地■西平马来酸盐对体外糖尿病并发抑郁症大鼠海马神经元变性损伤及凋亡的保护作用

Protective effect of dizocilpine maleate on denaturation injury and apoptosis of hippocampal neurons in rats with diabetes mellitus with depression in vitro

目的研究地■西平马来酸盐(MK-801)对体外糖尿病并发抑郁症(DD)大鼠海马神经元变性损伤和凋亡的影响。方法海马神经元原代细胞取自受孕18 d SD大鼠胎鼠用皮质酮联合葡萄糖干预构建DD海马神经元细胞模型。将培养的海马神经元随机分为正常组、模型组、对照组和实验组。对照组给予体积分数10%的阳性药药物血清,实验组给予终浓度为10μmol·L-1的MK-801溶液,正常组与模型组均给予等量细胞培养液。造模干预18 h后,用四氮唑蓝(MTT)法检测海马神经元细胞活力,用尼氏染色检测海马神经元变性损伤情况,用Hoechst荧光染色检测海马神经元的凋亡情况,用高内涵细胞成像分析技术检测N-甲基-D-天冬氨酸受体(NR) 2种亚型NR2A和NR2B的蛋白表达。结果正常组、模型组及实验组海马神经元细胞活力分别为(100. 04±3. 26)%,(52. 55±2. 94)%和(65. 02±2. 96)%,NR2A平均荧光强度分别为(2038. 49±187. 03),(2378. 36±125. 99)和(1784. 81±102. 87),NR2B平均荧光强度分别为(1642. 52±122. 92),(1845. 79±108. 17)和(1453. 61±150. 80)。与正常组比较,模型组海马神经元细胞活力明显降低(P <0. 01),尼氏小体数量显著减少,神经元凋亡显著,NR2A和NR2B的蛋白表达量均明显升高(P <0. 01或P <0. 05);给予MK-801干预后可以逆转上述变化。结论 MK-801对体外DD大鼠海马神经元变性损伤及凋亡具有保护作用,其作用机制与下调NR2A和NR2B的蛋白表达量有关。

Objective To study the effects of dizocilpine maleate(MK-801)on degeneration injury and apoptosis of hippocampal neurons in rats with diabetes mellitus with depression(DD).Methods The primary cells of hippocampal neurons were obtained from SD rats foetuses at 18-day after conception.The corticosterone combined with glucose was used to construct the DD hippocampal neuron cell model.Cultured hippocampal neurons were randomly divided into normal,model,control and experimental groups.Control group was given a volume fraction of 10%positive drug serum.Experimental group was given a final concentration of 10μmol·L-1 MK-801 solution.Normal and model groups were given the same amount of culture solution.After 18 h of modeling inte-rvention,the cell viability of hippocampal neurons was detected bymethylcyclopentadienyl manganese tricarbonyl(MTT)assay,the degeneration injury of hippocampal neurons was detected by Nissl staining,the apoptosis of hippocampal neurons was detected by Hoechst fluorescence staining,and the protein expression of N-methyl-D-aspartate receptor 2 A(NR2 A)and NR2 B were detected by high-content cell imaging analysis technique.Results In the normal,model and experimental groups,the cell viability of hippocampal neurons were(100.04±3.26)%,(52.55±2.94)%and(65.02±2.96)%,the average fluorescence intensity of NR2A were(2038.49±187.03),(2378.36±125.99)and(1784.81±102.87),the average fluorescence intensity of NR2B were(1642.52±122.92),(1845.79±108.17)and(1453.61±150.80).Compared with the normal group,the cell viability of hippocampal neurons in the model group decreased significantly(P<0.01),the number of Nissl bodies decreased significantly,neuronal apoptosis was significant,and the protein expression of NR2A and NR2B increased significantly(P<0.01 or P<0.05);the above changes can be reversed after MK-801 intervention.Conclusion MK-801 has protective effects on degeneration and apoptosis of hippocampal neurons in DD rats in vitro,and its mechanism is related to down-regulation of NR2A and NR2B protein expression.