ArticlePDF AvailableLiterature Review

Dl-3-n-Butylphthalide (NBP):A Promising Therapeutic Agent for Ischemic Stroke



Background and objective: Stroke is a leading cause of morbidity and mortality in both developed and developing countries all over the world. The only drug for ischemic stroke approved by FDA is recombinant tissue plasminogen activator (rtPA). However, only 2-5% stroke patients receive rtPAs treatment due to its strict therapeutic time window. As ischemic stroke is a complex disease involving multiple mechanisms, medications with multi-targets may be more powerful compared with single-target drugs. Dl-3-n-Butylphthalide (NBP) is a synthetic compound based on l-3-n- Butylphthalide that is isolated from seeds of Apium graveolens. The racemic 3-n-butylphthalide (dl- NBP) was approved by Food and Drug Administration of China for the treatment of ischemic stroke in 2002. A number of clinical studies indicated that NBP not only improved the symptoms of ischemic stroke, but also contributed to the long-term recovery. The potential mechanisms of NBP for ischemic stroke treatment may target different pathophysiological processes, including anti-oxidant, antiinflammation, anti-apoptosis, anti-thrombosis, and protection of mitochondria et al. Conclusion: In this review, we have summarized the research progress of NBP for the treatment of ischemic stroke during the past two decades.
CNS & Neurological Disorders - Drug Targets
ISSN: 1871-5273
eISSN: 1996-3181
Send Ord ers for R eprints to
CNS & Neurological Disorders - Drug Targets, 2018, 17, 338-347
Dl-3-n-Butylphthalide (NBP): A Promising Therapeutic Agent for
Ischemic Stroke
Shan Wang1,#, Fei Ma1,#, Longjian Huang1, Yong Zhang1, Yuchen Peng1, Changhong Xing2,
Yipu Feng1, Xiaoliang Wang1,* and Ying Peng1,*
1State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chi-
nese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; 2Departments of Radiol-
ogy and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
Received: April 02, 2018
Revised: May 27, 2018
Accepted: June 08 , 2018
Abstract: Background and Objective: Stroke is a leading cause of morbidity and mortality in both
developed and developing countries all over the world. The only drug for ischemic stroke approved by
FDA is recombinant tissue plasminogen activator (rtPA). However, only 2-5% stroke patients receive
rtPAs treatment due to its strict therapeutic time window. As ischemic stroke is a complex disease in-
volving multiple mechanisms, medications with multi-targets may be more powerful compared with
single-target drugs. Dl-3-n-Butylphthalide (NBP) is a synthetic compound based on l-3-n-
Butylphthalide that is isolated from seeds of Apium graveolens. The racemic 3-n-butylphthalide (dl-
NBP) was approved by Food and Drug Administration of China for the treatment of ischemic stroke in
2002. A number of clinical studies indicated that NBP not only improved the symptoms of ischemic
stroke, but also contributed to the long-term recovery. Th e potential mech anisms of NBP fo r ischemic
stroke treatment may target different pathophysiological processes, including anti-oxidant, anti-
inflammation, anti-apoptosis, anti-thrombosis, and protection of mitochondria et al.
Conclusion: In this review, we have summarized the research progress of NBP for the treatment of
ischemic stroke during the past two decades.
Keywords: Dl-3-n-butylphthalide, ischemic stroke, cerebral microcirculation, neuroprotection, mitochondria, apoptosis, oxida-
tive stress.
As one of the leading causes of morbidity and mortality,
stroke places a great deal of economic burden on patients,
their relatives as well as the entire society [1]. In China, ac-
cording to the World Bank Data, there will be about 31.77
million stroke patients by 2030 with the cost of as much as
$40.0 billion per year [2]. Approximately 87% of strokes are
ischemic in nature [3]. Recombinant tissue plasminogen
activator (rtPA), a thrombolytic that restores blood flow to
the ischemic brain, remains to be the only specific medica-
tion approved by the FDA for clinical management in acute
ischemic stroke (AIS). Unfortunately, due to its limited
*Address correspondence to this author at the Pharmacology Department,
Institute of Materia Medica, Chinese Academy of Medical Sciences & Pe-
king Union Medical College, No.1, Xiannongtan Street, Xicheng District,
Beijing 100050, China; Tel: +86-10-63165173; Fax: +86-10-63017757;
E-mail: and Tel: +86-10-63165330; Fax: +86-10-
63165330; E-mail:
#Shan Wang and Fei Ma contributed equally to this work.
therapeutic time window, rtPA is only provided to a minority
of patients (2%-5%) [4]. Although scientists attempted to
develop novel medications for ischemic stroke, a large num-
ber of clinical trials failed, including thrombolytic agents,
antiplatelet agents, anticoagulants, modulators of excitatory
amino acids, modulators of calcium influx, metabolic activa-
tors, anti-edema agents, inhibitors of leukocyte adhesion,
free radical scavengers, promoters of membrane repair and
so on [2, 5].
The neurovascular unit is not only an anatomical con-
struct but also serves as a functional unit for the interactions
between neurons, glial cells and blood vessels [6]. The con-
cept of neurovascular unit has been used not only in the in-
vestigation of acute stroke pathophysiology, but also ex-
tended to dissect the delayed phases of stroke recovery. In
addition, this conception emphasized that the brain injury of
ischemic stroke should be regarded as an integral structure to
be protected, and neuroprotectants with multi-targets might
be a promising option for ischemic stroke. In this context, an
ideal therapeutic target for stroke would be one that rescues
1996-3181/18 $58.00+.00 © 2018 Bentham Science Publishers
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 339
neurovascular signaling and is active in multiple brain cell
types. However, some researchers believe that the first step
of neuroprotective strategy should be revascularization, in
which case, neuroprotectants could enter the salvageable
tissues and then exert their neuroprotective effects more effi-
ciently. Briefly, for the treatment of ischemic stroke,
neuroprotective strategy should be comprehensive which
includes revascularization, using neuroprotectants, and the
long-term recovery of neurofunction [7]. Dl-3-n-
Butylphthalide (NBP) may be one of these candidates.
NBP is a synthetic chiral compound based on l-3-n-
butylphthalide which is originally isolated from seeds of
Apium graveolens. Its chemical name is ()3-butyl-3H-2-
benzonfuran-1-one (chemical formula: C12H14O2), and mo-
lecular weight is 190.24 (Fig. 1). NBP is a colorless or light
yellow viscous liquid with a celery odor. In 1995, NBP was
first time used to treat ischemic stroke by Feng et al. [8]. In
the preclinical studies, NBP showed protective effects
against ischemic stroke in various animal models [9-15].
Treatment with NBP (20 mg·kg-1, i.p.) following 15 minutes
of middle cerebral artery occlusion (MCAO) in rat models
significantly reduced infarct volume by 87% below the con-
trol value and improved neurological deficits [13]. Similar
effects were also obtained even administrating with NBP (80
mg·kg-1, p.o., b.i.d.) after 2 hours of MCAO in mouse mod-
els as well as stroke-prone spontaneously hypertensive rat
models [10, 14]. The underlying mechanisms of protective
action of NBP might be involved in increasing regional
blood flow [9, 16], ameliorating brain edema and blood-
brain barrier damage [11], reducing the chance of thrombosis
[17, 18], improving microcirculation in arterioles [10],
improving energy metabolism [8], decreasing oxidative
damage [19], preventing the inflammatory response [20], and
reducing neuronal apoptosis [21, 22]. In this paper, the
pharmacokinetics, clinical trial and pharmacological
mechanisms of NBP on ischemic stroke treatment would be
Fig. (1). Structure of dl-NBP.
Pharmacokinetics studies in human revealed that the peak
plasma concentration of NBP was achieved at approximately
0.75 hours after an oral administration of 200 mg NBP
(t.i.d.) soft capsules, with mean value of 514 ng·ml-1. In addi-
tion, the average AUC0-∞ value of NBP was 864 ng·h·ml-1,
and the mean t1/2 is 5.33 hours [23]. After being absorbed,
NBP primarily undergoes hydroxylation on the n-butyl side
chain and C-3, and forms four principal metabolites, includ-
ing 10-keto-NBP (M2), 3-hydroxy-NBP (M3-1), 10-
hydroxy-NBP (M3-2), and NBP-11-oic acid (M5-2). No
parent drug of NBP was detected in the urine or feces, and
the urinary metabolites accounted for about 81.6% of the
administrated dosage [23-25]. Actually, while NBP is effec-
tive, out of hydrophobicity, its bioavailability being as low
as 15%, which to some extent, limits the application of NBP
capsule [26]. Given this problem, potassium 2-(1-
hydroxypentyl)-benzoate (PHPB), a pro-drug of NBP, which
could entirely change into NBP in the body, has been devel-
oped. With extremely high solubility in water, PHPB has
better bioavailability than NBP [27]. Currently, PHPB is in
phase II-III clinical studies in China, and its clinical applica-
tion might be achieved in the near future. Safety studies in
human showed that the incidence of serious and non-serious
adverse events was similar between the NBP (25 mg, i.v.,
b.i.d. or, 200 mg, p.o., b.i.d.) treated group and placebo
group [24, 26] except that NBP had mild hepatotoxicity [23].
In different stages of clinical trials (II, III, IV), the incidence
of the elevated alanine aminotransferase of NBP in patients
ranged from 3.0% to 17.5% [26, 28, 29]. However, this ef-
fect was recoverable and it has been suggested that the ele-
vated alanine aminotransferase might be dose-related [26].
As a result, during the administration period of NBP, it is
necessary to regularly monitor the liver function.
The randomized, double-blinded, placebo-control, multi-
center clinical trials suggested that NBP might be a safe and
effective treatment for ischemic stroke [24, 26, 30]. In a
clinical trial, 573 patients were administrated with NBP (25
mg, i.v., b.i.d. or 200 mg, p.o., t.i.d.) within 48 hours of onset
of ischemic stroke for 90 days. According to the measure-
ments of modified Rankin scale, NBP might be more effec-
tive and safer than thromboxane A2 (TXA2) synthase inhibi-
tor ozagrel and aspirin, especially for those patients with
moderate severity of ischemic stroke [26]. In another trial
with an enrollment of 60 patients within 12 h of AIS, a sig-
nificant improvement of National Institutes of Health Stroke
Scale (NIHSS) scores at 21-day after stroke onset was ob-
served in NBP-treated group compared with Cerebrolysin
[24]. NBP (200 mg, p.o., t.i.d.) also elevated the number of
circulating endothelial progenitor cells in a study including
170 patients with AIS [30]. Furthermore, NBP seemed to
improve the long-term recovery of stroke-related disability,
such as walking, limb motor, language, sensor, thinking and
memory [26]. With the accumulating evidences, NBP has
been approved as a novel drug for ischemic stroke by the State
Food and Drug Administration of China since 2002. Up to
now, several clinical trials of NBP are ongoing on Cere-
brovascular Occlusion (NCT02594995), AIS (NCT02905565
and NCT02149875), Vascular Cognitive Impairment no De-
mentia (NCT02993367), AD (NCT02711683), and Restenosis
(NCT01405248). The phase II clinical trial of NBP softgel
capsules for ischemic stroke patients began in the United
State (NCT02905565) in 2017.
