Neuroprotection of Botch in experimental intracerebral hemorrhage in rats

Abstract

Notch1 maturation participates in apoptosis and inflammation following intracerebral hemorrhage (ICH). It has been reported that Botch bound to and blocked Notch1 maturation. Here we estimated the role of Botch in ICH-induced secondary brain injury and underlying mechanisms. Experimental ICH model was induced by autologous arterial blood injection in Sprague-Dawley rats, and cultured primary rat cortical neurons were exposed to oxyhemoglobin to mimic ICH in vitro. Specific small interfering RNAs and expression plasmids encoding wild type Botch and Botch with Glu115Ala mutation were exploited. The protein levels of Botch and Notch1 transmembrane intracellular domain (Notch1-TMIC) were increased within brain tissue around hematoma. Botch overexpression led to an increase in unprocessed Notch1 full-length form accompanied by a significant decrease in Notch1-TMIC, while Botch knockdown resulted in an approximately 1.5-fold increase in Notch1-TMIC. There were increased cell apoptosis, necrosis and neurobehavioral deficits after ICH, which was inhibited by Botch overexpression and enhanced by Botch knockdown. Double immunofluorescence showed a colocalization of Botch and Notch1 in the trans-Golgi. Overexpression of wild type Botch, but not Botch E115A mutant, led to an increase in the interaction between Botch and Notch1, reduced the formation and the nuclear localization of Notch1 intracellular domain, and attenuated cell apoptosis and inflammation. In conclusion, Botch exerts neuroprotection against neuronal damage via antagonizing the maturation of Notch1 in Glu115-denpendent manner. However, neuroprotection mediated by endogenous Botch is not enough to reverse ICH-induced secondary brain injury.

Full text

Oncotarget1
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Neuroprotection of Botch in experimental intracerebral
hemorrhage in rats
Binbin Mei1,*, Haiying Li1,*, Juehua Zhu2,*, Junjie Yang3, Ziying Yang3, Zunjia Wen1,
Xiang Li1, Haitao Shen1, Meifen Shen1 and Gang Chen1
1Department of Neurosurgery and Brain and Nerve Research Laboratory, The First Afliated Hospital of Soochow University,
Suzhou, Jiangsu Province, China
2Department of Neurology, The First Afliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
3Institute for Cardiovascular Science and Department of Cardiovascular Surgery of The First Afliated Hospital, Soochow
University, Suzhou, Jiangsu Province, China
*These authors have contributed equally to this work
Correspondence to: Gang Chen, email: nju_neurosurgery@163.com
Meifen Shen, email: meifenshen@suda.edu.cn
Keywords: intracerebral hemorrhage, secondary brain injury, Neuron, Botch, Notch1
Received: July 05, 2017 Accepted: August 04, 2017 Published: August 24, 2017
Copyright: Mei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited.
ABSTRACT
Notch1 maturation participates in apoptosis and inammation following
intracerebral hemorrhage (ICH). It has been reported that Botch bound to and blocked
Notch1 maturation. Here we estimated the role of Botch in ICH-induced secondary
brain injury and underlying mechanisms. Experimental ICH model was induced by
autologous arterial blood injection in Sprague-Dawley rats, and cultured primary
rat cortical neurons were exposed to oxyhemoglobin to mimic ICH in vitro. Specic
small interfering RNAs and expression plasmids encoding wild type Botch and Botch
with Glu115Ala mutation were exploited. The protein levels of Botch and Notch1
transmembrane intracellular domain (Notch1-TMIC) were increased within brain
tissue around hematoma. Botch overexpression led to an increase in unprocessed
Notch1 full-length form accompanied by a signicant decrease in Notch1-TMIC, while
Botch knockdown resulted in an approximately 1.5-fold increase in Notch1-TMIC.
There were increased cell apoptosis, necrosis and neurobehavioral decits after ICH,
which was inhibited by Botch overexpression and enhanced by Botch knockdown.
Double immunouorescence showed a colocalization of Botch and Notch1 in the trans-
Golgi. Overexpression of wild type Botch, but not Botch E115A mutant, led to an
increase in the interaction between Botch and Notch1, reduced the formation and the
nuclear localization of Notch1 intracellular domain, and attenuated cell apoptosis and
inammation. In conclusion, Botch exerts neuroprotection against neuronal damage
via antagonizing the maturation of Notch1 in Glu115-denpendent manner. However,
neuroprotection mediated by endogenous Botch is not enough to reverse ICH-induced
secondary brain injury.
INTRODUCTION
As the second most common and deadliest type of
stroke, intracerebral hemorrhage (ICH) is becoming an
important public health problem [1, 2], which is associated
with severe disability and high mortality. Although there is
signicant progress in clinical treatment, the outcomes of
current treatment strategies for ICH are still not satisfying
[3, 4].
The neuroprotective gene 7 (NPG7) is also known
as Chac, cation transport regulator homolog 1, and
is identied in functional screen for neuroprotective
www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2017

