Li Li, Klebe Damon, Doycheva Desislava, McBride Devin W, Krafft Paul R, Flores Jerry, Zhou Changman, Zhang John H, Tang Jiping
Department of Anatomy & Histology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China; Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA.
Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA.
Exp Neurol. 2015 Jan;263:141-9. doi: 10.1016/j.expneurol.2014.10.004. Epub 2014 Oct 18.
Granulocyte-colony stimulating factor (G-CSF), a growth factor, has known neuroprotective effects in a variety of experimental brain injury models. Herein we show that G-CSF administration attenuates neuronal apoptosis after neonatal hypoxia-ischemia (HI) via glycogen synthase kinase-3β (GSK-3β) inhibition. Ten day old Sprague-Dawley rat pups (n=157) were subjected to unilateral carotid artery ligation followed by 2.5h of hypoxia or sham surgery. HI animals received control siRNA, GSK-3β siRNA (4 μL/pup), G-CSF (50 μg/kg), G-CSF combined with 0.1 or 0.4 nM G-CSF receptor (G-CSFR) siRNA, phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin (86 ng/pup), or DMSO (vehicle for Wortmannin). Pups were euthanized 48 h post-HI to quantify brain infarct volume. G-CSFR, activated Akt (p-Akt), activated GSK-3β (p-GSK-3β), Cleaved Caspase-3 (CC3), Bcl-2, and Bax were quantified using Western blot analysis and the localizations of each was visualized via immunofluorescence staining. Neuronal cell death was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Our results showed p-GSK-3β increased after HI until its peak at 48 h post-ictus, and both GSK-3β siRNA and G-CSF administration reduced p-GSK-3β expression, as well as infarct volume. p-GSK-3β and CC3 were generally co-localized in neurons. Furthermore, G-CSF increased p-Akt expression and the Bcl-2/Bax ratio and also decreased p-GSK-3β and CC3 expression levels in the ipsilateral hemisphere, which were all reversed by G-CSFR siRNA, Wortmannin, and GSK-3β siRNA. In conclusion, G-CSF attenuated caspase activation and reduced brain injury by inhibiting GSK-3β activity after experimental HI in rat pups. This neuroprotective effect was abolished by both G-CSFR siRNA and Wortmannin.
粒细胞集落刺激因子（G-CSF）是一种生长因子，在多种实验性脑损伤模型中具有已知的神经保护作用。在此我们表明，给予G-CSF可通过抑制糖原合酶激酶-3β（GSK-3β）减轻新生大鼠缺氧缺血（HI）后的神经元凋亡。10日龄的Sprague-Dawley幼鼠（n = 157）接受单侧颈动脉结扎，随后进行2.5小时的缺氧处理或假手术。HI动物接受对照小干扰RNA（siRNA）、GSK-3β siRNA（4μL/幼鼠）、G-CSF（50μg/kg）、G-CSF与0.1或0.4 nM G-CSF受体（G-CSFR）siRNA联合使用、磷脂酰肌醇3-激酶（PI3K）抑制剂渥曼青霉素（86 ng/幼鼠）或二甲基亚砜（DMSO，渥曼青霉素的溶剂）。幼鼠在HI后48小时安乐死，以量化脑梗死体积。使用蛋白质印迹分析对G-CSFR、活化的Akt（p-Akt）、活化的GSK-3β（p-GSK-3β）、裂解的半胱天冬酶-3（CC3）、Bcl-2和Bax进行定量，并通过免疫荧光染色观察各自的定位。使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法测定神经元细胞死亡情况。我们的结果显示，HI后p-GSK-3β升高，直至发作后48小时达到峰值，GSK-3β siRNA和给予G-CSF均降低了p-GSK-3β的表达以及梗死体积。p-GSK-3β和CC3通常在神经元中共定位。此外，G-CSF增加了p-Akt的表达以及Bcl-2/Bax比值，还降低了同侧半球中p-GSK-3β和CC3的表达水平，而G-CSFR siRNA、渥曼青霉素和GSK-3β siRNA均逆转了这些作用。总之，在幼鼠实验性HI后，G-CSF通过抑制GSK-3β活性减轻了半胱天冬酶激活并减少了脑损伤。G-CSFR siRNA和渥曼青霉素均消除了这种神经保护作用。