The cerebral circulatory system plays a vital part in keep-
ing an optimal cerebral blood flow (CBF) for the brain’s needs
of nutrients and oxygen [31]. Ischemic stroke is characterized
by luminal obstruction and reduced blood flow to the brain
[32]. Hence, restoring reperfusion via thrombolysis, intravas-
cular clot removal, or vasodilation is associated with an im-
proved outcome. NBP might be an effective drug to increase
340 CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 Wang et al.
3.1. Improve Vasodilation
Administration with NBP (10 mg·kg-1, i.p.) after 10 min-
utes of MCAO in rat models, the regional CBF of NBP
groups were significantly increased by 107%, 211% and
370% above the control value respectively after 60, 120 and
180 minutes of MCAO [33]. The action of NBP on regional
CBF was mainly related to the vasodilation induced by ele-
vation of nitric oxide (NO), a strong vasodilator generated by
nitric oxide synthase (NOS) in endothelial cells, neurons and
glial cells. NBP (1 or 10 μmolL-1) significantly increased
the activity of NOS and the production of extracellular NO
in bovine aortic endothelial cells and bovine cerebral endo-
thelial cells, which might improve the cerebral microcircula-
tion and restore the supply of oxygen and nutrients to
ischemic hemisphere [34].
3.2. Inhibit Platelet Aggregation and Thrombosis Forma-
After ischemia, a great deal of arachidonic acid (AA) is
produced by phospholipid decomposing by PLA2 and me-
tabolized into Prostaglandin I2 (PGI2) and TXA2 by cy-
clooxygenase. PGI2 is a vasodilator with the effect of anti-
platelet aggregation, while TXA2 can activate the aggrega-
tion of platelet and induce vasoconstriction. The disturbance
of the balance between PGI2 and TXA2 is related to some
pathological process, such as thrombogenesis and angio-
spasm [35]. After 5 minutes and 120 minutes of MCAO in
rats, treatment with NBP (10 or 20 mg·kg-1, i.p.) could down-
regulate the expression of PLA2 and depress the release of
AA in the cerebral cortex by about 60% below the control
value [36]. Moreover, post-treatment with NBP (10 or 20
mg·kg-1, i.p.) markedly elevated the ratio of PGI2/TXA2 after
reperfusion in rats, which might have beneficial effects on
the impaired microcirculation in post-ischemic brain tissues
[18]. In addition, NBP was a potent antiplatelet drug. Pre-
incubated with 300 μmolL-1 NBP could prevent the aggre-
gation of the platelets via inhibiting TXA2 synthesis and in-
creasing the level of cAMP in a dose-dependent manner in
human platelets [17]. Apart from that, experiments in rats
also confirmed the antithrombotic and antiplatelet activity of
NBP, and the decrease of 5-HT release from platelets might
be involved in this effect [37].
3.3. Regulate Angiogenesis
Vascular endothelial growth factor (VEGF) is the most
potent factor that stimulates angiogenesis and vasculogenesis
[38]. Besides VEGF, the basic fibroblast growth factor
(bFGF) and transforming growth factor-β1 (TGF-β1) are
also important angiogenic factors [39-41]. In a cold-induced
ischemic stroke model of stroke-prone renovascular hyper-
tensive rats, pretreatment with NBP (80 mg·kg-1, p.o.) mark-
edly reduced the attack incidence and the infarct volume and
increased the perfused microvessels [10]. NBP significantly
up-regulated the expressions of VEGF and bFGF in the hip-
pocampus andVEGF and TGF-β1 levels in the peri-
infarcted area of different models [42-44]. Lu et al. found
that NBP-induced in vitro angiogenesis was mediated by the
up-regulation of FGF-2 expression and the activations of
both ERK1/2 and PI3K/Akt-eNOS signaling pathways [45].
All these findings demonstrated that NBP might improve
angiogenesis after cerebral ischemia.
The blood brain barrier (BBB) is an essential factor in the
homeostatic regulation of the brain microenvironment [46].
Structurally, the BBB comprises endothelium and surrounding
astrocytes, pericytes and perivascular microglia [47]. BBB is
a highly selective permeability barrier which controls the
flux of some molecules between the blood and central nerve
system. After cerebral ischemia, the function of all
constituents of the BBB, including endothelial cells, astrocytes
and pericytes, was influenced [48]. The pinocytotic vesicles in
the endothelial cells were increased and the tight junction
proteins were deranged, which enhanced the permeability of
BBB and damaged its integrity [49].
NBP reduced IgG extravasation after focal cerebral
ischemia in a dose-dependent manner. Furthermore, ultra-
structure damage of capillaries and brain edema were re-
markably alleviated after post-treatment with NBP in a
reperfusion-induced BBB damage in rats (10 or 20 mg·kg-1,
i.p.) [11]. Additionally, NBP (10 μmolL-1) could protect
endothelial cells against oxidative/nitrosative stress, mito-
chondrial damage and subsequent cell death triggered by
oxygen glucose deprivation (OGD) [50]. It has been well
demonstrated that activation of Rho A led to the increase of
BBB permeability and cerebral edema [51]. NBP (40 mg kg-1,
p.o.) inhibited Rho A protein expression in the cerebral
cortex around focal cerebral infarction, consequently reduced
BBB damage and cerebral edema in rats after focal cerebral
infarction [52]. One latest study revealed the protective
effect of post-treatment with NBP (60 mg kg-1, p.o.) on the
structural integrity and the functional stability of BBB in rats
exposed to CO through the increases of Zonula occludens-1
(ZO-1) and claudin-5, two important molecules in tight
junction complexes [53].
The primary physiological function of mitochondria is to
generate adenosine triphosphate (ATP) through oxidative
phosphorylation by the electron transport chain [54], so mi-
tochondria is a major target in hypoxic or ischemic injury
[55]. NBP has been shown to improve mitochondrial func-
tions through several different aspects.
5.1. Increase Mitochondrial Complexes
Most cellular energy is produced through oxidative phos-
phorylation, a process catalyzed by a series of respiratory
enzyme complexes (complexes I–V) located in the inner
membrane of mitochondria. Xiong et al. found that complex
IV suffered a sharp decrease from 0.167 mmolkg-1min-1 to
0.09 mmolkg-1min-1 following one hour of MCAO in rats
and came back to the normal level after reperfusion for three
hours. The reduction of complex IV triggered by ischemia
could be markedly attenuated in the presence of NBP (5 mg
kg-1 or 10 mg kg-1, i.p., 10 minutes before ischemia) [56]. In
primary cultured neurons, exposure to 1 μmolL-1 NBP could
completely avert the disruption of complex IV induced by 6-
hour hypoglycemia and hypoxia [56]. In addition, the levels
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 341
of phosphocreatine and ATP were efficiently elevated by
1.5- and 2-fold after NBP (150 mg kg-1 or 200 mg kg-1, s.c.)
treatment in complete brain ischemia mouse model [8].
Hence, we inferred that NBP might directly act on mito-
chondria to upregulate the activity of respiratory enzyme
complexes and the synthesis of intracellular energy.
5.2. Protect the Structure and Function of Mitochondrial
Two major factors in the physiologic function of mito-
chondriamitochondrial membrane potential (MMP) and
mitochondrial membrane fluidity (MMfu), were impaired by
oxygen-glucose deprivation in both human umbilical vein
endothelial cells and primary cultured neurons. However, 10
μmolL-1 NBP effectively suppressed OGD-induced HIF-1α
enhancement [50]. Xiong et al. had also demonstrated that
NBP (5 mg kg-1 or 10 mg kg-1, i.p., 10 minutes before
ischemia) could inhibit the decrease of MMfu in MCAO rats,
and NBP reversed the augment of microviscosity of mito-
chondrial membrane. Additionally, NBP could also prevent
mitochondria from swelling, fracturing as well as vacuolat-
ing [57].
5.3. Elevate Mitochondrial ATPase Activity
ATPase plays an important role in intracellular ionic ho-
meostasis and would be strongly perturbed by ischemia. The
activities of mitochondrial Ca2+-ATPase, Na+, K+-ATPase
and Mg2+-ATPase were sharply cut down due to cerebral
ischemia in rats, while NBP (5 mg kg-1 or 10 mg kg-1 or 20
mg kg-1, i.p., 10 minutes before ischemia) effectively ele-
vated the activities of Ca2+-ATPase and Na+, K+-ATPase
compared to the vehicle group respectively [19]. This effect
was confirmed by in vitro study that NBP (0.1 μmolL-1)
could maintain the level of ATPase in primary cultured neu-
rons subjected to OGD [57].
Reactive oxygen species (ROS) are essential signaling
molecules that play a pivotal role in maintaining physiologi-
cal cell functions. ROS can regulate the activities of protein
kinase pathways including receptor tyrosine kinases, protein
kinase C (PKC), and mitogen-activated protein kinases
(MAPK) as well as key transcription factors such as activator
protein-1 (AP-1) and nuclear factor-kappa-B (NF-kB) [58].
In the pathophysiology of cerebral ischemia, oxidative stress
initiates a series of complicated biochemical cascades and
finally accelerates the malignant progression of ischemic
stroke [59]. ROS are excessively produced during cerebral
ischemia or reperfusion, and oxidize cellular components
like lipids, proteins and DNA resulting in cellular damage
and death [60, 61]. Increased production of ROS results in
dysfunction of mitochondria, including the breakup of mito-
chondrial inner membrane integrity, the mitochondrial depo-
larization, the inhibition of mitochondrial electron transfer
chain, the opening of the mitochondrial permeability transi-
tion pore, and disruption of the intracellular calcium homeo-
stasis [62-66]. Besides, ROS itself also triggers the apoptotic
signaling pathway following ischemic damage [67, 68]. Mul-
tiple antioxidant effects of NBP after ischemia have been
6.1. Enhance Anti-oxidant Enzymes Activities
Physiologically, ROS are continuously developed, but
are balanced by endogenous antioxidant defense mecha-
nisms. The homeostasis would be ruined by brain ischemia.
