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Journal of Ethnopharmacology 140 (2012) 64–72

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Journal of Ethnopharmacology

j ournal homepage: www.elsevier.com/locate/jethpharm

Synergistic protective effect of astragaloside IV–tetramethylpyrazine against

cerebral ischemic-reperfusion injury induced by transient focal ischemia

a,1 b,1 c a a a,∗

Jiehong Yang , Jinhui Li , Jing Lu , Yuyan Zhang , Zhenhong Zhu , Haitong Wan

Institute of Cardio-Cerbrovascular Diseases, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China

Department of Chinese Medicine & Rehabilitation, Second Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, Zhejiang 310009, China

Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Astragaloside IV and tetramethylpyrazine have been extensively used in

Received 1 February 2011

the cardio-cerbrovascular diseases of medicine as a chief ingredient of glycoside or alkaloid formulations

Received in revised form 4 December 2011

for the treatment of stroke and myocardial ischemia diseases.

Accepted 13 December 2011

Aim of the study: To investigate the effects of astragaloside IV (ASG IV) and tetramethylpyrazine (TMPZ)

Available online 21 December 2011

on cerebral ischemia-reperfusion (IR) injury model in rat model.

Materials and methods: Rats were randomly divided into the following ﬁve groups: sham group, IR group

Keywords:

and treatment group including ASG IV, ASG IV–TMPZ and nimodipine treatment. The therapeutic effect

Apoptosis 18

was evaluated by micro-positron emission tomography (Micro-PET) using F-ﬂuoro-2-deoxy-d-glucose.

Astragaloside IV

The neurological examination, infarct volume and the levels of oxidative stress- and cell apoptosis-related

Ischemia reperfusion

Oxidative stress molecules were assessed.

Tetramethylpyrazine Results: Micro-PET imaging showed that glucose metabolism in the right hippocampus was signiﬁcantly

Positron emission tomography decreased in the IR group compared to the sham group (P < 0.01). ASG IV and ASG IV–TMPZ treatments

reversed the decreased glucose metabolism in the model group (P < 0.05 and P < 0.01, respectively). IR

induced the increase of Caspase-3 mRNA levels, MDA content and iNOS activity, but it caused the decrease

of SOD activity and Bcl-2 expression compared the sham group (P < 0.01). ASG IV–TMPZ and ASG IV

reversed the IR-induced changes of these parameters, i.e. the down regulation of Caspase-3 mRNA, MDA

content and iNOS activity, and the up regulation of SOD activity and Bcl-2 expression (P < 0.05).

Conclusion: This study showed that ASG IV–TMPZ played a pivotal synergistic protective role against focal

cerebral ischemic reperfusion damage in a rat experimental model.

1. Introduction compared with single puerarin use in IR model (Wan et al.,

2008). This protective effect of chuanxiongzine-puerarin on cere-

Cerebral ischemia-reperfusion (IR) injury often causes irre- bral ischemia was assumed to be mediated by the inhibition of

versible brain damage, and the cascade of events leading to neu- free radicals formation, and other mechanisms. However, it has

ronal injury and death in brain ischemia includes interruption of the found that glucose metabolism in the brain is closely related

cerebral blood ﬂow, the depletion of energy stores excitotoxicity, to neuronal activity (Chugani et al., 1991), we used F labeled

free radical-induced neuronal damage, inflammation, apoptosis, 2-deoxy-2-fluoro-d-glucose ( F-FDG) that reflects the state of glu-

and necrosis. We have demonstrated that chuanxiongzine- cose metabolism (Rohren et al., 2004) to evaluate the effects both

puerarin treatment exhibited signiﬁcant neuroprotective effect ASG IV and TMPZ on the cerebral ischemia-reperfusion injury.

As we all know, B-cell lymphoma/leukemia-2 genes (Bcl-2), a

protooncogene, plays an important role in inhibiting apoptosis and

relieving cell death. Over-expression of Bcl-2 can enhance the cells

Abbreviations: IR, ischemia-reperfusion; ASG IV, astragaloside IV; TMPZ,

that observed by the majority of resistance to cytotoxic factors.

tetramethylpyrazine; SOD, superoxide dismutase; MDA, malondialdehyde; iNOS,

18 18 This discovery led to the recognition of apoptosis in a variety of

induced nitric oxide synthase; F-FDG, F-ﬂuoro-2-deoxy- -glucose; PET, positron

emission tomography; ELISA, enzyme-linked immunosorbent assay; TTC, triphenyl- signal transduction pathways, and provided the access road or the

tetrazolium chloride. intersection point of regulation by Bcl-2. In addition, Bcl-2 over-

