抑制 JNK 通路对蛛网膜下腔出血大鼠海马区神经细胞自噬与学习记忆功能的影响

摘要

Objective To investigate inhibition of JNK pathway on autophagy and learning function after subarachnoid hemorrhage in rat hippocampus nerve cell.Methods 36 healthy male SD rats were randomly divided into three groups,namely the Sham group (n =12),the SAH group (n =12),the JNK group (n=12).Intracranial arterial puncture method was used to generate the SAH model.Sham group was only operation but without arterial perforation;SP600125 group where rats were given intraventricular injection of 10 μL SP600125 solution at the concentration of 3 μg/μL 30 min before modeling applications rat ster-eotaxic instrument.Based on the successful model,Morris water maze method was used to observe the im-pact on learning function in rats (including place navigation test and spatial probe test);Haematoxylin-eo-sin (HE)method was used to observe and evaluate the variation of morphology and cell number;both im-munohistochemistry and western-blot were used to quantitatively analyze the variation of Beclin-1,LC3-ⅡJNK and p-JNK pathway protein.Results Passive avoidance latency (PLA)of SAH group was observed to be significantly extended compared with sham group (P <0.05);the number of crossing the platform was shown to be significantly reduced relative to sham group (P < 0.05);Compared with SAH group, PLA of SP600125 group was found to be significantly shortening (P <0.05)and the number of crossing the platform was displayed to be increased (P <0.05);Compared with Sham group,the morphology of neurons were irregular whose number was presented to be significantly reduced (P < 0.05 ).The expression of Beclin-1,LC3-Ⅱ and p-JNK was observed to be significantly up-regulated than that of sham group (P <0.05).Compared with SAH group,abnormality of nucleus was shown to remarkably mitigated and the number of cells with normal arrangement was shown to be pronouncedly increased (P <0.05);The expression of Beclin-1,LC3-Ⅱ and p-JNK was shown to be markedly down-regulated (P < 0.05). Conclusion Inhibition of JNK pathway was shown to be able to have a positive effect on the protection of hippocampal neurons after SAH.The underlying mechanism might be associated with over-activation of JNK signaling pathway,thereby resulting in moderate activation of autophagy.%目的：探讨抑制 c-Jun 氨基末端激酶（c-Jun N-terminal ki-nase，JNK）信号通路对蛛网膜下腔出血（subarachnoid hemorrhage，SAH）大鼠海马区神经细胞自噬及其学习记忆功能的影响。方法成年雄性 Sprague-Dawley 大鼠36只（体质量350～450 g），随机分成正常对照组（Sham 组）（n ＝12）、蛛网膜下腔出血组（SAH组）（n ＝12）和 JNK 抑制剂蛛网膜下腔出血组联合 JNK 抑制剂组（SP6001250组）（n ＝12）3组。使用血管内穿刺法制作 SAH 模型，Sham 组只进行手术，不行血管穿刺，SP6001250组在造模前30 min 应用大鼠脑立体定位仪给予脑室注射3μg／μL 的 SP600125溶液10μL。造模成功后24 h 应用 Morris 水迷宫实验观察大鼠学习记忆功能（包括定位航行实验与空间探索实验）；HE 染色法观察各组大鼠海马区神经元的形态和数量；免疫组织化学染色以及 Western-Blot 法对自噬标记蛋白（Beclin-1）、微管相关蛋白（LC3-Ⅱ）以及 JNK 通道表达蛋白 p-JNK 进行定性定量检测。结果与 Sham 组比较，SAH 组的逃避潜伏期（passive avoidance latency，PLA）延长（P ＜0．05），穿越平台次数减少（P ＜0．05）；与 SAH 组相比较，SP600125组的 PLA 缩短（P ＜0．05），穿越平台次数增加（P ＜0．05）；与 Sham 相比，SAH 组海马区神经细胞形态不规则，排列紊乱，数量减少（P ＜0．05）；Beclin-1、LC3-Ⅱ表达水平均较高（P ＜0．05）；p-JNK 表达上升（P ＜0．05）。与 SAH 组相比，神经细胞细胞核异常现象减轻，形态排列正常的数目增多（P ＜0．05）；Beclin-1、LC3-Ⅱ表达水平下降显著（P ＜0．05）；p-JNK 表达下降（P ＜0．05）。结论抑制JNK 通路对 SAH 后在保护海马区神经元细胞同时对学习功能具有积极作用，其机制可能与抑制 JNK 信号通路的激活过度从而适度激活自噬有关。