The primary sources of ROS in the setting of cerebral ische-
mia/reperfusion are the mitochondrial respiratory chain,
NAPDH oxidases, and xanthine oxidase (XO) [69]. In a
four-vessel occlusion model of rats, NBP (20 or 40,
i.p., 20 min before ischemia) dose-dependently inhibited the
accumulation of purine metabolites including hypoxanthine,
the substrates of XO, which showed the most dramatic de-
creasing from the peak 5.41 to 1.24 nmolml-1 [70]. Consis-
tently, NBP (0.1 μmolL-1) significantly inhibited the activity
of XO and decreased the formation of superoxide anion
compared with the vehicle group in vitro [71]. In addition,
NBP (20, i.p.) enhanced the activities of two en-
dogenous antioxidants L-Glutathione (GSH) and superoxide
dismutase (SOD), and decreased the content of malondialde-
hyde (MDA) in MCAO rats [19, 71].
6.2. Increase the Expression of Nrf2
The nuclear factor erythroid 2-related factor 2 (Nrf2) is a
key regulator of antioxidative defense responses and an im-
portant protective-survival factor in central nervous system
[72, 73]. Under normal condition, Nrf2 binds the cytosolic
protein Keap1 in an inactive state. After activated by redox
stimulation, Nrf2 translocates to the nucleus and binds anti-
oxidant response elements (ARE) [74]. Nrf2-ARE binding
can initiate transcription of hundreds of protective genes,
including SOD, thioredoxin (TRX), glutathione peroxidase
(GPX), NAD(P)H quinine oxidoreductase (NQO1), and
heme oxygenase (HO-1) [75]. The activation of Nrf2 can
exert neuroprotective effects against permanent cerebral
ischemia [76], whereas the lack of Nrf2 results in cortical
astrocytes and neurons more susceptible to oxidative stress
[77]. Compared with ischemia/reperfusion (I/R) group in
rats, the expression of Nrf2 in the cortex nuclear fraction was
significantly higher in the NBP (60 mg kg-1, p.o.) group,
demonstrating that NBP treatment induced Nrf2 activation
and subsequent nuclear localization [78]. In a carbon monox-
ide (CO)-induced brain damage rat model, the expressions of
Keap1, Nrf-2, and NQO-1 were up-regulated after 1, 3 and 7
day of NBP (60 mg kg-1, p.o.) administration compared with
the CO poisoning group [79].
After cerebral ischemia, apoptosis is triggered through
different signal pathways. The extrinsic pathway is triggered
by the involvement of death receptor, which initiates a sig-
naling cascade regulated by caspase-8 activation. By con-
trast, the intrinsic pathway is engaged when various apop-
totic stimuli activate the release of cytochrome c from mito-
chondria. Both pathways eventually lead to the activation of
caspase-3, degrading cellular proteins that are essential to
maintain cell survival and integrity [80]. In the penumbra
region of MCAO mice, NBP (100 mg kg-1, i.p., two hours
before and immediately after ischemia) treatment remarkably
decreased the release of apoptosis inducing factor (AIF) and
cytochrome c from cytosol to the mitochondria, and down-
regulated caspase-9 and caspase-3 by 33% and 43% respec-
342 CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 Wang et al.
tively [21]. In addition, NBP (40 mg kg-1, i.p., immediately
after I/R) markedly increased the ratio of Bcl-2 (anti-
apoptosis)/Bax (pro-apoptosis) in the hippocampus of Mon-
golian gerbil after global cerebral ischemia and reperfusion
damage [81]. These data illustrated that NBP treatment
might inhibit mitochondria-dependent cellular apoptotic cas-
cade after ischemia and reperfusion.
JNKs, members of MAPK family, can be activated by
many insults including ischemia [82]. Once activated, JNKs
phosphorylate a variety of transcription factors and cellular
proteins that are related to apoptosis including Bcl-2, P53,
and others [83]. Wen et al. found that NBP (15 mgkg-1, p.o.,
t.i.d., 20 min after I/R) decreased the apoptosis of CA1 py-
ramidal neurons in the I/R-induced brain injury in rats by
preventing phosphorylation of JNK and the activations of its
two important downstream proteins, FasL and c-jun [84].
NBP (75 mgkg-1, q.d., p.o.) treatment also down-regulated
pro-apoptotic signaling mediated by phospho-JNK and p38
in I/R injury of rats [85].
The activations of calcineurin and calpain changed the
structure of chromosome, activated the endonuclease, and
induced DNA fragmentation and the apoptosis of ischemic
neurons [86]. Interestingly, NBP (20, i.p., 10 min
before ischemia) inhibited the activities of calcineurin and
calpain in focal cerebral ischemia rats [87], and NBP (10
μmolL-1) prevented DNA fragmentation and attenuated
morphological changes in rat cortical neurons after hypoxia
and hypoglycemia [88].
Fig. (2). Possible targets of NBP on multiple factors that might be involved in stroke. NBP can improve rCBF through the effect of vasodila-
tion with the elevation of NO, PGI2 and the decrease of TXA2 which could also prevent the platelet aggregation. NBP is also beneficial for
cerebral circulation mainly by up-regulating VEGF, bFGF, TGF-β1 with the effect of angiogenesis, these effects could in turn protect BBB.
During ischemic stroke, mitochondria is being damaged, NBP can restore it through improving the activity of respiratory enzyme complexes,
protecting the structure and function of mitochondrial membrane, as well as maintaining the level of ATPase. Ischemic stroke could induce
oxidative stress by increasing ROS, RNS and MDA levels while decreasing SOD and NOS activities, leading to apoptosis as well as inflam-
mation. However, NBP can modulate these processes by decreasing oxidative stress, up-regulating anti-apoptotic protein Bcl-2, down-
regulating pro-apoptotic proteins Bax, Bad, and Bid expression, inhibiting levels of TNF-α, IL-1β, IL-6, and Nrf2, NF-κB, JNK might also
be involved in these effects. In addition, NBP could decrease the releases of glutamate and intracellular calcium. Up arrows denote enhance-
ment, down arrows denote inhibition. BBB, blood-brain barrier; ROS, reactive oxygen species; bFGF, basic fibroblast growth factor; MMP,
mitochondrial membrane potential; MMfu, mitochondrial membrane fluidity; PLA2, phospholipase A2; TXA2, thromboxane A2; VEGF,
vascular endothelial growth factor; HGF, hepatocyte-growth factor; SOD, superoxide dismutase; MDA, malondialdehyde; GSH,
L-Glutathione; NO, nitric oxide; Nrf2, the nuclear factor erythroid 2-related factor 2; ARE, antioxidant response elements; TGF-β1, trans-
forming growth factor-β1.
ETC complex IV
Cyt c
platelet aggregation
ROS/RNS damage
Ino Hyp Xan
Endoplasmic reticulum
Caspase 9, 3
Bd-2 Cyt c Apaf-1
Ke ap1
stroke brain
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 343
Recently, autophagy has been found to be crucial in the
process of cellular apoptosis as it can degrade and clean up
damaged mitochondria [89, 90]. NBP (10 μmolL-1, begin-
ning of the reperfusion) could inhibit ischemia-induced neu-
roamyloidogenesis by down-regulating autophagy and sup-
pressing the activation of NF-kB pathway in neuroblastoma
2a/amyloid precursor protein 695 cells by 6 h OGD/12 h
reperfusion, indicating that autophagy might be one of the
potential mechanisms of the protective effects of NBP [91].
Inflammation plays an important role in the pathogenesis
of ischemic stroke. After ischemia onset, circulating leuko-
cytes adhere to vessel walls, migrate and accumulate into
ischemic brain tissue, subsequently release pro-inflammatory
mediators (e.g. TNF-α, IL-1), which will exacerbate cerebral
injury [92]. Cell adhesion molecules such as selectins and
intercellular adhesion molecule 1 (ICAM-1) play a vital role
in the infiltration of leukocytes [93-95]. The expressions of
ICAM-1, P-selectin and E-selectin were up-regulated on the
endothelial cell surface in ischemic brain [20]. And the level
of TNF-α in the ischemic cortex was also elevated in rat fo-
cal ischemic stroke models [96]. Administration of NBP (20
mgkg-1, i.p., 10 minutes and 60 minutes after MCAO) inhib-
ited the increases of both ICAM-1 and TNF-α in a MCAO
model, and NBP (0.01 μmolL-1) attenuated the neutrophil-
endothelial cells adhesion induced by TNF-α or IL-1 [20,
97]. TLR4/NF-κB is another important inflammation-related
signaling pathway in ischemic stroke. In a cerebral IR-
induced rat model, NBP (4.5 mgkg-1, i.p., q.d., after IR)
suppressed the activation of TLR4, the phosphorylation of
NF-kB, and subsequently the elevation of pro-inflammatory
cytokines, including TNF-a, IL-1, IL-6 and IL-18 [98].
Glutamate is an endogenous excitatory neurotransmitter
in central nervous system. During ischemia, the failure of
Na+, K+-ATPase resulting from ATP depletion leads to the
decrease of Na+ and K+ gradients and the depolarization of
presynaptic membranes. Sustained cell membrane depolari-
zation causes increased glutamate release from vesica and
decreased glutamate uptake by presynaptic membranes. Ac-
cumulation of glutamate in the synaptic cleft, ultimately,
results in neuronal swelling or necrosis [99]. 5-HT, an im-
portant vasoactive substance, modulates the post-synaptic
action of excitatory amino acids (EAA) by impacting the
activity of N-methyl-D-aspartate (NMDA) [100, 101]. After
global ischemia, the release of glutamate and 5-HT in hippo-
campus and striatum were markedly elevated [102]. NBP (10
μmolL-1) decreased the release of glutamate and 5-HT by
28.9% and 45.3% respectively in cultured cortical neurons
induced by hypoglycemia/hypoxia [103].
After ischemia, glutamate released from neurons and glia
activated the NMDA receptor and then induced the influx of
Ca2+, which is a key activator of ischemic cell death path-
ways [104-106]. Besides, Ca2+ reuptake into the endoplasmic
reticulum by the SERCA-ATPase is impaired due to the
scarce supplies of ATP, whereas Ca2+ release from intracel-
lular stores through the ryanodine receptor is enhanced
[107]. The elevation of intracellular Ca2+ causes the disor-
dered activation of a wide range of enzyme systems. These
enzymes and their metabolic products, such as oxygen free
radicals, cause mitochondrial damage, cytoskeleton break-
down as well as cell important components detrition, such as
proteins, lipids, and DNA [108-110]. NBP (10 μmolL-1)
attenuated the elevation of intracellular calcium induced by
thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-
ATPase in neurons. Nevertheless, NBP had no effect on in-
creased intracellular calcium induced by elevated K+, indi-
cating that NBP might only reduce calcium release from
intracellular stores [111]. Consistent with these results, NBP
(100 μmolL-1) was found to have inhibitory effect on the
release of intracellular calcium that was initiated by norepi-
nephrine in the rat isolated tail artery, but it exerted no effect
on the constriction when extracellular calcium was added
NBP has been widely used for treating acute ischemic
stroke in China and shows a good clinical effect. Moreover,
the phase II clinical trial of NBP softgel capsules in ischemic
stroke patients has started in United State in 2017. This will
be a randomized, double-blind, placebo-controlled, add-on to
standard-of-care study with a primary objective to assess the
safety of NBP treatment in patients with mild to moderate
acute ischemic stroke, and the results are looked forward to.
