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Project Details
Project Number :
777611
Molecular regulations of reactive nitrogen species
inhibitors for preventing thrombolysis-induced blood
Project Title(English) :
brain barrier disruption and hemorrhagic
transformation in experimental cererbal ischemia-
reperfersion models 
活性氮自由基抑制劑對缺血性中風溶栓治療腦出血並
Project Title(Chinese) :
發症的分子調節研究 
Principal Investigator(English) :
Prof Shen, Jiangang 
Principal Investigator(Chinese) :
Department :
School of Chinese Medicine
Institution :
The University of Hong Kong
E-mail Address :
[email address] 
Tel :
25890439 
Co - Investigator(s) :
Prof Cheung, Raymond Tak Fai
Prof Chung, Sookja Kim
Prof Liu, Ke Jian
Dr Siu, Chung Wah
Prof Yang, Dan
Panel :
Biology & Medicine
Subject Area :
Medicine, Dentistry & Health
Exercise Year :
2011 / 12
Fund Approved :
890,000
Project Status :
Completed
Completion Date :
30-9-2014
Project Objectives :
Objective 1: To test the hypothesis that RNS
inhibitors can alleviate t-PA-induced MMPs
activations, BBB disruption and hemorrhagic
transformation in cerebral ischemia-reperfusion
injury
Objective 2: To test the hypothesis that cav-1 is a
critical target protein of the RNS inhibitors on
reducing the BBB disruption and hemorrhagic
transformation in the experimental ischemic stroke
model with thrombolytic treatment
Objective 3: To test the effects of a natural
antioxidant on the production of RNS, the
expression of cav-1, the activations of MMPs, the
BBB permeability and hemorrhagic transformation
in the experimental ischemic stroke model with the
delayed t-PA treatment.
1 of 10

Abstract as per original application
Stroke is the leading cause of disability and the
(English/Chinese):
secondary killer in human diseases worldwide.
Tissue-plasminogen activator (t-PA) is the only
FDA-approved thrombolytic drug for acute ischemic
stroke. Early recanalization after t-PA infusion can
improve patient outcome. However, systemic
thrombolysis with t-PA administered beyond the 3
hour therapeutic window after ischemic stroke carries
a potential risk of hemorrhagic transformation and
potentiates neuronal damage. The activation of
matrix metalloproteinases (MMPs), a proteolytic
zinc-containing enzyme family, and the induction of
blood brain barrier (BBB) hyper-permeability are
critical pathological processes in hemorrhagic
transformation during thrombolytic treatment for
acute ischemic stroke. Rebuilding circulation by
thrombolysis is accompanied by cerebral ischemia-
reperfusion injury. Ischemia-reperfusion produces
free radicals including reactive oxygen species
(ROS) and reactive nitrogen species (RNS). As the
major components of RNS, nitric oxide (NO) and
peroxynitrite (ONOO-) can activate MMPs, attack
tight junction-associated proteins and increase BBB
permeability. Since RNS are simultaneously present
in the t-PA-treated ischemic brains, it is important to
address the questions whether the MMPs activations,
the BBB disruption and hemorrhagic transformation
are due to the actions of t-PA itself, RNS or the
synergistic effects of RNS and t-PA, and how the
MMPs activations and related molecular cascades
are induced in the process. Our studies revealed that
caveolin-1, a 22 kDa integral membrane protein, can
inhibit RNS production, MMPs activations and BBB
disruption, whereas over-production of NO from the
ischemic brains can down-regulate caveolin-1,
activate MMPs and increase BBB permeability.
Several synthesized and natural RNS inhibitors
revealed to inhibit the MMPs activations and reduce
BBB permeability in an experimental ischemic stroke
animal model with t-PA treatment. Following this line,
we will test the hypothesis that the RNS inhibitors
can up-regulate caveolin-1 expression, inhibit MMPs
activations reduce BBB permeability, and prevent
hemorrhagic transformation in t-PA treated ischemic
stroke animal model. If the hypothesis can be
validated, we logically expect that the antioxidant
therapy for inhibiting RNS production can be used to
reduce the risk of hemorrhagic transformation and
extend the therapeutic time window of t-PA for acute
ischemic stroke. Subsequently, the antioxidant
therapy can be an add-on therapeutic strategy with
t-PA treatment for acute ischemic stroke patients. As
t-PA is the only thrombolytic drug clinically used for
acute ischemic stroke patients and antioxidants are
commonly used healthy supplements, the study will
highlight their therapeutic value for stroke treatment.
Therefore, the study will be highly innovative and has
a multitude of application potentials.
2 of 10

