W-7对惊厥持续状态幼鼠海马GRP78表达及神经元凋亡的影响

Effects of W-7 on the expression of GRP78 and neuronal apoptosis in immature rat hippocampus after status convulsion

摘要:

目的：探讨钙调蛋白抑制剂W-7 对幼年大鼠惊厥持续状态（SC）后 GRP78 表达及神经元凋亡的影响。方法：将19～21日龄Sprague-Dawley大鼠117只随机分为生理盐水对照（NS）组、惊厥持续状态（SC）组、W-7 预处理（W-7）组；各组再按不同时间点分4 h、24 h、48 h 3个亚组（n=13）。应用氯化锂匹鲁卡品建立大鼠SC模型，W-7组在制模前15 min予尾静脉注射W-7。逆转录-聚合酶链反应（RT-PCR）和免疫组织化学法检测每组各时间点大鼠海马GRP78 RNA 和蛋白的表达情况；原位末端标记法（TUNEL）检测海马CA1区的神经元凋亡情况。结果：SC组幼年大鼠海马24 h GRP78 mRNA 的表达量较 NS 组显著升高（P<0.01），SC组24 h和48 h GRP78 蛋白表达量较NS 组相应时间点也显著升高（P<0.01）；W-7组24 h和48 h的GRP78 mRNA及蛋白表达量较SC和NS组明显升高（P<0.05或P<0.01）；SC组在24 h 、48 h海马CA1区TUNEL阳性细胞数（21.0±2.5，29.4±2.8）显著高于NS组（7.1±1.4，7.3±1.6；P<0.01）；W-7组在24 h、48 h海马 CA1 区 TUNEL 阳性细胞数（15.0±2.5，20.0±2.9）较SC组显著降低（P<0.01），但仍显著高于 NS 组（P<0.01）。结论：钙调蛋白抑制剂 W-7 可通过上调 GRP78 的表达，减轻神经元凋亡, 具有脑保护作用。

Abstract:

OBJECTIVE: To investigate the effects of the calmodulin inhibitor W-7 on the expression of the key marker of ERS GRP78 and neuronal apoptosis in the immature rat hippocampus after status convulsion (SC). METHODS: One hundred and seventeen male Sprague-Dawley rats aged 19-21 days were randomly divided into three groups: normal saline control (control), SC with and without W-7 pretreatment. Each of the 3 groups was further subdivided into subgroups sacrificed at 4, 24 and 48 hrs. SC model was prepared using lithium-pilocarpine. GRP78 mRNA expression in the hippocampus was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). GRP78 protein was ascertained by immunohistochemistry. Neuronal apoptosis was observed with TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: The expression of GRP78 mRNA was significantly increased in the non-pretreated SC group compared with the control group 24 hrs after injection of saline or lithium-pilocarpine (P<0.01), and the expression of GRP78 protein also increased markedly in the seizure group compared with the control group 24 and 48 hrs after the injection (P<0.01). The expression of GRP78 mRNA and protein in the W-7 pretreatment group was significantly higher than both the control and the non-pretreated seizure groups 24 and 48 hrs after injection. The TUNEL positive cells in the hippocampus CA1 in the non-pretreated SC group 24 and 48 hrs after injection (21.0±2.5 and 29.4±2.8, respectively) were increased compared to the control group (7.1±1.4 and 7.3±1.6, respectively; P<0.01). W-7 pretreatment decreased TUNEL positive cells to 15.0±2.5 and 20.0±2.9 at 24 and 48 hrs after injection compared to the non-pretreated seizure group (P<0.01), but the number of TUNEL positive cells in the W-7 pretreatment group remained significantly greater than in the control group (P<0.01). CONCLUSIONS: W-7 may up-regulate the expression of GRP78 and reduce the number of apoptotic neurons, thus provides a neuroprotective effect against brain damage following SC.