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Introduction
Cerebral ischemia is accompanied by a marked
inflammatory process, which is initiated by higher levels of
expression of cytokines, adhesion molecules, and other
inflammatory mediators, including nitric
oxide[1,2]. Recent studies have demonstrated that nitric oxide (NO) and pro-inflammatory
cytokines released by microglial cells, which act as resident
macrophage-like cells in the brain, are partly responsible for
neuronal cell death. NO levels are associated directly with
the development of brain injury in strokes and other
neuropathological disorders in
humans[3,4].
Following acute ischemic or hypoxic injury to the brain,
over-entry of Ca2+ into cells causes the activation of nitric
oxide synthase (NOS), which catalyzes an enzymatic reaction,
leading to the synthesis of nitric
oxide[5-7]. Three kinds of
distinct NOS isoforms have been identified, including
neuronal nNOS, endothelial eNOS, and an inducible isoform,
iNOS, originally isolated from macrophages. nNOS and eNOS
are constitutively expressed and calcium-dependent, whereas
iNOS is expressed in response to various inflammatory stimuli,
and its activity is independent of intracellular calcium
concentrations. NO can be neuroprotective or neurotoxic
during cerebral ischemia, depending on the NOS isoform
involved. eNOS produces NO with beneficial effects
(vasodilation, inhibition of platelet aggregation and
polymorphonuclear neutrophil
adhesion)[7-9], whereas NO overproduction by nNOS or iNOS during
ischemia is cytotoxic. Based on these findings, it thought that the NO-synthases
could be attractive targets for treating cerebral
ischemia-induced neuronal damage[10,11].
Scutellarin, a flavonoid, is the major active ingredient
extracted from Erigeron breviscapus Hand Mazz, a plant used
in Chinese herbal medicine, which is a
Ca2+-channel-blocking agent used for the clinical treatment of cerebrovascular
disorders. Studies have demonstrated the protective effects
of scutellarin on brain injury induced by cerebral
ischemia/reperfusion (I/R) through interaction with a wide variety of
targets because of its anti-oxidative and anti-inflammatory
actions, and its ability to attenuate neuronal
damage[12,13]. It is presumed that these effects might be related to the effects
of scutellarin on the NO synthases. Therefore, the objective
of the present study was to elucidate the effects of scutellarin
on the expression of the NOS isoforms (iNOS, eNOS, nNOS)
in a model of cerebral I/R in rats. Moreover, the key
angiogenic molecules, vascular endothelial growth factor (VEGF)
and basic fibroblast growth factor (bFGF), were also studied.
Materials and methods
Chemicals and drugs Scutellarin was supplied by Yuxi
Pharmaceuticals (Kunming, China). The purity of this compound
was more than 96% and it was dissolved in saline before use.
2,3,5-Triphenyltetrazolium chloride (TTC,
No 20010201) was obtained from the Shanghai Chemical Agent Company. All other
chemicals and solvents were of analytical grade.
Animal treatment and administration Male
Sprague-Dawley rats (Grade II, Certificate
No 19-050 ), weighing 230-280 g, were obtained from the Experimental Animal Center of
Tongji Medical College. Rats were housed at a constant
temperature of 22 ºC under a 12 h light-dark cycle with free
access to food and drinking water. Rats were divided into 5
groups. The sham-operated and vehicle-treated I/R groups
were pretreated with 0.5 mL/kg ig saline for 7 d before ischemia,
and the scutellarin-treated I/R groups were pretreated with
25, 50, 75 mg/kg ig scutellarin for 7 d before ischemia.
Cerebral I/R procedure Rats were anesthetized with
chloral hydrate (300 mg/kg, ip). Brain I/R injury was induced by a
middle cerebral artery occlusion (MCAO) as described
previously[14]. Briefly, the right common carotid artery, external
carotid artery (ECA) and internal carotid artery (ICA) were
isolated via a ventral midline incision. A 50 mm length of
monofilament nylon suture (f 0.22-0.24 mm), with its tip
rounded by heating near a flame, was introduced into the ECA
lumen and advanced into the ICA for a distance of
18-20 mm in order to block the origin of the MCA. The body
temperature of the rats was maintained at 36.5-37.5 ºC during the
surgical procedure with an infra red heat lamp. Sham-operated
animals were not exposed to I/R. After 2 h of ischemia, the
nylon suture was withdrawn to establish reperfusion. After
arousal from anesthesia, the rats were returned to the cages.
