Angioedema and Hemorrhage After 4.

5-Hour tPA (Tissue-

Type Plasminogen Activator) Thrombolysis Ameliorated by
T541 via Restoring Brain Microvascular Integrity
Qing-Fang Chen, MD; Yu-Ying Liu, MD; Chun-Shui Pan, PhD; Jing-Yu Fan, MD; Li Yan, MD;
Bai-He Hu, PhD; Xin Chang, PhD; Quan Li, PhD; Jing-Yan Han, MD, PhD

Background and Purpose—tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic
agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation
beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion
injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time
window.
Methods—Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed
by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were
observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage,
cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then.
An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate
effect of T541 on tight junctions and F-actin in the presence of tPA.
Results—tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate,
whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema
and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1,
occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile,
ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and
IV declined, whereas malondialdehyde and 8-Oxo-2′-deoxyguanosine increased and F-actin arrangement disordered. All
the insults after tPA treatment were attenuated by addition of T541 dose dependently.
Downloaded from http://ahajournals.org by on November 7, 2023

Conclusions—The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage
and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-
brain barrier is likely attributable to its effects observed.
Visual Overview—An online visual overview is available for this article.   (Stroke. 2018;49:2211-2219. DOI: 10.1161/
STROKEAHA.118.021754.)
Key Words: basement membrane ◼ oxidative stress ◼ reperfusion injury ◼ thrombosis ◼ tight junctions

I schemic stroke is an emergency condition in clinic, charac-

terized by a sudden loss of blood flow to an area of brain
because of blockage of thrombus. A timely and appropriate
management is crucial for the outcome of the patients present-
ing with ischemic stroke. tPA (tissue-type plasminogen activa-
tor) is the currently used intravenous thrombolytic agent for
acute ischemic stroke.1 Although tPA treatment is proved to be
safe, its efficiency is greatly restricted by the short time window
of 3 to 4.5 hours after onset of symptoms. The major challenge
for tPA treatment is hemorrhagic transformation (HT), particu-
larly for the patients who received a delayed administration of
tPA.2 Thus, to extent the feasibility of tPA, an adjunctive rem-
edy is needed to counteract HT after tPA treatment.
Blood-brain barrier (BBB) disruption underlies HT after
tPA treatment. Several structures participate in formation
of BBB, including endothelial cells tight junction (TJ) and
adherens junction (AJ), basement membrane, pericyte, and
gliacyte end feet.3 The ischemia and reperfusion (I/R) after

Received March 9, 2018; final revision received June 20, 2018; accepted July 12, 2018.
From the Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing, China (Q.-F.C.,
J.-Y.H.); Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China (Q.-F.C., Y.-Y.L., C.-S.P., J.-Y.F., L.Y., B.-H.H.,
X.C., Q.L., J.-Y.H.); Key Laboratory of Microcirculation (Q.-F.C., Y.-Y.L., C.-S.P., J.-Y.F., L.Y., B.-H.H., X.C., Q.L., J.-Y.H.) and Key Laboratory of Stasis
and Phlegm (Q.-F.C., Y.-Y.L., C.-S.P., J.-Y.F., L.Y., B.-H.H., X.C., Q.L., J.-Y.H.), State Administration of Traditional Chinese Medicine of the People’s
Republic of China, Beijing; State Key Laboratory of Core Technology in Innovative Chinese Medicine, Beijing (Q.-F.C., Y.-Y.L., C.-S.P., J.-Y.F., L.Y.,
B.-H.H., X.C., Q.L., J.-Y.H.); and Beijing Microvascular Institute of Integration of Chinese and Western Medicine (Q.-F.C., Y.-Y.L., C.-S.P., J.-Y.F., L.Y.,
B.-H.H., X.C., Q.L., J.-Y.H.).
The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.118.021754.
Correspondence to Jing-Yan Han, MD, PhD, Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking
University, 38 Xueyuan Rd, Beijing 100191, China. Email hanjingyan@bjmu.edu.cn
© 2018 American Heart Association, Inc.
Stroke is available at https://www.ahajournals.org/journal/str DOI: 10.1161/STROKEAHA.118.021754

