中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (4): 721-727.doi: 10.4103/1673-5374.247476

• 原著:脊髓损伤修复保护与再生 • 上一篇    下一篇

雷帕霉素激活自噬和Akt信号通路实现对大鼠脊髓损伤的神经保护作用

  

  • 出版日期:2019-04-15 发布日期:2019-04-15
  • 基金资助:

    国家自然科学基金资助（81401004）；浙江省医疗卫生技术发展项目（2015-KY1001-036）

Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling

Xi-Gong Li  , Jun-Hua Du, Yang Lu, Xiang-Jin Lin   

  1. Department of Orthopedic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
  • Online:2019-04-15 Published:2019-04-15
  • Contact: Xi-Gong Li, MD, PhD, lixigong@zju.edu.cn.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81401004 (to XGL); the Medical and Health Technology Development Program of Zhejiang Province of China, No. 2015-KY1001-036 (to XGL).

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摘要：

雷帕霉素可增加自噬活性，也能激活Akt磷酸化并在缺血再灌注损伤模型中可发挥抑制细胞凋亡作用，但是否同时通过激活Akt磷酸化途径对脊髓损伤产生神经保护作用相关报道较少。因此，实验设计以雷帕霉素干预SCI模型大鼠，观察其是否同时触发自噬和Akt信号传导，发挥神经保护作用。浙江省实验动物中心提供的成年SD大鼠采用钳夹力30 g钳夹动脉瘤夹制作脊髓损伤模型大鼠，先采用雷帕霉素1 mg/kg腹腔内注射，并分别用自噬抑制因子3-甲基腺嘌呤2.5 mg/kg及Akt抑制剂IV 1 μg/kg腹腔内注射。以Western blot，免疫荧光染色，TUNEL染色观察神经细胞自噬分子Beclin 1，凋亡相关分子Bcl-2，Bax，细胞色素c，caspase 3以及Akt信号的表达。发现如下现象：（1）雷帕霉素可抑制损伤脊髓组织mTOR表达，同时上调Beclin 1和p-Akt表达；（2）雷帕霉素可阻止损伤脊髓组织bcl-2表达下降，降低脊髓损伤24 h后Bax，细胞色素 c及caspase 3表达水平，减少损伤脊髓组织凋亡神经细胞数量；（3）3-MA及Akt抑制剂IV干预能分别抑制损伤脊髓组织Beclin-1及p-Akt的表达，并降低雷帕霉素对凋亡神经细胞的保护作用；（4）上述结果表明，雷帕霉素对脊髓损伤大鼠的神经保护作用可通过激活自噬和Akt信号通路2个途径的变化而协同实现。

orcid: 0000-0002-7972-6298(Xi-Gong Li)

关键词: 脊髓损伤, 雷帕霉素, 哺乳动物类雷帕霉素靶蛋白, 自噬, Beclin 1, 3甲基腺嘌呤, 急性脊髓损伤, 凋亡, Bax, mTOR, Akt, 神经再生

Abstract:

Rapamycin treatment has been shown to increase autophagy activity and activate Akt phosphorylation, suppressing apoptosis in several models of ischemia reperfusion injury. However, little has been studied on the neuroprotective effects on spinal cord injury by activating Akt phosphorylation. We hypothesized that both effects of rapamycin, the increased autophagy activity and Akt signaling, would contribute to its neuroprotective properties. In this study, a compressive spinal cord injury model of rat was created by an aneurysm clip with a 30 g closing force. Rat models were intraperitoneally injected with rapamycin 1 mg/kg, followed by autophagy inhibitor 3-methyladenine 2.5 mg/kg and Akt inhib¬itor IV 1 μg/kg. Western blot assay, immunofluorescence staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay were used to observe the expression of neuronal autophagy molecule Beclin 1, apoptosis-related molecules Bcl-2, Bax, cytochrome c, casp ase-3 and Akt signaling. Our results demonstrated that rapamycin inhibited the expression of mTOR in injured spinal cord tissue and up-reg¬ulated the expression of Beclin 1 and phosphorylated-Akt. Rapamycin prevented the decrease of bcl-2 expression in injured spinal cord tissue, reduced Bax, cytochrome c and caspase-3 expression levels and reduced the number of apoptotic neurons in injured spinal cord tissue 24 hours after spinal cord injury. 3-Methyladenine and Akt inhibitor IV intervention suppressed the expression of Beclin-1 and phosphorylated-Akt in in¬jured spinal cord tissue and reduced the protective effect of rapamycin on apoptotic neurons. The above results indicate that the neuroprotective effect of rapamycin on spinal cord injury rats can be achieved by activating autophagy and the Akt signaling pathway.

Key words: nerve regeneration, rapamycin, mammalian target of rapamycin, mTOR, autophagy, Beclin 1, 3-methyladenine, acute spinal cord injury, apoptosis, Bax, Akt, neural regeneration