G-CSF promotes autophagy and reduces neural tissue damage after spinal cord injury in mice
G-CSF promotes autophagy and reduces neural tissue damage after spinal cord injury in mice
Yuji Guo 0
Shangming Liu 0
Xianghong Zhang 0
Liyan Wang 0
Jiangang Gao 1
Aiqing Han 2
Aijun Hao 0
0 Key Laboratory of the Ministry of Education for Experimental Teratology, Department of Histology and Embryology, Shandong University School of Medicine , Jinan , China
1 Institute of Developmental Biology, College of Life Science, Shandong University , Jinan , China
2 Department of Obstetrics, Maternal and Children Health Hospital of Jinan City, Jinan, China Professor A Hao, Key Laboratory of the Ministry of Education for Experimental Teratology, Department of Histology and Embryology, Shandong University School of Medicine , 44
3 , Wenhua Xi Road, Jinan, Shandong 250012 , China
Granulocyte colony-stimulating factor (G-CSF) was investigated for its capacity to induce autophagy and related neuroprotective mechanisms in an acute spinal cord injury model. To accomplish this goal, we established a mouse spinal cord hemisection model to test the effects of recombinant human G-CSF. The results showed that autophagy was activated after spinal cord injury and G-CSF appears to induce a more rapid activation of autophagy within injured spinal cords as compared with that of non-treated animals. Apoptosis as induced in mechanically injured neurons with G-CSF treatment was enhanced after inhibiting autophagy by 3-methyladenine (3-MA), which partially blocked the neuroprotective effect of autophagy as induced by G-CSF. In addition, G-CSF inhibited the activity of the NF-?B signal pathway in neurons after mechanical injury. We conclude that G-CSF promotes autophagy by inhibiting the NF-?B signal pathway and protects neuronal structure after spinal cord injury. We therefore suggest that G-CSF, which rapidly induces autophagy after spinal cord injury to inhibit neuronal apoptosis, may thus provide an effective auxiliary therapeutic intervention for spinal cord injury. Laboratory Investigation (2015) 95, 1439-1449; doi:10.1038/labinvest.2015.120; published online 2 November 2015
Spinal cord injury (SCI) destroys cellular structures causing cell
death and inflammation. The initial stages of SCI instigate a
progressive cascade of secondary injuries, which exacerbate the
extent of this destruction.1?3 In specific, apoptosis represents a
major component of such secondary injuries and an
impediment to functional recovery after SCI.4,5 In addition to
apoptosis, recent findings have revealed that autophagy
represents another mechanism involved with regulating
programmed cell death after SCI.6,7
Autophagy has an important role in the degradation of
cytoplasmic constituents via the autophagosomal?lysosomal
pathway.8,9 This pathway provides a means through which
damaged organelles, toxic agents and long-lived, unwanted
proteins are degraded and recycled, thereby maintaining
cellular homeostasis. Abnormalities in autophagy have been
implicated in SCI, as well as in many chronic
neurodegenerative diseases, such as Alzheimer?s disease, Parkinson?s
disease and amyotrophic lateral sclerosis.10?12 In animal
models of these conditions, boosting autophagy appears to
retard the progression of the disease by facilitating removal of
misfolded proteins that have a pathogenic role. However, it
has also been reported that activation of autophagy induced
cell death in a myocardial ischemia and reperfusion model.13
Moreover, in traumatic brain injury and cerebral ischemia
autophagy has been shown to be involved in the induction of
cell death.14,15 Whether autophagy is protective or
detrimental for neural tissue after SCI remains to be determined.
Accordingly, a better understanding of autophagy?s role in
SCI will help identify new targets for treatment.