2211
 2212  Stroke  September 2018

thrombolytic intervention per se imposes an impact on BBB
integrity. Delayed tPA treatment exaggerates I/R injury and
causes excessive degradation of the proteins of TJ, AJ, and
basement membrane and bleeding because of thrombolytic
activity, which amplifies BBB disruption.4 Paradoxically, the
thrombolytic activity is required for the clinic use of tPA.
Thus, a promising strategy to attenuate HT after tPA treatment
is to target I/R injury.
T541 is a Chinese compound medicine consisting of total
Astragalus saponins (AS), total salvianolic acids (SAAs),
and total Panax notoginseng saponins (PNS) in a ratio of
AS:SAA:PNS=5:4:1. The potential to attenuate myocardial
I/R injury through regulation of energy metabolism has been
demonstrated by our laboratory for astragaloside IV5—the
main component of AS—and ginsenoside Rb1,6 ginsenoside
Rg1,7 and notoginsenoside R18—the main components of
PNS and SAA.9 Buyang Huanwu Decoction—a compound
Downloaded from http://ahajournals.org by on November 7, 2023
Chinese medicine containing AS as an ingredient—has been
reported to enhance energy metabolism and maintain BBB
integrity in mouse I/R model.10
In view of the available results, we speculated that T541
is potentially able to lessen BBB disruption and attenuate HT
after tPA treatment by upregulating energy metabolism. This
study was aimed to test this speculation.
Methods
Data that support the findings of this study are available from the cor-
responding author on reasonable request.
Carotid Artery Thrombosis Model
and Animal Treatment
Male C57BL/6 N mice (21±2 g) were obtained from the Animal
Center of Peking University Science Center (Beijing, certificate
No. SCXK 2006-0008). Experiment handling was approved by

Figure 1. T541 improves carotid thrombolysis and cerebral blood flow in mice receiving tPA (tissue-type plasminogen activator) treatment. A, Carotid artery
thrombus (bar=200 μm). B, Statistical analysis of thrombus area. C, Brain surface blood perfusion images. D, Statistical analysis of cerebral blood flow in
the right middle cerebral artery domination. Data are normalized with baseline. tPA and T541 treated at 4.5 hours after FeCl3. *P<0.05 vs sham, †P<0.05 vs
thrombus+T541-20, ‡P<0.05 vs thrombus+tPA, #P<0.05 vs thrombus.
 Chen et al   T541 Ameliorated Hemorrhage After tPA   2213

Figure 2. Survival rate, neurological scores, and cerebral edema are improved at 24 hours after drug treatment. A, Survival rate. B, Neurological deficit scores
tested with modified neurological severity score (mNSS) and 15-point neurological evaluation scale (NES). Normal mice exhibited 0 points in mNSS and 15 points
Downloaded from http://ahajournals.org by on November 7, 2023

in NES. C, Evans blue leakage in brain tissue sections, FITC-labeled albumin leaked out of cerebral venules, transmission electron microscope and scanning elec-
tron microscope images showing perivascular edema in the penumbra cortex (bars in rows b through f are 50, 2, 0.5, 20, and 5 μm, separately). D, Statistical anal-
ysis of Evans blue content extravasated into brain tissue, albumin leakage showed as fluorescence intensity ratio of the fluorescent intensity in interstitial tissue/the
fluorescent intensity in cerebral venule (I/V), average opening microvascular numbers of 5 ×500 magnification SEM fields chosen randomly, and hemisphere water
content. *P<0.05 vs sham, †P<0.05 vs thrombus+T541-20, ‡P<0.05 vs thrombus+tPA (tissue-type plasminogen activator), #P<0.05 vs thrombus.

the Committee on the Ethics of Animal Experiments of Peking
University Health Science Center (LA2016308). Carotid artery
thrombosis model was established as described.11 In short, ani-
mal was anesthetized by intraperitoneal injection of sodium pen-
tobarbital (2%; 45 mg/kg), and right common carotid artery was
isolated and wrapped with 1-mm wide filter paper infiltrated with
10% FeCl3 for 3 minutes to induce thrombus formation, which
was replaced by saline in sham group. Carotid artery thrombosis
was detected over time as described below, and only those ani-
mals whose maximum thrombus blocked >80% artery diameter at
4.5 hours after ischemia onset were used for further experiments.
Animals were then randomly divided into different groups and
treated with tPA (10 mg/kg; Boehringer Ingelheim, Inc, Germany)
with/without T541 (5, 10, or 20 mg/kg; Tasly Pharmaceutical,
Co, Ltd, Tianjin, China; see Figure I in the online-only Data
Supplement for details).
To study the effect of different components of T541 on the cere-
bral injury, single component groups of AS, SAA, and PNS and
2-component groups of AS+SAA, AS+PNS, and SAA+PNS were
included (Table I in the online-only Data Supplement). All the groups
in vivo were generated by random number table.
Cell Culture and Hypoxia/Reoxygenation In Vitro
Human brain microvascular endothelial cells (CRL-3245;
American Type Culture Collection, Manassas, VA) were cul-
tured under 1% O2 for 4.5 hours, followed by reoxygenation for
3 hours to establish hypoxia/reoxygenation model. tPA 20 μg/mL
with/without T541 (400, 40, 4, 0.4, or 0.04 μg/mL) was added
just before reoxygenation (see Table II in the online-only Data
Supplement for details). No difference was observed in cell viabil-
ity between control and control+T541 groups (40, 4, and 0.4 μg/
mL) except for control+T541 400 μg/mL (Figure II in the online-
only Data Supplement).
Dynamic Assessment of Carotid Artery
Thrombosis and Cerebral Blood Flow
Carotid artery thrombosis was showed by acridine red labeled plate-
lets. Cerebral blood flow (CBF) was determined by a laser Doppler
perfusion imager (PeriScan PIM 3–1143; Jarfalla, Sweden).
Both variables were recorded at baseline, 10 minutes, 4.5 hours,
5.5 hours (1 hour after drug treatment), 6.5 hours (2 hours after
drug treatment), and 28.5 hours (24 hours after drug treatment)
after FeCl3 challenge, respectively (Figure I in the online-only
Data Supplement). For evaluation of albumin leakage from cere-
bral venules, FITC-labeled albumin (50 mg/kg; Sigma-Aldrich,
St. Louis) was infused via femoral vein after craniotomy at 24
hours after treatment. The albumin leakage from cerebral venules
was observed using an upright intravital fluorescent microscopy
(BX51WT; Olympus, Japan) and expressed as I/V (I, the fluores-
cent intensity in interstitial tissue; V, the fluorescent intensity in
cerebral venule).
Neurological Deficit Evaluation
Twenty-four hours post-reperfusion, neurological scores were evalu-
ated according to modified neurological severity scores12 and 15-point
 2214  Stroke  September 2018
Downloaded from http://ahajournals.org by on November 7, 2023