Behavioral testing and measurement of infarct area
After 24 h reperfusion, the neurological deficit score of each rat
was obtained according to Longa¡¯s
method[14] by a single experimenter, who was blinded to the experimental treatment
groups. The neurological findings were scored on a 5-point
scale: no neurological deficit=0, failure to extend right paw
fully =1, circling to right=2, falling to right=3, did not walk
spontaneously and had depressed levels of
conscious-ness=4. Then the rats were anesthetized with 10% chloral
hydrate (350 mg/kg) ip and subsequently decapitated. The
brains were removed for measurement of infarct volume by
using the TTC staining method. Five thin sections were
selected from the thick slices at 2 mm intervals (from the
anterior 5 mm to the anterior 13 mm) to determine the infarct
areas. The slices were immersed in 2% triphenyltetrazolium
chloride in saline and incubated at 37 ºC for 20 min, and then
fixed with 10% formaldehyde (Sigma) neutral buffer solution
(pH 7.4). At that time, the infarct tissue was unstained,
whereas the normal part was stained red. Using a
computerized image analysis system (NIH Image, Version 1.61), the
infarct areas on each slice were summed and multiplied by
slice thickness to give the infarct volume, and then expressed
as the percentage of infarction per ipsilateral hemisphere.
Evaluation of permeability of blood-brain barrier
The integrity of the blood-brain barrier (BBB) was investigated
using Evans blue (EB) dye extravasation, according to the
method of Matsuo et al[15]. Briefly, after 6-h reperfusion, the
rats were treated with EB dye (2% in saline, 3 mg/kg iv).
After 45 min, the rats were anesthetized with 10% chloral
hydrate (350 mg/kg ip) and then the rats¡¯ chests were
subsequently opened. Physiological saline was perfused through
the left ventricle until a colorless perfusion fluid was
obtained from the right atrium. The cranial vault was opened,
and the brain was removed, weighed (wet tissue) and placed
in a 50% trichloroacetic acid solution. After homogenization
and centrifugation, the supernatant (extracted dye) was
diluted with ethanol (1:3) and its fluorescence was determined
(excitation at 620 nm and emission at 680 nm) with a
luminescence spectrometer (Hitachi, Tokyo, Japan). Calculations of
the amount of EB dye in the tissue were based on a linear
standard curve and were expressed per gram of tissue.
Determination of total NOx content in brain
tissue At the end of 2 h ischemia and 24 h reperfusion, the rats were
decapitated and the ischemic hemispheres were removed for
assay of the NO level in ischemic brain tissue. The levels of
metabolic products (NO2 and
NO3) in vivo were determined by using a chemiluminescent NO detector (Siever 280i) as
described previously[16].
Western blot analysis Western blot analysis was performed
after 24 h reperfusion. The rats¡¯ brains were removed and the
ischemic hemispheres were used for assay of the protein
expression of iNOS, eNOS, nNOS, VEGF and bFGF. The
hippocampus and the cortex were quickly isolated and rinsed in
sterilized water on ice, and then stored at -80 °C until use. Protein
determination was performed according to the Lowry method.
The obtained protein samples were subjected to 15% sodium
dodecylsulfate-polyacrylamide gel electrophoresis, using
7.5%-15% polyacrylamide gel, and electrotransferred to
polyvinyli-dene difluoride filter (PVDF) membranes. To reduce
non-specific binding, the PVDF was blocked for 2 h at room temperature
with 5% non-fat milk in phosphate-buffered saline (PBS). Then
membranes were incubated overnight at 4 °C with the primary
antibodies for iNOS (anti-rabbit iNOS mouse monoclonal
antibody, 1:200 dilution, Santa Cruz), eNOS (anti-human eNOS
rabbit polyclonal antibody, 1:200 dilution, Affinity Bioreagents),
nNOS (anti-human nNOS rabbit polyclonal antibody, 1:200
dilution, Sanying Biotechnology), VEGF (antihuman VEGF rabbit polyclonal antibody, 1:200 dilution, Santa Cruz) or bFGF
(anti-human bFGF rabbit polyclonal antibody, 1:500 dilution,
Santa Cruz), respectively. After incubation with the antibodies,
the membranes were washed with PBS-Tween-20 (PBS-T: 10
mmol/L phosphate buffer, pH 7.4, 150 mmol/L NaCl, 0.05%
Tween 20) for 30 min and incubated in the relevant horseradish
peroxidase-conjugated secondary antibody (1:600 dilution) for
30 min. The membranes were washed again with PBS-T and
immunoreactive protein bands were visualized using the
enhanced chemiluminescence detection system.