Granulocyte colony-stimulating factor (G-CSF), produced
by monocytes, fibroblasts and endothelial cells, is a low
molecular weight glycoprotein.16 G-CSF binds with a specific
receptor, G-CSF receptor (G-CSFR) and promotes the
proliferation and differentiation of granulocyte hemopoietic
progenitor to protect neutrophils from apoptosis. G-CSFR is
present within the CNS17 and increasing evidence has
revealed that G-CSF may inhibit neuronal apoptosis
associated with neural injury/disease and, in this way, promote
neuroprotective effects.18?20 In particular, G-CSF has been
shown to exert beneficial effects via an anti-autophagic
mechanism in cardiomyopathic hamster models.21 Based
upon these findings, the potential for G-CSF to function as a
neuroprotectant in acute SCI attracted our attention.
Therefore, in this report, we examined whether G-CSF affects
autophagy of neurons and the locomotor recovery process
after SCI. To accomplish this goal, we first established a
mouse spinal cord hemisection model. These mice were then
injected with recombinant human G-CSF (rhG-CSF) and
examined for the activation of autophagy at different time
points under conditions with or without G-CSF treatment
in vivo. In addition, the possible pathway of G-CSF
neuroprotection after neuronal mechanical injury in vitro
MATERIALS AND METHODS
Fifty-four female Kunming mice (30 ? 5 g) obtained from the
Laboratory Animal Center at Shandong University were used
in this experiment. Mice were bred and housed under
standard laboratory conditions at 23 ?C with an alternating
12-h light?dark cycle and free access to a commercial diet. All
animal experiments were approved by the Shandong
University Animal Care Committee.
Spinal Cord Hemisection
Spinal cord hemisection was performed as described
previously.22,23 Briefly, mice were anesthetized with 10%
chloral hydrate. Dorsal laminectomy was performed to expose
spinal cord segments T9?T11 using a superficial vein at T5?T6
as a landmark. The dura was incised (1 mm) at the midline of
T10. A complete hemisection of the right hemicord at T10 was
performed with the tip of iridectomy scissors. After surgery,
the muscles and skin were sutured in layers and an antibiotic
(Gentamicin, 1000 u) was administered subcutaneously.
Paralysis of the right hindlimb as assessed at 1 day after spinal
cord hemisection indicated that the surgery was successful and
an effective model was generated. Each mouse received
manual bladder manipulation twice daily until recovery of
sphincter control was present.
The fifty-four mice were divided randomly into two groups, a
G-CSF-treated group and an injury control group. The
G-CSF-treated group was injected subcutaneously with
rhG-CSF (50 ?g/kg day, Chugai Pharmaceutical, Tokyo, Japan)
for 3 consecutive days after spinal cord hemisection.24 The
injury control group was injected subcutaneously with an
equal volume of phosphate-balanced solution (PBS) for 3
consecutive days after spinal cord hemisection. A sample of six
mice were used for each time point as assessed at 1, 3, 5 or
7 days post-injury, with three mice used for immunohistological
studies and three for western blots. Another group of six mice
were used for electron microscopy as evaluated at 5 days
post-injury. The animals were also assessed for motor function
at specific time points post-SCI, and subsequently killed for
histological or biochemical measurements.
Spinal cords were isolated from adult Kunming mice as
described previously.25 Briefly, mice were killed and the spinal
cord was isolated in D-Hank?s balanced salt solution. Spinal
cord tissue was then dissociated into a cell suspension by
mechanical disruption and trypsinization (0.125% trypsin
and 0.02% ethylenediaminetetraacetic acid). Cells were
seeded at a density of 1 ? 106 cells/ml onto
poly-L-lysinecoated glass coverslips or tissue culture dishes in serum-free
Neurobasal-A medium (containing 2% B27). After 7 days in
culture, the cells were used for mechanical scratching.