Figure 3. T541 ameliorates endothelial cell junction injury after tPA (tissue-type plasminogen activator) treatment in vivo and in vitro. A, Representative immu-
nofluorescence images of occludin around the endothelial cells with marker von Willebrand factor (blue, nucleus; green, von Willebrand factor; red, occludin)
(bars in rows a and b are 10 and 5 μm). B, Western blot and analysis of zonula occludens-1 (ZO-1), VE-cadherin, occludin, and junctional adhesion mol-
ecule-1 (JAM-1) in penumbra cortex. *P<0.05 vs Sham, †P<0.05 vs thrombus+T541-20, ‡P<0.05 vs thrombus+tPA, #P<0.05 vs thrombus. C, Representative
images of JAM-1 at the periphery of cerebral microvascular endothelial cell after hypoxia/reoxygenation (H/R) in vitro (blue, nucleus; red, JAM-1) (bars in rows
a and b are 25 and 5 μm). D, Western blot and statistical analysis of VE-cadherin, claudin-5, and JAM-1 in cerebral microvascular endothelial cell after H/R.
*P<0.05 vs control, †P<0.05 vs H/R+T541-40, ‡P<0.05 vs H/R+tPA, #P<0.05 vs H/R.

neurological evaluation scale13 (see Tables III and IV in the online-
only Data Supplement for detailed criteria).
Assessment of Cerebral Infarction and Hemorrhage
Mice under anesthesia underwent cardiac perfusion 24 hours after
drug treatment with precooled PBS at 4°C. Brains were cut into
5 slices of 1 mm in thickness. Infarction size was assessed by 2%
2,3,5-triphenyltetrazolium chloride staining. Hemoglobin content
was measured using a hemoglobin colorimetric assay kit (Cayman
Chemical, MI).
Determination of Cerebral Microvascular
Injury and Energy Metabolism
Assessment of Evans blue extravasation and water content, vascular
morphology inspection by electron microscopy, and measurement of
TJ and basement membrane proteins using Western blot and immu-
nofluorescence microscopy were undertaken at 24 hours. Energy
metabolism was evaluated by the content of AMP, ADP, and ATP
and activity of respiratory chain complexes using ELISA kits (see the
online-only Data Supplement).
Data Analysis
Data were showed as mean±SEM and statistically analyzed using
2-way ANOVA (thrombus area, CBF, neurological deficit score, cell
viability) or 1-way ANOVA (the others) with Bonferroni post hoc
correction. P<0.05 was considered statistically significant.
Results
T541 Accelerates the Decrease in Carotid Artery
Thrombi and Recovers CBF After tPA Treatment
Dynamic images of carotid artery thrombosis in different
groups and related quantification are displayed in Figure 1A
and 1B. As noticed, thrombus occurred obviously at 10 minutes
after FeCl3 stimulation. As a result, carotid distal blood flow fell
to <20% of baseline at 15 minutes after ischemia onset (Figure
III in the online-only Data Supplement). The thrombi persisted
until 4.5 hours without significant changes in size if not inter-
vened. Treatment with tPA reduced the thrombus size in a time-
dependent manner with significant reduction being found at 24
hours after treatment. The FeCl3-induced thrombosis is a com-
plex process involving multiple factors. Collagen-dependent
aggregation of platelets is generally accepted as a major contrib-
utor. In addition, fibrin network is also reported to participate in
platelet recruitment, and the composition of the thrombus may
change over time.14 We have stained the thrombus by Martius
scarlet blue fibrin staining, showing that the thrombi consisted
of fibrin fibers (blue) 3 hours after FeCl3 stimulation, which
further increased in amount 4.5 hours after FeCl3 stimulation
(Figure IV in the online-only Data Supplement). This may be
the reason why tPA has an effect of thrombolysis at 4.5 hours.
 Chen et al   T541 Ameliorated Hemorrhage After tPA   2215
Downloaded from http://ahajournals.org by on November 7, 2023