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proteins [5]. Currently, the NPG7 is renamed Botch
(Blocks Notch), which was widely expressed in multiple
organs, including brain. Botch could prevent cell surface
presentation of Notch1 by inhibiting the S1 furin-like
cleavage of Notch1 and maintain Notch1 in the immature
full-length form (Notch1-FL) [6]. The Notch signaling
pathway is an evolutionarily conserved intercellular
signaling pathway in most multicellular organisms
[7]. As a member of Notch signaling pathway, there is
increasing evidence that Notch1 signaling involves in
neuropathological events including inammatory central
nervous system disease, brain and spinal cord trauma,
ischemic stroke and hemorrhagic stroke [8–11]. Immature
Notch1 is processed by cleavage by a furin-type protease
to form a mature heterodimeric receptor in which one
polypeptide becomes divided into the Notch1 extracellular
domain (NECD) and the transmembrane intracellular
domain (TMIC) [12–14]. The TMIC upon ligand binding
to the NECD will undergo S2 and S3 cleavage to generate
intracellular domain (NICD) [15, 16], which could
translocate into the nucleus. In nucleus, NICD converts
the C-promoter binding factor-1 (CBF-1) complex from
a transcriptional repressor to a transcriptional activator
resulting in expression of Notch target genes [16, 17], and
then ultimately participate in differentiation, proliferation,
apoptosis and inammation [18–21].
These ndings suggested that targeting Botch may
provide novel insights into the suppression of the adverse
reactions of Notch1 in neuropathological events. However,
the relationship between Botch and Notch1 and the effect
of Botch in ICH-induced secondary brain injury (SBI)
remain obscure. Therefore, the aim of present study was
to identify the roles of Botch and Notch1 in ICH-induced
SBI in rats and the potential mechanisms, and assess the
therapeutic potential of Botch following ICH.
RESULTS
ICH increased the protein levels of Botch and
Notch1-TMIC in brain tissue around hematoma
An experimental ICH model was established in
Sprague–Dawley (SD) rats (Figure 1A and 1B). To detect
the protein levels of Botch and Notch1in brain tissue
around hematoma after ICH, a time course study was
performed by western blot assay both in vivo and in vitro.
The results demonstrated that the protein levels of Botch
in brain tissue surrounding hematomas was signicantly
increased from 6 h after ICH and reached a peak level
at 48 h (Figure 1C). And the level of Notch1-TMIC
increased with time, peaked at 72 h, while Notch1-FL
was only detected in sham group (Figure 1D). Similarly,
the trend of the protein levels of Botch and Notch1
in cultured primary neurons in vitro was consistent
with that in vivo data (Figure 1E and 1F). Additionally,
double immunouorescence assay further veried the
ICH-induced increase in the protein levels of Botch and
Notch1-TMIC in neurons (Figure 2A and 2B).
Effects of overexpression and knockdown of
Botch on the protein levels of Botch and the
maturation of Notch1 in neurons under ICH
insults both in vivo and in vitro
To identify the effects of Botch on the maturation
of Notch1, overexpression and knockdown of Botch
by plasmid and siRNA transfection were implemented.
The transfection efciency of all the three siRNAs was
veried by western blot analysis (data not shown), and the
most efcient one (siRNA 2) was used in the following
study. Western blot analysis further veried the efciency
of overexpression and knockdown of Botch in neurons
both under normal condition (Figure 3A) and under
OxyHb insults (Figure 3B and 3C). A contrasting result
was appeared in the protein levels of Notch1: Botch
overexpression led to a vastly increase in the levels of
unprocessed Notch1-FL accompanied by a signicant
decrease in processed Notch1-TMIC (Figure 3D). And,
Botch knockdown resulted in an approximately 1.5-fold
increase in Notch1-TMIC (Figure 3E). Consistent with the
in vitro data, the vivo western blot assay showed a similar
trend (Figure 4).
Effects of overexpression and knockdown of
Botch on neuronal injury under ICH insults both
in vivo and in vitro
To evaluate the effects of overexpression and
knockdown of Botch on cell apoptotic and necrosis,
terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) staining, uoro-jade B (FJB) staining
and lactate dehydrogenase (LDH) activity assay were
performed both in vivo and in vitro. Compared with sham/
normal group, a signicant increase in the apoptotic index
was observed in ICH/ OxyHb group, which was attenuated
by Botch overexpression and exacerbated by Botch
knockdown (Figure 5A and 5B). Similarly, FJB-positive
cell in the brain samples signicantly increased in the ICH
group compared with sham group, which was signicantly
decreased by Botch overexpression and increased by
Botch knockdown (Figure 6A). Consistently, LDH activity
assay showed the same trend (Figure 6B).
Effects of overexpression and knockdown of
Botch on neurological behavior neurological
behavior after experimental ICH
To identify the effects of Botch intervention on
neurological behavior, behavioral activity was examined
in all groups. Compared with the sham group, the rats after
induction of ICH showed severe neurological behavior
impairment, which was signicantly alleviated with