中風是人類疾病的第二位殺手,t-PA是FDA批准的唯
一溶栓劑,用於缺血性中風治療。早期溶栓能提高病
人的治療效果,而中風3小時後的溶栓治療存在腦出血
的危險性。研究表明,髓質金屬蛋白酶(MMP)的活
化以及血腦屏障高通透性是溶栓治療腦出血的重要環
節,同時溶栓以及重建血流供應可產生活性氧和活性
氮自由基而造成腦缺血再灌注損傷。一氧化氮和過氧
亞硝基是活性氮的重要組成部分,它們能活化MMP,
攻擊緊密連接蛋白,增加血腦屏障通透性。由於t-PA
和活性氮均能造成上述病理變化,因此,我們提出溶
栓治療所致腦出血究竟是t-PA的直接作用的結果還是
活性氮自由基所致的問題。我們的實驗證據顯示活性
氮自由基清除劑能抑制t-PA引起的MMP的活化,減少
血腦屏障通透性。因此,我們設計有關實驗以測試是
否活性氮自由基清除劑能減少t-PA引起的血腦屏障破
壞和腦出血的形成。如果該假說能成立,抗氧化治療
就能作為t-PA溶栓治療的輔助療法,以減少溶栓併發
症。由於t-PA是目前唯一的溶栓藥物,而抗氧化劑也
是常用的保健品,本研究為抗氧化劑提供了新的治療
方向。
Realisation of objectives:
Objective 1. (1)We used mice brain microvascular
endothelial cells (BMECs) and investigated the
synergistic effect of tissue plasminogen activator
(t-PA) and RNS on cell death under oxygen and
glucose deprivation (OGD) condition, which mimics
cerebral ischemia-reperfusion injury in vitro. (2)We
conducted in vivo animal experiments by using SD
rat middle cerebral artery occlusion (MCAO)-induced
cerebral ischemia-reperfusion injury model. Using
this model, we investigated the effect of early and
delayed t-PA treatment on hemorrhagic
transformation, 3-NT formation and MMPs activation.
Then we tested the effect of FeTMPyP, a
representative peroxynitrite decomposition catalyst,
on hemorrhagic transformation, MMP-9/-2 activation
and neurological function in ischemic stroke with
delayed t-PA treatment. Objective 2. (1) We
investigated the role of caveolin-1 in mediating
MMP-9 activation, tight junction disruption and blood
brain barrier (BBB) damage in SD rat MCAO model.
We further compared the BBB damage in wild type
and caveolin-1 knockout mice in MCAO model. (2)
We investigated the role of caveolin-1 in regulating
MMP-9 by caveolin-1 knockdown in cultured BMEC.
(3) We tested the effect of NG-nitro-L-arginine methyl
ester [L-NAME, a non-selective nitric oxide synthase
(NOS) inhibitor] on caveolin-1 expression, MMP-9
activation, tight junctions’ expression and BBB
damage in both wild type and caveolin-1 knockout
mice. (4) We investigated Calycosin-7-O-β-
D-glucoside, an isoflavones derived from
AstragaliRadix. We evaluated its effect on nitric
oxide/caveolin-1/matrix metalloproteinases pathway
mediated BBB damage and brain injury in rat MCAO
model. Objective 3 (1) We developed
diarylamine-based fluorogenic probes for detection of
peroxynitrite. (2) We investigated the direct
scavenging effect of baicalin on peroxynitrite by using
3 of 10