Mechanical Injury Model in Neuronal Cultures
Scratch insult was performed on cultured spinal cord neurons
as described previously.26 Cell bodies and processes were cut
mechanically with a cataract knife. In order to standardize the
damage within each well, a self-made cardboard painting grid
with 2 mm intervals was placed beneath the transparent
plastic plates. The blade was then moved slowly and gently
along the gridlines to induce scratching. For one group,
G-CSF (100 ng/ml) was then added into the cultures (G-CSF
group). In a second group, 2 mM of 3-methyladenine (3-MA,
Sigma-Aldrich, St Louis, MO, USA) was administered at
10 min before mechanical injury followed by 100 ng/ml
G-CSF after mechanical injury (3-MA group). The third
group served as the control and received an equal volume of
saline as that of the G-CSF administration of the injury
For NF-?B inhibitor test, we added 100 ng/ml G-CSF into
culture system (G-CSF group). In another group, we added
10 ?M BAY11-7082 (NF-?B inhibitor, Sigma-Aldrich, St
Louis, MO, USA) into culture system after mechanical injury
(BAY11 group). The third group served as the control and
received an equal volume of saline after mechanical injury
For immunohistological assays, mice were perfused
transcardially with ice-cold 4% paraformaldehyde in 0.1 M phosphate
buffer (pH = 7.2) at 1, 3, 5 or 7 days after spinal cord
hemisection. The spinal cords were removed and post-fixed
in the same fixative. Fifteen-micrometer sections were
prepared on a cryostat. For western blot assays, mice were
killed at 1, 3, 5 or 7 days after spinal cord hemisection, spinal
cords were quickly frozen in liquid nitrogen and maintained
at ? 80 ?C.
To assess LC3B protein expression in lesioned tissue,
immunohistochemistry was performed following standard
methods. Sections were exposed for 30 min at room
temperature to normal goat serum (10% in
phosphatebuffered saline, pH 7.3) with 0.2% Triton X-100 for blocking
of the sections. The sections were then incubated with
primary antibodies against rabbit LC3B (1:100; Cell Signaling,
Danvers, MA, USA) at 4 ?C overnight followed by staining
with an ABC kit (Vector Laboratories, Burlingame, CA, USA).
The sections were counterstained with hematoxylin.
Double Immunofluorescence Staining
For determination of LC3B localization within spinal cords,
double immunofluorescence staining was performed following
standard procedures. Briefly, sections were subjected to normal
goat serum (10% in PBS, pH 7.3) with 0.2% Triton X-100 for
30 min at room temperature. The sections were then incubated
with primary antibodies against mouse MAP2 (1:200;
Millipore, Billerica, MA, USA) or GFAP (1:200; Abcam,
Cambridge, MA, USA) and primary antibodies against rabbit
LC3B (1:100; Cell Signaling) at 4 ?C overnight followed by
staining with fluorescence-conjugated goat anti-mouse/rabbit
IgG (1:100; Sigma-Aldrich,) for 1 h at 37 ?C. Nuclei were
counterstained with 4?,6-diamidino-2-phenylindole (DAPI)
(1:1000; Invitrogen, Carlsbad, CA, USA). Adjacent sections
were single stained immunohistochemically for MAP2, GFAP
or LC3B in order to validate the immunofluorescence labeling.
Fluorescence images were collected using a Leica microscope.
Terminal Deoxynucleotidyl Transferase-Mediated dUTP
Nick-End Labeling (TUNEL) Assay
TUNEL staining was performed following the manufacturer?s
instructions (KeyFEN, NJ, CHN). Briefly, sections were
incubated with 0.1% Triton X-100 in 0.1% sodium citrate.
After rinsing, sections were incubated in TdT Reaction
Mixture for 1 h at 37 ?C in a humidified chamber and then
counterstained with DAPI for 10 min. Fluorescence images
were collected for analysis using a Leica microscope and
counted in 30 fields. Sections near the lesion epicenter (from
300 ?m rostral to 300 ?m caudal) were excluded from
histological assessment, as tissue destruction was too severe
to count neurons precisely. Cell counting included the area
from either 300 to 800 ?m rostral or 300 to 800 ?m caudal to
the lesion epicenter as previously described.19 The examiners
were blinded as to the treatment condition of the mice.