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Figure 1: The protein levels of Botch and Notch1-TMIC in brain tissue around hematoma and cultured neurons
following ICH and OxyHb treatment. (A) Representative whole brains and autologous arterial blood injection ICH model. (B)
Schematic representation of the suitable region taken for assay. (C, D) Western blot analysis and quantication of the protein levels of Botch
and Notch1 in brain tissue around hematoma. (E, F) Western blots analysis and quantication of the protein levels of Botch and Notch1 in
cultured neurons. In (C-F), mean values for sham or normal group were normalized to 1.0. (C) N.S. indicates no signicant difference, p =
0.1609, 3 h vs sham; * p = 0.0174, 6 h vs sham; *p = 0.0113, 12 h vs sham; **p = 0.0036, 24 h vs sham; ***p < 0.001, 48 h vs sham; **p
= 0.0088, 72 h vs sham; #p = 0.0157, 48 h vs 24 h; #p = 0.0142, 48 h vs 72 h. (D) N.S. indicates no signicant difference, p = 0.2497, 3h
vs sham; *p = 0.0141, 6 h vs sham; *p = 0.0106, 12 h vs sham; **p = 0.0046, 24 h vs sham; **p = 0.0018, 48 h vs sham; ***p < 0.001, 72
h vs sham; #p = 0.0141,72 h vs 48 h. (E) **p = 0.0019, 24h vs normal; ***p < 0.001, 48 h vs normal; **p = 0.0013, 72 h vs normal; ##p
= 0.0065, 48 h vs 24 h; #p = 0.0248, 48 h vs 72 h. (F) **p = 0.0047, 24 h vs normal; **p = 0.0011, 48 h vs normal; **p = 0.0018, 72 h vs
normal; #p = 0.0484, 72 h vs 48 h.

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Botch overexpression treatment, yet more pronounced
neurological defect was observed in Botch knockdown
treatment group (Figure 6C).
Botch antagonized the cleavage and maturation
of Notch1 in Glu115 dependent manner
To elucidate the underlying mechanisms of Botch-
induced neuroprotection after ICH, expression vectors
encoding wild type Botch and Botch with E115A mutation
were prepared, and the immunouorescence and co-
immunoprecipitation (Co-IP) assay were performed.
The vitro multiple immunouorescence assay found that
Botch and Notch1 colocalized within the trans-Golgi
(Figure 7A). The in vivo co-immunoprecipitation (Co-IP)
experiment showed that there was an interaction between
wild type Botch and Notch1-FL, but not Notch-TMIC.
Notably, E115A mutant almost completely inhibit the
interaction between Botch and Notch1-FL (Figure 7B).
Immunouorescence assay was implemented
to describe the effects of Botch overexpression on the
release of the NICD. Results showed that more NICD
was released and translocated to the nucleus in the
control group compared with the normal group, and wild
type GFP-Botch overexpression decreased the levels of
NICD in the nucleus, whereas E115A mutant GFP-Botch
overexpression did not have the effect (Figure 7C).
Effects of wide type Botch and mutant Botch
overexpression on apoptosis and inammation in
cultured neurons exposed to OxyHb
To further ascertain the effects of wide type Botch
and mutant Botch overexpression on apoptosis and
inammation, the protein levels of cleaved caspase-3 in
neurons and the concentrations of IL-6, IL-1β and TNF-α
in cell culture supernatant were detected by western
blot analysis and enzyme-linked immunosorbent assay
(ELISA), respectively. As shown in Figure 8A, wild
type GFP-Botch overexpression suppressed the OxyHb-
induced caspase-3 activation, however, this effect was
abolished by E115A mutant. And we found that there
were increased concentrations of IL-6, IL-1β and TNF-α
in the culture supernatant of OxyHb group compared to
normal group, which was signicantly reduced by wild
type GFP-Botch overexpression. However, GFP-Botch-
E115A mutation overexpression did not have this effect
(Figure 8B).
Figure 2: The protein levels of Botch and Notch1 in neurons around hematoma following ICH treatment. (A, B) Double
immunouorescence analysis was performed with Botch/Notch1 antibodies (green) and neuronal marker (NeuN, red), and nuclei were
uorescently labeled with DAPI (blue). Arrows point to Botch/Notch1-positive neurons, scale bar = 100 μm. The relative uorescent
intensity of Botch/Notch1 in neurons was quantied. In (A, B), mean values for sham group were normalized to 1.0. (A) **p = 0.0046, 48
h vs sham. (b) **p = 0.0025, 72 h vs sham.

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Figure 3: Effects of overexpression and knockdown of Botch on the protein levels of Botch and the maturation of
Notch1 in cultured neurons. (A) Transfection efciency of Botch expression plasmid and siRNA in cultured neurons under normal
condition. (B, C) Transfection efciency of Botch expression plasmid and siRNA in cultured neurons exposed to OxyHb. (D, E) Effects of
overexpression and knockdown of Botch on the protein levels of Notch1 in neurons under OxyHb insults. In (A-E), mean values for normal
group were normalized to 1.0. (A) ## p = 0.0022 vs. vector; && p = 0.0024 vs. ctr-siRNA. (B) **p = 0.0090 vs normal; # p = 0.0109 vs
OxyHb + vector. (C) **p = 0.0011 vs normal; N.S. indicates no signicant difference; ##p = 0.0015 vs OxyHb + ctr-siRNA. (D) **p =
0.0019 vs normal; ## p = 0.0032 vs OxyHb + vector. (E) **p = 0.0017 vs normal; ## p=0.0021 vs OxyHb + ctr-siRNA.