Apart from the potent pharmacological actions in ischemic
stroke, NBP also showed beneficial effects in multiple neu-
rodegenerative diseases, such as Alzheimer’s disease (AD)
[113-116], dementia [117-119], Parkinson’s disease [118,
120, 121], and Amyotrophic lateral sclerosis (ALS) [122]. A
number of studies demonstrated that NBP could affect the
two important pathophysiological aspects of ischemic stroke,
improving the condition of cerebral circulation and neuro-
protection by acting on multiple active targets (Fig. 2). Al-
though NBP has been considered as a multi-target drug, the
in-depth study of its target has been ongoing. The latest re-
search found NQO1, p53 and indoleamine 2,3-dioxygenase
(IDO) might be direct binding target of NBP [123]. The pre-
clinical and clinical studies of NBP could provide useful
clues for developing more potent and comprehensive thera-
peutic strategies for ischemic stroke. However, further study
will be needed to understand the precise molecular mecha-
nisms in the future.
AD = Alzheimer’s Disease
AIF = Apoptosis-Inducing Factor
AIS = Acute Ischemic Stroke
ALS = Amyotrophic Lateral Sclerosis
ARE = Antioxidant Response Elements
BBB = Blood-Brain Barrier
bFGF = Basic Fibroblast Growth Factor
CIR = Cerebral Ischemia-reperfusion
344 CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 Wang et al.
CO = Carbon Monoxide
COPD = Chronic Obstructive Pulmonary Disease
EAA = Excitatory Amino Acids
HGF = Hepatocyte-Growth Factor
ICAM-1 = Intercellular Adhesion Molecule-1
I/R = Ischemia/Reperfusion
GSH = L-Glutathione
6-BBP = 6-Bromo-3-n-butylphthalide
LPS = Lipopolysaccharide
MCA = Middle Cerebral Artery
MCAO = Middle Cerebral Artery Occlusion
MDA = Malondialdehyde
MMfu = Mitochondrial Membrane Fluidity
MMP = Mitochondrial Membrane Potential
NBP = Dl-3-n-Butylphthalide
NIHSS = National Institutes of Health Stroke Scale
NMDA = N-methyl-D-aspartate
NOS = Nitric Oxide Synthase
NO = Nitric Oxide
Nrf2 = The Nuclear Factor Erythroid 2-related Factor 2
OGD = Oxygen-glucose Deprivation
PHPB = 2-(1-Hydroxypentyl)-benzoate
PLA2 = Phospholipase A2
PGI2 = Prostaglandin I2
ROS = Reactive Oxygen Species
rtPAs = Recombinant Tissue Plasminogen Activators
SOD = Superoxide Dismutase
TGF-β1 = Transforming Growth Factor-β1
TXA2 = Thromboxane A2
VEGF = Vascular Endothelial Growth Factor
The authors all agreed with the publication of this article.
The authors declare no conflict of interest, financial or
This project was supported by the grants from National
Natural Sciences Foundation of China (No. 81373387,
81473200 and 81673420), CAMS Innovation Fund for
Medical Sciences (No. 2017-I2M-2-004), and Beijing Key
Laboratory of New Drug Mechanisms and Pharmacological
Evaluation Study (BZ0150).
[1] Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ,
Cushman M, et al. Executive Summary: Heart disease and stroke
statistics-2016 Update: A report from the American Heart Associa-
tion. Circulation 2016; 133: 447-54.
[2] Chen XL, Wang KW. The fate of medications evaluated for
ischemic stroke pharmacotherapy over the period 1995–2015. Acta
Pharm Sin B 2016; 6: 522-30.
[3] Benjamin EJ, Blaha MJ, Chiuve SE, et al. Heart disease and stroke
statistics-2017 update: A report from the American Heart Associa-
tion. Circulation 2017; 135: e146-e603.
[4] Roth JM, editor Recombinant tissue plasminogen activator for the
treatment of acute ischemic stroke. Baylor University Medical Cen-
ter Proceedings; 2011: Taylor & Francis.
[5] Kidwell CS, Liebeskind DS, Starkman S, Saver JL. Trends in acute
ischemic stroke trials through the 20th century. Stroke 2001; 32:
[6] Terasaki Y, Liu Y, Hayakawa K, et al. Mechanisms of neurovascu-
lar dysfunction in acute ischemic brain. Curr Med Chem 2014; 21:
[7] Xiong XY, Liu L, Yang QW. Refocusing Neuroprotection in Cere-
bral Reperfusion Era: New challenges and strategies. Front Neurol
2018; 9: 249.
[8] Feng YP, Hu D, Zhang LY. Effect of DL-butylphthalide (NBP) on
mouse brain energy metabolism in complete brain ischemia in-
duced by decapitation. Yao Xue Xue Bao 1995; 30: 741-4.
[9] Yan CH, Feng YP, Zhang JT. Effects of dl-3-n-butylphthalide on
regional cerebral blood flow in right middle cerebral artery occlu-
sion rats. Zhongguo Yao Li Xue Bao 1998; 19: 117-20.
[10] Liu CL, Liao SJ, Zeng JS, et al. dl-3n-butylphthalide prevents
stroke via improvement of cerebral microvessels in RHRSP. J Neu-
rol Sci 2007; 260: 106-13.
[11] Chong ZZ, Feng YP. dl-3-n-butylphthalide attenuates reperfusion-
induced blood-brain barrier damage after focal cerebral ischemia in
rats. Zhongguo Yao Li Xue Bao 1999; 20: 696-700.
[12] Peng Y, Zeng X, Feng Y, Wang X. Antiplatelet and antithrombotic
activity of L-3-n-butylphthalide in rats. J Cardiovasc Pharmacol
2004; 43: 876-81.
[13] Liu XG, Feng YP. Protective effect of dl-3-n-butylphthalide on
ischemic neurological damage and abnormal behavior in rats sub-
jected to focal ischemia. Yao Xue Xue Bao 1995; 30: 896-903.
[14] Lin JF, Feng YP. Effect of dl-3-n-butylphthalide on delayed neu-
ronal damage after focal cerebral ischemia and intrasynaptosomes
calcium in rats. Yao Xue Xue Bao 1996; 31: 166-70.
[15] Zhang LY, Feng YP. Effect of dl-3-n-butylphthalide (NBP) on life
span and neurological deficit in SHRsp rats. Yao Xue Xue Bao
1996; 31: 18-23.
[16] Chong Z, Feng Y. Protective effects of dl-3-n-butylphthalide on
changes of regional cerebral blood flow and blood-brain barrier
damage following experimental subarachnoid hemorrhage. Chin
Med J (Engl) 1998; 111: 858-60.
[17] Ye J, Zhai L, Zhang S, et al. DL-3-n-butylphthalide inhibits platelet
activation via inhibition of cPLA2-mediated TXA2 synthesis and
phosphodiesterase. Platelets 2015; 26: 736-44.
[18] Chong ZZ, Feng YP. Effects of dl-3-n-butylphthalide on produc-
tion of TXB2 and 6-keto-PGF1 alpha in rat brain during focal cere-
bral ischemia and reperfusion. Zhongguo Yao Li Xue Bao 1997;
18: 505-8.
[19] Dong GX, Feng YP. Effects of NBP on ATPase and anti-oxidant
enzymes activities and lipid peroxidation in transient focal cerebral
ischemic rats. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2002; 24:
[20] Xu HL, Feng YP. Inhibitory effects of chiral 3-n-butylphthalide on
inflammation following focal ischemic brain injury in rats. Acta
Pharmacol Sin 2000; 21: 433-8.
[21] Li J, Li Y, Ogle M, et al. DL-3-n-butylphthalide prevents neuronal
cell death after focal cerebral ischemia in mice via the JNK path-
way. Brain Res 2010; 1359: 216-26.
[22] Chang Q, Wang XL. Effects of chiral 3-n-butylphthalide on apop-
tosis induced by transient focal cerebral ischemia in rats. Acta
Pharmacol Sin 2003; 24: 796-804.
[23] Diao X, Deng P, Xie C, et al. Metabolism and pharmacokinetics of
3-n-butylphthalide (NBP) in humans: The role of cytochrome
P450s and alcohol dehydrogenase in biotransformation. Drug Me-
tab Dispos 2013; 41: 430-44.
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 345
[24] Xue LX, Zhang T, Zhao YW, Geng Z, Chen JJ, Chen H. Efficacy
and safety comparison of DL-3-n-butylphthalide and Cerebrolysin:
Effects on neurological and behavioral outcomes in acute ischemic
stroke. Exp Ther Med 2016; 11: 2015-20.
[25] Diao X, Ma Z, Wang H, et al. Simultaneous quantitation of 3-n-
butylphthalide (NBP) and its four major metabolites in human
plasma by LC-MS/MS using deuterated internal standards. J Pharm
Biomed Anal 2013; 78-79: 19-26.
[26] Cui LY, Zhu YC , Gao S, et al. Ninety-day administration of dl-3-n-
butylphthalide for acute ischemic stroke: A randomized, double-
blind trial. Chin Med J (Engl) 2013; 126: 3405-10.
[27] Li J, Xu SF, Peng Y, Feng N, Wang L, Wang XL. Conversion and
pharmacokinetics profiles of a novel pro-drug of 3-n-
butylphthalide, potassium 2-(1-hydroxypentyl)-benzoate, in rats
and dogs. Acta Pharmacol Sin 2018; 39: 275-85.
[28] Cui L, Li S, Zhang W, et al. Effects of dl-3-butylphthalide soft
capsules on treatment of acute ischemic stroke: Multi-center, ran-
domized, double-blind, double-dummy and aspirin-control study.
Zhonghua Shen Jing Ke Za Zhi 2008; 41: 727-30.
[29] Cui L, Liu X, Zhu Y, et al. Effects of dl-3-butylphthalide on treat-
ment of acute ischemic stroke with moderate symptoms: a multi-
center, randomized, double-blind, placebo-control trial. Chin J
Neurol 2005; 38: 251-4.
[30] Zhao H, Yun W, Zhang Q, et al. Mobilization of circulating endo-
thelial progenitor cells by dl-3-n-butylphthalide in acute ischemic
stroke patients. J Stroke Cerebrovasc Dis 2016; 25: 752-60.
[31] Hu X, De Silva TM, Chen J, Faraci FM. Cerebral vascular disease
and neurovascular injury in ischemic stroke. Circ Res 2017; 120:
[32] Donnelly J, Budohoski KP, Smielewski P, Czosnyka M. Regulation
of the cerebral circulation: Bedside assessment and clinical impli-
cations. Crit Care 2016; 20: 129.