At 5 days after spinal cord hemisection, the mice were
overdosed by an intraperitoneal injection of 100 mg/kg
sodium pentobarbital. The mice were perfused transcardially
with normal saline, followed by 2% paraformaldehyde and
2.5% glutaraldehyde in cacodylate buffer. For electron
microscopic analyses, spinal cord segments containing the
injured site were removed and post-fixed in 2.5%
glutaraldehyde at 4 ?C overnight. The spinal cords were then cut
with a sharp razor blade and serial 1 mm transverse slices
surrounding the injury site were removed. These tissue slices
were post-fixed in 1% osmium tetroxide for 1 h at 4 ?C, rinsed
in PBS, dehydrated in a graded series of alcohol and
propylene oxide solutions and embedded in Epon. Blocks
showing a predominantly transverse orientation of the injured
spinal cords were selected from toluidine-blue-stained thick
sections. Ultrathin (70 nm) sections were prepared on an
ultramicrotome (Ultracut R, Leica, Heerbrugg, Switzerland)
with a diamond knife, stained with uranyl acetate and lead
citrate and viewed using an electron microscope (JEM-1200,
JOEL, Tokyo, Japan).
Crystal Violet Staining
Crystal violet staining was performed following standard
methods at 5 days after spinal cord hemisection. Sections
were incubated with 0.1% cresyl violet solution
(SigmaAldrich) for 5?10 min, then differentiated in 95% ethyl
alcohol for 2?30 min and assessed microscopically.
Monodansylcadaverine (MDC) staining was also performed
using a procedure similar to that described previously.27
Neurons were fixed with 4% PFA for 10 min at 37 ?C at
24 h after mechanical injury, and then incubated for 10 min at
37 ?C with 50 ?M MDC, an autofluorescent compound
that labels autophagic vacuoles. Fluorescence images were
collected using a Leica microscope.
Western Blot Analysis
Spinal cord tissue was washed with cold PBS and lysed in cold
lysis buffer containing 10 mM Tris?HCl, pH 8.0, 240 mM
NaCl, 5 mM EDTA, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 1% Triton X-100, 1 mM sodium
vanadate, and 1 g/ml of leupeptin, pepstatin and aprotinin.
Cell lysates were incubated on ice for 30 min and then
centrifuged at 12 000 r.p.m. for 10 min at 4 ?C. The
supernatant was collected and protein content assayed
colorimetrically. Ten micrograms of total proteins was loaded
onto a 10% gradient polyacrylamide gel, electrophoretically
transferred to a polyvinylidene difluoride membrane and
probed with rabbit anti-LC3B antibody (1:1000; Cell
Signaling) and mouse anti-NF-?B p65 (1:1000; Cell Signaling).
Monoclonal anti-?-actin (1:1000; Sigma-Aldrich) was
used as an internal control. Secondary antibodies were
horseradish peroxidase conjugated to goat anti-mouse IgG
(1:5000; Sigma-Aldrich). The membranes were developed
using an ECL detection system (Pierce, Rockford, IL, USA).
The intensity of bands was determined using the Gel pro 4.0
The Basso Mouse Scale (BMS) for locomotion was used to
assess the degree of motor dysfunction after SCI.28,29 All mice
were gently handled and allowed to walk in an open field to
acclimatize them to the apparatus for several days before
induction of SCI. The mice were tested before injury, to
ensure that they demonstrated equivalent baseline scores. On
each post-SCI day, the mice were observed for 4 min by two
independent observers who were blinded as to the treatment
group. Scores were assigned for each hindlimb and averaged
for each day. The data analyses were performed on
Data presented in the text and figures are expressed as the
mean ? s.d. for at least three experiments. The data were
analyzed using the Student?s t-test. The differences were
considered statistically significant when the P-value was
G-CSF Promoted the Improvement of Neuronal Structure and Locomotor Recovery After SCI
Morphological changes of neurons were observed by crystal
violet staining and electron microscopy near the epicenter at
5 days after spinal cord hemisection. Crystal violet staining of
the dorsal horn area revealed shrunken neurons and fewer
Nissl bodies in the control group as compared with that in
G-CSF-treated group (Figures 1a and b).