Mass spectroscopy. We further investigated the
peroxynitrite scavenging effect of baicalin in human
SH-SY5Y neuroblastoma cells under the challenges
of 3-morpholinosydnonimine (SIN-1, a peroxynitrite
donor). Finally, we investigated the effects of baicalin
on 3-nitrotyrosine formation in rat MCAO brains. (3)
We studied the effects of baicalin on reducing
neurotoxicity induced by peroxynitrite. We further
investigated baicalin’s effect on brain infarct volume
and neuronal apoptosis in rat MCAO model. (4) We
investigated the effect of baicalin on attenuating
hemorrhagic transformation, improving neurological
outcome, reducing mortality rate, inhibiting MMP-9
activation, and preserving tight junction
ZO-1expression in rat MCAO model with delayed
t-PA treatment. (5) We investigated Sodium
Danshensu (SDSS), a representative active
compound from salvia miltiorrhiza. We evaluated the
potential effects of SDSS on scavenging peroxynitrite
(ONOO-) and protecting neuronal cells from ischemia
reperfusion injury in human SH-SY5Y cells in vitro
and MCAO model in vivo.
Summary of objectives addressed:
Percentage
Objectives
Addressed achieved
Objective 1: To test the
hypothesis that RNS
inhibitors can alleviate
t-PA-induced MMPs
activations, BBB
1.
Yes
100%
disruption and
hemorrhagic
transformation in
cerebral ischemia-
reperfusion injury
Objective 2: To test the
hypothesis that cav-1 is
a critical target protein of
the RNS inhibitors on
reducing the BBB
2. disruption and
Yes
100%
hemorrhagic
transformation in the
experimental ischemic
stroke model with
thrombolytic treatment.
Objective 3: To test the
effects of a natural
antioxidant on the
production of RNS, the
expression of cav-1, the
activations of MMPs, the
3.
Yes
100%
BBB permeability and
hemorrhagic
transformation in the
experimental ischemic
stroke model with the
delayed t-PA treatment.
4 of 10

Research Outcome
Major findings and research outcome:
1. We developed a highly sensitive probe for
peroxynitrite detection in vitro and in vivo In this
study, we developed diarylamine-based fluorogenic
probes for detection of peroxynitrite which only
generate fluorescence in presence of peroxynitrite
without cross-reaction with other free radicals like
hydroxyl radical. The results have been published in
J Am Chem Soc, 136(33), 11728-11734. 2.
Peroxynitrite plays an important role in mediating
delayed tissue plasminogen activator (t-PA)
treatment induced hemorrhagic transformation (HT)
in ischemic brains via regulating MMPs activation
The study proves that early t-PA reduces 3-NT (a
peroxynitrite biomarker) formation, while delayed
t-PA treatment exacerbates 3-NT formation in
ischemic brains and induces HT. FeTMPyP, a
representative peroxynitrite decomposition catalyst,
significantly reduces HT via inhibiting MMP-9/-2
activation. These results have been published in
CNS Neurosci Ther 21(7):585-590, 2015. 3.
Caveolin-1 plays an important role in mediating blood
brain barrier damage in ischemic stroke via
regulating MMPs activity We found that caveolin-1
was down regulated in ischemic stroke, leading to
activation of MMPs, disruption of tight junction ZO-1
and subsequently BBB damage. L-LAME treatment
partially restored the expression of caveolin-1 and
down regulated MMP-9 activity, preserved tight
junction ZO-1, thus protected blood brain barrier.
These results have been published in J Neurochem.
120(1):4-6, 2012. 4. Calycosin-7-O-β-D-glucoside
regulates nitric oxide/caveolin-1/matrix
metalloproteinases pathway and reduced BBB
damage in ischemic stroke The study found that
Calycosin-7-O-β-D-glucoside inhibited nitric oxide,
secured expression of caveolin-1 and inhibited
MMP-9/-2 activity in rat MCAO model and in cultured
BMECs, hence, reduced brain infarct volume and
BBB damage in vivo and attenuated BMECs death in
vitro. These results have been published in J
Ethnopharmacol, 155(1), 692-701, 2014. 5. Baicalin
could directly scavenge peroxynitrite and reduce
neuronal apoptosis in ischemic stroke The study
found that baicalin, a flavonoid compound isolated
from Scutellaria baicalensis G, could directly
scavenge peroxynitrite in vivo and in vitro. Moreover,
baicalin attenuated peroxynitrite induced
neurotoxicity in vitro and reduced the brain infarct
volume in vivo. These results have been published in
Journal of Ethnopharmacology 150(1):116-124, 2013.
6. Baicalin co-treatment significantly reduced
hemorrhagic transformation induced by delayed t-PA
treatment in ischemic stroke, via inhibiting
peroxynitrite mediated MMP-9 activation The study
found that baicalin co-treatment with t-PA significantly
attenuated HT, ameliorated brain edema, reduced
mortality rate, and improved the neurological
5 of 10

