Keap1-tat小肽降低缺血后大鼠海马CA1区神经元氧化应激损伤和空间学习记忆缺陷

Keap1-tat peptide attenuates oxidative stress damage in hippocampal CA1 region and learning and memory deficits following global cerebral ischemia

目的:设计Keap1-tat小肽,并探讨其对全脑缺血后大鼠海马CA1区神经元的保护作用及空间学习记忆功能的影响。方法:制作大鼠四动脉结扎全脑缺血模型,随机分为假手术sham组、sham+Keap1-tat组、缺血再灌注组(ischemia/reperfusion,I/R)、Keap1-tat处理组和溶剂对照组(vehicle)。Keap1-tat处理组大鼠于缺血前30 min于侧脑室微量注射30、50、100μg Keap1-tat,溶剂对照组侧脑室注射等体积(5μL)0.9%(质量分数)Na Cl溶液(生理盐水),焦油紫染色观察海马CA1区神经元的损伤;4-羟基壬烯醛(4-hydroxy-2-noneal,4-HNE)、8-羟基脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,8-OHd G)共聚焦技术观察海马CA1区神经元氧化应激损伤;Morris水迷宫观察大鼠的空间学习和记忆功能。结果:与sham组相比,vehicle和I/R组大鼠海马CA1区存活神经元数量显著减少,而Keap1-tat处理组可显著降低海马CA1区神经元损伤,且50μg剂量组存活的神经元最多;Keap1-tat处理组与vehicle组相比,4-HNE和8-OHd G阳性反应显著降低,且找到水下平台需要的时间显著减少,移走水下平台后,该组大鼠在原平台所在象限探索的时间较vehicle组显著延长。结论:Keap1-tat小肽可能通过降低缺血再灌注诱导的氧化应激而降低CA1区神经元损伤,并改善缺血后大鼠的学习记忆功能。

Objective：To design Keapl-tat peptide and explore its neuroprotective role on hipocampal CA1 neuron, as well as the effect on spacial learning and memory function following global cerebral isehe- mia. Methods： Adult male Sprague Dawley （SD） rats were subjected to global cerebral ischemia （GCI） by four-vessel occlusion for 15 rain and randomly divided into five groups： sham, sham ＋ Keapl-tat, is- chemia/reperfusion （L/R）, Keapl-tat peptide- and vehicle-administrated groups. For Keapl-tat or vehi- cle groups, the rats were treated with Keapl-tat （30, 50, 100 μg in 5 μL 0.9% saline） or the same vo- lume vehicle by intraeerebroventricular injection （icy） 30 rain prior to ischemia. Cresyl violet staining was used to observe the surviving neurons and 4-hydroxy-2-noneal （4-HNE） and 8-hydroxy-2＇-deox- yguanosine （8-OHdG） immunostaining were used to detect the change of markers response to oxidative stress in hippocampal CA1 region. The spatial learning and memory function of the rats was evaluated using Morris water maze. Results： Compared with sham group, the number of surviving neurons in isehe- mia-reperfusion and vehicle groups significantly decreased in the hippocampal CA1 region （P 〈 0.05 ） , while administration of Keapl-tat significantly decreased the damage following GCI （P 〈 0.05 ） , and the dose of 50μg existed the most effective neuroprotective role. Furthermore, immunostaining intensity of 4- HNE and 8-OHdG, markers of oxidative stress damage attenuated by Keapl-tat peptide as compared with vehicle group in CA1 region. Of significant interest, the time of finding underwater platform in Keapl-tat group animals was significantly short, and after removing the platform, the probe time of Keapl-tat group animals in the original quadrant where the platform was significantly increased compared with that of vehi- cle and I/R group animals （P 〈0.05 ）. Conclusion： Keapl-tat peptide can effectively attenuate neuro- nal damage in hippocampal CA1 region and improve learning and memory function, which might bedue tothe attenuation of oxidative stress caused by GCI.