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This report aims to describe the oxidative damage profile in brain of presenilin1 and presenilin2 conditional double knockout mice (dKO) at both early and late age stages, and to discuss the correlation between oxidative stress and the Alzheimer-like phenotypes of dKO mice.
The protein level of Aβ42 in dKO cortex and free 8-OHdG level in urine were measured by ELISA. Thiobarbituric acid method and spectrophotometric DNPH assay were used to determine the lipid peroxidation and protein oxidation in cortex, respectively. SOD and GSH-PX activities were assessed by SOD Assay Kit-WST and GSH-PX assay kit, separately.
Significant decrease of Aβ42 was verified in dKO cortex at 6 months as compared to control mice. Although lipid peroxidation (assessed by MDA) was increased only in dKO cortex at 3 months and protein oxidation (assessed by carbonyl groups) was basically unchanged in dKO cortex, ELISA analysis revealed that free 8-OHdG, which was an indicator of DNA lesion, was significantly decreased in urine of dKO mice from 3 months to 12 months. Activities of SOD and GSH-PX in dKO and control cortices showed no statistical difference except a significant increase of GSH-PX activity in dKO mice at 9 months.
Oxidative damage, especially DNA lesion, was correlated with the neurodegenerative symptoms that appeared in dKO mice without the deposition of Aβ42. Triggers of oxidative damage could be the inflammatory mediators released by activated microglia and astrocytes.
观察3, 6, 9, 12月龄presenilin1和presenilin2双敲除小鼠(dKO)脑组织氧化损伤情况,探讨氧化损伤与presenilins功能丧失所引起的阿尔茨海默病样症状之间的关系。
酶联免疫反应(ELISA)检测dKO小鼠大脑皮层中Aβ42水平和尿液中8-羟基脱氧鸟苷(8-hydroxy-2-deoxyguanosine, 8-OHdG)水平; 硫代巴比妥酸法检测脂质过氧化水平, 分光光度计测定DNPH法检测蛋白质损伤水平; 试剂盒法检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性。
与对照组相比, dKO小鼠大脑皮层中Aβ42水平显著降低。 同时, 除了在3月龄dKO小鼠大脑皮层中发现脂质过氧化水平有显著增加外, 蛋白质损伤在各年龄段dKO小鼠中均没有明显变化。 ELISA检测结果显示尿液中游离的8-OHdG水平在各年龄段的dKO小鼠中均显著降低。 SOD和GSH-PX活性均无明显变化, 只有9月龄dKO小鼠中的GSH-PX活性有显著增高。
氧化损伤特别是DNA损伤参与dKO小鼠神经退行性症状的发生过程; 由于dKO小鼠大脑皮层中没有Aβ42的沉积, 推测造成氧化损伤的原因可能是由活化的小胶质细胞和星形胶质细胞所释放的免疫因子介导的。