∗

Corresponding author at: Institute of Cardio-Cerbrovascular Diseases, Zhejiang

expression can reduce the oxygen free radicals and lipid peroxide

Chinese Medical University, 548 Binwen Road, Hangzhou, Zhejiang 310053, China.

formation. It inhibits the trans-membrane calcium ﬂow. Further-

Fax: +86 0571 86613712.

more, it has found that Bcl-2 has a neuroprotective roles against

E-mail addresses: [email protected] (J. Li), [email protected] (H. Wan).

These authors contributed equally to this work. ischemic brain injury through the reduction of neuronal apoptosis

J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72 65

(Han et al., 2002), and decrease free radical induced by neuronal ﬁlament thread (SP 184, 4-0) was inserted into the right exter-

damage (Ginsberg, 2001). nal carotid artery and was carefully maneuvered into the internal

ASG IV, a glycoside of cycloartane-type triterpene isolated from carotid artery. After 60 min of MCAO, the nylon ﬁlament was taken

the root of Astragalus membranaceus (Fisch). Bunge, which used out and the blood was reﬂowed. The rats in sham group were

extensively in traditional Chinese medicine, it helps to speed heal- prepared by the MCAO method except inserting the nylon ﬁla-

ing and treat diabetes mellitus (Liu et al., 2004; Shao et al., 2004). ment thread. The sham group and IR group rats were administered

Also it helps to improve the functioning of the lungs, adrenal the same dose of sterile saline. The dose of the drugs in treat-

glands and the gastrointestinal tract. Whats more, it could increase ment groups were as follows: ASG IV was 20 mg/kg and TMPZ was

metabolism, promote healing of wounds and injuries, and reduce 10 mg/kg in ASG IV–TMPZ group in order, ASG IV was 20 mg/kg in

fatigue (Balch, 2006). ASG IV has been reported to decrease the level single ASG IV group, and nimodipine was used by 10 mg/kg body

of malondialdehyde which is an indicator of lipid peroxidation, and weight. All the drugs were intraperitoneally injected for each group

increase the levels of the antioxidant enzymes glutathione peroxi- with the same volume (2 mL) at 0, 12 h, 1 d, 2 d, 3 d, till to 7 d after

dase, superoxide dismutase; moreover, it decreased expression of reperfusion.

occludin and zonae occludens-1, the tight junction proteins in brain

micro-vascular endothelial cells (Luo et al., 2004; Qu et al., 2009).

2.4. Neurologic evaluation

TMPZ, a type of Chinese herb also used for the treatment of ischemic

cerebral vascular disorders (Ho et al., 1989).

A neurological examination was performed after 24-h of cere-

In this study, the neuronal activity, antioxidant, and anti-

bral IR according to the method described by Longa et al. (1989). The

apoptosis potential effects of ASG IV–TMPZ were mainly inves-

neurological ﬁndings were scored on a 5-point scale: rats with no

tigated in a rat model of transient cerebral ischemia-reperfusion

neurologic deﬁcit scored 0, rats with (failure to extend left forepaw

induced by middle cerebral artery occlusion (MCAO).

fully) a mild focal neurologic deﬁcit scored 1, rats with (circling

to the left) a moderate focal neurologic deﬁcit scored 2, rats with

2. Materials and methods

(falling to the left) a severe focal deﬁcit scored 3. Rats with a score

of 4 did not walk spontaneously and had a depressed level of con-

2.1. Animals sciousness.

Male Sprague–Dawley rats weighing 280 ± 20 g, were individ-

ually maintained in an accredited animal husbandry facility that 2.5. Micro-PET imaging

was approved by the Animal Center of Zhejiang Chinese Medical

University (Laboratory animal certiﬁcate: scxk 2008-0115) with A total of 15 rats with cerebral IR injury were imaged by micro-

a 12-h light/dark environment. A total of 30 rats were randomly positive emission tomography (micro-PET) using F-FDG. Rats

divided into ﬁve groups: sham operation group, cerebral IR group, were maintained on 2% isoﬂurane in oxygen, and then injected F-

treatment groups including ASG IV, ASG IV–TMPZ and nimodipine FDG from tail vein. After an uptake period of 30 min, the rat was

treatment). Animal care and surgical procedure were performed in placed in a spread prone position and scanned with the micro-PET

accordance with the National Institute of Health Guide for the Care R4 (Concorde Microsystems, Knoxville, TN, USA), which consists