outcomes. Moreover, baicalin co-treatment reduced
3-NT formation, attenuated MMP-9 activation and
preserved ZO-1 expression in ischemic brains with
delayed t-PA treatment. The manuscript is in
preparation.
Potential for further development of the research Based on the achievements of our studies, we expect
and the proposed course of action:
following potential directions for further studies: (1) To
further explore the molecular mechanism of how t-PA
reduced or exacerbated 3-NT formation at different
treatment time points in ischemic stroke model; (2) To
compared the effect of baicalin with baicalein on
attenuating hemorrhagic transformation induced by
delayed t-PA treatment in ischemic stroke; (3) To
investigate the potential roles of peroxynitrite in
mediating hyperglycemia induced hemorrhagic
transformation in ischemic stroke. Due to the
limitation of funding and research period, we could
not achieve the goals. We plan to seek for more
funds from RGC GRF grants or other funding
sources to continue our studies in this direction.
Layman's Summary of
Stroke is a leading cause of disability in human
Completion Report:
diseases and is one of the major disease burdens
worldwide. Tissue plasminogen activator is the only
FDA approved agent for acute ischemic stroke
treatment, with limited time window of 4.5 hours.
Treatment beyond the time window significantly
induces hemorrhagic transformation (HT), which is a
severe complication resulting in worsened outcomes.
Thus, developing approaches to reduce HT is timely
important. With the support of RGC GRF grant, we
conducted a series of experiments to test our
hypothesis that peroxynitrite plays an important role
in mediating hemorrhagic transformation induced by
delayed t-PA treatment in ischemic stroke. We
demonstrate that peroxynitrite is increased in
ischemic brains by delayed t-PA treatment and
scavenging peroxynitrite significantly reduces
hemorrhagic transformation. Caveolin-1 may be the
downstream of peroxynitrite, and mediated blood
brain barrier (BBB) damage in ischemic stroke.
Baicalin is a natural compound isolated from
Traditional Chinese Medicine. Baicalin directly
scavenges peroxynitrite, significantly reduces
hemorrhagic transformation and improves the overall
outcomes. Therefore, peroxynitrite is an important
target for reducing hemorrhagic transformation in
ischemic stroke with delayed t-PA treatment. This
project yields 7 original articles, 2 articles in
preparation, 6 review articles and trained 5
postgraduate students.
Research Output
Peer-reviewed journal publication(s)
Year of
arising directly from this research project :
Author(s)
Title and Journal/Book
Publication
(* denotes the corresponding author)
Gu Y, Zheng Caveolin-1 regulates
2012
GQ, Xu MJ, nitric oxide mediated
Li Y, Chen
matrix
6 of 10

metalloproteinases
activity and blood-brain
XM, Zhu
barrier permeability in
WZ, Tong Y, focal cerebral ischemia
Chung SK, and reperfusion injury.
Liu KJ,
Journal of
Shen JG* 
Neurochemistry
120(1):147-156, 2012 
Reactive nitrogen
species: Dual roles for
blood brain barrier
disruption and brain
2012
Shen JG* 
repairs in cerebral
ischemia and
reperfusion injury. Acta
Biophysica Sinica 2012,
28(4) 295-306 
Targeting reactive
nitrogen species: A
Chen XM,
promising therapeutic
Chen HS,
strategy for cerebral
2013
Xu MJ,
ischemia-reperfusion
Shen JG* 
injury. Acta
Pharmacologica Sinica
34(1):67-77, 2013 
Insights into
mechanisms of blood
Brain barrier
permeability: Roles of
free radicals, matrix
Shen JG*,
2014
metalloproteinases and
Gu Y. 
caveolin-1. Systems
Biology of Oxidative
Stress. (Eds.Ismail
Laher),Springer pp
2049-2067 
Baicalin can scavenge
Xu MJ,
peroxynitrite and
Chen X, Gu ameliorate endogenous
Y, Peng T,
peroxynitrite-mediated
Yang D,
2013
neurotoxicity in cerebral
Chang R,
ischemia-reperfusion
So KF, Liu
injury. J
KJ, Shen
Ethnopharmacol,
JG* 
150(1), 116-124. 
Secondary metabolites
Yang LP,
of Genus Astragalus :
Shen JG*,
Structure and biological
2013
Xu WC,Li J, activity update.
Jiang JQ*.  Chemistry & Biodiversity
10: 1004-1054, 2013 
Site-2 protease
responds to oxidative
Gu Y, Lee
2014
stress and regulates
W, Shen J*.  oxidative injury in
mammalian cells, Sci
7 of 10