Figure 4. T541 alleviates basement membrane injury and cerebral hemorrhage injury 24 hours after tPA (tissue-type plasminogen activator) treatment. A,
Images of hemorrhage, infarct size, and immunofluorescence images of collagen IV and laminin in penumbra cortex (blue: nucleus; red: row b, collagen
IV; row c, laminin), (bar=25 μm). B, Western blot of collagen IV and laminin in cerebral penumbra cortex. C, Statistical analysis of hemisphere hemoglo-
bin content, cerebral infarct area, and Western blot analysis of collagen IV and laminin. *P<0.05 vs sham, †P<0.05 vs thrombus+T541-20, ‡P<0.05 vs
thrombus+tPA, #P<0.05 vs thrombus.

No effect on the thrombus size was observed when treated
with T541 alone over time. However, there is significantly
difference in thrombus area between thrombus+tPA and
thrombus+tPA+T541 groups in all dosage from 1 hour after treat-
ment to 24 hours, indicating that T541 strengthened the thrombo-
lytic effect of tPA significantly in a dose-dependent manner.
CBF in ischemic hemisphere was recorded continuously
(Figure 1C and 1D), which showed that CBF decreased sig-
nificantly at 10 minutes and maintained by 4.5 hours if not
intervened. Neither T541 nor tPA alone had effect on the
reduced CBF at any time point examined. Treatment with
tPA+T541 restored CBF in a dose- and time-dependent man-
ner, whereas at 24 hours, tPA+T541 5 mg/kg, tPA+T541 10
mg/kg, and tPA+T541 20 mg/kg increased carotid thrombo-
lytic rates to 64.40±1.76%, 85.38±0.64%, and 98.76±0.41%
of sham level, respectively, which were significantly higher
than that in tPA alone (45.53±5.07%; Table V in the online-
only Data Supplement).
We quantified the area of carotid thrombus and CBF at 4.5
and 24 hours after drug treatment, respectively, showing that
carotid recanalization rate is correlated with CBF (R2=0.956;
P=0.003; Table VI in the online-only Data Supplement; Figure
V in the online-only Data Supplement).
T541 Elevates Survival Rate and Improves
Neurological Scores
The survival rate of animals was recorded over time, and
neurological scores were assessed 24 hours after drug treat-
ment (Figure 2A and 2B). Interestingly, except for Sham
and Sham+T541 groups, the animals in thrombus group had
the highest survival rate but poorest neurological scores as
compared with other groups. The survival rate of tPA treat-
ment group at 4.5 hours was 50.0%, comparable with clini-
cal data15—a phenomenon likely attributable to HT (Figure
VI in the online-only Data Supplement), which was improved
by adding T541 at 20 mg/kg (81.8%). Importantly, addition
of T541 at 20 mg/kg to tPA treatment group significantly
improved neurological scores. On the contrary, addition of
T541 at 5 or 10 mg/kg to tPA treatment group did not reveal
significant effect on survival rate at 24 hours, though it did
improve neurological scores. This result suggests the dose of
T541 as an important determinant in clinic outcome.
T541-20 Protects BBB From Breakdown After tPA
Treatment
Noting that T541 at 20 mg/kg benefited both survival rate
and neurological scores, we assessed the effect of it on BBB
 2216  Stroke  September 2018

Figure 5. The effect of T541 and its ingredients on energy metabolism disturbance and oxidative stress injury after I/R caused by thrombosis and thrombolysis
Downloaded from http://ahajournals.org by on November 7, 2023

or hypoxia/reoxygenation (H/R). A, Representative immunofluorescence images of F-actin and average F-actin length in cerebral microvascular endothelial cells
after H/R in vitro (blue, nucleus; red, F-actin) (bars in rows a and b are 25 and 10 μm). *P<0.05 vs control, †P<0.05 vs H/R+T541-20, ‡P<0.05 vs H/R+tPA (tissue-
type plasminogen activator), #P<0.05 vs H/R. B, Western blot and statistical analysis of ATP5D (ATP synthase subunit) in cortex 24 hours after drug treatment. C,
The contents of ATP, ADP, and AMP; ratio of ADP/ATP and AMP/ATP; malondialdehyde (MDA) and 8-Oxo-2′-deoxyguanosine (8-OHdG); and activity of complex
I, II, and IV in cortex 24 hours after drug treatment. AS indicates total Astragalus saponins; PNS, total Panax notoginseng saponins; and SAA, total Salvianolic
acids. mOD indicates milli-optical density. *P<0.05 vs sham, †P<0.05 vs thrombus+T541-20, ‡P<0.05 vs thrombus+tPA, #P<0.05 vs thrombus.

