摘要
目的 观察幼年大鼠惊厥持续状态（status convulsion,SC）后脑组织海马中内质网分子伴侣葡萄糖调节蛋白78（glucose regulated protein,GRP78）、半胱氨酸门冬氨酸特异性蛋白酶12（Caspase-12）的表达及神经细胞凋亡的变化,并探讨依达拉奉（edaravone,ED）对三者的影响.方法 将195只SD幼年雄性大鼠随机分为生理盐水对照组（NS组）、惊厥持续状态组（SC组）和依达拉奉预处理组（ED组）,每组65只,各组均按SC后处死时间点分为4、12、24、48、72 h五个亚组,每组13只.采用氯化锂-匹鲁卡品化学点燃法制备幼年大鼠SC模型.应用逆转录多聚酶链反应（RT-PCR）法检测GRP78 mRNA和Caspase-12 mRNA的表达,免疫组化法检测大鼠海马中GRP78和Caspase-12 蛋白表达情况,TUNEL法检测神经细胞凋亡数的变化.结果 （1）免疫组化结果显示SC组幼年大鼠海马中GRP78（12 h组0.1480±0.0164,24 h组0.1682±0.0114,48 h组0.1540±0.0102）和Caspase-12（12 h组0.1325±0.0165,24 h组0.1794±0.0213,48 h组0.1525±0.0423,72 h组0.1309±0.0199）表达增强,与NS组（GRP78：12 h组0.1214±0.0147,24 h组0.1272±0.0177,48 h组0.1260±0.0157;Caspase-12：12 h组0.1050±0.0121,24 h组0.1041±0.0151,48 h组0.1058±0.0222,72 h组0.1036±0.0186）比较差异有统计学意义（P＜0.01或＜0.05）;与SC组比较,ED组GRP78（12 h组0.1550±0.0131,24 h组0.1886±0.0154,48 h组0.1721±0.0151）表达明显增强,而Caspase-12（12 h组0.1211±0.0184,24 h组0.1545±0.0205,72 h组0.1085±0.0219）表达明显降低,差异均有统计学意义（P＜0.01或P＜0.05）;（2）RT-PCR法检测结果显示GRP78 mRNA和Caspase-12 mRNA表达趋势与蛋白基本相似;（3）SC组在惊厥12 h（11.41±2.37）时间点海马CA1区TUNEL阳性细胞数已显著高于NS组（P＜0.01）,48 h（28.78±5.11）达峰,而ED组TUNEL阳性细胞数在12～72 h时间点（12 h组8.98±2.22,24 h组13.09±2.54,48 h组20.57±4.89）均较SC组显著下降（P＜0.01或P＜0.05）,但仍高于NS组（12 h组6.22±1.50,24 h组6.57±1.61,48 h组6.72±1.14）（P＜0.01或P＜0.05）,在4 h时间点三组（NS组6.29±1.49,SC组6.61±1.71,ED组5.75±1.41）间TUNEL阳性细胞数差异无统计学意义（P＞0.05）.结论 SC后大鼠海马GRP78和Caspase-12的表达增强,ED可上调匹鲁卡品致（癎）大鼠海马GRP78和下调Caspase-12的表达,并使神经细胞凋亡数减少;提示ED对SC引起的脑损伤可能有保护作用.
Objective To observe the expression of GRP78 （glucose regulated protein, GRP78）,Caspase-12 and the change of neuron apoptosis in the juvenile rat hippocampus after status convulsivus （ SC）, and to explore the effect of edaravone on them. Methods One hundred and ninety-five juvenile male Sprague-Dawley（SD） rats were randomly divided into normal saline control group （NS group）, status convulsivus group（SC group）and edaravone treatment group（ ED group）. Each group was further divided into five subgroups in different executed time points after SC. The rats in status convulsivus group were kindled into epilepsy by lithium-pilocarpine method. Expression of GRP78 mRNA and caspase-12 mRNA was detected with reverse transcription-polymerase chain reaction （RT-PCR） method. Expressions of GRP78and caspase-12 protein were detected with immunohistochemical methods. The neuron gpoptosis was observed by TdT-mediated dUTP nick end labeling （TUNEL）. Results （1） Measured by immunohistochemistry the value of OD of GRP78 （ 0. 1480 ± 0. 0164,0. 1682 ± 0. 0114, and 0. 1540 ±0. 0102,respectively, 12 h-48 h points ） and caspase-12 （ 0. 1325 ± 0. 0165,0. 1794 ± 0. 0213,0. 1525 ±0. 0423, and 0. 1309 ± 0. 0199, respectively, 12 h-72 h points）positive cells in the SC group increased, there was a significant difference compared with NS group （GRP78： 0. 1214 ± 0. 0147,0. 1272 ± 0. 0177, and 0. 1260 ±0. 0157, respectively, 12 h-72 h points. Caspase-12：0. 1050 ±0. 0121,0. 1041 ± 0. 0151,0. 1058±0. 0222, and 0. 1036 ± 0. 0186, respectively, 12 h-72 h points ） （ P 〈 0. 01, or P 〈 0. 05 ）. By ED intervention GRP78 （ 0. 1550 ± 0. 0131, 0. 1886 ± 0. 0154, and 0. 1721 ± 0. 0151, respectively, 12 h-48 h points） positive cells value of the OD increased as compared with SC group （P 〈 0. 01, or P 〈 0. 05 ）. and caspase-12（0. 1211 ±0.0184,0. 1545 ±0.0205,and 0. 1085 ± 0.0219,respectively, 12 h,24 h and 72 h points） positive cells value of the A decreased as compared with SC group （ P 〈 0. 01, or P〈0.05）.（2）Measured by RT-PCR,the expression of GRP78 mRNA and easpsse-12 mRNA trend was similar to protein.（3） The TUNEL positive cells in hippocampus CA1 of SC group （ 11.41 ± 2. 37 ） were more than that of NS group after the SC 12 h（ P 〈 0. 01 ）, reached its highest level at 48 h（28. 78 ± 5.11）, after the intervention with edaravone （ 8.98 ± 2. 22, 13.09 ± 2.54 and 20. 57 ± 4. 89, respectively, 12 h-48 h points ）, TUNEL positive cells showed a significant drop in SC group at 12 h-48 h time points （P〈0.01,or P〈0.05）, but still significantly higher than that of the NS group（6. 22 ± 1.50,6. 57 ± 1.61 and 6. 72 ± 1.14 ,respectively）（ P 〈 0. 01, or P 〈 0. 05 ）, at the 4 h time point （ NS group 6.29 ± 1.49, SC group 6. 61 ± 1.71, ED group 5. 75 ± 1.41 ） among the three groups, no significant difference in TUNEL positive cells was found （ P =0. 759）. Conclusions The expression of GRP78 and caspase-12 increased after SC. Edaravone increased expression of GRP78 and decreased expression of caspase-12 in hippocampus rat with pilocarpine-induced seizures, reduced the number of neuronal apoptosis. These results suggest that edaravone may have protective effect against the hippocampal damage caused by status convulsivus.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2011年第1期53-59,共7页
Chinese Journal of Pediatrics
