Li Guang-Qian, Wang Hai-Ping, Jiang Chun-Ming
Department of Neurology, The Sixth Hospital Affiliated to Zhejiang University of Traditional Chinese Medicine, Hangzhou Children's Hospital, Hangzhou 310014, China.
Zhonghua Er Ke Za Zhi. 2011 Jan;49(1):53-9.
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 convulsive (SC), and to explore the effect of edaravone on them.
One hundred and ninety-five juvenile male Sprague-Dawley (SD) rats were randomly divided into normal saline control group (NS group), status convulsive 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 convulsive 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 GRP78 and caspase-12 protein were detected with immunohistochemical methods. The neuron apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL).
(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 caspase-12 mRNA trend was similar to protein. (3) The TUNEL positive cells in hippocampus CA(1) 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).
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 convulsive.
观察惊厥持续状态（SC）后幼鼠海马中葡萄糖调节蛋白78（GRP78）、半胱天冬酶 - 12（Caspase - 12）的表达及神经元凋亡的变化，并探讨依达拉奉对其的影响。
将195只雄性幼龄Sprague - Dawley（SD）大鼠随机分为生理盐水对照组（NS组）、惊厥持续状态组（SC组）和依达拉奉治疗组（ED组）。每组在SC后的不同处死时间点再分为5个亚组。惊厥持续状态组大鼠采用锂 - 匹罗卡品法点燃癫痫。采用逆转录 - 聚合酶链反应（RT - PCR）法检测GRP78 mRNA和半胱天冬酶 - 12 mRNA的表达。采用免疫组织化学方法检测GRP78和半胱天冬酶 - 12蛋白的表达。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）观察神经元凋亡情况。
（1）免疫组织化学检测显示，SC组GRP78（12 h - 48 h各时间点OD值分别为0.1480±0.0164、0.1682±0.0114、0.1540±0.0102）和半胱天冬酶 - 12（12 h - 72 h各时间点OD值分别为0.1325±0.0165、0.1794±0.0213、0.1525±0.0423、0.1309±0.0199）阳性细胞数增加，与NS组（GRP78：12 h - 72 h各时间点OD值分别为0.1214±0.0147、0.1272±0.0177、0.1260±0.0157。半胱天冬酶 - 12：12 h - 72 h各时间点OD值分别为0.1050±0.0121、0.1041±0.0151、0.1058±0.0222、0.1036±0.0186）相比有显著差异（P < 0.01或P < 0.05）。经依达拉奉干预后，与SC组相比，GRP78（12 h - 48 h各时间点OD值分别为0.1550±0.0131、0.1886±0.0154、0.1721±0.0151）阳性细胞OD值升高（P < 0.01或P < 0.05），半胱天冬酶 - 12（12 h、24 h和72 h各时间点OD值分别为0.1211±0.0184、0.1545±0.0205、0.1085±0.0219）阳性细胞OD值降低（P < 0.01或P < 0.05）。（2）RT - PCR检测显示，GRP78 mRNA和半胱天冬酶 - 12 mRNA的表达趋势与蛋白相似。（3）SC后12 h，SC组海马CA(1)区TUNEL阳性细胞（11.41±2.37）多于NS组（P < 0.01），48 h时达到最高水平（28.78±5.11），依达拉奉干预后（12 h - 48 h各时间点分别为8.98±2.22、13.09±2.54、20.57±4.89），SC组12 h - 48 h时间点TUNEL阳性细胞显著下降（P < 0.01或P < 0.05），但仍显著高于NS组（分别为6.22±1.50、6.57±1.61、6.72±1.14）（P < 0.01或P < 0.05），在4 h时间点（NS组6.29±1.49，SC组6.61±1.71，ED组5.75±1.41），三组TUNEL阳性细胞无显著差异（P = 0.759）。
SC后GRP78和半胱天冬酶 - 12表达增加。依达拉奉可增加匹罗卡品诱导癫痫大鼠海马中GRP78的表达，降低半胱天冬酶 - 12的表达，减少神经元凋亡。这些结果表明依达拉奉可能对惊厥持续状态所致海马损伤具有保护作用。