breakdown after tPA treatment by test of Evans blue extravasa-
tion, albumin leakage, cerebral microvascular ultrastructure, and
cerebral water content (Figure 2C and 2D). Impressively, all the
results pointed toward a conclusion that BBB was disrupted by
tPA treatment, and this disruption was protected by addition of
T541 at 20 mg/kg. Morphological examination by transmission
electron microscope and scanning electron microscope further
confirmed this result showing a less perivascular edema in the
ischemic penumbra of tPA+T541-20 group compared with tPA-
alone group. Perivascular edema compressed capillaries, leading
to lower cerebral perfusion, which has been reversed by T541.
T541 Maintains the Integrity of Endothelial
Cell Junctions After tPA Treatment
Endothelial cell TJ is an essential contributor to BBB. We
thus first explored the effect of T541 on endothelial cell TJ
by immunofluorescence in vivo and in vitro. tPA treatment
caused a discontinuity of occludin staining around the periph-
ery of endothelial cells, implying an impaired TJ (Figure 3A).
Western blot showed that the impaired TJ integrity including
the decreased expression of zonula occludens-1 and junctional
adhesion molecule-1 in addition to occludin (Figure 3B). The
decrease in the expression of VE-cadherin found by Western
blot implied involvement of AJ as well in BBB breakdown after
tPA treatment, consistent with the recognition that both TJ and
AJ participate in maintaining BBB integrity. In vitro study using
hypoxia/reoxygenation model further consolidated this notion
(Figure 3D). Moreover, in vitro study revealed a decreased
expression and disarrangement of JAM-1 in the cells after tPA
treatment (Figure 3C). Dramatically, all aforementioned altera-
tions caused by tPA were protected by addition of T541, high-
lighting the beneficial role of T541 in maintaining BBB integrity.
T541 Attenuates HT and Infarction After tPA
Treatment
tPA treatment elicited an obvious HT and infarction in the
mice with thrombi, which were attenuated significantly by
addition of T541 (Figure 4A and 4C; Figures VII through XII
in the online-only Data Supplement).
tPA treatment-caused cerebral infarction was likely attrib-
utable to cell apoptosis via increasing the ratio of Bcl-2 (B-cell
lymphoma 2)/Bax (Bcl-2-associated X protein). which was
protected by addition of T541 both in vivo and in vitro (Figure
XIII in the online-only Data Supplement). Hemorrhage after
tPA treatment implied disruption of endothelium barrier, to
which basement membrane acted as a major contributor. As
 Chen et al   T541 Ameliorated Hemorrhage After tPA   2217

expected, basement membrane of microvasculature was found
disrupted in thrombus+tPA group, concurrent with a reduction
of collagen IV and laminin—the 2 major constituents of base-
ment membrane (Figure 4). Of note, the tPA-evoked change
in basement membrane was prevented by addition of T541.
T541 Attenuates Energy Metabolism Disturbance
and Oxidative Stress After tPA Treatment
We next assessed the energy metabolism and oxidative
stress in different conditions, both of which are closely
related to mitochondria function. TJs and AJs maintained
BBB structure by combining with ATP-affinity cytoskel-
eton F-actin.16 F-actin was depolymerized in endothelial
cells after hypoxia/reoxygenation tPA and reassembled
when adding T541 (Figure 5A). It may be the consequence
of ATP5D (ATP synthase subunit) low expression and
ATP deficiency. It was corroborated by Western blot and
ELISA tests of cortex (Figure 5B and 5C). Thrombolysis
followed by tPA treatment resulted in a disturbed energy
metabolism, which was improved by T541. Moreover, tPA
combined with AS+PNS group inhibited ADP and AMP
increase; and tPA combined with any group, including AS
or PNS, inhibited AMP/ATP increase.
The oxidative stress implicated high level of malondial-
dehyde and 8-Oxo-2′-deoxyguanosine; low activities of mito-
chondria complex I, II, and IV in tPA-treated ischemia cortex
were recovered T541 at 20 mg/kg. Interestingly, addition
Downloaded from http://ahajournals.org by on November 7, 2023
of tPA with SAA or AS+SAA reduced the high content of
8-Oxo-2′-deoxyguanosine; and tPA with SAA+PNS recov-
ered the activity of complex I.
Each ingredient of T541 contributed as separated role in
mitochondria protection. AS and PNS were found beneficial
in restoring energy metabolism and SAA efficient in oxidative
stress. However, none of the ingredients alone or in combi-
nation of any 2 worked comparably with T541. This result
further suggests the superiority of including multiple compo-
nents in the medicine.
T541 Increases the Activity of Plasmin and
Decreases the Activity of MMPs After tPA Treatment
We assessed the activity and content of plasmin and MMP
(matrix metalloproteinase)-2/3/9 in plasma and cortex
(Figure 6). The results showed that tPA increased the activity
of plasmin and MMPs in plasma in animals with thrombus.
Administration of T541 together with tPA further increased
the activity of plasmin, whereas AS, SAA, and PNS each
alone or in combination had no effect. On the contrary,
T541+tPA decreased the activity of MMPs compared with
tPA alone. AS, SAA, and PNS each alone or in combination
exhibited attenuating effect on activity of MMPs as well but
to different degree depending on the MMP concerned. These
results suggest that T541 may enhance the activity of plasmin
to assist in thrombolysis and decreased the activity of MMPs
to protect BBB.