Rep, 4:6268 
Herbal Medicines for
Ischemic Stroke:
Gu Y, Chen Combating Inflammation
2014
J, Shen J*  as Therapeutic Targets,
J Neuroimmune
Pharmacol, 9(3):313-39 
Peng T,
Wong NK,
Molecular imaging of
Chen X,
peroxynitrite with
Chan YK,
HKGreen-4 in live cells
2014
Ho DH, Sun and tissues, J Am Chem
Z, Hu JJ,
Soc, 136(33),
Shen J,
11728-11734 
El-Nezami
H, Yang D* 
Xingmiao
Chen,
Pros and Cons of
Hansen
Current Approaches for
Chen,
Detecting Peroxynitrite
2014
Ruixia
and Their Applications,
Deng,
Biomedical journal,
Jiangang
37(3), 120 
Shen* 
Calycosin-7-O-β-
D-glucoside regulates
nitric oxide /caveolin-
1/matrix
Fu S, Gu Y, metalloproteinases
Jiang JQ,
pathway and protects
2014
Chen X, Xu blood-brain barrier
M, Chen X, integrity in experimental
Shen J*. 
cerebral ischemia-
reperfusion injury, J
Ethnopharmacol,
155(1), 692-701 
Momordica charantia
polysaccharides could
Gong J, Sun protect against cerebral
F, Li Y, Zhou ischemia/reperfusion
X, Duan Z,
injury through inhibiting
2015
Duan F,
oxidative stress
Zhao L,
mediated c-Jun
Chen H, Qi N-terminal kinase 3
S, Shen J*  signaling pathway,
Neuropharmacology, 91,
123-134 
Peroxynitrite
Decomposition Catalyst
Chen HS,
Reduces Delayed
Chen XM,
Thrombolysis-induced
Feng JH,
2015
Hemorrhagic
Liu KJ, Qi
Transformation in
SH, Shen
Ischemia-reperfused Rat
JG* 
Brains, CNS
neuroscience and
8 of 10

therapeutics
21(7):585-590 
Recognized international conference(s)
Conference
in which paper(s) related to this research
Month/Year/City
Title
Name
project was/were delivered :
Danshensu is a
novel
peroxynitirte
17th
scavenger and
International
has
Biophysics
10/2011/Beijing neuroprotective
Congress &12th
effects against
Chinese
cerebral
Biophysics
ischemia-
Congress 
reperfusion
injury. 
Baicalin protects The 11th
neural cells
Meeting of the
against cerebral Consortium for
08/2012/Macau ischemia
Globalization of
reperfusion injury Chinese
by scavenging
Medicine 
peroxynitrite. 
The 1st
Reactive
International
nitrogen species: Chinese
Dual roles for
Symposium on
blood brain
Free Radical
08/2012
barrier disruption Research, the
/Lanzhou
and brain repairs 5th Symposium
in cerebral
for Three
ischemia and
Distracts of
reperfusion
Cross-straits on
injury 
Free Radical
Research 
Current progress
in medical
The 9th
imaging
National
diagnosis and
Integrated
therapeutic
Western and
08/2012/Xiamen strategies for
Chinese
stroke:
Medical
Opportunities
Congress in
and challenges
Neurology. 
for Chinese
medicine 
Caveolin-1 is a
XXVth
crucial molecular International
target in blood-
Symposium on
brain-barrier
Cerebral Blood
(BBB) disruption Flow,
05/2013
and infarction
Metabolism and
/Shanghai
enlargement
Function& XIth
during cerebral
International
ischemia-
Conference on
reperfusion
Quantification
injury 
of Brain
9 of 10


Function with
PET 
Caveolin-1
phosphorylation
plays an
important role in
inhibition of
XXI World
oxygen-glucose-
09/2013/Vienna
congress of
deprivation-
neurology 
induced
endothelial cell
apoptosis via
regulating Stat3
pathway 
Nitric
International
oxide/Caveolin-1/ Conference for
MMP pathway: a Microcirculation
novel therapeutic and the 13th
strategy for drug Annual
discovery from
Conference of
herbal medicine
09/2013/Beijing
the Professional
targeting blood- Committee for
brain-barrier
Microcirculation,
disruption during Chinese
cerebral
Association of
ischemia-
Integrative
reperfusion
Medicine 
injury 
Other impact
Patent: Diarylamine-Based Fluorogenic Probes for
(e.g. award of patents or prizes,
Detection of Peroxynitrite Inventors: Dan Yang, Tao
collaboration with other research institutions,
Peng, Jiangang Shen, Xingmiao Chen. US
technology transfer, etc.):
application or PCT International Application No.
13/754,499.
SCREEN ID: SCRRM00542
10 of 10