␥

and Use of Laboratory Animals (NIH Publications No. 80-23) revised of a 15-cm diameter ring of 96 position-sensitive -ray scintilla-

in 1996. Formal approval to conduct the experiments was obtained tion detectors, providing a 10.8-cm trans axial, and a 7.8-cm axial

from the University Animal Subjects Review Board. All efforts ﬁeld of view with image resolution at less than 1.8 mm. Brieﬂy, we

were made to minimize the number of animals used and their injected 22.2–25.9 MBq FDG into the tail vein of the rat and put

suffering. it back in a quiet room for 30 min for glucose uptake. Ten-minute

static acquisition was performed in 3D mode. Corrections for dead

time, random scattering, and attenuation were performed. Micro-

2.2. Drugs and reagents

PET image studies were reconstructed using a statistical maximum

a posteriori probability algorithm. The ratio of the regions of

The SOD kit (Lot No. 20090928) and MDA kit (Lot No. 20090928)

interest (ROIs) in the temporal lobe, apical lobe, frontal lobe to

were provided by Nanjing Jiancheng Biological Engineering Insti-

cerebellum was calculated for each scan using ASIPro 6.0.5.0 soft-

tute (Nanjing, China). Rat iNOS ELISA kit (Lot No. 501199) was

ware (CTI Concorde Microsystems, LLC proﬁle in Knoxville, TN,

bought from Shanghai Ximei Biotechnology Ltd. (Shanghai, China).

USA) that is matched to 3D Micro-PET images at the Medical PET

Ligustrazine hydrochloride injection was purchased from Tri-

Center of Zhejiang University. These ROIs were drawn and calcu-

lion Pharmaceutical Co., Ltd., Harbin (Lot No. 090923A, Harbin,

lated by the author who had extensive experience in manual ROIs

Heilongjiang, China), nimodipine injection was purchased from

deﬁnition.

Bayer Healthcare AG (Leverkusen, Germany, Lot No. BXBSTX1

13), and ASG IV was prepared by semi-preparative HPLC agi-

␮

lent 1200 (diameter of 30 mm, length of 100 mm, 5 m particle 2.6. Triphenyltetrazolium chloride assessment

size), Biological and Pharmaceutical Engineering Laboratory, Zhe-

jiang Medical University, Zhejiang, China). F-FDG was produced Rats were anesthetized, killed by rapid decapitation, and

by the Department of Nuclear Medicine, Second Afﬁliated Hospi- the brain was collected at 7 d after reperfusion. We improved

◦

tal, Zhejiang University College of Medicine, Hangzhou, Zhejiang, the methods and refrigerated the brains at −20 C, then sliced

China. into 2-mm thick coronal section and stained with 2% 2,3,5-

◦

triphenyltetrazolium chloride solution for 10 min at 37 C and ﬁxed

◦

in 4% paraformaldehyde at 4 C overnight (Bederson et al., 1986).

2.3. MCAO rat modeling and administration

After ﬁxation these brain slices were scanned using a ﬂat-bed scan-

ner. Infarct volume was quantiﬁed with the professional image

1% pentobarbital sodium solution (40 mg/kg) was used with

analysis software. Infarct volumes were calculated by summation

an intraperitoneal injection to the rats (Belayev et al., 1996). We

of the infarct areas in six brain slices and integrated by thickness

exposed the right common artery, external carotid artery and inter-

(2 mm) slightly.

nal carotid artery, respectively, under sterile conditions. A nylon

66 J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72

Table 1

2.7. Determination of oxidative stress and iNOS

Values of ﬁve groups of neurological deﬁcit after reperfusion.

After MCAO under anesthesia, the rats were decapitated 7 d later Group n Neurologic score

◦

and the brains were quickly taken out, and stored at −80 C. The

Sham group 10 0.20 ± 0.42

brain tissue was homogenated and the supernatant was collected Model group 10 3.40 ± 0.52

to assay SOD with xanthine oxidase method, MDA with thiobarbi- Astragaloside IV group 10 2.40 0.51

***

Astragaloside IV–tetramethylpyrazine 10 1.80 ± 0.42

turic acid method, and iNOS with ELISA method according to the

Nimodipine group 10 1.60 ± 0.51

manufacturers’ instruction.