[33] Yan CH, Zhang JT, Feng YP. Effect of dl-3-n-butylphthalide on
striatum cerebral blood flow in normal and middle cerebral artery
occlusion rats. Chinese J Pharmacol Toxicol 1998; 12: 36-9.
[34] Xu HL, Feng YP. Effects of 3-n-butylphthalide on production of
vasoactive substances by cerebral and aortic endothelial cells.
Zhongguo Yao Li Xue Bao 1999; 20: 929-33.
[35] Murohara T, Horowitz JR, Silver M, et al. Vascular endothelial
growth factor/vascular permeability factor enhances vascular per-
meability via nitric oxide and prostacyclin. Circulation 1998; 97:
[36] Chong ZZ, Feng YP. Effects of DL-3-n-butylphthalide on arachi-
donic acid release and phospholipase A 2 mRNA expression in
cerebral cortex after middle cerebral artery occlusion in rats. Yao
Xue Xue Bao 2000; 35: 561-5.
[37] Xu HL, Feng YP. Effects of 3-n-butylphthalide on thrombosis
formation and platelet function in rats. Yao Xue Xue Bao 2001; 36:
[38] Lapi D, Colantuoni A. Remodeling of Cerebral Microcirculation
after Ischemia-Reperfusion. J Vasc Res 2015; 52: 22-31.
[39] Fisher M, Meadows ME, Do T, et al. Delayed treatment with intra-
venous basic fibroblast growth factor reduces infarct size following
permanent focal cerebral ischemia in rats. J Cereb Blood Flow Me-
tab 1995; 15: 953-9.
[40] Leker RR, Soldner F, Velasco I, Gavin DK, Androutsellis-
Theotokis A, McKay RD. Long-lasting regeneration after ischemia
in the cerebral cortex. Stroke 2007; 38: 153-61.
[41] Ponce CC, de Lourdes Lopes Ferrari Chauffaille M, Ihara SS, Silva
MR. Increased angiogenesis in primary myelofibrosis: latent trans-
forming growth factor-beta as a possible angiogenic factor. Rev
Bras Hematol Hemoter 2014; 36: 322-8.
[42] Li QF, Kong SY, Deji QZ, He L, Zhou D. Effects of dl-3-n-
butylphthalide on expression of VEGF and bFGF in rat brain with
permanent focal cerebral ischemia. Sichuan Da Xue Xue Bao Yi
Xue Ban 2008; 39: 84-8.
[43] Liao SJ, Lin JW, Pei Z, Liu CL, Zeng JS, Huang RX. Enhanced
angiogenesis with dl-3n-butylphthalide treatment after focal cere-
bral ischemia in RHRSP. Brain Res 2009; 1289: 69-78.
[44] Jiang Y, Sun L, Xuan X, Wang J. Impacts of N-Butylphthalide on
expression of growth factors in rats with focal cerebral ischemia.
Bosn J Basic Med Sci 2016; 16: 102-7.
[45] Lu XL, Luo D, Yao XL, et al. dl-3n-Butylphthalide promotes angi-
ogenesis via the extracellular signal-regulated kinase 1/2 and phos-
phatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase sig-
naling pathways. J Cardiovasc Pharmacol 2012; 59: 352-62.
[46] Borlongan CV, Rodrigues AA, Jr., Oliveira MC. Breaking the
barrier in stroke: what should we know? A mini-review. Curr
Pharm Des 2012; 18: 3615-23.
[47] Ballabh P, Braun A, Nedergaard M. The blood-brain barrier: an
overview: structure, regulation, and clinical implications. Neuro-
biol Dis 2004; 16: 1-13.
[48] Kaur C, Ling EA. Blood brain barrier in hypoxic-ischemic condi-
tions. Curr Neurovasc Res 2008; 5: 71-81.
[49] Brouns R, Wauters A, De Surgeloose D, Marien P, De Deyn PP.
Biochemical markers for blood-brain barrier dysfunction in acute
ischemic stroke correlate with evolution and outcome. Eur Neurol
2011; 65: 23-31.
[50] Li L, Zhang B, Tao Y, et al. DL-3-n-butylphthalide protects endo-
thelial cells against oxidative/nitrosative stress, mitochondrial dam-
age and subsequent cell death after oxygen glucose deprivation in
vitro. Brain Res 2009; 1290: 91-101.
[51] Kawasaki K, Yano K, Sasaki K, et al. Correspondence between
neurological deficit, cerebral infarct size, and Rho-kinase activity
in a rat cerebral thrombosis model. J Mol Neurosci 2009; 39: 59-
[52] Hu J, Wen Q, Wu Y, Li B, Gao P. The effect of butylphthalide on
the brain edema, blood-brain barrier of rats after focal cerebral in-
farction and the expression of Rho A. Cell Biochem Biophys 2014;
69: 363-8.
[53] Bi M, Zhang M, Guo D, et al. N-Butylphthalide Alleviates Blood-
Brain Barrier Impairment in Rats Exposed to Carbon Monoxide.
Front Pharmacol 2016; 7: 394.
[54] Chan PH. Mitochondrial dysfunction and oxidative stress as deter-
minants of cell death/survival in stroke. Ann N Y Acad Sci 2005;
1042: 203-9.
[55] Ham PB, 3rd, Raju R. Mitochondrial function in hypoxic ischemic
injury and influence of aging. Prog Neurobiol 2017; 157: 92-116.
[56] Xiong J, Feng Y. Effects of butylphthalide on the activities of
complexes of the mitochondrial respiratory chain. Yao Xue Xue
Bao 1999; 34: 241-5.
[57] Xiong J, Feng Y. The protective effect of butylphthalide against
mitochondrial injury during cerebral ischemia. Yao Xue Xue Bao
2000; 35: 408-12.
[58] Chen H, Yoshioka H, Kim GS, et al. Oxidative stress in ischemic
brain damage: mechanisms of cell death and potential molecular
targets for neuroprotection. Antioxid Redox Signal 2011; 14: 1505-
[59] Gilgun-Sherki Y, Rosenbaum Z, Melamed E, Offen D. Antioxidant
therapy in acute central nervous system injury: Current state. Phar-
macol Rev 2002; 54: 271-84.
[60] Chan PH. Reactive oxygen radicals in signaling and damage in the
ischemic brain. J Cereb Blood Flow Metab 2001; 21: 2-14.
[61] Sugawara T, Chan PH. Reactive oxygen radicals and pathogenesis
of neuronal death after cerebral ischemia. Antioxid Redox Signal
2003; 5: 597-607.
[62] Cojocaru IM, Cojocaru M, Sapira V, Ionescu A. Evaluation of
oxidative stress in patients with acute ischemic stroke. Rom J In-
tern Med 2013; 51: 97-106.
[63] Chrissobolis S, Miller AA, Drummond GR, Kemp-Harper BK,
Sobey CG. Oxidative stress and endothelial dysfunction in cere-
brovascular disease. Front Biosci 2011; 16: 1733-45.
[64] Szeto HH. Mitochondria-targeted cytoprotective peptides for
ischemia-reperfusion injury. Antioxid Redox Signal 2008; 10: 601-
[65] Eisenberg T, Buttner S, Kroemer G, Madeo F. The mitochondrial
pathway in yeast apoptosis. Apoptosis 2007; 12: 1011-23.
[66] Burwell LS, Brookes PS. Mitochondria as a target for the cardio-
protective effects of nitric oxide in ischemia-reperfusion injury.
Antioxid Redox Signal 2008; 10: 579-99.
[67] Broughton BR, Reutens DC, Sobey CG. Apoptotic mechanisms
after cerebral ischemia. Stroke 2009; 40: e331-9.
[68] Manzanero S, Santro T, Arumugam TV. Neuronal oxidative stress
in acute ischemic stroke: sources and contribution to cell injury.
Neurochem Int 2013; 62: 712-8.
[69] Rodrigo R, Fernandez-Gajardo R, Gutierrez R, et al. Oxidative
stress and pathophysiology of ischemic stroke: Novel therapeutic
opportunities. CNS Neurol Disord Drug Targets 2013; 12: 698-714.
[70] Hu D, Huang XX, Feng YP. Effect of dl-3-n-butylphthalide (NBP)
on purine metabolites in striatum extracellular fluid in four-vessel
occlusion rats. Yao Xue Xue Bao 1996; 31: 13-7.
346 CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 Wang et al.
[71] Chong ZZ, Feng YP. Effect of 3-n-butylphthalide on reperfusion
induced lipid peroxidation following cerebral ischemia in rats and
superoxide radical formation in vitro. J Chinese Pharmac Sci1999;
8: 95-9.
[72] Zhao X, Sun G, Zhang J, et al. Transcription factor Nrf2 protects
the brain from damage produced by intracerebral hemorrhage.
Stroke 2007; 38: 3280-6.
[73] Eftekharzadeh B, Maghsoudi N, Khodagholi F. Stabilization of
transcription factor Nrf2 by tBHQ prevents oxidative stress-
induced amyloid beta formation in NT2N neurons. Biochimie
2010; 92: 245-53.
[74] Zhang DD. Mechanistic studies of the Nrf2-Keap1 signaling path-
way. Drug Metab Rev 2006; 38: 769-89.
[75] Bhakkiyalakshmi E, Sireesh D, Ramkumar KM. Redox Sensitive
Transcription via Nrf2-Keap1 in Suppression of Inflammation.
Immunity and Inflammation in Health and Disease: Elsevier; 2017.
p. 149-61.
[76] Wang B, Cao W, Biswal S, Dore S. Carbon monoxide-activated
Nrf2 pathway leads to protection against permanent focal cerebral
ischemia. Stroke 2011; 42: 2605-10.
[77] Lee JM, Calkins MJ, Chan K, Kan YW, Johnson JA. Identification
of the NF-E2-related factor-2-dependent genes conferring protec-
tion against oxidative stress in primary cortical astrocytes using
oligonucleotide microarray analysis. J Biol Chem 2003; 278:
[78] Hua K, Sheng X, Li TT, et al. The edaravone and 3-n-
butylphthalide ring-opening derivative 10b effectively attenuates
cerebral ischemia injury in rats. Acta Pharmacol Sin 2015; 36: 917-
[79] Li Q, Cheng Y, Bi M, et al. Effects of N-butylphthalide on the
activation of Keap1/Nrf-2 signal pathway in rats after carbon mon-
oxide poisoning. Environ Toxicol Pharmacol 2015; 40: 22-9.
[80] Broughton BR, Reutens DC, Sobey CG. Apoptotic mechanisms
after cerebral ischemia. Stroke 2009; 40: e331-e9.
