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

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4h、24h、48h3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15min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GRP78RNA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24hGRP78mRNA的表达量较NS组显著升高(P<0.01),SC组24h和48hGRP78蛋白表达量较NS组相应时间点也显著升高(P<0.01);W-7组24h和48h的GRP78mRNA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24h、48h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24h、48h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。

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（P0.01）,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（P0.01）.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;P0.01）.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（P0.01）,but the number of TUNEL positive cells in the W-7 pretreatment group remained significantly greater than in the control group（P0.01）.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.