Values are mean SD.

P < 0.01; signiﬁcantly higher compared with values of sham operation group.

2.8. RNA isolation and real-time quantitative PCR P < 0.01; signiﬁcantly lower compared with values of model group.

***

P < 0.01; signiﬁcantly lower compared with values of model group and astraga-

loside IV group.

The rats were decapitated after 24 h of cerebral I/R injury, the

brains were taken out and the cortex was dissected from the

ipsilateral and contralateral hemispheres, separately. The samples

◦

−

were immediately stored at 80 C for further use. Caspase-3 2.10. Statistical analysis

gene (forward, 5 -CTGGACTGCGGTATTGAGAC-3 , 20 bp; reverse,

5 -CCGGGTGCGGTAGAGTAAGC-3 , 20 bp, GenBank Accession All data are showed as mean SEM and are accompanied

␤

No. NC005115.2) and -actin (forward, 5 -CCGTAAAGACCTC- by the number of observations. Student’s unpaired t-test was

TATGCCAACA-3 , 23 bp; reverse 5 -TAGGAGCCAGGGCAGTAATC-3 , used to determine the differences between two groups. In more

␤

20 bp, GenBank Accession No. EF156276.1). -Actin was used as than 2 groups, the data were assessed by ANOVA using the

an internal control for all experiments. Total RNA was extracted SPSS 13.0 (SPSS Inc., Chicago, IL, USA) statistical package pro-

with Trizol reagent according to the manufacturer’s recommended gram. Signiﬁcant differences were accepted when P < 0.05 or

procedures. About 100 mg of total RNA were segregated by Two P < 0.01.

Step RT-PCR kit (Cat No. BSB05MI, Hangzhou Bayer Technology Co.,

Ltd.) was reverse transcribed using an M-MLV First Strand cDNA

3. Results

Synthesis (Taq DNA Polymerase Kit, Lot 00043711, Fermentas

Revert Aid , Lithuania MBI). Quantitative PCR was performed

3.1. Effects on neurological deﬁcit

using MJ Mini personal Termal (BIO-RAID Ltd., American); Reac-

tion system: iQ SYBR Gene surpmix 12 ␮L, upstream primer 1 ␮L,

After cerebral IR injury, neurological deﬁcit score of the model

downstream primer 1 ␮L, cDNA template 1 ␮L, starile water 10 ␮L,

group was signiﬁcantly higher than that of the sham group

␮ ◦

total 25 L; reaction conditions: pre-incubation at 95 C 3 min;

(P < 0.01), whereas all the three treatment groups were lower than

◦ ◦

incubation at 95 C 10 s, incubate at 61 C 50 s; plate read; go to

the model group (P < 0.01) (Table 1).

◦ ◦

line 2 for 40 cycles; melting curve from 65 C to 95 C, read every

◦

0.5 C, hold 5 s. All experiments were performed three times, and

3.2. Effects on brain glucose metabolism

used quantitative analysis software processed data. Through the

−Ct

relative quantity (RQ = 2 ) calculate the target gene (calibra-

The glucose metabolism in brain was signiﬁcantly reduced in

tor) and compare the differences in gene expression by multiples

the hippocampus region of the model group compared with that of

of the reference factor. The ␤-actin gene as a restricted reference

the sham operation group after cerebral IR injury (P < 0.01). Glu-

materials, each sample Ct = Ct (target gene) − Ct (␤-actin), and

cose metabolism in injury brain while being treated with ASG

the Ct = Ct (target gene) − Ct (calibrator).

IV and ASG IV–TMPZ, was increased signiﬁcantly (P < 0.05 and

P < 0.01, respectively), in the area compared with the model group.

2.9. Immunohistochemistry Whereas, glucose metabolism of the ASG IV–TMPZ group was sig-

niﬁcantly recovered in the right area of compared with the ASG IV

The sections were stained with ENVision (Dako-Cytomation, group (P < 0.05), demonstrating a visible therapeutic effect of ASG

Glostrup, Denmark) two-step strategy and high-temperature anti- IV–TMPZ (Fig. 1). The F-FDG uptake of focal ischemic lesion in two

␮

gen retrieval. Parafﬁn-embedded sections with 5- m thickness treatment groups was higher than in the model group (Table 2), of

were deparafﬁnized twice, 10 min each in 100% xylene, and then which the ASG IV–TMPZ group showed best glucose metabolism

dehydrated with 100% ethanol for 5 min, 95% for 3 min, and 80% recovery.