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Figure 4: Effects of overexpression and knockdown of Botch on the protein levels of Botch and the maturation of
Notch1 in brain tissue around hematoma. (A) Transfection efciency of Botch expression plasmid and siRNA in rat brains. (B, C)
Transfection efciency of Botch expression plasmid and siRNA in brains of ICH rats. (D, E) Effects of overexpression and knockdown of
Botch on the protein levels of Notch1 in brain tissue under ICH insults. In (A-E), mean values for sham group were normalized to 1.0. (A)
# p = 0.0202 vs vector, && p = 0.0017 vs ctr-siRNA. (B) **p = 0.0039 vs sham, # p = 0.0128 vs ICH + vector. (C) *p = 0.0168 vs sham;
# p = 0.0305 vs ICH + ctr-siRNA. (D) **p = 0.0050 vs sham; ## p = 0.0023 vs ICH + vector. (E) **p = 0.0033 vs sham; ## p = 0.0032 vs
ICH + ctr-siRNA.

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DISCUSSION
Our present study showed the roles of Botch and
Notch1 in the pathophysiological progression of SBI in
a rat ICH model. First, results showed elevated levels of
Botch and Notch1-TMIC in brain tissue around hematoma
after ICH and cultured neuron exposed to OxyHb, while
Notch1-FL was only detected in sham group. As the
transmembrane and intracellular domain, Notch1-TMIC is
a S1-cleaved mature form of Notch1 and is the dominant
form of Notch1 [6]. Under ICH condition, the increased
Notch1-TMIC and undetected Notch1-FL implied that
Notch1 may be involved in the pathological process of
ICH-induced SBI. Furthermore, there was an interaction
between Botch and Notch1-FL, which could inhibit
Notch1 mature in an E115-independent manner.
As previously reported [6, 22], Botch blocks Notch1
signaling through inhibition of the furin-like cleavage
of Notch1 via its γ-glutamyl cyclotransferase (GGCT)-
like activity, whereas Botch-E115A did not have this
activity. So, E115 might be a key amino acid for Botch
enzymatic function. As shown in Figure 7, Botch and
Notch1 colocalized within the trans-Golgi, where Notch
is processed by S1 cleavage by a furin-like protease
[23]. Markedly, under ICH condition, wild type Botch
interacted with the Notch1-FL, but not the Notch1-TMIC,
while Botch E115A mutant lost this effect. The result was
consistent with the previous research [6].
Figure 5: Effects of overexpression and knockdown of Botch on apoptosis in brain tissue around hematoma and
cultured neurons exposed to OxyHb. (A) Double staining for TUNEL (green) and DAPI (blue) in vivo. Arrows point to TUNEL-
positive cells. Scale bar = 60 μm. Percentage of TUNEL-positive cells was shown. **p = 0.0058 vs sham; ## p = 0.0060 vs ICH + vector;
& p = 0.0110 vs ICH + ctr-siRNA. (B) Double staining for TUNEL (green) and DAPI (blue) in vitro. Arrows point to TUNEL-positive cells
in cultured neurons. Scale bar = 100 μm. Percentage of TUNEL-positive cells in cultured neurons was shown. **p = 0.0012 vs normal; #
p=0.0153 vs OxyHb + vector; && p = 0.0052 vs OxyHb + ctr-siRNA.

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A series of secondary cascade of injury following
ICH are referred to as SBI, the molecular and cellular
mechanisms underlying the SBI are complicated,
apoptosis and inammation might play a momentous
role in SBI pathology [19, 24, 25]. The activation of
Notch1 results in the sequential proteolytic cleavage
of the Notch1 receptor, which releases NICD into the
nucleus. NICD in turn activates downstream gene
transcription, eventually participates in the pathologic
progress of SBI after ICH [26]. In our study, wild
type Botch overexpression reduced the release and
translocate to the nucleus of NICD, and attenuated cell
apoptosis and inammation, however, which was not
statistically signicant change with E115A mutant Botch
overexpression. Noticeably, the variation tendency of
NICD was consistent with inammatory and apoptosis
index, indicating further that the maturation of Notch1
participates in ICH-induced SBI, while Botch confers
neuroprotection against SBI. Taken together, we further
demonstrated that E115 was required for Botch to
interferes with Notch1 maturation by interacting with
Notch1.
The inactive form of Notch1, before it reaching
the plasma membrane, is rstly cleaved by a furin-like
convertase in the trans-Golgi network to yield an active,
ligand-accessible form. Ultimately, multiple cleavage
processes result in the release and translocate to the nucleus
of NICD, which subsequently activates transcription
of downstream target genes and eventually involved in
promoting cell apoptosis and inammation following ICH.
The cleavage and creation of the heterodimeric form of
Notch1 occur in the Golgi apparatus, where Notch1 and
Botch interact. Botch physical binds to the full-length
unprocessed NECD interferes with the S1 furin-like
cleavage of Notch1, whereas Botch-E115A is devoid of
activity. Overall, as the negative regulator of ICH-induced
SBI, Botch acts by maintaining Notch in an immature
inactive form, which is dependent on the critical site E115.
The mechanisms map is shown in Figure 9.
This study focused on the Botch and its possible
role in neuroprotection following ICH. Botch, NPG7,
was identied in functional screen for neuroprotective
proteins, and there are previous studies on the correlation
between Botch and the Notch1 signaling pathway
Figure 6: Effects of overexpression and knockdown of Botch on cell necrosis and neurological behavior under ICH
and OxyHb insults. (A) FJB staining showing the effects of Botch intervention on neuronal necrosis. Arrows point to FJB-positive cells
in the brain. Scale bar = 60 μm. Quantication of the FJB staining as shown. **p = 0.0020 vs sham; #p = 0.0286 vs ICH + vector; && p =
0.0015 vs ICH + ctr-siRNA. (B) LDH assay of cell culture supernatants. **p = 0.0027 vs normal; ## p = 0.0071 vs OxyHb + vector; &p =
0.0136 vs OxyHb + ctr-siRNA. (C) Neurological behavior scores. *** p < 0.0001; ### p = 0.0004; && p = 0.0013.

