雷帕霉素增强自噬表达对大鼠急性脊髓损伤后神经细胞凋亡影响的实验研究

Experimental study on effects of rapamycin-enhanced autophagy on neuronal apoptosis in acute spinal cord injure in rats

为研究雷帕霉素增强的自噬表达对大鼠脊髓损伤(spinal cord injury,SCI)后神经细胞凋亡的影响,将成年SD大鼠(n=20/组)分为椎板切开术组(假手术组)、单纯脊髓损伤组和脊髓损伤+雷帕霉素治疗组(1.0mg/kg,单次,腹腔注射),采用硬膜外施加动脉夹实现大鼠脊髓损伤。使用Western blotting测定脊髓损伤后组织中自噬蛋白的表达,HE染色及电镜观察损伤后的神经细胞,TUNEL法观察神经细胞凋亡并测定凋亡蛋白,免疫荧光法检测线粒体自噬反应,并通过BBB评分对大鼠的运动功能进行评分。结果表明,雷帕霉素组的LC3和Beclin1蛋白表达显著,高于假手术组和脊髓损伤组。相对脊髓损伤组,雷帕霉素组在显微观察下脊髓坏死减轻,炎性细胞数量减少,存活神经元数目较多;TUNEL阳性细胞明显减少;线粒体中LC3蛋白表达更显著;BBB评分显著增高。雷帕霉素能减少急性脊髓损伤后神经元细胞的凋亡,可能是由于雷帕霉素增强了神经元细胞的自噬,损伤后神经细胞可能的自我保护机制是细胞自噬。

To investigate the effects of rapamycin-enhanced autophagy on neuronal apoptosis in rat model of acute spinal cord injury（SCI）.Adult Sprague-Dawley（SD）rats（n=20/group）were divided into lamina incision group（sham operation group）,pure SCI group,SCI plus rapamycin treatment group（1 mg/kg,single time,intraperitoneal injection）.The SCI in rats was achieved by applying an extradural artery clamp.Neuronal autophgy and apoptosis,mitochondrial autophagy were examined using Western blotting,TUNEL and immunofluorescence staining.Injured spinal cord tissues were observed under electron microscopy.Functional assessment was performed using the BBB scoring system.The results show that rapamycin treatment results in higher expression of autophgic markers LC3 and Beclin1,less number of TUNEL-possitive cells,higher LC3 protein expression in mitochondria,less inflammatory reaction in the injured spinal cord tissue.And higher BBB score is observed in rapamycin-treated rats with SCI.In conclusion,rapamycin may have protective effects in acute spinal cord injury by increasing autophagy activity and reducing apoptosis.