,时间为3d,然后取脑片分别置于含6-羟多巴胺或6-羟多巴胺+同型半胱氨酸的人工脑脊液中孵育2h。免疫组织化学方法观测各组大鼠黑质酪氨酸羟化酶阳性细胞体及突起的变化。用硫代巴比妥酸法、黄嘌呤氧化酶法、Claiborne法分别检测各组大鼠丙二醛、超氧化物歧化酶、过氧化氢酶。结果:每组实验动物均达到实验目的,全部进入结果分析。①酪氨酸羟化酶免疫组织化学结果:6-羟多巴胺对照组与6-羟多巴胺+同型半胱氨酸对照组酪氨酸羟化酶阳性细胞数目减少,突起严重缺失,有突起的酪氨酸羟化酶阳性神经元占所有酪氨酸羟化酶阳性神经元的比例降低,并有无定型模糊的背景染色。而经过褪黑素干预的6-羟多巴胺+褪黑素组与6-羟多巴胺+同型半胱氨酸+褪黑素组脑片的酪氨酸羟化酶阳性细胞数目相对较多,细胞膜较为完整,突起缺失相对较少,有突起的酪氨酸羟化酶阳性神经元占所有酪氨酸羟化酶阳性神经元的比例明显增多。差异有显著性(P<0.01)。②丙二醛、超氧化物歧化酶、过氧化氢酶检测结果:6-羟多巴胺+同型半胱氨酸对照组及6-羟多巴胺+同型半胱氨酸组+褪黑素组与6-羟多巴胺对照组及6-羟多巴胺+褪黑素组相比丙二醛值高,超氧化物歧化酶、过氧化氢酶值低,差异有显著性(P<0.01)。6-羟多巴胺对照组与6-羟多巴胺+褪黑素组比较丙二醛值高,超氧化物歧化酶、过氧化氢酶值低,差异有显著性(P<0.01);6-羟多巴胺+同型半胱氨酸对照组与6-羟多巴胺+同型半胱氨酸+褪黑素组比较丙二醛值高,超氧化物歧化酶、过氧化氢酶值低,差异有显著性(P<0.01)。结论:同型半胱氨酸加重离体帕金森病模型动物的多巴胺能神经元损伤,其机制可能与氧化应激反应有关。褪黑素能减轻同型半胱氨酸的氧化应激反应。

AIM： To observe the influence of homocysteine on model of Parkinson disease in vitro and the protective effect of melatonin. METHODS： The experiment was conducted in the Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from September 2003 to February 2004. Rat models of Parkinson disease in vitro were induced according to the methods in the reference. Twelve healthy female SD rats of 2 months old were divided into 4 groups with 3 rats in each： 6-hydroxydopamine （6- OHDA） control group, 6-OHDA＋ melatonin group, 6-OHDA＋homocysteine control group, 6-OHDA ＋homocysteine＋ melatonin group. Rats in the melatonin and control groups were treated with intraperitoneal injection of melatonin （10 mg/kg） or saline of the same volume for 3 days, and then brain slices were prepared and incubated in artificial cerebrospinal fluid containing 6-OHDA or 6-OHDA ＋homocysteine for 2 hours respectively. The immolunohistochemical technique was used to observe the changes of tyroxine hydroxylase （TH）-stained neurons, including cell, bodies and dendrites in the substantia nigra. The content of malondialdehyde （MDA）, activities of superoxide dismutase （SOD） and catalase were detected with thiobarbituric acid-reacting substances, xanthine oxidase and Cllaiborue methods respectively. RESULTS： All the rats were involved in the analysis of results. （1） Results of TH immunohistochemistry： In the 6-OHDA control group and 6- OHDA＋homocysteine control group, the numbers of TH-stained neurons were decreased, dendrites were severely fragmented and truncated, the proportion of TH-stained neurons with dendrites to all the TH-stained neurons was decreased, and had unfixed-form vague staining. But in the 6- OHDA＋melatonin group and 6-OHDA ＋homocysteine ＋melatonin group, there were relatively more TH-stained neurons, more complete cell membrane, relatively fewer deletion of dendrites, and the proportion of TH- stained neurons with dendrites to all the TH-stained neurons was obviously increase, there were significant differences （P 〈 0.01）. （2） Detected results of MDA, SOD and catalase： As compared with the and 6-OHDA＋melatonin group, the MDA content was significantly higher, the activities of SOD and catalase were significantly lower in the 6-OHDA ＋homocysteine control group and 6-OHDA＋homocysteine＋melatonin group （P. 〈 0.01）. The MDA content was significantly higher, activities of SOD and catalase were significantly lower in the 6-OHDA control group than in the 6-OHDA ＋ melatonin group （P 〈 0.01）, The MDA content was .significantly. higher, activities of SOD and catalase were significantly lower in the 6-OHDA ＋ homocysteine control group than in the 6-OHDA＋homocysteine＋melatonin group （P 〈 0.01）. CONCLUSION： Homocysteine can aggravate the injury of dopaminergic neuons in animal models of Parkinson disease, and the mechanism may be associated with the anti-oxidative stress. Melatonin can ameliorate anti- oxidative stress of homocysteine.