解剖学报

解剖学报 ›› 2017, Vol. 48 ›› Issue (4): 381-386.doi: 10.16098/j.issn.0529-1356.2017.04.003

• 神经生物学 • 上一篇    下一篇

排斥性导向分子a抑制剂6FNⅢ对大鼠大脑皮质神经元缺血再灌注后自噬的作用

谢扉 秦新月* 张融融 李敏   

  1. 重庆医科大学附属第一医院神经内科，重庆市神经病学重点实验室，重庆 400016
  • 收稿日期:2016-11-03 修回日期:2016-12-15 出版日期:2017-08-06 发布日期:2017-08-06
  • 通讯作者: 秦新月 E-mail:qinxy20011@sina.com
  • 基金资助:
    国家自然科学基金面上项目

Effect of repulsive guidance molecule a specific inhibitor 6FNⅢ on cortex neuron autophagy in rats after cerebral ischemia/reperfusion injury

XIE Fei QIN Xin-yue* ZHANG Rong-rong LI Min   

  1. Department of Neurology, the First Affiliated Hospital of Chongqing Medical University，Chongqing Key Laboratory of Neurology, Chongqing 400016, China
  • Received:2016-11-03 Revised:2016-12-15 Online:2017-08-06 Published:2017-08-06
  • Contact: QIN Xin-yue E-mail:qinxy20011@sina.com
  • Supported by:
    National natural science foundation of China

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摘要：

目的 探讨排斥性导向分子a(RGMa)特异性抑制剂6FNⅢ对大鼠大脑皮质神经元缺血再灌注引发的自噬及对神经功能恢复的影响。 方法 雄性成年SD大鼠立体定位侧脑室注射不同浓度6FNⅢ后建立大脑中动脉闭塞（MCAO）/再灌注模型，Western blotting检测RGMa下游分子脑衰反应调节蛋白-2（CRMP-2）蛋白表达水平，确定最佳6FNⅢ干预浓度。将72只雄性成年SD大鼠随机分成4组：假手术组（sham）、脑缺血再灌注组（I/R）、生理盐水干预组（I/R+NS）、6FNⅢ干预组（I/R+6FNⅢ），每组18只。缺血2h再灌注后24h，采用神经功能缺损评分评估各组大鼠神经功能恢复情况；Western blotting检测自噬相关蛋白LC3Ⅱ/Ⅰ水平；免疫荧光双标检测LC3在神经元内的表达情况；透射电子显微镜观察自噬体的形成情况。 结果 根据各浓度6FNⅢ干预组CRMP-2表达，确定中浓度（0.5 g/L）为本实验的最适浓度。缺血再灌注后24h，I/R组较sham组神经功能评分降低(P＜0.05)、自噬相关蛋白LC3Ⅱ/Ⅰ水平升高(P＜0.05)、自噬体形成增多；I/R+6FNⅢ组较I/R组神经功能评分增高(P＜0.05)、自噬相关蛋白LC3Ⅱ/Ⅰ水平降低(P＜0.05)、自噬体形成减少；而I/R+NS组与I/R组以上各项均无统计学意义(P＞0.05)。 结论 RGMa特异性抑制剂6FNⅡ可在缺血再灌注急性期抑制自噬发生，促进神经功能恢复。

关键词: 排斥性导向分子a, 6FNⅢ, 脑缺血再灌注, 自噬, 免疫印迹法, 大鼠

Abstract:

Objective To determine the effect of repulsive guidance molecule a (RGMa) specific inhibitor 6FNⅢ on cerebral ischemia/reperfusion(I/R) injury-induced autophagy and neurological function. Methods Different concentrations of 6FNⅢ were injected into the right lateral ventricles of adult male Sprague-Dawley rats before I/R surgery. The most proper concentration of 6FNⅢ was assessed by Western blotting. The rats were randomly divided into sham operation group（sham）,cerebral I/R group（I/R）,I/R plus normal saline group（I/R+NS）,I/R plus 6FNⅢ intervention group（I/R+6FNⅢ）, 18 rats per group. NS and 6FNⅢ were injected into the right lateral ventricle of the rats before I/R surgery. The blood was occluded for 120 minutes, followed by 24 hours reperfusion. Neurological function was evaluated. The expression of LC3Ⅱ/Ⅰ in the brain tissue was detected by Western blotting and immunohistochemical assay. Transmission electron microscope was used to observe the autophagosome formation. Results According to the expression of CRMP-2 , the optimum concentration of 6FNⅢ was 0.5 g/L. 6FNⅢ treatment improved neurological deficits(P<0.05), decreased LC3Ⅱ/Ⅰ levels(P<0.05), and attenuated autophagosome formation at 24 hours after cerebral I/R injury. Conclusion RGMa-specific inhibitor 6FNⅢ may protect the brain against strokeinduced injury through inhibiting autophagy.

Key words: Repulsive guidance molecule a, 6FNⅢ, Cerebral ischemia/reperfusion, Autophagy, Western blotting, Rat