Ultrastructural analysis showed clear degeneration within
portions of the myelin sheath with layers of myelin sheath
being separated and isolated from the neuraxis in the injured
group and the neuraxis was swollen. In the G-CSF group, the
myelin sheath was relatively dense with some minor
separation of layers being present within portions of the
myelin sheath (Figure 1c).
To evaluate the effects of G-CSF upon behavioral responses
after acute SCI, BMS scores were assessed at 1?7 days after
spinal cord hemisection. Averages of BMS scores at the
different time points are summarized in Figure 1d. BMS
scores were significantly increased in the G-CSF group versus
those in the injury control group at 5 days after spinal cord
hemisection. BMS scores of the G-CSF group varied around
4, with occasional plantar stepping being observed. At 7 days
after SCI, scores were about 6 with mice in G-CSF group
showing frequent episodes of coordinated plantar stepping.
By contrast, only dorsal stepping and occasional plantar
stepping at 7 days after spinal cord hemisection were present
within the injury control group.
G-CSF Induced Activation of Autophagy After SCI
To examine whether autophagy was activated in response to
SCI, immunohistochemical staining and western blots for
LC3B and electron microscopic analysis in the peri-lesioned
spinal cord tissue were performed at 1, 3, 5 and 7 days after
spinal cord hemisection. Immunohistochemical staining
showed that cells expressing LC3B were increased on the
injured versus contralateral side on each of the days sampled
after spinal cord hemisection (Figures 2a and j).
LC3Bpositive cells were mainly located in the gray matter of the
injured side and diminished as a function of distance from the
injury site. The number of LC3B-positive cells was relatively
higher at 3 and 5 days than at the other time points
(Figures 2a and j). Ultrastructural analysis revealed that
autophagosomes, containing cytoplasmic material and
wrapped by double-membrane structures, were visible in
the damaged neurons at 5 days after SCI (Figure 2k).
To detect whether G-CSF treatment modified autophagy
activation after SCI, western blots for LC3B in the
perilesioned spinal cord tissue were performed at 1, 3, 5 and
7 days after spinal cord hemisection. LC3B expression was
observed at an earlier time point (1 h) in the G-CSF versus
that of the control group (4 h) (data not shown). Levels of
LC3B protein were significantly increased in the G-CSF group
as compared with that in the control group during the first
5 days post-lesion, with maximal differences being observed
at 3 and 5 days after spinal cord hemisection (Figures 2l and
m). Although LC3B expression at 7 days remained increased,
these levels failed to differ significantly from that of the
control group (Figures 2l and m). In this way, G-CSF
treatment appears to advance the onset time for initiating
autophagy and maintains increased levels for longer durations
in the injured spinal cord. Notably, we found that LC3B
expression persisted for 1 month after SCI (data not shown).
These findings suggesting that G-CSF may induce a rapid
onset and prolonged activation of autophagy in response
Autophagy was Present Mainly in Neurons After SCI
To identify LC3B-positive cells within the injured spinal cord,
tissues were double stained with LC3B and MAP2/GFAP at
5 days after SCI. LC3B-positive punctuated dots were sparse
on the injured side of spinal cord (Figure 3a). LC3B was
expressed within neurons co-labeled with the neuronal
marker, MAP2 (Figure 3b). Very few astrocytes expressed
LC3B (Figure 3c).
Autophagy Activation Inhibited Apoptosis After Injury
To examine whether autophagy affected apoptosis after
injury, double staining for LC3B and TUNEL was performed
at 5 days after spinal cord hemisection. Near the site of injury,
elevated amounts of TUNEL-positive and LC3B-positive cells
were detected. Moreover, some TUNEL-positive cells were
also LC3B positive, a finding that was most frequently
observed in G-CSF-treated mice (Figures 4a and d). Notably,
most nuclei of LC3B-positive cells that were TUNEL positive
were round and not shrunken or fragmented, as is typical of
apoptotic nuclei (Figures 4a and d). The expression of LC3B
and morphology of these nuclei suggest that in at least some
of the injured neurons autophagy may have protected these
neurons from apoptosis after injury. In fact, more
TUNELpositive cells were present in the injured mice without G-CSF
treatment than in that of G-CSF-treated mice (Figure 4e).