for 5 min. After 2–5 min soaking in distilled water, the sections

were put into the pressure cooker ﬁlled with 1000 mL of boil- 3.3. Triphenyltetrazolium chloride evaluation

ing sodium citrate buffer (pH 6.0) and heated under pressure for

2 min after steaming and then cooled. The sections were rinsed The infarct volume in the model group was signiﬁcantly

for 2–3 min with phosphate-buffered saline. Then the primary increased than the sham group (P < 0.01). In contrast, the ASG

rabbit-anti-rat Bcl-2 monoclonal antibody, (Wuhan Boster Biolog- IV–TMPZ group showed the smallest infarct volume (0.182 ± 0.019,

ical Technology, Ltd., Hubei, China) was applied overnight at room P < 0.01), followed by the ASG IV group (0.213 ± 0.028, P < 0.01)

temperature. After 2–5 min soaking in distilled water, and 2–3 min and the nimodipine group (0.195 ± 0.014, P < 0.01), compared

rinsing in PBS for three times, the sections were incubated with with the model group after treatment, as shown in Fig. 2 and

secondary antibody [goat-anti-rabbit polyclonal antibody (Wuhan Table 3.

◦

Boster Biological Technology, Ltd., Hubei, China)] for 2 h at 37 C

between 2–3 min rinsings in PBS for 3 times, with diaminoben-

3.4. Drugs attenuated oxidative stress and determination of iNOS

zidine chromagen added, soaking in distilled water, hematoxylin

stained 2–5 min differentiated with 1% hydrochloric acid alco-

The SOD activity was signiﬁcantly decreased in the model group

hol, and dehydrated, transparent, blue-based, coverslipped with

(P < 0.01) compared with the sham group; whereas MDA level and

glycerin, the sections were observed and examined under a light

iNOS content was increased (P < 0.01). Although MDA and iNOS

microscope.

content in treatment group was decreased signiﬁcantly compared

J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72 67

Fig. 1. FDG-PET images of activity of hibateral hippocampus regions glucose metabolism. Sham group (A), model group (B), astragaloside IV group (C), astragaloside

IV–tetramethylpyrazine group (D), nimodipine group (E).

with the model group (P < 0.01). In the ASG IV–TMPZ (P < 0.01) and 3.5. Expression of Caspase-3

nimodipine groups (P < 0.01), SOD activity was signiﬁcantly higher

than in the ASG IV group, and the MDA and iNOS content was sig- The expression of Caspase-3-mRNA in the model group was

niﬁcantly decreased compared with the ASG IV group as shown in signiﬁcantly increased compared with the sham group (P < 0.01).

Table 4. Caspase-3 expression in the ASG IV (P < 0.05), ASG IV–TMPZ

Table 2

Effects of different groups on brain glucose metabolism (nCi/cc) in hibateral hippocampus.

Group n Left Right

Sham operation 8 7501.67 ± 24.906 7023.33 ± 50.123

Model 8 7407.68 ± 51.160 4473.67 ± 86.002

Astragaloside IV 8 7481.00 ± 93.258 5443.00 ± 52.000

**,w

Astragaloside IV–tetramethylpyrazine 8 7417.00 ± 68.462 6514.00 ± 22.338

Nimodipine 8 7412.67 ± 18.502 6584.33 ± 85.471

Values are mean SD.

P < 0.01; signiﬁcantly lower compared with values of sham operation group.

P < 0.01; signiﬁcantly higher compared with values of model group.

P < 0.01; signiﬁcantly higher compared with values of astragaloside IV group.

68 J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72

Fig. 2. 2,3,5-Triphenyltetrazolium chloride (TTC) staining to delineate rat brain infarcts of middle cerebral artery occlusion-induced focal cerebral ischemia. Sham group (A),

model group (B), astragaloside IV group (C), astragaloside IV–tetramethylpyrazine group (D), nimodipine group (E).

Table 3

(P < 0.01), however, nimodipine groups (P < 0.01) was decreased

Infarct volume of different groups in brain cortex sections after cerebral IR with TTC

signiﬁcantly compared with the model group. Although Caspase-

staining.