Figure 6. The effect of T541 and its ingredients on plasmin and MMPs (matrix metalloproteinase) 24 hours after drug treatment. The activity and content of
plasmin, MMP-2/3/9 in plasma (A) and in cortex (B). AS indicates total Astragalus saponins; PNS, total Panax notoginseng saponins; and SAA, total Salviano-
lic acids. *P<0.05 vs sham, †P<0.05 vs thrombus+T541-20, ‡P<0.05 vs thrombus+tPA (tissue-type plasminogen activator), #P<0.05 vs thrombus.
 2218  Stroke  September 2018
Discussion
tPA is the sole management recommended for patients pre-
senting with ischemic stroke. However, the risk of HT, par-
ticularly when applied later than 4.5 hours after onset of
ischemia, limits the application of tPA in clinic.17 In line with
the clinic observation, the present study revealed that admin-
istration of tPA at 4.5 hours after onset of ischemia resulted
in severe cerebral hemorrhage in mice, accompanied with
cerebral infarction and a lower survival rate. Interestingly,
tPA+T541 at 20 mg/kg significantly improved the throm-
bolysis efficiency and outcome of mice with thrombus, with
reducing hemorrhage and infarction and elevating neuro-
logical scores. This result highlights T541 as a promising
adjunctive therapy for tPA.
Hemorrhage after tPA treatment results from BBB disrup-
tion. Several structures, including endothelial cells TJ, AJ, and
basement membrane, participate in formation of BBB.18 BBB
disruption initiates in ischemia, in which energy metabolism
fails because of insufficient supply of oxygen and nutrition
and impairment of mitochondria function.19 Energy metabo-
lism failure leads to a cascade of consequence, such as ATP
depletion, a decrease in Na+-K+ATPase activity,20 a rise in
intracellular osmotic pressure, lactic acidosis, and disarrange-
ment of F-actin, which together with induction of protease,
that is, MMPs, contribute to BBB disruption.21,22 Delayed
administration of tPA prolongs the ischemic impact on BBB
and provokes reperfusion injury that further exaggerates the
impairment of BBB by release of oxygen peroxide and apop-
tosis signaling from dysfunctional mitochondria.23 Obviously,
Downloaded from http://ahajournals.org by on November 7, 2023
mitochondria play a key role in the initiation and progression
of BBB breakdown in I/R injury,24,25 and strategy able to pro-
tect mitochondria is expected to attenuate BBB disruption
and hemorrhage after tPA treatment.26 The result of the pres-
ent study shows that T541 may work as such strategy, as evi-
denced by its potential to restore mitochondria structure and
function and maintain the expression of TJ and AJ proteins and
collagen IV and laminin.
The finding of the present study is predicable consider-
ing our previously published results attenuating myocardial
I/R injury.6–9 For example, 3,4-dihydroxyl-phenyl lactic acid,
which was contained in SAA, significantly ameliorated the
I/R-induced myocardial damage and apoptosis.27 In this study,
AS and PNS were found beneficial in inhibiting ADP/AMP
energy metabolism disorders. SAAs were efficient in 8-Oxo-
2′-deoxyguanosine content and complex I activity recovery.
However, none of them worked as good as T541. This result
likely suggests a necessity and superiority to include all the
ingredients in T541, at least for the setting tested. The rea-
son for this is at present unknown. Nonetheless, more study
is required to elucidate the detailed mechanisms responsible
for the effects of T541 observed, particularly the signaling
pathway for each of the ingredients and any likely interaction
between them.
There were only male mice used in this study because of
the fear of estrogen interference and limited funding. Further
study is needed to clarify whether sex difference exists in the
effect of T541.
In conclusion, our study demonstrated that tPA admin-
istration at 4.5 hours after thrombosis exhibited a rising risk
of angioedema and hemorrhage with undesirable outcomes,
