尿酸对帕金森病模型大鼠多巴胺能神经元氧化应激的影响

Effects of uric acid on oxidative stress of dopaminergic neurons of rat model of Parkinson＇s disease

目的探讨尿酸对6羟多巴胺（6OHDA）诱导的SD大鼠帕金森病体外模型多巴胺（DA）能神经元氧化应激损伤的影响。方法取孕12～14dSD大鼠中脑原代细胞进行培养。实验分3组：（1）对照组：原代培养细胞；（2）6-OHDA组：原代培养细胞加6OHDA；（3）尿酸组：不同浓度尿酸（5、50、100、250、500μmol／L）分为5个亚组。培养第5天开始加尿酸干预，持续作用5d。于第10天加50μmlol／L的6OHDA，作用2h，培养第10天收集细胞。经酪氨酸羟化酶（TH）免疫细胞化学染色，3-（4，5-二甲基噻唑-2）-2，5二苯基四氮唑溴盐（MTT）法检测细胞活力；通过流式细胞仪，用罗丹明123（Rh123）检测线粒体膜电位（△Фm）。结果对照组TH阳性（TH^＋）细胞[（296．8±42．5）个／ml]较多，突起较长，部分密集成网状；6OHDA组TH^＋细胞[（92．8±19．7）个／ml]明显减少，突起较短，部分有断裂；5、50、100、250、500μmol／L尿酸组TH^＋细胞数（96．5±20．1、115．5±30．0、152．5±26．7、205．0±48．2、230．1±22．5）个／ml，较对照组减少，但明显多于6-OHDA组，并且同尿酸浓度成正相关（F—13．94，P〈0．05）。与对照组比较，6OHDA组细胞活力明显下降（F=90．19，P〈0．05）；5、50、100、500μmol／L尿酸组细胞活力较对照组下降（F=14．56，P〈0．05），但高于6OHDA组（F=40．96，P〈O．05）。250μmol／L尿酸组细胞活力与对照组比较，差异无统计学意义（F=1．27，P〉0．05）。对照组△Фm（30．05±5．88）％，与6-OHDA组（23．67±2．72）比较明显下降（F=6．30，P〈0．05）；而尿酸各组与6OHDA组比较，均升高；其中100、250μmol／L尿酸组△Фm（36．91±2．44）％、（38．08±2．90）％高于对照组（F=4．62，P〈0．05）。结论尿酸可减少6-OHDA对神经元的毒性作用，提高细胞活力，稳定细胞膜电位，表明尿酸能通过抗氧化应激活性发挥其对多巴胺能神经元的保护作用。

Objective To study the effects of uric acid （UA） on oxidative stress of dopaminergie neurons of rat model of Parkinson＇s disease induced by 6-hydroxydopamine （6-OHDA）. Methods Mesencephalic neurons in culture were prepared from embryonic Sprague-Dawley rat of 12 14 days. Various groups were categorized as follows： control group, 60HDA group and UA group which was sub divided into five subgroups according to different concentrations of UA （5, 50, 100, 250 and 500 μmol/L）. Cultures were added with UA from the 5th day to the 10th day. At the 10th day, the cultures were co treated with the toxin 6-OHDA （50 μmol/L） for 2 h, the cells were collected. The dopaminergic neurons were identified by tyrosine hydroxylase （ TH ） immunocytochemistry. The rate of cell viability was evaluated by MTT assay. By measuring the intracellular rbodamine 123 fluorescence density, mitochondrial membrane potential （△Фm） was evaluated. Results There was more TH-positive （TH-） neurons in the control group （296.84- 42.5）, moreover, some cross linked network structure could be found. As compared with the control group, there were less TH^＋ neurons in 6-OHDA group （92.8 ± 19.7, F= 75.26, P〈0.05）. More TH^＋ neurons were found in 5, 50, 100, 250 and 500μmol/L UA groups （96.5±20.1,115.5±30.0, 152.5±26.7, 205.0±48.2 and 230.1±22.5） compared with 6-OHDA group （F=10.72, P〈 0. 05）. Furthermore, there was a positive relationship of TH^＋ neuron number with concentration of UA （F=13.94, P〈0.05）. The rate of cell viability of neurons in 5, 50, 100 and 500 μ mol/L UA groups increased compared with 6-OHDA group （F=40.96, P〈0. 05）. There was no significant difference in cell viability of neurons between 250 μmol/L UA group and the control group （F= 1.27, P〈0. 05）. As compared with 6-OHDA group, the percentages of △Фm increased in UA groups （F= 8.82, P〈0.05）. The percentages of △Фm were higher in 100 and 250 μmol/L UA group than in control group （F = 4.62, P 〈 0.05）. Conclusions UA can reduce the neurotoxic effect of 6-OHDA, increase the viability of the cell and maintain mitochondrial membrane potential. The results suggest that UA shows a neuroprotective effect on dopaminergic neurons by anti-oxidative stress.