3-MA Partially Blocks the Neuroprotective Activity of
G-CSF in Cultured Neurons
To investigate the possibility that autophagy is involved in the
neuroprotective effect of G-CSF on neurons, we established a
neuronal mechanical injury model in these cultured neurons.
The specific inhibitor of autophagy, 3-MA, was then added in
the presence of G-CSF in this model to determine whether the
neuroprotective effect of G-CSF was altered after inhibiting
the activation of autophagy. MDC staining revealed that
autophagy was induced in neurons when they were treated
with G-CSF after mechanical injury (Figure 5a). LC3B
immunofluorescence staining indicated that the expression
of LC3B was significantly increased in the G-CSF group as
compared with that of the control group (Figure 5b).
Moreover, we found more TUNEL-positive cells in the
3-MA-treated mechanically injured neurons than that in the
G-CSF group (Figure 5c). These results show that the
neuroprotective activity of G-CSF in injured neurons was
partially blocked after inhibiting autophagy by 3-MA.
NF-?B is Involved in G-CSF-Induced Activation of
Autophagy After Mechanical Injury of Cultured Neurons
We next determined whether the NF-?B signaling pathway
was involved in G-CSF-induced autophagy in neurons in this
mechanical injury model, like that observed in the
cardiomyopathic hamster model.21 Therefore, we examined NF-?B
activation in mechanically injured neurons treated with
G-CSF. Western blot analysis revealed that G-CSF treatment
significantly reduced the expression of NF-?B p65 in neurons
after mechanical injury (Figures 6a and b).
To further determine the role of NF-?B in the SCI-induced
autophagy, we added BAY11-7082 (NF-?B inhibitor) to
neuronal mechanical injury model and examined the LC3B
expression in neurons after mechanical injury. Western blot
results showed that the expression of LC3B was upregulated
after treatment of NF-?B inhibitor (Figures 6c and d).
Compared with the expression of LC3B in G-CSF group,
there was no significant difference. These results show that
inhibiting NF-?B pathway may mimic the autophagy
activation by G-CSF in injured neurons.
After SCI, spinal cord tissue is in a state of stress, which
results in apoptosis of neurons at the injury site. Autophagy is
generally maintained at a low level in cells but is activated to
promote cell survival through the degradation of excess or
damaged organelles and proteins during conditions of stress.
Recently, it has been reported that autophagy has a key role in
injured spinal cord tissue.6,30,31 In this study, we report that
autophagy is activated at the injury site after SCI. LC3B
expression is increased, beginning at 4 h and peaking at 7 days
after SCI. The time course of this LC3B expression is quite
similar to that of apoptosis after SCI. Moreover, we found
that this LC3B expression is upregulated mainly in neurons.
In line with our results are the findings that LC3B is expressed
mainly in neurons after early CNS damage.15,32,33 It is
noteworthy that G-CSF can induce autophagy activation in
the injured spinal cord, which contributes to promote
improvement in neuronal structure and locomotor recovery
after SCI. G-CSF is one example of a hematopoietic growth
factor with a low molecular weight. It can pass through the
blood?brain barrier to enter the CNS.34,35 Moreover,
accumulating evidence has shown that G-CSF also has
important non-hematopoietic functions in the CNS.36,37 We
reported previously that G-CSFR was expressed specifically
within neurons of the spinal cord38 and in this study we find
that autophagy also occurs in these neurons. The induction of
autophagy activation in the injured spinal cord by G-CSF may
begin as early as at 1 h after SCI and this autophagy response
in G-CSF-treated mice is slightly higher than that of the
non-treated animals. One possible explanation for this effect
of G-CSF is an elevation in LC3B levels that reach maximal
values in response to G-CSF treatment. This upregulation of
LC3B may maximize and prolong autophagy activity in
neurons after treatment with G-CSF.