Statistical analysis Data are expressed as mean±SD and
analyzed by using Microsoft Excel 2002. Statistical analyses
were performed by using Student¡¯s t-test.
P<0.05 was considered significant.
Results
Effects of scutellarin on the infarct area, neurological
score and the permeability of the blood-brain barrier
Scutellarin (50 or 75 mg/kg) significantly reduced the infarct
area and ameliorated the neurological deficit
(P<0.05 or P<0.01 vs vehicle-operated group) (Table 1). The EB content
of brain tissue after I/R for sham-operated,
vehicle-operated and scutellarin-operated groups (25, 50, or 75 mg/kg) was
3.83±1.03, 8.45±1.67, 7.45±1.77, 5.02±1.12, and 4.45±1.05,
respectively (Figure 1). There was a significant increase in the
permeability of the BBB in rats in the vehicle-treated group
compared with the sham-treated group (P<0.01). Scutellarin
(50 or 75 mg/kg) obviously inhibited the increased EB content
induced by cerebral I/R, and there was no obvious difference
between the 2 doses.
Effects of scutellarin on total NOx production
After cerebral I/R, total NOx production, as determined by NOx
content in the ischemic brain hemispheres, was markedly
increased in the vehicle rats (4.87±0.90) compared with the
sham-treated rats (1.83±0.34) (P<0.01, Figure 2). Total NOx
production in rats pretreated with scutellarin at
concentrations of 50 or 75 mg/kg (3.01±0.68, 2.31±0.48), were
significantly reduced compared with the vehicle-operated group
(P<0.05 or P<0.01, respectively) (Figure 2).
After 24 h reperfusion, the expression levels of iNOS,
eNOS and nNOS were detected in the hippocampus and in
the cortex, with molecular masses of 130, 140, 160 kDa,
respectively. In the vehicle-treated group, the expression
levels of the 3 NO synthases in the hippocampus and in the
cortex markedly increased after cerebral I/R
(P<0.01 or P<0.05, Figure 3A, 3B, lane 2). Densitometric analysis
showed that the protein levels of eNOS and iNOS in the
scutellarin-treated (50 or 75 mg/kg) rats were 368.0±70.3%
and 278.0%±56.6% in the hippocampus (Figure 3A, lanes
3, 4), and 198.1%±19.2% and 148.3%±17.6% in the cortex
(Figure 3B, lane 3, 4) for iNOS; and 469.0%±40.5% and
523.0%±67.3% in the hippocampus (Figure 3A, lanes 3, 4),
and 188.3%±31.2% and 234.2%±37.8% in the cortex
(Figure 3B, lanes 3, 4) for eNOS, respectively. The data
indicate that scutellarin downregulated the expression of
iNOS and simultaneously upregulated that of eNOS as
compared with the vehicle-operated group (P<0.05 or
P<0.01, respectively), whereas there was no difference in nNOS
expression in the hippocampus or the cortex between the
vehicle-treated and scutellarin-treated rats.
Immunoblot analysis showed single bands with molecular
masses of approximately 39 and 18 kDa, which correspond to
VEGF and bFGF in the hippocampus and in the cortex,
respectively. The bands obtained from the vehicle-operated
I/R rats (VEGF: 278.2%±43.4% in the hippocampus and
256.7%±36.8% in the cortex; bFGF: 432.2%±50.4% in the
hippocampus and 289.4%±49.7% in the cortex) (Figure 3C, 3D,
lane 2) were stronger than those from the sham group rats
(VEGF: 111.9%±24.2% in the hippocampus and 123.9%±
16.1% in the cortex; bFGF: 98.8%±15.4% in the hippocampus
and 98.8%±10.9% in the cortex) (Figure 3C, 3D, lane 1,
P<0.01). Scutellarin at doses of 50 or 75 mg/kg (Figure 3C, 3D, lanes 3,
4) significantly decreased the expression of VEGF and bFGF
in the hippocampus and in the cortex, as compared with the
vehicle-operated group (P<0.05 or
P<0.01). When rats were pretreated with scutellarin at a concentration of 50 or 100
mg/kg, the VEGF protein levels in the hippocampus were
189.8%±35.4% and 123.5%±30.1%, whereas in the cortex they
were 178.2%±22.1% and 145.7%±11.9%, respectively; bFGF
protein levels in the hippocampus were 212.9%±33.2% and
134.5%±19.1%, while in the cortex they were 212.9%±30.4%
and 145.6%±17.6%, respectively.
