title: G-CSF ameliorates neuronal apoptosis through GSK-3β inhibition in neonatal hypoxia-ischemia in rats.
abstract: 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.
authors.affiliation: 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.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA.
authors.forename: Damon
authors.initials: D
authors.lastname: Klebe
authors.affiliation: Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA.
authors.forename: Desislava
authors.initials: D
authors.lastname: Doycheva
authors.affiliation: Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA.
authors.forename: Devin W
authors.initials: DW
authors.lastname: McBride
authors.affiliation: Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Krafft
authors.affiliation: Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA.
authors.forename: Jerry
authors.initials: J
authors.lastname: Flores
authors.affiliation: Department of Anatomy & Histology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
authors.forename: Changman
authors.initials: C
authors.lastname: Zhou
authors.affiliation: Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA; Departments of Anesthesiology and Neurosurgery, Loma Linda University School of Medicine, Loma Linda, CA, USA.
authors.forename: John H
authors.initials: JH
authors.lastname: Zhang
authors.affiliation: Department of Physiology & Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, USA. Electronic address: jtang@llu.edu.
authors.forename: Jiping
authors.initials: J
authors.lastname: Tang
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism* / Q000473:pathology; D020287:In Situ Nick-End Labeling; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
chemical_list: D018696:Neuroprotective Agents; D034741:RNA, Small Interfering; D016179:Granulocyte Colony-Stimulating Factor; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3
keywords: Apoptosis; GSK-3β; Granulocyte-colony stimulating factor (G-CSF); Hypoxia–ischemia (HI); Neonatal; PI3K