Interestingly, we also find that most nuclei of
LC3Bpositive cells that are TUNEL positive are round and not
shrunken or fragmented, suggesting that in at least some of
these injured neurons autophagy may have an important
protective role by inhibiting apoptosis at this early stage of
SCI, a conclusion supported by Kanno et al.7 The role of
autophagy activation during SCI remains a matter of debate.
Many investigators regard autophagy as exerting a
cytoprotective function that involves a mechanism for recycling
injured cells and reducing damage against cell death.15,39?41
However, others have reported that autophagy mainly has a
destructive role by inducing cell death in CNS injury.42,43 To
address this issue, we treated mice with 3-MA at 10 min
before neuronal mechanical injury. Our results show that
3-MA significantly increases TUNEL-positive neurons at 24 h
after SCI. These results indicate that apoptosis in the injured
neurons is aggravated after inhibiting autophagy by 3-MA.
Therefore, the neuroprotection of G-CSF may, in part, be
related to a rapid onset activation of autophagy.
Most importantly, our results show that G-CSF inhibits
activity of NF-?B signal pathway in neurons after mechanical
injury. Although NF-?B-induced responses are most
commonly associated with immunological and inflammatory
processes, the role of NF-?B in pathological neuronal
functions has not been well established. It has been shown
that SCI can inhibit the production of protective i?B, promote
NF-?B within the nucleus44 and NF-?B activation will in turn
aggravate effects of an injury and neuronal degeneration.45
NF-?B activation is also detected in cerebellar granule cells
after exposure to glutamate.46 Moreover, in these studies an
inhibition of NF-?B activation prevented neuronal cell
death.47 Autophagy is associated with a particular type of
cellular death and has an important role in the degradation of
cytoplasmic constituents in the autophagosomal?lysosomal
pathway.8,9 In this report, we highlight the significance of the
NF-?B family member p65 to control autophagy induced by
G-CSF. Consistent with this postulate are the findings that
NF-?B activation mediates repression of autophagy in tumor
necrosis factor-alpha-treated Ewing sarcoma cells and
inhibition of NF-?B results in an enhancement of ischemia-induced
autophagy.48,49 However, it has also been reported that p65
binds to the promoter of the essential autophagic gene BECN1
and regulates its expression in response to ceramide and
tamoxifen treatment.50 Inhibition of NF-?B leads to decreased
expression of three autophagy-related genes?ATG101, ATG7
and GABARAPL1.51 Taken together, activation of NF-?B and
autophagy represent two processes involved in the regulation
of cell death. Further studies are needed to establish the
possible cross-talk between these two signaling pathways.
A wealth of evidence suggests that initial (within hours)
inflammatory responses contribute to later stages of CNS
injury and result in a worsening of the neurological
outcome.52,53 A rapid induction of autophagy to injury may
contribute to maintenance of local microenvironmental
homeostasis and alleviation of neuronal damage. We
therefore suggest that G-CSF, which induces autophagy and applies
as soon as possible after SCI, would inhibit neuronal
apoptosis and thus provide an effective auxiliary therapeutic
intervention for SCI.
This work is supported by the National Natural Science Foundation of China
(grant no. 81100919); special financial grant from the China postdoctoral
science foundation (2014T70647); postdoctoral science foundation of
Shandong province (201203050); the Scientific Research Foundation for
Returned Scholars, Ministry of Education of China (21300005451001);
Shangdong Province Young and Middle-Aged Scientists Research Awards
Fund (BS2010YY041); Natural Science Foundation of Shandong Province
(2012GSF11842; ZR2011HL070, ZR2015HM030).
DISCLOSURE/CONFLICT OF INTEREST
The authors declare no conflict of interest.
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