which was related to BBB disruption because of energy
depletion and oxidative stress. All the insults after tPA treat-
ment can be ameliorated by addition of T541, suggesting
T541 as a new strategy that helps extend thrombolysis time
window in clinic.
Acknowledgments
Dr Chen performed the research, analyzed the data, and wrote the
manuscript; Drs Liu and Li contributed to animal experiments; Dr
Pan contributed to cell experiments and Western blotting; Dr Yan con-
tributed to immunochemistry staining; Drs Hu and Chang contrib-
uted to electron microscope experiments; Drs Fan and Han revised
the manuscript; Dr Han designed and funded the research, interpreted
the data, and finally approved the submission of this manuscript. All
authors have read and agreed with the manuscript.
Sources of Funding
This study was supported, in part, by the Production of New Medicine
Program of Ministry of Science and Technology of the People’s
Republic of China (2013ZX09402202) and State Key Laboratory of
Core Technology in Innovative Chinese Medicine (20170034).
Disclosures
None.
References
1. Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis
NC, Becker K, et al; American Heart Association Stroke Council. 2018
guidelines for the early management of patients with acute ischemic
stroke: a guideline for healthcare professionals from the American Heart
Association/American Stroke Association. Stroke. 2018;49:e46–e110.
doi: 10.1161/STR.0000000000000158
2. Sandercock P, Wardlaw JM, Lindley RI, Dennis M, Cohen G, Murray
G, et al; IST-3 Collaborative Group. The benefits and harms of intrave-
nous thrombolysis with recombinant tissue plasminogen activator within
6 h of acute ischaemic stroke (the third international stroke trial [ist-
3]): a randomised controlled trial. Lancet. 2012;379:2352–2363. doi:
10.1016/S0140-6736(12)60768-5
3. Thomsen MS, Routhe LJ, Moos T. The vascular basement membrane
in the healthy and pathological brain. J Cereb Blood Flow Metab.
2017;37:3300–3317. doi: 10.1177/0271678X17722436
4. Niego B, Medcalf RL. Plasmin-dependent modulation of the blood-brain
barrier: a major consideration during tPA-induced thrombolysis? J Cereb
Blood Flow Metab. 2014;34:1283–1296. doi: 10.1038/jcbfm.2014.99
5. Tu L, Pan CS, Wei XH, Yan L, Liu YY, Fan JY, et al. Astragaloside IV
protects heart from ischemia and reperfusion injury via energy regula-
tion mechanisms. Microcirculation. 2013;20:736–747. doi: 10.1111/
micc.12074
6. Cui YC, Pan CS, Yan L, Li L, Hu BH, Chang X, et al. Ginsenoside Rb1
protects against ischemia/reperfusion-induced myocardial injury via
energy metabolism regulation mediated by RhoA signaling pathway. Sci
Rep. 2017;7:44579. doi: 10.1038/srep44579
7. Li L, Pan CS, Yan L, Cui YC, Liu YY, Mu HN, et al. Ginsenoside
Rg1 ameliorates rat myocardial ischemia-reperfusion injury by modu-
lating energy metabolism pathways. Front Physiol. 2018;9:78. doi:
10.3389/fphys.2018.00078
8. He K, Yan L, Pan CS, Liu YY, Cui YC, Hu BH, et al. ROCK-dependent
ATP5D modulation contributes to the protection of notoginsen-
oside NR1 against ischemia-reperfusion-induced myocardial injury.
Am J Physiol Heart Circ Physiol. 2014;307:H1764–H1776. doi:
10.1152/ajpheart.00259.2014
9. Tang H, Pan CS, Mao XW, Liu YY, Yan L, Zhou CM, et al. Role of
NADPH oxidase in total salvianolic acid injection attenuating ischemia-
reperfusion impaired cerebral microcirculation and neurons: implication
of AMPK/Akt/PKC. Microcirculation. 2014;21:615–627. doi: 10.1111/
micc.12140
10. Chen HJ, Shen YC, Shiao YJ, Liou KT, Hsu WH, Hsieh PH, et al.
Multiplex brain proteomic analysis revealed the molecular therapeutic
 Chen et al   T541 Ameliorated Hemorrhage After tPA   2219
effects of Buyang Huanwu Decoction on cerebral ischemic stroke mice.
PLoS One. 2015;10:e0140823. doi: 10.1371/journal.pone.0140823
11. Owens AP III, Lu Y, Whinna HC, Gachet C, Fay WP, Mackman N.
Towards a standardization of the murine ferric chloride-induced carotid