Discussion
In the present study, we demonstrated that scutellarin
(at doses of 50 or 75 mg/kg) significantly reduced infarct
volume, ameliorated the neurological deficit and reduced the
permeability of the BBB after cerebral I/R. Therefore, the
conclusions obtained from the above observations were that
scutellarin has protective effects for the neuronal damage
induced by cerebral I/R in rats.
Evidence has accumulated that NO produced both
before and after cerebral ischemia may be an important factor in
the pathogenesis of neuronal ischemic injury. NO is a
signaling molecule that regulates many biological processes in
the brain. The present paper also investigated the effects of
scutellarin on the total NOx content in rat brain tissues after
cerebral I/R. Our results showed that total NOx production, as
determined by NOx content, in the ischemic brain hemispheres
was markedly increased in cerebral I/R rats, which indicates
that NO regulates the severity of cerebral ischemic injury.
However, NOx content markedly decreased in brain tissues
after treatment with scutellarin at doses of 50 or 75 mg/kg.
Numerous studies have been conducted regarding the
differential roles of NOS isoforms and their temporal NO
production in the pathogenesis of ischemic brain
injury[17-19]. eNOS-derived NO is thought to be beneficial for promoting
collateral circulation and microvascular flow, whereas nNOS-
and iNOS-derived NO is detrimental in the ischemic brain.
NO is a nontoxic agent and acts as a second messenger in
normal brain; however, in the presence of
O2-, NO reacts with
O2- to form ONOO- or nitrogen dioxide
(NO2-), causing injury to the mitochondrial electron transport system,
resulting in neuronal damage. In addition, excess NO stimulates
ADP ribosyltransferase and binds closely to iron-sulfur
centers of enzymes, including enzymes involved in the
mitochondrial electron transport chain and the tricarboxylic acid
cycle (TCA), and DNA[20-22]. In view of the detrimental and beneficial roles of NOS isoforms in ischemic brain injury,
further investigations into the effect of scutellarin on the
expression of NOS isoforms (iNOS, eNOS, nNOS) both in the
hippocampus and in the cortex after cerebral I/R were also
performed in the present study. We found that expression of the
NO-synthases in the hippocampus and cortex all markedly
increased after cerebral I/R in the vehicle-operated group, a
similar finding to those of previously
studies[22-26]. Scutellarin at doses of 50 and 75 mg/kg downregulated iNOS expression
and upregulated eNOS expression, which partly account for
its protective effect on brain damage induced by cerebral I/R.
Increasing evidence has shown that some angiogenic
molecules, including VEGF and bFGF, increase in
concentration after cerebral I/R. VEGF is an angiogenesis and
vascular permeability factor that undergoes transcriptional and
post-transcriptional induction by hypoxia; it couples hypoxia
to angiogenesis in diverse tissues, including the
brain[23-26]. VEGF may also play an important
role in the vascular response to cerebral ischemia, because
ischemia stimulates VEGF expression in the brain, which
promotes the formation of new cerebral blood
vessels[27,28]. It is thought that eNOS
is involved in mediating the angiogenic molecules (VEGF
and bFGF). Both factors induced eNOS expression, so eNOS
may be a downstream messenger in their angiogenic action.
In the present study we also investigated the expression of
the angiogenic molecules VEGF and bFGF in the
hippocampus and in the cortex. In agreement with the results of
previous reports[29,30], our data showed that the expression of VEGF
and bFGF in the hippocampus and cortex were upregulated in
the vehicle-operated group after cerebral I/R. Scutellarin
downregulated the expression of VEGF and bFGF. Further
study is needed to shed light on the mechanisms involved in
the effect of scutellarin on the expression of eNOS
(upregula-tion) and VEGF (downregulation) in I/R brain tissue.
In conclusion, scutellarin alleviated hippocampal
neuronal dysfunction after cerebral I/R. This alleviation was
accompanied by the effects of the molecular features of NOS
isoforms and angiogenic molecules. These findings
suggest that scutellarin exerts neuroprotective effects on brain
injury induced by cerebral I/R, which allows a better
understanding regarding the potential clinical therapeutic use of
scutellarin.
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