[81] Hai W, Yang Y, Wang YL, Nie YX. The effect of dl-3n-
butylphthalide on the neurons in the hippocampus of mongolian
gerbil and the expression of p-ERK, Bcl-2 and Bax after global
cerebral ischemia and reperfusion damage. Chin J Clini-
cians(Electronic Edition) 2015: 1157-62.
[82] Mujaibel LM, Kilarkaje N. Mitogen-activated protein kinase sig-
naling and its association with oxidative stress and apoptosis in
lead-exposed hepatocytes. Environ Toxicol 2015; 30: 513-29.
[83] Zhang S, Qi Y, Xu Y, et al. Protective effect of flavonoid-rich
extract from Rosa laevigata Michx on cerebral ischemia-
reperfusion injury through suppression of apoptosis and inflamma-
tion. Neurochem Int 2013; 63: 522-32.
[84] Wen XR, Tang M, Qi DS, et al. Butylphthalide Suppresses Neu-
ronal Cells Apoptosis and Inhibits JNK-Caspase3 Signaling Path-
way After Brain Ischemia /Reperfusion in Rats. Cell Mol Neuro-
biol 2016; 36: 1087-95.
[85] Yan RY, Wang SJ, Yao GT, Liu ZG, Xiao N. The protective effect
and its mechanism of 3-n-butylphthalide pretreatment on cerebral
ischemia reperfusion injury in rats. Eur Rev Med Pharmacol Sci
2017; 21: 5275-82.
[86] Shioda N, Fukunaga K. Functional roles of constitutively active
calcineurin in delayed neuronal death after brain ischemia. Ya-
kugaku Zasshi 2011; 131: 13-20.
[87] Dong GX, Feng YP. Effects of 3-N-butylphthalide on cortical
calcineurin and calpain activities in focal cerebral ischemia rats.
Yao Xue Xue Bao 2000; 35: 790-2.
[88] Dong G, Feng Y. Hypoxia/hypoglycemia-induced apoptosis of rat
cortical neurons is prevented by dl-3-butylphthalide. Yao Xue Xue
Bao 1999; 34: 176-80.
[89] Levine B, Klionsky DJ. Development by self-digestion: molecular
mechanisms and biological functions of autophagy. Dev Cell 2004;
6: 463-77.
[90] Kubli DA, Gustafsson AB. Mitochondria and mitophagy: The yin
and yang of cell death control. Circ Res 2012; 111: 1208-21.
[91] Zhang T, Wang H, Li Q, Huang J, Sun X. Modulating autophagy
affects neuroamyloidogenesis in an in vitro ischemic stroke model.
Neuroscience 2014; 263: 130-7.
[92] Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke:
Role of inflammatory cells. J Leukoc Biol 2010; 87: 779-89.
[93] Yilmaz G, Granger DN. Cell adhesion molecules and ischemic
stroke. Neurol Res 2008; 30: 783-93.
[94] Lindsberg PJ, Carpen O, Paetau A, Karjalainen-Lindsberg ML,
Kaste M. Endothelial ICAM-1 expression associated with inflam-
matory cell response in human ischemic stroke. Circulation 1996;
94: 939-45.
[95] Lakhan SE, Kirchgessner A, Hofer M. Inflammatory mechanisms
in ischemic stroke: therapeutic approaches. J Transl Med 2009; 7:
[96] Stins MF, Gilles F, Kim KS. Selective expression of adhesion
molecules on human brain microvascular endothelial cells. J Neu-
roimmunol 1997; 76: 81-90.
[97] Xu HL, Feng YP. Effects of 3 n butylphthalide on neutrophil endo-
thelial cell adhesion and endothelial cell injury induced by an-
oxia/reoxygenation, interleukin 1 and tumor necrosis factor α 1.
Chinese J Pharmacol Toxicol 1999; 4: 281-4.
[98] Zhang P, Guo ZF, Xu YM, Li YS, Song JG. N-Butylphthalide
(NBP) ameliorated cerebral ischemia reperfusion-induced brain in-
jury via HGF-regulated TLR4/NF-kappaB signaling pathway.
Biomed Pharmacother 2016; 83: 658-66.
[99] Arundine M, Tymianski M. Molecular mechanisms of glutamate-
dependent neurodegeneration in ischemia and traumatic brain in-
jury. Cell Mol Life Sci 2004; 61: 657-68.
[100] Nedergaard S, Engberg I, Flatman JA. The modulation of excita-
tory amino acid responses by serotonin in the cat neocortex in vitro.
Cell Mol Neurobiol 1987; 7: 367-79.
[101] Reynolds JN, Baskys A, Carlen PL. The effects of serotonin on N-
methyl-D-aspartate and synaptically evoked depolarizations in rat
neocortical neurons. Brain Res 1988; 456: 286-92.
[102] Globus MY, Wester P, Busto R, Dietrich WD. Ischemia-induced
extracellular release of serotonin plays a role in CA1 neuronal cell
death in rats. Stroke 1992; 23: 1595-601.
[103] Chong ZZ, Feng YP. Effects of NBP on the release of glutamate
and 5-HT from cultured neurons subjected to hypoglyce-
mia/hypoxia. Chinese Pharmac J 1999; 34: 589-91.
[104] Kristian T, Siesjo BK. Calcium in ischemic cell death. Stroke 1998;
29: 705-18.
[105] Tymianski M, Tator CH. Normal and abnormal calcium homeosta-
sis in neurons: a basis for the pathophysiology of traumatic and
ischemic central nervous system injury. Neurosurgery 1996; 38:
[106] Chung JW, Ryu WS, Kim BJ, Yoon BW. Elevated calcium after
acute ischemic stroke: association with a poor short-term outcome
and long-term mortality. J Stroke 2015; 17: 54-9.
[107] Kalogeris T, Baines CP, Krenz M, Korthuis RJ. Cell biology of
ischemia/reperfusion injury. Int Rev Cell Mol Biol 2012; 298: 229-
[108] Abe K, Aoki M, Kawagoe J, et al. Ischemic delayed neuronal
death. A mitochondrial hypothesis. Stroke 1995; 26: 1478-89.
[109] Halliwell B. Reactive oxygen species and the central nervous sys-
tem. J Neurochem 1992; 59: 1609-23.
[110] Kukreja RC, Kontos HA, Hess ML, Ellis EF. PGH synthase and
lipoxygenase generate superoxide in the presence of NADH or
NADPH. Circ Res 1986; 59: 612-9.
[111] Xiong J, Feng YP. Effects of 3-n-butylphthalide on the increase of
intracellular calcium in neurons subjected to hypoxia and hypogly-
cemia and its mechanisms. Yao Xue Xue Bao 1999; 12: 893-7.
[112] Lu X, Feng Y. Effect of dl-3-n-butylphthalide on isolated tail artery
contraction of rat induced by potassium chloride and norepineph-
rine. Chinese J Pharmacol Toxicol 1995; 10: 113-5.
[113] Peng Y, Xu S, Chen G, Wang L, Feng Y, Wang X. l-3-n-
Butylphthalide improves cognitive impairment induced by chronic
cerebral hypoperfusion in rats. J Pharmacol Exp Ther 2007; 321:
[114] Peng Y, Xing C, Xu S, et al. L-3-n-butylphthalide improves cogni-
tive impairment induced by intracerebroventricular infusion of
amyloid-beta peptide in rats. Eur J Pharmacol 2009; 621: 38-45.
[115] Peng Y, Sun J, Hon S, et al. L-3-n-butylphthalide improves cogni-
tive impairment and reduces amyloid-beta in a transgenic model of
Alzheimer's disease. J Neurosci 2010; 30: 8180-9.
[116] Peng Y, Hu Y, Xu S, et al. L-3-n-butylphthalide reduces tau phos-
phorylation and improves cognitive deficits in AbetaPP/PS1-
Alzheimer's transgenic mice. J Alzheimers Dis 2012; 29: 379-91.
[117] Huai Y, Dong Y, Xu J, et al. L-3-n-butylphthalide protects against
vascular dementia via activation of the Akt kinase pathway. Neural
Regen Res 2013; 8: 1733-42.
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 347
[118] Xiong N, Huang J, Chen C, et al. Dl-3-n-butylphthalide, a natural
antioxidant, protects dopamine neurons in rotenone models for
Parkinson's disease. Neurobiol Aging 2012; 33: 1777-91.
[119] Wang F, Chen H, Sun XJ, Ke ZJ. Improvement of cognitive defi-
cits in SAMP8 mice by 3-n-butylphthalide. Neurol Res 2014; 36:
[120] Huang JZ, Chen YZ, Su M, et al. dl-3-n-Butylphthalide prevents
oxidative damage and reduces mitochondrial dysfunction in an
MPP(+)-induced cellular model of Parkinson's disease. Neurosci
Lett 2010; 475: 89-94.
[121] Koppula S, Kumar H, More SV, Kim BW, Kim IS, Choi DK. Re-
cent advances on the neuroprotective potential of antioxidants in
experimental models of Parkinson's disease. Int J Mol Sci 2012; 13:
[122] Feng X, Peng Y, Liu M, Cui L. DL-3-n-butylphthalide extends
survival by attenuating glial activation in a mouse model of
amyotrophic lateral sclerosis. Neuropharmacology 2012; 62: 1004-
[123] Wang Y, Qi W, Zhang L, et al. The novel targets of DL-3-n-
butylphthalide predicted by similarity ensemble approach in com-
bination with molecular docking study. Quant Imaging Med Surg
2017; 7: 532-6.
DISCLAIMER: The above article has been published in Epub (ahead of print) on the basis of the materials provided by the author. The Edito-
rial Department reserves the right to make minor modifications for further improvement of the manuscript.
PMID: 29895257
... Interleukin 4 receptor IL- 6 Interleukin-6 LIFR ...
... Despite advances in understanding the pathophysiology of ischemic stroke, successful treatment remains a major challenge in clinical medicine. Reperfusion with recombinant tissue plasminogen activator (rtPA) remains the only pharmacological therapy approved by the Food and Drug Administration (FDA) to treat ischemic stroke [6]. Although the fact that some progress has been made in drug and mechanical thrombolytic recanalization therapy to help patients recover from ischemic stroke in the past few decades, the treatment options for ischemic stroke are still limited. ...
