BACKGROUND Hypoxic-ischemic encephalopathy (HIE) is an important cause of permanent damage to the central nervous system, associated with long-lasting neurological disabilities and neurodevelopmental impairment in neonates. Granulocyte-colony stimulating factor (G-CSF) has been shown to have neuroprotective activity in a variety of experimental brain injury models and G-CSF is a standard treatment in chemotherapeutic-induced neutropenia. The underlying mechanisms are still unclear. The mTOR (mammalian target of rapamycin) signaling pathway is a master regulator of cell growth and proliferation in the nervous system. However, the effects of G-CSF treatment on the mTOR signaling pathway have not been elucidated in neonates with hypoxic-ischemic (HI) brain injury. Our study investigated the neuroprotective effect of G-CSF on neonates with hypoxic-ischemic (HI) brain injury and the possible mechanism involving the mTOR/p70S6K pathway. METHODS Sprague-Dawley rat pups at postnatal day 7 (P7) were subjected to right unilateral carotid artery ligation followed by hypoxic (8% oxygen and balanced nitrogen) exposure for 2.5 h or sham surgery. Pups received normal saline, G-CSF, G-CSF combined with rapamycin or ethanol (vehicle for rapamycin) intraperitoneally. On postnatal day 9 (P9), TTC staining for infarct volume, and Nissl and TUNEL staining for neuronal cell injury were conducted. Activation of mTOR/p70S6K pathway, cleaved caspase-3 (CC3), Bax and Bcl-2 and cytokine expression levels were determined by western blotting. RESULTS The G-CSF treated group was associated with significantly reduced infarction volume and decreased TUNEL positive neuronal cells compared to the HI group treated with saline. The expression levels of TNF-α and IL-1ß were significantly decreased in the G-CSF treated group, while IL-10 expression level was increased. The relative immunoreactivity of p-mTOR and p-p70S6K was significantly reduced in the HI group compared to sham. The HI group treated with G-CSF showed significant upregulated protein expression for p-mTOR and p-p70S6K levels compared to the HI group treated with saline. Furthermore, G-CSF treatment increased Bcl-2 expression levels and decreased CC3 and Bax expression levels in the ipsilateral hemispheres of the HI brain. The effects induced by G-CSF were all reversed by rapamycin. CONCLUSION Treatment with G-CSF decreases inflammatory mediators and apoptotic factors, attenuating neuroinflammation and neuronal apoptosis via the mTOR/p70S6K signalling pathway, which represents a potential target for treating HI induced brain damage in neonatal HIE.

中文翻译：

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G-CSF 通过 mTOR/p70SK6 信号通路减轻新生儿缺氧缺血大鼠模型的神经炎症和神经元凋亡。

背景缺氧缺血性脑病 (HIE) 是中枢神经系统永久性损伤的重要原因，与新生儿的长期神经功能障碍和神经发育障碍有关。粒细胞集落刺激因子 (G-CSF) 已在各种实验性脑损伤模型中显示出具有神经保护活性，并且 G-CSF 是化疗引起的中性粒细胞减少症的标准治疗方法。其潜在机制仍不清楚。mTOR（哺乳动物雷帕霉素靶点）信号通路是神经系统中细胞生长和增殖的主要调节因子。然而，G-CSF 治疗对 mTOR 信号通路的影响尚未在缺氧缺血 (HI) 脑损伤的新生儿中阐明。我们的研究调查了 G-CSF 对缺氧缺血性 (HI) 脑损伤新生儿的神经保护作用以及涉及 mTOR/p70S6K 通路的可能机制。方法 出生后第 7 天 (P7) 的 Sprague-Dawley 大鼠幼崽进行右侧单侧颈动脉结扎，然后进行缺氧（8% 氧气和平衡氮气）暴露 2.5 小时或假手术。幼崽腹膜内接受生理盐水、G-CSF、G-CSF 联合雷帕霉素或乙醇（雷帕霉素载体）。在出生后第 9 天 (P9)，对梗塞体积进行 TTC 染色，对神经元细胞损伤进行 Nissl 和 TUNEL 染色。通过蛋白质印迹确定 mTOR/p70S6K 通路的激活、裂解的 caspase-3 (CC3)、Bax 和 Bcl-2 以及细胞因子表达水平。结果与用盐水处理的HI组相比，G-CSF处理组与显着减少的梗塞体积和减少的TUNEL阳性神经元细胞相关。G-CSF治疗组TNF-α和IL-1β的表达水平显着降低，而IL-10的表达水平升高。与假手术相比，HI 组中 p-mTOR 和 p-p70S6K 的相对免疫反应性显着降低。与用盐水处理的 HI 组相比，用 G-CSF 处理的 HI 组显示出 p-mTOR 和 p-p70S6K 水平的蛋白质表达显着上调。此外，G-CSF 治疗增加了 Bcl-2 表达水平并降低了 HI 大脑同侧半球的 CC3 和 Bax 表达水平。G-CSF 诱导的作用均被雷帕霉素逆转。