MP对大鼠急性脊髓损伤后的神经保护作用及自噬的影响

Effects of MP on neuroprotective effect and autophagy in rats with acute spinal cord injury

目的了解甲基强的松龙(methylprednisolone,MP)对大鼠急性脊髓损伤后的神经保护作用及自噬的影响。方法采用改良Allen′s重物打击法建立大鼠急性脊髓损伤模型,将75只大鼠随机分为3组:假手术组、手术组和MP组,每组25只。MP组在急性脊髓损伤模型成功后立即经尾静脉注射MP(30 mg/kg),假手术组在打开椎板后不损伤脊髓。分别于损伤后4 h、12 h、1 d、3 d和7 d,采用Basso Beattie Bresnahan(BBB)运动功能评分;RT-PCR方法检测损伤的脊髓组织中炎症因子IL-1βm RNA和IL-6 m RNA的表达情况。于损伤后第3 d取脊髓组织用免疫荧光染色法检测自噬相关蛋白Beclin-1和caspass-3在各组的表达情况;用Tunel法检测各组凋亡情况。结果假手术组4 h、12 h、1 d、3 d和7 d,BBB分值分别为:19.8±0.8、19.1±0.4、20.5±0.5、20.4±0.5和20.1±0.5;MP组各个时间点BBB分值分别为:1.2±0.1、5.8±0.3、7.2±0.3、9.4±0.4和12.3±1.1;手术组各个时间点BBB分值分别为:0.6±0.1、0.7±0.1、2.2±0.2、2.5±0.2和3.3±0.2;假手术组大鼠的活动不受限,MP组的BBB评分值在各个时间点均高于手术组,差异有统计学意义(P<0.05)。MP组中IL-1βm RNA和IL-6 m RNA的表达在大鼠急性脊髓损伤后12 h、1 d和3 d相对表达量分别为0.43±0.07、0.39±0.08、0.46±0.03和0.26±0.04、0.17±0.03、0.28±0.02,与手术组相比明显降低,差异有统计学意义(P<0.05)。假手术组Beclin-1阳性神经细胞数量为:51.0±2.0,MP组数量为235.0±10.0,手术组数量为430.0±25.0;假手术组Caspase-3阳性细胞数量为18.0±2.0,手术组为:45.0±4.0,MP组为76.0±3.0,假手术组凋亡细胞数量为:14.0±2.0,手术组为51.0±3.0,MP组为75.0±3.0,与手术组相比,在急性脊髓损伤后3天MP组中Beclin-1阳性细胞数目、caspass-3的表达和凋亡细胞数目都明显降低,差异有统计学意义(P<0.05)。结论应用甲基强的松龙降低炎症反应,显著抑制大鼠急性脊髓损伤神经细胞自噬活动,降低凋亡相关蛋白的表达,减少神经细胞的凋亡从而对大鼠急性脊髓损伤后神经元细胞有保护作用。

[Objective]To investigate the effects of methylprednisolone（MP） on neuroprotective effects and autophagy in rats with acute spinal cord injury.[Methods] The model of acute spinal cord injury was established by modified Allen＇s weight method,and 75 rats were randomly divided into three groups：sham operation group,operation group and MP group,25 rats in each group. The MP group was injected with MP（30 mg/kg） immediately after the success of the acute spinal cord injury model,and the sham operation group did not injure the spinal cord after opening the lamina. At the fourth hour,the 12 th hour,the first day,the third day and seventh day after injury,the Basso Beattie Bresnahan（BBB） movement function score was performed. The expression of inflammatory cytokines 1β（IL-1β） mRNA and IL-6 mRNA in injured spinal tissues was detected by RT-PCR. The expression of autophagy associated protein beclin-1 and caspas-3 in each group was detected by immunofluorescence staining on the third day after the injury. Apoptosis was detected by Tunel method. [Results] At the fourth hour,the twelfth hour,the first day,the third day and seventh day after injury,BBB score in sham operation group was respectively 19.8 ±0.8,19.1 ±0.4,20.5 ±0.5,20.4 ±0.5 and 20.1 ±0.5,that in MP group was respectively 1.2 ±0.1,5.8 ±0.3,7.2 ±0.3,9.4 ±0.4 and 12.3 ±1.1,and that in operation group was respectively 0.6 ±0.1,0.7 ±0.1,2.2 ±0.2,2.5 ±0.2 and 3.3 ±0.2. The activities of rats in sham operation group were not restricted.BBB score values of MP group at all time points were higher than that of operation group,and the difference was statistically significant（P〈0.05）. The expression of IL-1β mRNA and IL-6 mRNA in MP group at the twelfth hour,the first day and the third day after injury were respectively 0.43 ±0.07,0.39±0.08,0.46±0.03 and 0.26 ±0.04,0.17±0.03,0.28±0.02,which were significantly lower than those in operation group（P〈0.05）. The number of beclin-1 positive neurons in sham operation group,MP group and operation group was respectively 51.0 ±2.0,235.0 ±10.0 and 430.0 ±25.0,the number of caspase-3 positive cells in sham operation group,MP group and operation group was respectively 18.0 ±2.0,45.0 ±4.0 and 76.0 ±3.0,while the number of apoptotic cells in sham operation group,MP group and operation group was respectively 14.0 ±2.0,51.0 ±3.0 and 75.0 ±3.0.Compared with the operation group,the number of beclin-1 positive cells,the expression of caspas-3 and the number of apoptotic cells decreased significantly on the third day after the acute spinal cord injury, and the differences were statistically significant（P〈0.05）. [Conclusion] Application of methyl prednisone can reduce inflammatory reaction,inhibit autophagy of nerve cells in rats with acute spinal cord injury,reduce the expression of apoptosis related proteins,decrease the apoptosis of neural cells,so as to protect the neuron cells of rats after acute spinal cord injury.