缺血预处理通过增强自噬水平及抑制自噬性细胞死亡保护脊髓神经
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国家自然科学基金资助(81401807);无锡市科技立项课题(MD201304)


schemic preconditioning improves autophagy and suppresses autophagic cell death in rat spinal cord following ischemia-reperfusion
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    摘要:

    目的:探讨缺血预处理保护脊髓神经的具体机制。方法:50只成年SD大鼠随机分为假手术组(A组5只)-缺血再灌注组(B组20只)-缺血预处理组(C组5只)-缺血预处理+缺血再灌注组(D组20只)。A组动物麻醉后仅切开腹腔后关腹;B组和D组采用左肾下方0.5 cm处腹主动脉夹闭法夹闭0.5 h后松开,再灌注3-6-12-24 h,其中D组在缺血再灌注损伤前行缺血5 min-再灌注5 min缺血预处理,共3次;C组仅行3次缺血预处理。造模成功后,利用HE染色和免疫组化评估各组神经元的存活率,透射电镜观察自噬活性,免疫组化和蛋白印迹分析自噬标志物LC3-Ⅱ和自噬相关蛋白Beclin-1的表达情况。结果:缺血再灌注组中,LC3-Ⅱ蛋白在再灌注3 h后显著升高达到峰值后明显下降,24 h后几乎无表达;Beclin-1蛋白于再灌注后3 h开始升高且在24 h达到峰值;再灌注24 h后实验动物表现出明显的脊髓损伤症状,脊髓神经细胞存活率明显降低。缺血预处理+缺血再灌注组中,LC3-Ⅱ蛋白表达从再灌注3 h开始升高并持续至再灌注24 h;Beclin-1蛋白在再灌注24 h后才开始升高;与缺血再灌注组相比,缺血预处理+缺血再灌注组脊髓神经元死亡率明显降低。结论:缺血预处理通过维持缺血再灌注后神经细胞自噬水平并抑制自噬性细胞死亡从而保护脊髓神经细胞。

    Abstract:

    Objective: To explore the neuroprotective mechanism of ischemic preconditioning in spinal core ischemia. Methods:Fifty adults rats were randomly assigned to 4 groups: the sham group receiving anesthesia and surgical preparation(group A, n=5),the ischemia/reperfusion (I/R)group adminstrated to 0.5 h ischemia followed by 3,6,12,or 24 h reperfusion (group B, n=20),the IPC group receiving three cycles of 5 min ischemia followed by 5 min of reperfusion (group C, n=5),and the IPC+Ⅰ/R group (group D, n=20). HE and immunohistochemical staining were used to evaluate the spinal neuron. Autophagic activity was investigated by electron microscopy and immunohistochemistry. The autophagosome marker LC3-Ⅱ and the autophagy-associated protein Beclin-1 were detected by Western blot. Results: In the Ⅰ/R group,LC3-Ⅱ was significantly elevated after 3 h of reperfusion,but declined significantly by 24 h. At 24 h,rats in the I/R group exhibited extensive spinal damage and decreased neuronal survival. In the IPC+Ⅰ/R group, neuronal death was reduced and expression of LC3-Ⅱ sustained throughout the 24 h reperfusion period. Conclusion:Ischemic preconditioning can sustain the beneficial effects of autophagic lysosomal degradation during Ⅰ/R by inhibiting autophagic cell death.

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顾 军,殷 建,殷照阳,江 涛,杨海源,黄 瑶,恽 波,凡 进.缺血预处理通过增强自噬水平及抑制自噬性细胞死亡保护脊髓神经[J].南京医科大学学报(自然科学版),2015,(11):1504-1510

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  • 收稿日期:2015-06-17
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  • 在线发布日期: 2015-11-30
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