雌二醇在大鼠脊髓损伤模型中抗自噬作用的研究

目的 探讨自噬在雌二醇干预严重脊髓损伤(SCI)过程中的作用。 方法 选取72只SD雌性大鼠,体重为200~220 g。采用随机数字表法分为3组,每组24只。其中2组(模型组和干预组)采用动脉夹(15 g闭合力)建立大鼠严重脊髓损伤夹闭模型。干预组在脊髓损伤前1周即开始隔天肌注苯甲酸雌二醇,持续到安乐死之前。为比较高剂量雌二醇的疗效,脊髓损伤后7、14、21、28 d采用BBB(Basso,Beattie,Bresnahan)评分评估大鼠运动功能。实时荧光定量PCR和蛋白印记检测脊髓损伤后3 d自噬标志物表达变化。免疫组化法检测损伤后3 d LC3在运动神经元中的免疫活性。 结果 高剂量雌二醇明显改善大鼠脊髓损伤后运动功能的恢复,损伤后14、21、28 d各时间点对应的BBB评分较模型组高。脊髓损伤术后3 d,模型组的LC3、Beclin-1表达高于假手术组,雌二醇干预治疗在mRNA和蛋白水平上抑制其表达。与之相反,自噬底物P62在模型组较假手术组相比有明显降低。然而,给予雌二醇治疗后,P62表达显著增加。免疫组化结果显示脊髓损伤后运动神经元中增强的LC3免疫活性被高剂量雌二醇所抑制。 结论 雌二醇在脊髓损伤过程中的神经保护效应在一定程度上与抑制过度自噬相关。 

Anti-autophagy role of estradiol after spinal cord injury in rats

Objective To investigate the role of autophagy in estradiol intervention for severe acute spinal cord injury (SCI). Methods A total of 72 adult female Sprague-Dawley rats,weighed 200-220 g,were randomly divided into 3 groups (n=24).Severe SCI models were established with an artery clip (15 g closing force) in model group and intervention group.The intervention group received an intramuscular injection of estradiol benzoate every other day one week before injury and last into sacrifice.The Basso,Beattie,Bresnahan (BBB) open field locomotor rating scale were performed on 7,14,21 and 28 days after SCI to evaluate the functional recovery of locomotion.Real-time PCR and Western blot analysis were used to detect the expression of autophagy marker and Immunohistochemical staining for LC3 was performed using spinal cord sections obtained on days 3 after SCI. Results High dose of estradiol significantly improved locomotor recovery in vivo;the BBB scores in the intervention group were higher than that in the model group on 14,21,and 28 days after spinal cord injury.The expression of LC3 and Beclin-1 in model groups were higher than sham group at 3 days after SCI;E2 treatment inhibited the expression at both mRNA and protein level.Conversely,the expression levels of autophagy substrates protein P62 in the model group was significantly reduced when compared with the sham group.However,the expression was increased when treated with estradiol.Immunohistochemical result indicated that the enhanced immunoreactivity for LC3 in motor neurons after SCI was repressed when treated with high dose of estradiol. Conclusion The neuroprotective effects of E2 in SCI is related to the restraint of excessive autophagy.