甲基强的松龙对大鼠脊髓损伤后神经细胞凋亡的影响及其机理

Effect and mechanism of methylprednisolone on inducing neural cell apoptosis after spinal cord transection in rat

目的:研究甲基强的松龙(MP)对大鼠横断性脊髓损伤(SCI)后神经细胞凋亡的影响及其作用机理。方法:60只成年Wistar大鼠随机分成正常对照组、脊髓损伤组和MP治疗组,每组20只,MP治疗组在横断T10脊髓组织后30min经尾静脉给予MP治疗,损伤组和对照组未予任何治疗。MP治疗组和脊髓损伤组大鼠于脊髓损伤后8h、24h、3d和1周取材,采用透射电镜、TUNEL染色观察细胞凋亡情况,采用免疫组织化学染色观察Fas、半胱氨酸蛋白酶(caspase)-8和caspase-3在SCI前后的变化情况,采用改良Tarlov评分方法观察大鼠后肢运动功能。结果:SCI后24h大鼠后肢运动功能逐渐恢复,MP治疗组大鼠的后肢运动功能恢复优于损伤组,7d后尤其明显。TUNEL染色和电镜检查证实SCI后8h即有凋亡细胞出现,其中既有神经元也有胶质细胞;3d凋亡细胞数达到高峰;7d仍有凋亡细胞存在,但已明显减少;各时间点治疗组凋亡细胞数量明显少于损伤组(P<0.05)。治疗组和损伤组在SCI后8h可以检测到少量的Fas和caspase-8阳性神经元及胶质细胞,Fas和caspase-8的表达在SCI后3d达到高峰,7d表达下降,各时间点治疗组的Fas和caspase-8灰度值明显大于损伤组,二者有显著性差异(P<0.05)。治疗组和损伤组caspase-3的表达在SCI后8h均逐渐增加(与对照组相比),7d达到高峰,治疗组灰度值大于损伤组,但二者无显著性差异。结论:MP能抑制大鼠脊髓横断性脊髓损伤后的神经细胞凋亡,但不能推迟细胞凋亡出现的高峰时间;MP抑制细胞凋亡的途径可能通过非特异性抑制Fas和caspase-8的表达来实现。

Objective：To define the effect and mechanism of methylprednisolone （MP） on the activation of caspase-3 and Caspase-8 in the lesioned area following SCI.Method：Forty rats with complete transection of the thoracic spinal cord were collected,half of them received MP within half an hour post-injury and were then sacrificed at 8,24h,3 and 7 days thereafter,while the sham （20 rats） group only received laminectomy without MP.The expression of caspase-3 and Caspase-8 in nerve cells were detected using the method of immunohistochemistry,and apoptotic cells were identified using TUNEL staining and electron microscopy in both neuronal and glial cells.The motor function of hinder limb was evaluated using modified Tarlov method.Result：Apoptotic cells including neuronal and glial cells emerged at 8h after SCI,the maximal apoptosis occured at 3 days after SCI.Caspase-8-like enzyme activity increased significantly at days 3 （P〈0.05）,whereas caspase-3-1ike enzyme activity increased at day 7（P〈0.05） in rats after SCI.The gray degree of Caspase-8 and Fas in MP group were higher than in transection group,and there was significant deference between MP group and transection group.Conclusion：Mice treated with MP demonstrated a significant improvement of motor function and a reduction of apoptotic cell,the reduction of apoptotic cell is associated with the inhibition of Fas and Caspase-8,but MP can not delay the climax time of cell apoptosis.