Purα对匹罗卡品致痫大鼠海马DNA损伤的保护作用

Protective effects of Purer on rat hippocampus DNA damage induced by epilepsy

目的探讨Purα蛋白对匹罗卡品诱导癫痫大鼠海马所致DNA损伤的保护作用，以及Purα蛋白在DNA损伤修复中的作用。方法将Purα蛋白过表达的慢病毒和Purα siRNA慢病毒在立体定位仪指导下注入大鼠海马组织。注入后14d通过荧光切片和免疫印迹证实病毒已经在海马组织表达，然后用匹罗卡品腹腔注射诱导癫痫发作。癫痫发作1h后处死动物，分别取样进行免疫组织化学和免疫印迹检测与DNA损伤和修复。结果免疫组织化学结果表明，DNA损伤标志性蛋白γH2AX在大鼠CA1区的表达，各组选20张相同背景色的海马CA1区通过图像分析软件iPP6．0测定阳性细胞的平均光密度值；与空载组（0．40±0．11）和对照组（0．42±0．05）相比，Purα过表达组（0．15±0．10）免疫着色阳性细胞数目明显减少（P〈0．01）；而在Purα沉默组（0．68±0．06）明显增高（P〈0．01）。免疫印迹结果表明，在DNA损伤修复相关蛋白Parp-1的表达上：与空载组（0．93±0．11）和对照组（1．00±0．00）相比，Purα过表达组（0．17±0．09）的表达明显降低（P〈0．01）；Purα沉默组的表达明显增高（P〈0．01）。结论癫痫发作早期可引起明显的DNA损伤，Purα蛋白对癫痫引起的DNA损伤有明显的保护作用。

Objective To investigate the protective effects of Purα protein on rat hippocampus DNA damage induced by epilepsy and the effects of Purer protein on the repair of DNA damage. Methods The Purα overexpressing and siRNA lentiviruses were packaged in vitro and the high tittered virion was injected into rat hippocampus guided by stereotaxic apparatus. 14 days later after the lentivirus injection, epileptic onset is induced by intraperitoneal injection of pilocarpine. The experimental animals were executed 1 hour after the epileptic onset and the hippocampus samples were collected for immunohistochemical staining and Western blotting assay was used to examine the pertinent protein expression to investigate the protective effects of Purα on DNA damage and repair. Results Immunohistochemical analysis demonstrated that γH2AX, a signal protein of DNA damage, expressed in rat hippocampal CA1 region. 20 slides of rat hippocampal CA1 region with the same background and position were chosen for γH2AX positive staining cell analysis with image analysis software iPP6. 0 and the cells with positive staining were selected to evaluate the average optical density. Compared with empty vector group （0. 40 ± 0. 11 ） and control group （0. 42 ± 0. 05 ）, the number of positive staining cells in Purα overexpression group （0. 15 ± 0. 05 ） was significantly decreased （ P 〈 0.01 ）, while the number increased in Purα silence group （ 0. 68 ± 0. 06 ） （ P 〈 0. 01 ）. Western blotting analysis showed, compared with empty vector group （0. 93 ± 0. 11 ） and control group （ 1.00±0. 00）, that the expression levels of proteins related to the DNA repair such as parp-1, was much lower in Purα overexpression group （0. 17 ± 0.09 ） , while it increased in Purer silence group （P 〈 0.01 ）. Conclusion The DNA damage can occur in the early stage of epilepsy onset, and Purα protein can protect the DNA damage caused by epilepsy and also participate in the repair process of DNA damage.