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[6, 22]. It is generally accepted that Notch1 plays a
critical role in many fundamental processes and in
a wide range of tissues, and it is not surprising that
aberrant gain or loss of Notch1 signaling components
has been directly linked to differentiation, proliferation,
inammation and apoptosis [13, 18–21, 27]. Yet, none
of these studies have elucidated whether Botch could
involve in the brain protection against ICH-induced SBI
through its suppression effects of Notch1. In the current
study, we found that Botch-induced neuroprotection
was associated with the inhibition of Notch1 signaling
and was dependent on the critical site E115. However,
endogenous Botch was inadequate to completely reverse
ICH-induced neuronal damage.
The current study has some limitations. In our
experiment, we used healthy adult male SD rats, which did
not mimic human high-risk populations maximally, such as
patients with cardiovascular diseases and the elderly. And,
different sex animals will be tested in our further study.
Besides, the time course study showed that there was a
difference in the peak time of protein levels of Botch and
Notch1. We chose 48 h for following researches. However,
whether there is other appropriate time to implement the
intervention requires further investigation. Additionally, the
remarkable rescue effect of Botch overexpression suggests
a powerful therapeutic potential of Botch, however, how to
better regulate the interaction between Botch and Notch1 is
not answered and need to explore further.
Figure 7: Botch antagonized the cleavage and maturation of Notch1 in Glu115 dependent manner. (A) Multiple
immunouorescence for GFP-Botch (green) and Notch1 (red) counterstained with Golgi (purple) was performed under indicated treatment
in vitro. Nuclei were uorescently labeled with DAPI (blue). Arrows point to the colocations of Botch and Notch1 in Golgi. (B) Co-IP
analysis of the interaction between GFP-Botch and Notch1 in vivo. (C) Immunouorescence analysis was performed with NICD antibodies
(green), and nuclei were uorescently labeled with DAPI (blue). Scale bar = 20 μm. The relative uorescent intensity of NICD in nucleus
was shown. ***p < 0.001 vs normal; # p = 0.0117 vs OxyHb + vector; N.S. indicates no signicant difference vs OxyHb + vector; && p
= 0.0040.

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Figure 8: Effects of wide type Botch and mutant Botch overexpression on the cell apoptosis and inammation in
cultured neurons. (A) The protein levels of cleaved caspase-3 were assessed by western blot analysis. ***p < 0.001 vs normal; ## p =
0.0064 vs OxyHb + vector; N.S. indicates no signicant difference vs OxyHb + vector; && p = 0.0023, OxyHb + WT vs OxyHb + MT. (B)
Concentrations of pro-inammatory cytokines (IL-6, IL-1β and TNF-α) in cell culture supernatants were assayed by ELISA. IL-6: ***p <
0.001 vs normal; ## p = 0.0019 vs OxyHb + vector; N.S. indicates no signicant difference vs OxyHb + vector; && p = 0.0037, OxyHb +
WT vs OxyHb + MT. IL-1β: ***p < 0.001 vs normal; ## p = 0.0047 vs OxyHb + vector; N.S. indicates no signicant difference vs OxyHb
+ vector; & p = 0.0271, OxyHb + WT vs OxyHb + MT. TNF-α: ***p < 0.001 vs normal; # p = 0.0119 vs OxyHb + vector; N.S. indicates
no signicant difference vs. OxyHb + vector; && p = 0.0031, OxyHb + WT vs OxyHb + MT.
Figure 9: Schematic representations of the roles of Botch and Notch1 in SBI following ICH and the underlying
mechanisms. Briey, Botch could signicantly reduce ICH-induced SBI by effectively antagonizing Notch1, maintaining Notch1 in the
immature full-length.

