激活Akt/mTOR/p70S6K信号通路对大鼠脊髓损伤后神经再生的影响

The effects of activated Akt/m TOR/p70S6K signaling pathway on the neural regeneration after spinal cord injury in rats

目的：探讨激活蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（m TOR）/p70核糖体S6蛋白激酶（p70S6K）信号通路对大鼠脊髓损伤（spinal cord injury,SCI）后神经再生及神经功能恢复的影响,为促进SCI修复提供分子学机制依据。方法：72只雌性SD大鼠建立轻型SCI模型后随机均分为激活组（Act组,SCI＋ATP）、对照组（Con组,SCI＋生理盐水）、阻断组（Int组,SCI＋ATP＋雷帕霉素）;24只大鼠仅打开椎板、不损伤脊髓,为假手术组（Sham组）。分别于术后1d、3d、7d、14d采用BBB运动功能评分评价各SCI组大鼠经不同治疗后的神经功能,并采用Western blot检测各组在术后各时间点脊髓组织中Akt、p-Akt、m TOR、p-m TOR、p70S6K、p-p70S6K、Nestin、Neu N蛋白表达情况,免疫组化检测各组在术后各时间点的Nestin和Neu N表达情况。结果 ：各SCI组术后1d BBB评分为3～5分,之后逐渐增加,术后1d和3d各组间BBB评分无显著性差异（P〉0.05）,Act组在SCI后7d和14d时的BBB评分明显高于Con与Int组（P〈0.05）。各SCI组在术后各时间点脊髓组织中Akt、m TOR及p70S6K磷酸化水平均明显高于Sham组（P〈0.05）,Act组的磷酸化水平在术后各时间点均明显高于Con组及Int组（P〈0.05）,术后7d时磷酸化水平增高最为显著（P〈0.01）。各SCI组大鼠在术后各时间点的Nestin表达水平均高于Sham组（P〈0.05）,Act组的表达水平较Con组、Int组显著性增高（P〈0.05）、Nestin阳性细胞数显著性多于Con组与Int组（P〈0.05）。各SCI组在术后各时间点的Neu N表达均显著性低于Sham组（P〈0.01）,各SCI组Neu N的表达在术后1d、3d无显著性差异（P〉0.05）;Act组在术后7d和14d时的Neu N表达水平明显高于Con组及Int组（P〈0.05）,Neu N阳性细胞数明显多于Con组与Int组（P〈0.05）。结论：激活Akt/m TOR/p70S6K信号通路可显著增加SCI后Nestin及Neu N的表达,促进SCI后神经恢复和功能改善,此信号通路是治疗SCI的重要干预环节。

Objectives： To examine the impacts of the protein kinase B（Akt）/mammalian target of rapamycin（m TOR）/p70 ribosomal S6 protein kinase（p70S6K） signaling transduction pathway on neural regeneration and functional recovery after spinal cord injury（SCI） in adult rats, and to provide the molecular mechanism for promoting the SCI repair. Methods： A total of 72 female Sprague-Dawley rats of minimal injury model was randomly divided into the following groups： activation group（Act group, SCI plus ATP）, control group（Con group, SCI plus saline） and interruption group（Int group, SCI plus ATP and rapamycin）. The sham-operated group（Sham group） including 24 rats undergoing laminectomy without SCI. The BBB locomotor rating scale was used to evaluate the animal′s locomotor performance after different treatments were administered at 1, 3,7, and 14 days following SCI. The changes of Akt, p-Akt, m TOR, p-m TOR, p70S6 K, p-p70S6 K, Nestin and Neu N expressions in spinal cord tissues of all groups were detected by using Western blot analysis at all experimental time points after different disposals were administered following surgery, and the immunostaining was further performed for detecting the Nestin and Neu N in all groups after surgery. Results： Locomotor function assessment showed the BBB scores were 3-5 in the animals with SCI at 1d after injury, which gradually exhibited partial recovery of locomotor function after 1d following injury. No significant differences in BBB scores were observed among the three SCI groups at 1d and 3d after injury（P〈0.05）. At 7d and 14 d after injury, the rats in the Act group exhibited significantly higher BBB score compared with the rats in the Con and Int group（P〈0.05）. Western blot showed prominently elevated phosphorylation level of Akt, m TOR and p70S6 K in spinal cord tissues of all SCI groups at all experimental time points after surgery compared with the sham-operated group（P〈0.05）. The phosphorylation level of this signaling pathway in the Act group was significantly elevated by exogenous administration of ATP compared with the Con and Int group at all experimental time points after surgery（P〈0.05）, and the elevated level was particularly evident at 7d after SCI（P〈0.01）. Prominently elevated levels of nestin were detected in the injured spinal cords of all SCI groups compared with the Sham group at 1, 3, 7 and 14 days after surgery（P〈0.05）. A marked upregulation of nestin was noted in the Act group following SCI compared with the Con and Int group（P〈0.05）, and the number of Nestin-positive cells in the Act group was more than that of the Con and Int group（P〈0.05）. Significantly decreased expression of Neu N in the SCI groups was noted compared with the Sham group after surgery（P〈0.01）, and no significant differences in the expression were observed among the three SCI groups at 1d and3 d after injury（P〉0.05）. The expression of Neu N in the SCI groups gradually increased after 7d following SCI.The injured spinal cords in the Act group showed faster increase of Neu N and significantly higher level of Neu N expression compared with those from rats in the Con and Int group at 7d and 14 d after SCI（P〈0.05）,and the number of Neu N-positive cells in the Act group was more than that of the Con and Int group respectively（P〈0.05）. Conclusions： Activated Akt/m TOR/p70S6 K signaling pathway can significantly increase the expression of Nestin and Neu N in the injured spinal cords after SCI and posses the potential of promoting neural progenitor cells（NPCs） proliferation and neural regeneration, which improves motor function and promotes neural repair following SCI, intervention to this signaling pathway activity should be considered as a potential therapeutic strategy.