G-CSF attenuates neuroinflammation and neuronal apoptosis via the mTOR/p70SK6 signaling pathway in neonatal Hypoxia-Ischemia rat model.

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. 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. 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. 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.

Dumbuya JS ，Chen L ，Shu SY ，Ma L ，Luo W ，Li F ，Wu JY ，Wang B ... -  《-》

G-CSF ameliorates neuronal apoptosis through GSK-3β inhibition in neonatal hypoxia-ischemia in rats.

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.

Li L ，Klebe D ，Doycheva D ，McBride DW ，Krafft PR ，Flores J ，Zhou C ，Zhang JH ，Tang J ... -  《-》

The Akt/mTOR/p70S6K Pathway Is Involved in the Neuroprotective Effect of Erythropoietin on Hypoxic/Ischemic Brain Injury in a Neonatal Rat Model.

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 erythropoietin (EPO) treatment on the mTOR signaling pathway have not been elucidated in neonates with hypoxic/ischemic (H/I) brain injury. We investigated the mechanism underlying the neuroprotective effect of EPO by analyzing the mTOR signaling pathway after H/I injury in a neonatal rat model. Seven-day-old rats were subjected to left carotid artery ligation and hypoxic exposure (8%) for 90 min (H/I). EPO at a dose of either 3,000 U/kg or a vehicle (V) was administered by intraperitoneal injection 0, 24 and 48 h after H/I. At 72 h after H/I (postnatal day 10), 2,3,5-triphenyltetrazolium chloride staining, myelin basic protein (MBP) immunofluorescence staining and Western blot analysis of the Akt/mTOR/p70S6K pathway were performed. Neuromotor behavioral tests included Rotarod challenge and cylinder rearing test 1 performed 3 and 6 weeks after H/I. EPO treatment resulted in significant offsetting of MBP depletion ipsilateral (p = 0.001) and contralateral (p = 0.003) to ligation. Western blot analysis showed that the relative immunoreactivity of phosphorylated (p)-Akt, p-mTOR and p-p70S6K ipsilateral to ligation was significantly decreased in the H/I+V group compared with the sham-operated groups. However, EPO treatment significantly upregulated Akt/mTOR/p70S6K signals ipsilateral to ligation compared to the H/I+V group. The behavior tests showed that EPO attenuates long-term impairment in Rotarod challenge and cylinder test performance from 3-6 weeks. This study demonstrates an underlying mechanism of the mTOR signaling pathway after EPO treatment, which is a potential target for treating H/I-induced brain injury.

Lee HJ ，Koh SH ，Song KM ，Seol IJ ，Park HK ... -  《-》

Rh-CSF1 Attenuates Oxidative Stress and Neuronal Apoptosis via the CSF1R/PLCG2/PKA/UCP2 Signaling Pathway in a Rat Model of Neonatal HIE.

Oxidative stress (OS) and neuronal apoptosis are major pathological processes after hypoxic-ischemic encephalopathy (HIE). Colony stimulating factor 1 (CSF1), binding to CSF1 receptor (CSF1R), has been shown to reduce neuronal loss after hypoxic-ischemia- (HI-) induced brain injury. In the present study, we hypothesized that CSF1 could alleviate OS-induced neuronal degeneration and apoptosis through the CSF1R/PLCG2/PKA/UCP2 signaling pathway in a rat model of HI. A total of 127 ten-day old Sprague Dawley rat pups were used. HI was induced by right common carotid artery ligation with subsequent exposure to hypoxia for 2.5 h. Exogenous recombinant human CSF1 (rh-CSF1) was administered intranasally at 1 h and 24 h after HI. The CSF1R inhibitor, BLZ945, or phospholipase C-gamma 2 (PLCG2) inhibitor, U73122, was injected intraperitoneally at 1 h before HI induction. Brain infarct volume measurement, cliff avoidance test, righting reflex test, double immunofluorescence staining, western blot assessment, 8-OHdG and MitoSOX staining, Fluoro-Jade C staining, and TUNEL staining were used. Our results indicated that the expressions of endogenous CSF1, CSF1R, p-CSF1R, p-PLCG2, p-PKA, and uncoupling protein2 (UCP2) were increased after HI. CSF1 and CSF1R were expressed in neurons and astrocytes. Rh-CSF1 treatment significantly attenuated neurological deficits, infarct volume, OS, neuronal apoptosis, and degeneration at 48 h after HI. Moreover, activation of CSF1R by rh-CSF1 significantly increased the brain tissue expressions of p-PLCG2, p-PKA, UCP2, and Bcl2/Bax ratio, but reduced the expression of cleaved caspase-3. The neuroprotective effects of rh-CSF1 were abolished by BLZ945 or U73122. These results suggested that rh-CSF1 treatment attenuated OS-induced neuronal degeneration and apoptosis after HI, at least in part, through the CSF1R/PLCG2/PKA/UCP2 signaling pathway. Rh-CSF1 may serve as therapeutic strategy against brain damage in patients with HIE.

Hu X ，Li S ，Doycheva DM ，Huang L ，Lenahan C ，Liu R ，Huang J ，Gao L ，Tang J ，Zuo G ，Zhang JH ... -  《-》

mTOR activates hypoxia-inducible factor-1α and inhibits neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia.

The mammalian target of rapamycin (mTOR) exerts neuroprotective effects under hypoxic or ischemic conditions. To explore whether mTOR participates in neuroprotective signaling through regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and neuronal apoptosis in developing rat brain with hypoxia-ischemia (HI), we operated on postnatal day 10 rats by ligating the common carotid artery followed by exposure to systemic hypoxia. Brains were collected at various intervals to detect the expression of mTOR, phosphorylated mTOR (p-mTOR), HIF-1α, VEGF and cleaved caspase 3 (CC3), using immunohistochemistry and Western blot analysis. We also used terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) to detect neuronal apoptosis. The p-mTOR protein expression increased at 2h after HI, peaked at 8h, lasted 24h, and then dropped to the basal level. Also, the expression of HIF-1α and VEGF was significantly enhanced and peaked at 8h after HI. Up-regulated expression of CC3 was observed at 2h, peaked at 24h, and lasted 72h after HI. Increased neuronal apoptosis is associated with reduced HIF-1α and VEGF expression. Furthermore, pretreatment with rapamycin, a mTOR specific inhibitor, significantly inhibited HIF-1α and VEGF protein after HI. The expression of CC3 and the number of TUNEL-positive cells were up-regulated at 8h and down-regulated at 24h after HI in the rapamycin-treated group. Our findings suggest that mTOR may participate in the regulation of HIF-1α, VEGF and neuronal apoptosis, serving neuroprotective functions after HI in developing rat brain.

Chen H ，Xiong T ，Qu Y ，Zhao F ，Ferriero D ，Mu D ... -  《-》