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In summary, this study showed a role of Botch in
inducing neuroprotection against ICH-induced SBI for
the rst time, which is mediated through antagonize the
cleavage and maturation of Notch1 in Glu115-dependent
manner. And the remarkable alleviation in the cell apoptosis
and inammation after Botch overexpression, suggesting
that therapies targeting Notch1 hold signicant promise
for the treatment and prevention of pathologic processes
characterized by ICH-induced brain injury, and Botch might
be good target for improving SBI after ICH. However,
neuroprotection mediated by endogenous Botch is not
enough to reverse ICH-induced secondary brain injury.
MATERIALS AND METHODS
Animals
The experiments were approved by the Ethics
Committee of the First Afliated Hospital of Soochow
University and were implemented in strict accordance with
the guidelines of the National Institutes of Health on the
care and use of animals. All adult male SD rats weighing
between 250 and 300 g were purchased from Animal
Center of Chinese Academy of Sciences, Shanghai, China.
The rats were housed in temperature- and humidity-
controlled animal quarters with a 12-h light/dark cycle.
Rat ICH model
Experimental ICH model was a modication of
the method as described previously [28, 29]. Firstly, rats
were anesthetized with 4% chloral hydrate (10 ml/kg,
Intraperitoneal injection), and xed in the prone position
to a stereotactic frame (Shanghai Ruanlong Science and
Technology Development Co., Ltd., Shanghai, China).
Rats were positioned with the anterior and posterior
fontanelles in the same horizontal plane. The median
scalp was shaved and sterilized, and a median incision
approximately 1 cm long was made. The periosteum was
stripped using a bone stripper, and the anterior fontanelle
and coronal suture exposed. A round hole (1.0 mm
diameter) was drilled using a dental drill (3.5 mm right
and 0.2 mm posterior to bregma) until the dural surface
was reached. 80 ul non-heparinized autologous arterial
blood were collected by cardiac puncture and slowly
injection at a rate of 16ul/min using a microinjector xed
on the stereotaxic apparatus, which entered vertically
approximately 5.5 mm along the hole, and maintained
in place for 5 min. Finally, the needle was removed and
the scalp sutured. A schematic representation of the brain
coronal sections for assay was shown in Figure 1A and 1B.
Cell culture
Primary rat cortical neurons were obtained and
cultured as described previously [30]. Briey, cortical
neurons were isolated from E16-18 rat embryos, and
treated with Trypsin-EDTA Solution for 5 min at 37°C.
Dissociated neurons were plated onto plates (Corning,
USA) precoated with 0.1 mg/ml poly-D-lysine (Sigma,
USA), cultured in Neurobasal-A medium supplemented
with 2% B-27 and 0.5 mM GlutaMAX™-I (all from
Invitrogen, Grand Island, NY), and maintained at
37°C under humidied conditions and 5% CO2 for
approximately 2 weeks. Half of the media were exchanged
for fresh media every two days.
Experimental design
The total experiments were composed of two
sections, and each part all included both in vivo and in vitro
experiments. In experiment 1, 42 rats (50 rats were used,
42 rats were survived after the surgery) were randomly
assigned to 7 groups of 6 rats each, a sham group, and 6
experimental groups arranged by time: 3, 6, 12, 24, 48 and
72 h after ICH. Then brain tissue samples were obtained
separately from rats for the time course study for the
protein levels of the Botch and Notch1. Then, to simulate
ICH in vitro, enriched neurons were divided into 4 groups:
normal, 24, 48, 72 h after exposed to OxyHb.
Based on the results of the time course study, 48h
after ICH or OxyHb treatment was exploited in experiment
2. First, another 84 rats were randomly divided into 7
groups: sham, ICH, ICH + vector, ICH + wild type Botch
overexpression (WT), ICH + Botch E115A overexpression
(MT), ICH + control-siRNA (ctr-siRNA), ICH + siRNA-
Botch. The brain cortex of rats was extracted for TUNEL
staining, FJB staining, Co-IP and western blot analysis for
the roles of Botch in ICH-induced SBI and the potential
mechanisms. Similarly, to explore the roles of Botch in
vitro, neurons were divided into 7 groups: normal, OxyHb
+ control, OxyHb + vector, OxyHb + Botch-WT, OxyHb
+ Botch-MT, OxyHb + ctr-siRNA, OxyHb + siRNA-
Botch. After these treatments, the total protein of the cells
was collected and stored at -80 °C until tested, and cells
for immunouorescence analysis were xed with 4 %
paraformaldehyde.
Antibodies
Notch1 antibody (ab27526), Chac1 antibody
(ab76386), Rb pAb to activated Notch1 (ab52301), Rb
mAb to NeuN (ab177487), Ms mAb to NeuN (ab104224)
were from Abcam. β-tubulin (sc-9014), TGN 38 (sc-
27680), normal rabbit IgG (sc-2027), normal mouse IgG
(sc-2025) and normal goat IgG (sc-2028) were from Santa
Cruz Biotechnology. Anti-Botch1/Chac1 (75-181) was
from Neuromab. Cleaved caspase-3 (D175) was from
Cell Signaling Technology. Protein A+G Agarose (P2012)
was from Beyotime. Secondary antibodies for western blot
analysis, including goat anti-rabbit IgG-HRP (sc-2004),
donkey anti-goat IgG-HRP (sc-2020), and goat anti-mouse