arterial thrombosis model. J Thromb Haemost. 2011;9:1862–1863. doi:
10.1111/j.1538-7836.2011.04287.x
12. García-Yébenes I, Sobrado M, Zarruk JG, Castellanos M, Pérez de la Ossa N,
Dávalos A, et al. A mouse model of hemorrhagic transformation by delayed
tissue plasminogen activator administration after in situ thromboembolic
stroke. Stroke. 2011;42:196–203. doi: 10.1161/STROKEAHA.110.600452
13. Garcia JH, Wagner S, Liu KF, Hu XJ. Neurological deficit and extent of
neuronal necrosis attributable to middle cerebral artery occlusion in rats.
Statistical validation. Stroke. 1995;26:627–634; discussion 635.
14. Eckly A, Hechler B, Freund M, Zerr M, Cazenave JP, Lanza F, et al.
Mechanisms underlying FeCl3-induced arterial thrombosis. J Thromb
Haemost. 2011;9:779–789. doi: 10.1111/j.1538-7836.2011.04218.x
15. Yaghi S, Eisenberger A, Willey JZ. Symptomatic intracerebral hemorrhage
in acute ischemic stroke after thrombolysis with intravenous recombinant
tissue plasminogen activator: a review of natural history and treatment.
JAMA Neurol. 2014;71:1181–1185. doi: 10.1001/jamaneurol.2014.1210
16. Jonckheere AI, Smeitink JA, Rodenburg RJ. Mitochondrial ATP syn-
thase: architecture, function and pathology. J Inherit Metab Dis.
2012;35:211–225. doi: 10.1007/s10545-011-9382-9
17. Yaghi S, Willey JZ, Cucchiara B, Goldstein JN, Gonzales NR, Khatri
P, et al; American Heart Association Stroke Council; Council on
Cardiovascular and Stroke Nursing; Council on Clinical Cardiology; and
Council on Quality of Care and Outcomes Research. Treatment and out-
come of hemorrhagic transformation after intravenous alteplase in acute
ischemic stroke: a scientific statement for healthcare professionals from
the American Heart Association/American Stroke Association. Stroke.
2017;48:e343–e361. doi: 10.1161/STR.0000000000000152
18. Komarova YA, Kruse K, Mehta D, Malik AB. Protein interactions at
endothelial junctions and signaling mechanisms regulating endothelial
Downloaded from http://ahajournals.org by on November 7, 2023
permeability. Circ Res. 2017;120:179–206. doi: 10.1161/CIRCRESAHA.
116.306534
19. Sun K, Fan J, Han J. Ameliorating effects of traditional Chinese medi-
cine preparation, Chinese materia medica and active compounds on
ischemia/reperfusion-induced cerebral microcirculatory disturbances
and neuron damage. Acta Pharm Sin B. 2015;5:8–24. doi: 10.1016/j.
apsb.2014.11.002
20. Weilinger NL, Maslieieva V, Bialecki J, Sridharan SS, Tang PL,
Thompson RJ. Ionotropic receptors and ion channels in ischemic neu-
ronal death and dysfunction. Acta Pharmacol Sin. 2013;34:39–48. doi:
10.1038/aps.2012.95
21. Suzuki Y, Nagai N, Umemura K, Collen D, Lijnen HR. Stromelysin-1
(MMP-3) is critical for intracranial bleeding after t-PA treatment of
stroke in mice. J Thromb Haemost. 2007;5:1732–1739. doi: 10.1111/j.
1538-7836.2007.02628.x
22. Sifat AE, Vaidya B, Abbruscato TJ. Blood-brain barrier protection as a
therapeutic strategy for acute ischemic stroke. AAPS J. 2017;19:957–
972. doi: 10.1208/s12248-017-0091-7
23. Kalogeris T, Baines CP, Krenz M, Korthuis RJ. Ischemia/reperfusion.
Compr Physiol. 2016;7:113–170. doi: 10.1002/cphy.c160006
24. Han JY, Li Q, Ma ZZ, Fan JY. Effects and mechanisms of compound
Chinese medicine and major ingredients on microcirculatory dysfunc-
tion and organ injury induced by ischemia/reperfusion. Pharmacol Ther.
2017;177:146–173. doi: 10.1016/j.pharmthera.2017.03.005
25. Jin Z, Wu J, Yan LJ. Chemical conditioning as an approach to ischemic
stroke tolerance: mitochondria as the target. Int J Mol Sci. 2016;17:351.
doi: 10.3390/ijms17030351
26. Ortolano S, Spuch C. tPA in the central nervous system: relations
between tPA and cell surface LRPs. Recent Pat Endocr Metab Immune
Drug Discov. 2013;7:65–76.
27. Wei XH, Liu YY, Li Q, Yan L, Hu BH, Pan CS, et al. Treatment with cardio-
tonic pills(®) after ischemia-reperfusion ameliorates myocardial fibrosis
in rats. Microcirculation. 2013;20:17–29. doi: 10.1111/micc.12002