神经生长因子和依达拉奉对缺血／再灌注损伤脑保护作用的比较研究

A comparative study on the protection effect of nerve growth factor and edaravone pretreatment against cerebral ischemia/reperfusion injury

目的比较神经生长因子（NGF）和自由基清除剂依达拉奉预处理对脑缺血／再灌注（I／R）损伤神经细胞的保护作用。方法取出生24h内的SD乳鼠脑皮质细胞，体外培养7d后分为对照组，药物损伤组，NGF10、50、100弘g／L预处理组及依达拉奉100μmol／L预处理组。各预处理组分别预处理24h后，给予200μmol／L谷氨酸（Glu）损伤0．5h，换正常培养液继续培养24h；培养9d时检测神经细胞存活率（四甲基偶氮唑盐（MTT）比色法]、乳酸脱氢酶（LDH）漏出率（分光光度法）、细胞凋亡率（流式细胞术）；用苏木素一伊红（HE）染色后在倒置相差显微镜下观察细胞形态变化，在透射电镜下观察细胞超微结构改变。结果与药物损伤组比较，NGF10、50、100pg／L预处理组和依达拉奉预处理组细胞存活率明显升高[（0．21±0．04）％、（0．23±0．04）％、（0．21±0．04）％、（0．24±0．04）％比（0．19±0．04）％]，LDH漏出率[（0．50±0．06）％、（0．46±0．07）％、（0．50±0．02）％、（0．43±0．06）％比（0．56±o．03）％]和细胞凋亡率[（10．77±1．07）％、（10．38±0．70）％、（13．34±0．57）％、（9．99±0．77）％比（14．52±0．77）％]均不同程度降低（P〈0．05或P〈0．01）；细胞形态受损程度和神经元超微结构病变均减轻。NGF3个浓度组中以50μg／L组效果最佳，且与依达拉奉组各指标差异无统计学意义。结论NGF和依达拉奉预处理脑皮质细胞24h对脑I／R损伤均有保护作用；50μg／LNGF和100μmol／L依达拉奉预处理效果最佳，且二者最佳浓度时疗效相当。

Objective To compare the protective effect of nerve growth/actor （NGF） and edaravone （a free radical scavenger） pretreatment against cerebral ischemia/reperfusion （I/R） injury to nerve cells. Methods Cortical neurons of Sprague-Dawley （SD） mouse aged shorter than 24 hours were cultured for 7 days, then they were randomly divided into control group, I/R group, NGF of 10, 50, 100μg/L pretreatment groups and 100μmol/L edaravone pretreatment group. In pretreatment groups the ceils were pretreated with drugs correspondingly. After culturing for 24 hours, glutamate of 200 μmol/L was given into the culture of all groups, except control group, for half an hour. Then culture medium in all groups were renewed with ordinary culture medium, and cultures were continued for 24 hours. The survival rate （by methyl thiazolyl tetrazolium assay）, the content of lactate dehydrogenase （LDH, by spectrometry） and the rate of apoptosis （by flow cytometric） were determined. The cellular shape and ultrastructure were observed by hematoxylin-eosin （HE） staining and electronic microscopy correspondingly. Results The survival rate of nerve cells in NGF groups and edaravone group was significantly higher than that in I/R group [（0. 21± 0. 04）%, （0. 23±0. 04）%, （0.21±0.04）%, （0.24±0.04）% vs. （0. 19±0. 04）%]. The content of LDH in culture medium and the rate of apoptosis in NGF groups and edaravone group were lower than those in I/R group （P〈0.05 or P〈0.01）. The release rate of LDH in each group was （0.50±0.06）%, （0.46± 0. 07 ） %, （0. 50 ± 0.02） %, （0.43 ± 0. 06）% vs. （0.56 ± 0. 03） %, respectively. The rate of apoptosis in each group was （10.77±1.07）%, （10.38±0.70）%, （13.34±0.57）%, （9.99±0.77）% vs. （14.52±0. 77）%, respectively. The cellular shape and ultrastructure of nerve cells in NGF groups and edaravone group were affected much less than that of I/R group. NGF of 50 μg/L pretreatment group gave the best effect among three groups. There was no significant difference between NGF 50 μg/L pretreatment group and edaravone pretreatment group. Conclusion NGF and edaravone pretreatment 24 hours before cerebral I/R give protective effects against cerebral I/R injury. The protective effects are best in NGF of 50 μg/L pretreatment group and edaravone pretreatment group, and there is no significant difference between them.