Full-text available
Ischemic stroke is a heterogeneous brain injury with complex pathophysiology and it is also a time sensitive neurological injury disease. At present, the treatment options for ischemic stroke are still limited. 6S-5-methyltetrahydrofolate-calcium (MTHF-Ca) is the calcium salt of the predominant form of dietary folate in circulation. MTHF-Ca has potential neuroprotective effect on neurocytes, but whether it can be used for ischemic stroke treatment remains unknown. We established zebrafish ischemic stroke model through photothrombotic method to evaluate the protective effect of MTHF-Ca on the ischemic brain injury of zebrafish. We demonstrated that MTHF-Ca reduced the brain damage by reducing motor dysfunction and neurobehavioral defects of zebrafish with telencephalon infarction injury. MTHF-Ca counteracted oxidative damages after Tel injury by increasing the activities of GSH-Px and SOD and decreasing the content of MDA. RNA-seq and RT-qPCR results showed that MTHF-Ca played a neuroprotective role by alleviating neuroinflammation, inhibiting blood coagulation, and neuronal apoptosis processes. Overall, we have demonstrated that MTHF-Ca has neuroprotective effect in ischemic stroke and can be used as a potential treatment for ischemic stroke.
... Stroke has high morbidity and may even be higher in further, the population of PSA will grow larger and larger, impacts more to patient's daily life and mental health. 31,32 It pushes us to pay more attention and medical resource to PSA's earlier identification and treatment. Following the guideline for cross-cultural adaption from WHO, we develop the CQAB adapted to environment and customs in China. ...
Full-text available
Purpose: The quick aphasia battery (QAB) was designed to evaluate language disorder from multi-dimension efficiently, which had been translated into several languages but lacked in Chinese. This study conducted cross-cultural adaption for the Chinese version and verified its psychometric properties. Material and methods: First, the Chinese Version of quick aphasia battery (CQAB) was adapted following WHO literature guidelines with steps of forward translation, expert panel, back-translation, pre-test, and interview, then develop the final version. Second, the psychometric properties tests were conducted in 128 post-stroke patients to identify if aphasia happens and verify the validity and reliability of CQAB. Results: The Cronbach's alpha coefficient of the CQAB is 0.962, test-retest reliability 0.849, and inter-rater reliability 0.998. Content validity 0.917, KMO 0.861, exploratory factor analysis extracted 2 factors named "language understanding" and "language program", cumulative variance contribution rate is 91.588% >50%. Calibration association validity 0.977. Sensitivity 0.977, specificity 0.932, with the optimal cutoff point is 8.86. Conclusion: The study supported CQAB, which adapted following standardized guidelines, is reliable and effective to assess language impairment in post-stroke patients.
Renal ischemia-reperfusion (I/R) injury leads to irreversible brain damage with serious consequences. Activation of oxidative stress and release of inflammatory mediators are considered potential pathological mechanisms. Butylphthalide (NBP) has anti-inflammatory and antioxidant effects on I/R injuries. However, it is unclear whether NBP can effectively mitigate renal I/R secondary to brain injury as well as its mechanism, which are the aims of this study. Both renal I/R injury rats and oxygen and glucose deprivation cell models were established and pre-intervened NBP. The Morris water maze assay was used to detect behavior. Hippocampal histopathology and function were examined after renal I/R. Apoptosis and tube-forming capacity of brain microvascular endothelial cells (BMVECs) were tested. Immunohistochemistry and Western blot were used to measure protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) pathway and NOD-like receptor C2 (NOD2)/Mitogen-activated protein kinases (MAPK)/Nuclear factor kappa-B (NF-κB) pathway. NBP treatment attenuated renal I/R-induced brain tissue damage and learning and memory dysfunction. NBP treatment inhibited apoptosis and promoted blood-brain barrier restoration and microangiogenesis. Also, it decreased oxidative stress levels and pro-inflammatory factor expression in renal I/R rats. Furthermore, NBP enhanced BMVECs’ viability and tube-forming capacity while inhibiting apoptosis and oxidative stress. Notably, the alleviating effects of NBP were attributed to Nrf2/HO-1 pathway activation and NOD2/MAPK/NF-κB inhibition. This study demonstrates that NBP maintains BBB function by activating the Nrf2/HO-1 pathway and inhibiting the NOD2/MAPK/NF-κB pathway to suppress inflammation and oxidative stress, thereby alleviating renal I/R-induced brain injury.
Full-text available
Objective To investigate the effect of butylphthalide combined with fasudil in the treatment of subarachnoid hemorrhage (SAH) with cerebral vasospasm (CVS) on inflammatory factors, cognitive function and vascular endothelial function. Methods It is a retrospective study in which a total of 104 patients with SAH with CVS admitted to Baoding First Central Hospital from July 2020 to February 2022 were selected and randomly divided into two groups by drawing lots. Patients in the control group were treated with basic symptomatic treatment, while those in the observation group were treated with butylphthalide soft capsule combined with fasudil hydrochloride injection on the basis of the control group. Before and after treatment, serum neuron specific enolase (NSE), tumor necrosis factor-α(TNF-α), interleukin-8(IL-8), hypersensitive C-reactive protein (CRP), Birmingham Cognitive Screen test (BCoS) score, serum soluble intercellular adhesion molecule-1(ICAM-1), serum endothelin-1(ET-1), vascular endothelial growth factor (VEGF) levels, and endothelium-dependent vasodilation function (FMD) in the two groups were compared. Results After treatment, the expression levels of NSE, TNF-α, IL-8 and CRP in the two groups were significantly decreased, and the expression levels of all indicators in the observation group were lower than that in the control group (p<0.05). After treatment, the scores of orientations, attention, memory, language, practice and action in the two groups were significantly increased, and the scores of all dimensions in the observation group were higher than those in the control group (p<0.05). After treatment, S-ICAM-1, ET-1, VEGF, FMD decreased in both groups, and all indicators of the observation group were lower than those of the control group, with statistically significant differences (p<0.05). Conclusion Butylphthalide combined with fasudil therapy was found as effective in reducing inflammatory factors, ameliorating cognitive function and vascular endothelial function in patients with subarachnoid hemorrhage complicated with cerebral vasospasm.
Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder of the central nervous system after Alzheimer's disease. The current understanding of PD focuses mainly on the loss of dopamine neurons in the substantia nigra region of the midbrain, which is attributed to factors such as oxidative stress, alpha-synuclein aggregation, neuroinflammation, and mitochondrial dysfunction. These factors together contribute to the PD phenotype. Recent studies on PD pathology have introduced a new form of cell death known as ferroptosis. Pathological changes closely linked with ferroptosis have been seen in the brain tissues of PD patients, including alterations in iron metabolism, lipid peroxidation, and increased levels of reactive oxygen species. Preclinical research has demonstrated the neuroprotective qualities of certain iron chelators, antioxidants, Fer-1, and conditioners in Parkinson's disease. Natural plant products have shown significant potential in balancing ferroptosis-related factors and adjusting their expression levels. Therefore, it is vital to understand the mechanisms by which natural plant products inhibit ferroptosis and relieve PD symptoms. This review provides a comprehensive look at ferroptosis, its role in PD pathology, and the mechanisms underlying the therapeutic effects of natural plant products focused on ferroptosis. The insights from this review can serve as useful references for future research on novel ferroptosis inhibitors and lead compounds for PD treatment.
Full-text available
Senkyunolides are a group of phthalide molecules that exist in Umbelliferae plants, with the highest abundance in Ligusticum chuanxiong Hort, a medicinal herb with therapeutic applications in various diseases such as coronary heart disease, migraine, and rheumatism. Modern research has highlighted the anti-inflammatory, analgesic, and cardiovascular-protective properties of senkyunolides. Recently, some novel effects, such as antibacterial, anti-osteoporosis, and anti-fibrosis, have been reported for senkyunolides, indicating their diverse biological activities. In addition, because of its high bioavailability and ability to cross the blood-brain barrier (BBB), particularly exemplified by senkyunolide I (SEI), holds promise for the treatment of brain diseases. Multiple signaling pathways have been related to the pharmacological activities of senkyunolides, such as the toll-like receptor 4/nuclear factor-kappa B (NF-κB) signaling, extracellular signal–regulated kinase (ERK) pathway, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase (JNK) pathways. Here, we review the research progress on the pharmacokinetics and pharmacological effects of senkyunolides, which are promising candidates for future drug development and have significant clinical value in the treatment of various diseases. Graphical abstract
Dl-3-n-butylphthalide (NBP) is a small-molecule drug used in the treatment of ischemic stroke in China, which is proven to ameliorate the symptoms of ischemic stroke and improve the prognosis of patients. Previous studies have shown that NBP accelerates recovery after stroke by promoting angiogenesis. In this study, we investigated the mechanisms underlying the angiogenesis-promoting effects of NBP in ischemic stroke models in vitro and in vivo. OGD/R model was established in human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), while the tMCAO model was established in mice. The cells were pretreated with NBP (10, 50, 100 µM); the mice were administered NBP (4, 8 mg/kg, i.v.) twice after tMCAO. We showed that NBP treatment significantly stimulated angiogenesis by inducing massive production of angiogenic growth factors VEGFA and CD31 in both in vitro and in vivo models of ischemic stroke. NBP also increased the tubule formation rate and migration capability of HUVECs in vitro. By conducting the weighted gene co-expression network analysis, we found that these effects were achieved by upregulating the expression of a hedgehog signaling pathway. We demonstrated that NBP treatment not only changed the levels of regulators of the hedgehog signaling pathway but also activated the transcription factor Gli1. The pro-angiogenesis effect of NBP was abolished when the hedgehog signaling pathway was inhibited by GDC-0449 in HUVECs, by Sonic Hedgehog(Shh) knockdown in HUVECs, or by intracerebroventricular injection of AAV-shRNA(shh)-CMV in tMCAO mice. Furthermore, we found that HUVECs produced a pro-angiogenic response not only to autocrine Shh, but also to paracrine Shh secreted by astrocytes. Together, we demonstrate that NBP promotes angiogenesis via upregulating the hedgehog signaling pathway. Our results provide an experimental basis for the clinical use of NBP.
Full-text available
Pathophysiological processes of stroke have revealed that the damaged brain should be considered as an integral structure to be protected. However, promising neuroprotective drugs have failed when translated to clinical trials. In this review, we evaluated previous studies of neuroprotection and found that unsound patient selection and evaluation methods, single-target treatments, etc., without cerebral revascularization may be major reasons of failed neuroprotective strategies. Fortunately, this may be reversed by recent advances that provide increased revascularization with increased availability of endovascular procedures. However, the current improved effects of endovascular therapy are not able to match to the higher rate of revascularization, which may be ascribed to cerebral ischemia/reperfusion injury and lacking of neuroprotection. Accordingly, we suggest various research strategies to improve the lower therapeutic efficacy for ischemic stroke treatment: (1) multitarget neuroprotectant combinative therapy (cocktail therapy) should be investigated and performed based on revascularization; (2) and more efforts should be dedicated to shifting research emphasis to establish recirculation, increasing functional collateral circulation and elucidating brain–blood barrier damage mechanisms to reduce hemorrhagic transformation. Therefore, we propose that a comprehensive neuroprotective strategy before and after the endovascular treatment may speed progress toward improving neuroprotection after stroke to protect against brain injury.