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IgG-HRP(sc-2005) were from Santa Cruz Biotechnology.
Secondary antibodies for immunouorescence, including
Alexa Fluor-488 donkey anti-rabbit IgG antibody
(A21206), Alexa Fluor-488 donkey anti-mouse IgG
antibody (A21202), Alexa Fluor-555 donkey anti-mouse
IgG antibody (A31570), Alexa Fluor-555 donkey anti-
rabbit IgG antibody (A31572), and Alexa Fluor-633
donkey anti-goat IgG antibody (A21082) were from life
technologies.
Construction of expression plasmids and site
directed mutagenesis
Specic expression plasmid of Botch was obtained
from Ribobio. For immunouorescence analysis, the
coding region of rat Botch cDNA was sub-cloned into
pEGFP-N2 expression vector to produce the pEGFPN2-
Botch construct. In addition, a rat Botch cDNA construct
with a mutation in a possible key site (Glu115) was
prepared. E115A mutant (Glu115 of Botch was mutated
to Ala) was also sub-cloned into the pEGFP-N2
expression vector as the wild-type Botch cDNA, which
allowed us to measure their location by uorescence
assay. All of the constructs were conrmed by DNA
sequencing.
Plasmid transfection in rat brain
The vivo plasmid transfection in rat brain was
performed according to the manufacturer’s instructions
for Entranster-in vivo DNA transfection reagent (Engreen,
18668-11-2). Firstly, 10 μL Entranster-in vivo DNA
transfection reagent was added to 5 μL plasmid or 5 μL
empty vector immediately. The solution was mixed for 15
min at room temperature. Finally, 15 μL Entranster-in vivo-
plasmid mixture was injected intracerebroventricularly at
48 h before ICH.
Transfection of siRNA in rat brain
According to the manufacturer’s instruction for
Entranster-in vivo RNA transfection reagent (Engreen,
18668-11-1), the transfection complex of siRNA was
prepared as follows. Briey, 5 nmol Botch siRNA and
5 nmol scramble siRNA were respectively dissolved in
66.5 μL DEPC RNase-free water. Then, 5 μL Entranster-
in vivo RNA transfection reagent and 5 ul normal saline
were added to 10 μL siRNA or 10 μL scramble siRNA
immediately. The solution was mixed for 15 min at room
temperature. Finally, 20 μL Entranster-in vivo-siRNA
mixture was injected intracerebroventricularly at 48 h
before ICH.
Specic siRNAs against Botch were obtained
from Ribobio. The knockdown efciency of all the
three siRNAs was separately tested by vitro western blot
analysis. The most efcient one was used in the following
study.
Botch siRNA sequences:
1. Sense: 5′GAGAGAAGCUGUGCUUGGU dTdT 3′
Antisense: 3′dTdT CUCUCUUCGACACGAACCA 5′
2. Sense: 5′CACUGAAGUACCUGAACGU dTdT 3′
Antisense: 3′dTdT GUGACUUCAUGGACUUGCA 5′
3. Sense: 5′CUAAGGAAGUCACCUUUUA dTdT 3′
Antisense: 3′dTdT GAUUCCUUCAGUGGAAAAU 5′
Transfection of plasmid and siRNA in cultured
neurons
Cultured neurons were transfected with indicated
expression vectors using Lipofectamine® 3000
Transfection Reagent (Invitrogen, L3000-015) or siRNAs
using Lipofectamine RNAi MAX (Invitrogen, 13778-075)
according to the manufacturer’s instructions. At 48 h after
transfection, neurons were treated with OxyHb for an
additional 48 h. Then neurons were harvested for further
analysis.
Neurological impairment
The effects of Botch intervention on behavioral
impairment were examined using a previously published
scoring system and monitored for appetite, activity, and
neurological defects [31].
Western blot analysis
In vivo, cortices were sampled 1 mm away from the
hematoma to avoid potential red blood cell contamination.
The brain samples and cells were collected and lysed in
ice-cold RIPA lysis buffer (P0013; Beyotime, Shanghai,
China). After centrifuge at 12,000 g for 10 min at 4°C,
the supernatants were collected. The protein concentration
was measured using the bicinchoninic acid kit (Beyotime,
P0010). The protein samples were loaded on SDS
polyacrylamide gel, separated, and electrophoretically
transferred to a polyvinylidene diuoride membrane
(IPVH00010; Millipore, Billerica, MA, USA). The
membrane was blocked with 5% non-fat milk for 2 h
at room temperature. Subsequently, the membrane was
probed with the primary antibody against Botch, Notch1,
and cleaved caspase-3 overnight at 4°C. The β- tubulin
was used as a loading control. Then they were incubated
in the appropriate HRP-conjugated secondary antibodies
for 1.5 h at room temperature and washed with PBST.
Finally, the protein bands were visualized using enhanced
chemiluminescence. The relative quantity of proteins
was analyzed using Image J and normalized to that of the
loading control.
Immunouorescence analysis
The vivo immunouorescence analysis was
prepared as follows. Briey, the brain samples were xed
in 4% paraformaldehyde, embedded in parafn, cut into 4

