Activation of Akt/mTOR/p70S6K signaling pathway limits excessive astrocytic responses after spinal cord injury in rats

doi:10.3969/j.issn.2095-4344.2015.05.008

Abstract

Abstract:

BACKGROUND: Most investigations have focused on nerve regeneration after spinal cord injury, but for how to inhibit excessive proliferation response of astrocytes after spinal cord injury and improve nerve regenerative environment is rarely reported.
OBJECTIVE: To examine the effects of the protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 protein kinase (Akt/mTOR/p70S6K) signal transduction pathway on excessive astrocytic responses after spinal cord injury in adult rats, and to provide the molecular mechanism evidence for improving nerve regenerative environment following spinal cord injury and repairing spinal cord injury．
METHODS: The minimal spinal cord injury model was produced in Spraque-Dawley rats. The rat models were divided into four groups: experiment group (receiving 7-day ATP treatment after modeling), control group (receiving 7-day saline treatment after modeling), interference group (receiving 7-day ATP and rapamycin treatment after modeling), and sham-operated group (receiving 7-day saline treatment after laminectomy). Using immunohistochemical staining and western blot analyses, we detected the changes of Akt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K, and glial fibrillary acidic protein expressions in spinal cord tissues after surgery. BBB locomotor rating scale was applied to evaluate the animal’s locomotor performance after different treatments were administered following spinal cord injury.
RESULTS AND CONCLUSION: The sham-operated group animals exhibited a low expression of the Akt/mTOR/p70S6K signaling pathway at the protein level, and the expressions increased following spinal cord injury. Prominently elevated levels of its components were observed in the ATP-treated group, whereas rapamycin suppressed the upregulations of the Akt/mTOR/p70S6K signaling molecules induced by ATP. Activated Akt/mTOR/p70S6K signaling pathway significantly attenuated expressions of glial fibrillary acidic protein in the injured spinal cord, limited excessive astrocytic responses, and elevated the BBB scores after spinal cord injury. Rapamycin inhibited the above effects induced by ATP. These findings suggest that ATP-mediated Akt/mTOR/p70S6K signaling pathway activation can inhibit excessive glial scar formation after spinal cord injury, and have the potential of improving the regenerative environment, motor function and promoting repair potential for spinal cord injury, so this signaling pathway should be considered as a potential therapeutic strategy for spinal cord injury.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: Sirolimus, Nervous System, Spine, Neuroglia

CLC Number:

R318

Hu Ling-yun, Zhang Jian-ying, Lin Hong, Gou Lin, Lin Tao, Li Wei, Liu Kang, Sun Zhen-gang . Activation of Akt/mTOR/p70S6K signaling pathway limits excessive astrocytic responses after spinal cord injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(5): 697-703.

Figures/Tables 2

2.1 实验动物数量分析实验分批进行，脱落动物在下一批随机分组补充。总共饲养104只SD大鼠，96只纳入实验，8只不纳入实验统计：3只死于造模过程中，5只死于后期膀胱破裂或泌尿系感染。 2.2 ATP激活脊髓损伤后脊髓组织中的Akt/mTOR/ p70S6K信号通路 Western blot检测结果发现，实验组的p-Akt、p-mTOR、p-p70S6K表达水平在脊髓损伤后7 d明显高于对照组以及假手术组(P < 0.01，图1)。应用mTOR的特异性抑制剂雷帕霉素后，发现雷帕霉素可显著抑制ATP所诱导的上述蛋白磷酸化水平上调(P < 0.01，图1)。mTOR的Ser2448位点磷酸化是体内、体外依赖Akt磷酸化的主要位点[5]，Thr389位点是mTOR在体内与体外激活p70S6K的主要位点[29]，且p70S6K的磷酸化依赖于mTOR，提示ATP诱导了受损脊髓组织中Akt/mTOR/ p70S6K信号通路的激活。 2.3 激活的Akt/mTOR/p70S6K信号对脊髓损伤后星形胶质细胞增生反应的影响免疫组织化学检测发现，在脊髓损伤后脊髓组织中的星形胶质细胞显著增生，胶质纤维酸性蛋白的表达水平明显高于假手术组(图2A)。在脊髓损伤后14 d，实验组的胶质纤维酸性蛋白表达水平低于对照组，而雷帕霉素阻碍了由ATP所诱导的此种效应。细胞计数发现实验组的胶质纤维酸性蛋白阳性细胞明显少于对照组与干预组(P < 0.05，图2B)，但实验组星形胶质细胞的增殖反应及胶质纤维酸性蛋白表达并未完全受到抑制。与实验组相比，对照组与干预组中的脊髓组织损伤附近可见粗厚与多支链的星形胶质细胞分布。 Western blot检测证实了免疫组化的发现，实验组的胶质纤维酸性蛋白表达水平明显低于对照组和干预组(P < 0.05，图3B)。与干预组相比，实验组胶质纤维酸性蛋白表达水平在术后时间段的增加趋势更为缓慢(图3A)。"

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