Full-text available
Background: DL-3-n-butylphthalide (NBP) is a drug for treating acute ischemic stroke, and may play a neuroprotective role by acting on multiple active targets. The aim of this study was to predict the target proteins of NBP in mammalian cells. Methods: The similarity ensemble approach search tool (SEArch), one of the commonly used public bioinformatics tools for target prediction, was employed in the experiment. The molecular docking of NBP to target proteins was performed by using the three-dimensional (3-D) crystal structure, substrate free. The software AutoDock Vina was used for all dockings. The binding targets of NBP were illustrated as 3-D and 2-D diagrams. Results: Firstly, the results showed that NBP bounded to the same binding site on NAD(P)H quinone oxidoreductases (NQO1) as the substrate FAD, leading to competitive inhibition for the catalytic site with -7.2 kcal/mol. This might break the 3-D structure of NQO1 and bring about P53 degradation, resulting in a decrease of p53-mediated apoptosis in ischemic brain cells. Secondly, NBP might exert its therapeutic effect on acute ischemic stroke via modulating indoleamine 2,3-dioxygenase (IDO) bioactivity after associating with it. NBP could alleviate the depression following ischemic stroke by inhibiting IDO. Thirdly, NBP might modulate the function of NADH-ubiquinone oxidoreductase by competitively embedding itself into this complex, further affecting mitochondrial respiration in cerebrovascular diseases as an anti-oxidant agent. Conclusions: Three potential target proteins of NBP were identified, which may provide a novel aspect for better understanding the protective effects of NBP on the nervous system at the molecular level.
Full-text available
Each year, the American Heart Association (AHA), in conjunction with the Centers for Disease Control and Prevention, the National Institutes of Health, and other government agencies, brings together in a single document the most upto-date statistics related to heart disease, stroke, and the factors in the AHA's Life's Simple 7 (Figure1), which include core health behaviors (smoking, physical activity [PA], diet, and weight) and health factors (cholesterol, blood pressure [BP], and glucose control) that contribute to cardiovascular health. The Statistical Update represents a critical resource for the lay public, policy makers, media professionals, clinicians, healthcare administrators, researchers, health advocates, and others seeking the best available data on these factors and conditions. Cardiovascular disease (CVD) and stroke produce immense health and economic burdens in the United States and globally. The Update also presents the latest data on a range of major clinical heart and circulatory disease conditions (including stroke, congenital heart disease, rhythm disorders, subclinical atherosclerosis, coronary heart disease, heart failure (HF), valvular disease, venous disease, and peripheral arterial disease) and the associated outcomes (including quality of care, procedures, and economic costs). Since 2006, the annual versions of the Statistical Update have been cited >20000 times in the literature. In 2015 alone, the various Statistical Updates were cited '4000 times.
Full-text available
Carbon monoxide (CO) poisoning is one of the most important health concerns and may result in neuropathologic changes and neurologic sequelae. However, few studies have addressed the correlation between CO poisoning and blood–brain barrier (BBB) impairment. In this study, we investigated the effects of N-butylphthalide (NBP) on the expressions of zonula occludens-1 (ZO-1), claudin-5 and aquaporin-4 (AQP-4) proteins in a CO poisoning rat model. The results indicated that the brain water content was obviously increased, and the tight junctions between endothelial cells were disrupted, resulting in significant cerebral edema and BBB dysfunction in a rat model of CO poisoning. Meanwhile, the ultrastructure of endothelial cells and pericytes was seriously damaged, and the expressions of ZO-1 and claudin-5 were decreased at an early stage (<7 days). NBP treatment could efficiently maintain the ultrastructural and functional integrity of BBB, alleviate cerebral edema. Besides, NBP could also markedly increase the levels of both ZO-1 and claudin-5 proteins compared with those in rats exposed to CO (P < 0.05), whereas NBP had no apparent regulatory effect on AQP-4 expression. Taken together, this study highlights the importance of ZO-1 and claudin-5 proteins in maintaining BBB ultrastructure and function after CO poisoning. NBP, as a novel treatment approach, may effectively inhibit the down-regulation of ZO-1 and claudin-5 proteins (but not AQP-4), thereby preserving the barrier function and reducing cerebral edema after CO poisoning.
Objective: To investigate the potential effect of 3-n-butylphthalide (NBP) pretreatment on the cerebral ischemia/reperfusion injury in rats and the relevant mechanism. Materials and methods: A total of 90 rats was divided into three groups: Sham operation group (Sham group), ischemia-reperfusion group (I-R group), and NBP pretreatment group (NBP group 75 mg·kg-1·d-1 gavage). Pre-treatment was given once a day within 1 week before establishing the rat model of cerebral ischemia-reperfusion injury. The middle cerebral artery occlusion (MACO) rat models were established with the improved Longa-Zea method on the 7th day after ischemia for 2 h and reperfusion for 24 h in all the rats. We detected the cerebral infarction, the pathologic change of brain, the apoptosis of nerve cell, the production levels of reactive oxygen species (ROS), the content of malonaldehyde (MDA) and the activity of superoxide dismutase (SOD), the water content and the permeability of blood-brain barriers (BBB). In addition, we also observed the expressions of mitogen-activated protein kinase (MAPK, p-38, JNK, ERK1/2) and cleaved caspase-3 in the hippocampus tissues. Results: Compared with Sham group, we discovered that NBP significantly reduced infarction area, cell apoptosis, BBB damage and water content. Further, we found that NBP could also decrease ROS and MDA, and increase SOD activity in brain tissues of rats with a cerebral ischemia-reperfusion injury. Moreover, results showed that NBP also inhibited the levels p38 and JNK. Conclusions: NBP protected the cerebral from I/R injury, providing ideas for the expansion of clinical adaptability of NBP and possible approaches for its application.
Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a novel pro-drug of 3-n-butylphthalide (dl-NBP) that is used to treat ischemic stroke. Currently, dl-PHPB is in phase II–III clinical trials in China. In this study, we investigated the conversion and pharmacokinetics profiles of dl-PHPB in vitro and in vivo. The conversion of dl-PHPB to dl-NBP was pH- and calcium-dependent, and paraoxonase was identified as a major enzyme for the conversion in rat plasma. The pharmacokinetics, tissue distribution and excretion of dl-PHPB were studied and compared with equal-molar doses of dl-NBP in rats and dogs. The in vivo studies showed that dl-PHPB could be quickly and completely converted to dl-NBP. The plasma concentration-time course of converted dl-NBP after intravenous dl-PHPB administration was nearly the same as that after equal-molar dl-NBP. The Cmax and AUC of dl-NBP after oral dl-PHPB administration in rats and dogs were higher by 60% and 170%, respectively, than those after oral dl-NBP administration. Analysis of the tissue distribution of dl-PHPB revealed that converted dl-NBP was primarily distributed in fat, the brain and the stomach. In the brain, the levels of dl-NBP were relatively higher after dl-PHPB treatment by orally than after treatment with equal-molar dl-NBP. Approximately 3%–4% of dl-NBP was excreted within 72 h after dosing with dl-PHPB or dl-NBP, but no dl-PHPB was detected in urine or feces excrements. Our results demonstrate that the conversion of dl-PHPB is fast after oral or intravenous administration. Furthermore, the bioavailability of dl-PHPB was obviously better than that of dl-NBP.
AIM: To study the effect of dl-3-n-butylphthalide (dl-NBP) on the function of mitochondrial respiratory chain and to elucidate the increasing effect of NBP on brain energy supply during cerebral ischemia. METHODS: Mitochondria were isolated from the brain of transient middle cerebral artery occluded (MCAO) rat and the activities of the four complexes of the respiratory chain were determined. RESULTS: The activity of complex IV was deeply decreased after 1 h-ischemia. It was back to normal level when treated with NBP (5 mg·kg -1 or 10 mg·kg -1 ip 10 min before ischemia). During the reperfusion period after ischemia, the activity of complex I was notably increased at 3 h, and that of complex II was decreased at 6 h. With NBP treatment, these altered activities also returned to normal level. In cultured neurons subjected to 6 h-hypoxia/hypoglycemia, the same increasing effect of NBP(d-, l-or dl-) on the activity of complex IV was also found, and d-NBP seemed to be more effective. CONCLUSION: NBP can act directly on complex IV to increase its activity. This action may play an important role in the increasing effect of NBP on brain energy supply during cerebral ischemia.
The consequences of cerebrovascular disease are among the leading health issues worldwide. Large and small cerebral vessel disease can trigger stroke and contribute to the vascular component of other forms of neurological dysfunction and degeneration. Both forms of vascular disease are driven by diverse risk factors, with hypertension as the leading contributor. Despite the importance of neurovascular disease and subsequent injury after ischemic events, fundamental knowledge in these areas lag behind our current understanding of neuroprotection and vascular biology in general. The goal of this review is to address select key structural and functional changes in the vasculature that promote hypoperfusion and ischemia, while also affecting the extent of injury and effectiveness of therapy. In addition, as damage to the blood-brain barrier is one of the major consequences of ischemia, we discuss cellular and molecular mechanisms underlying ischemia-induced changes in blood-brain barrier integrity and function, including alterations in endothelial cells and the contribution of pericytes, immune cells, and matrix metalloproteinases. Identification of cell types, pathways, and molecules that control vascular changes before and after ischemia may result in novel approaches to slow the progression of cerebrovascular disease and lessen both the frequency and impact of ischemic events.
N-Butylphthalide (NBP) has been known to have potential neuroprotective effects in Alzheimer's disease and stroke animal models. Hepatocyte-growth factor (HGF), with strong angiogenic properties, exerted protective role in brain injury. The present study was aimed to investigate the possible anti-inflammatory effects of NBP on the brain injury of rats with cerebral ischemia reperfusion (IR) and astrocytes activation induced by lipopolysaccharide (LPS) treatment. Our results showed that cerebral IR induced brain damage with down-regulation of HGF and astrocytes activation. NBP treatment significantly increased HGF expression and activated cMet/PI3K/AKT signaling pathway, stimulating mTOR activity and suppressing apoptosis in brain tissues. Also NBP inhibited pro-inflammatory cytokines expression, including IL-6, IL-1β, and TNFα, via TLR4/NF-κB suppression. Anti-HGF treatment enhanced TLR4 expression while HGF could suppress TLR4 activation and its down-streaming signals, attenuating inflammation finally. Notably, NBP up-regulated HGF and down-regulated TLR4 expression significantly in the astrocytes combined with the treatment of TLR4 inhibitor than the cells only treated with TLR4 inhibitor, suggesting that NBP could further suppress TLR4 activation, suggesting that NBP might impede TLR4 through up-regulating HGF expression. These results suggested that NBP treatment significantly ameliorated cerebral IR-induced brain injury by inhibiting TLR4/NF-κB-associated inflammation regulated by HGF.