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μm sections which were stained with primary antibodies
(diluted 1:100) and appropriate secondary antibodies
(1:500dilution). Normal rabbit IgG or normal mouse IgG
was used as a negative control (data not shown). Finally,
sections were observed by a uorescence microscope
(OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Japan),
and the relative uorescence intensity was analyzed by use
of Image J program.
The in vitro multiple labeling immunouorescence
analysis was also performed. In brief, cells were xed in 4
% paraformaldehyde for 15 min at room temperature and
blocked in 5 % bovine serum albumin (Biosharp, Hefei,
China) for 30 min, then incubated with primary antibody
overnight at 4 °C. Then, cells were washed with PBST for
three times and incubated with another antibody overnight
at 4 °C. Repeated the above process until the total primary
antibodies all used. Finally, cells were washed with PBST
and incubated with corresponding appropriate secondary
antibodies at 37 °C for 30-60 min. The uorescence
images were captured using a laser scanning confocal
microscope (ZEISS LSM 880, Carl Zeiss AG, Germany).
TUNEL staining
Cell apoptosis in brain tissue was detected by
TUNEL staining according to the manufacturer’s protocol
(DeadEnd Flurometric kit, Promega, WI, USA). Briey,
brain tissues were parafn embedded and sectioned, and
then heated and dewaxed. After dewaxed, the sections
were incubated in Triton X-100 for 8 min. Then the
sections were washed 3 times with PBS (5 min per wash),
and then incubated with TUNEL-staining at 37 °C for
1 h. Nuclei were stained with DAPI (Southern Biotech,
Birmingham, AL, U.S.) mounting medium after washed
3 times with PBS at room temperature. Finally, the
sections were visualized by a uorescence microscope
(OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Japan.).
To explore the extent of cell apoptosis, the apoptotic index
was dened as the percentage of TUNEL-positive cells
in each section. Cell apoptosis in enriched neurons was
also examined by TUNEL staining and the procedure was
similar to the above process, excluding the neurons was
xed in 4% paraformaldehyd, and incubated in Triton
X-100 for 2 min.
FJB staining
Cell necrosis in brain tissue was detected by FJB
staining. Brain sections were deparafnized, dehydrated,
incubated in 0.06% K permanganate for 10 min, then
rinsed in deionized water and immersed in FJB working
solution (0.1% acetic acid) for 20 min and dried in an
incubator (50-60 °C) for 10 min. Sections were cleared
in xylene and cover slipped with a non-aqueous, low-
uorescence, styrene-based mounting medium(DPX,
Sigma-Aldrich, MO, U.S.). The sections were visualized
by a uorescence microscope (OLYMPUS BX50/BX-
FLA/DP70; Olympus Co., Japan). The number of FJB-
positive cells in each section was calculated carefully per
sample, and cell counts from the section were averaged to
provide the mean value.
Assay of LDH activity
The level of LDH in cell culture supernatant was
quantied using LDH kit following the manufacturers’
instruction (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China). Firstly, the reaction wells included
standard wells, sample wells, control wells and empty
wells were created, and enough reagents for the number
of assays were prepared. Then the reaction mix was
performed and added into the relative groups. Finally, the
activity of LDH was measured immediately at OD 450 nm
on a microplate reader.
Co-immunoprecipitation (Co-IP) analysis
Co-IP analysis was performed as described
previously [32]. Firstly, the brain samples were lysed
in the ice-cold RIPA lysis buffer. And the lysate was
incubated with specic antibodies or normal IgG (negative
control) for 1 h at 4 °C with agitation. Then protein A+G
agarose beads were added to each immune complex and
the lysate-bead mixture was incubated overnight at 4°C
with rotary agitation. SDS-PAGE and immunoblotting
were then performed for further protein separation and
detection.
Assay of inammatory cytokines
The levels ofIL-6, IL-1β and TNF-α (Cloud-
Clone Corp.) in cell culture supernatant were quantied
using specic ELISA kits for rats according to the
manufacturers’ instructions. Firstly, determined wells for
diluted standard, blank and sample, and standard, blank
and samples were added to the plates and incubated for 1
h at 37 °C. Next, detection reagent was added to the each
well and incubated for 30min, followed by 90ul substrate
solution. Then reaction was stopped by adding 50ul of stop
solution. Finally, ran the microplate reader and conducted
measurement at 450 nm immediately.
Statistical analysis
Graphpad prism 6 was used for all statistical
analysis. Neurobehavioral scores were shown as median
with interquartile range. Frequency distribution for the
neurobehavioral score assay. The Mann-Whitney U test
was used to compare behavior and activity scores among
groups.
All the other data are presented as mean ± SD.
Data were analyzed by one-way ANOVA followed by

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either a Dunnett’s or a Sidak’s post hoc test, the former
for comparisons to a single control group, the latter for
comparisons with the preselected pairs of groups, P< 0.05
was considered statistically signicant.
Abbreviations
ICH, intracerebral hemorrhage; SBI, secondary
brain injury; SD, Sprague-Dawley; OxyHb,
oxyhemoglobin; Notch1-FL, Notch1 full-length form;
NECD, Notch1 extracellular domain; Noch1-TMIC,
Notch1 transmembrane intracellular domain; NICD,
Notch1 intracellular domain; NPG7, neuroprotective
gene 7; CBF-1, C-promoter binding factor-1; TUNEL,
Terminal Deoxynucleotidyl Transferase dUTP Nick
End Labeling; FJB, Fluoro-Jade B; LDH, Lactate
Dehydrogenase; GGCT, γ-glutamyl cyclotransferase. Co-
IP: Co-immunoprecipitation; WT: wide type GFP-Botch;
MT: E115A mutant GFP-Botch.
Author contributions
G.C. and M.S. conceived and designed the study,
including quality assurance and control. B.M., H.L. and
J.Z. performed the experiments and wrote the paper. J.Y.,
Z.Y. and Z.W. designed the study’s analytic strategy. H.S.
and X.L. helped conduct the literature review and prepare
the materials and methods section of the text. All authors
read and approved the manuscript.
ACKNOWLEDGMENTS
This work was supported by supported by the
Project of Jiangsu Provincial Medical Innovation Team
(CXTDA2017003), Jiangsu Provincial Medical Youth
Talent (QNRC2016728), Suzhou Key Medical Center
(Szzx201501), grant from the National Natural Science
Foundation of China (No. 81601011), the Natural Science
Foundation of Jiangsu Province (No. BK20160345,
BK20170363) Scientic Department of Jiangsu Province
(No. BL2014045), Suzhou Government (No. SYS201608,
and LCZX201601), Jiangsu Province (No. 16KJB320008).
CONFLICTS OF INTEREST
The authors declare no conicts of interest.
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