3-mRNA expression in both the ASG IV–TMPZ (P < 0.01) and the

Group n Infarct volume

nimodipine groups (P < 0.01) was lower than the ASG IV group

Sham operation group 8 0.000 ± 0.000 by real time quantitative PCR revealed the levels of expression of

Model group 8 0.315 0.031 Caspase-3-mRNA, as shown in Table 5.

Astragaloside IV group 8 0.245 ± 0.022

**,w

Astragaloside IV–tetramethylpyrazine 8 0.176 ± 0.014

Nimodipine group 8 0.206 ± 0.025

3.6. Expression of Bcl-2

Values are mean SD.

P < 0.01; signiﬁcantly higher compared with values of sham operation group. Bcl-2 immunoreactivity of neural was quantiﬁed by light

P < 0.01; signiﬁcantly lower compared with values of model group.

microscopy and a computerized image analysis system. Expres-

P < 0.01; signiﬁcantly lower compared with values of astragaloside IV group.

sion of Bcl-2 immunoreactivity was reduced in the model group

J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72 69

Table 4

Determination of drugs on the activity of SOD, contents of MDA and iNOS.

Group n SOD (U/mg) MDA (nmol/mg) iNOS (␮mol/L)

Sham operation group 8 51.89 ± 1.52 2.25 ± 0.08 6.73 ± 0.27

** * *

Model group 8 32.54 ± 0.84 5.52 ± 0.28 26.20 ± 1.66

www *** ***

Astragaloside IV group 8 39.05 ± 1.36 3.45 ± 0.10 16.76 ± 0.90

w ww ww

Astragaloside IV–tetramethylpyrazine 8 45.33 ± 1.64 2.92 ± 0.21 14.05 ± 1.28

www *** ***

Nimodipine group 8 45.63 ± 2.63 2.67 ± 0.12 11.80 ± 1.25

Values are mean SD.

MDA, malondialdehyde; SOD, superoxide dismutase; iNOS, inducible nitric oxide synthase.

P < 0.01; signiﬁcantly higher compared with values of sham operation group.

P < 0.01; signiﬁcantly lower compared with values of sham operation group.

***

P < 0.01; signiﬁcantly lower compared with values of model group.

P < 0.01; signiﬁcantly higher compared with values of model group and astragaloside IV group.

P < 0.01; signiﬁcantly lower compared with values of model group and astragaloside IV group.

www

P < 0.01; signiﬁcantly higher compared with values of model group.

Fig. 3. B-cell lymphoma 2 (Bcl-2) immunostained tissue (magniﬁcation 200×) of middle cerebral artery occlusion-induced focal cerebral ischemia in rats. Sham group (A),

model group (B), astragaloside IV group (C), astragaloside IV–tetramethylpyrazine group (D), nimodipine group (E).

70 J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72

Table 5

after full recovery from anesthesia. The neurologic deﬁcit scores

Real time-qPCR revealed the levels of Caspase-3 relative quantitative analysis after

for rats in the ﬁve groups are presented in Table 1.

reperfusion.

In our study, effects of both ASG IV and ASG IV–TMPZ produced

−Ct

Group n 2 of Caspase-3 mRNA

a signiﬁcant reduction in the infarct volume compared with MCAO-

Sham operation group 6 0.53 ± 0.033 induced focal cerebral ischemia model of stroke in rats. In addition

Model group 6 0.95 0.070 to study the effect of reduce the volume of cerebral infarction,

Astragaloside IV group 6 0.88 ± 0.049

ASG IV–TMPZ signiﬁcantly inhibited the cerebral infarct induced by

***

Astragaloside IV–tetramethylpyrazine 6 0.80 ± 0.043

*** MCAO compared with ASG IV, as demonstrated by the neurologic

Nimodipine group 6 0.67 ± 0.030

deﬁcit score.

Values are mean ± SD. 18

In previous study, we have used F-FDG micro-PET to detect

Caspase-3; RT-qPCR, real time quantitative PCR.

* many kinds of cerebrovascular diseases because of the consump-

P < 0.01; signiﬁcantly higher compared with values of sham operation group.

P < 0.05; signiﬁcantly lower compared with values of model group. tion of large amounts of glucose as an energy source. Micro-PET

***

P < 0.01; signiﬁcantly lower compared with values of model group and astraga- has the large advantage to monitor the glucose metabolism non-

loside IV.

invasively and assess the early effects for cerebrovascular disease

therapy (Lubec et al., 2000; Cherry and Gambhir, 2001; Zhao et al.,

2009). In our study, we investigated glucose metabolic activity of

(P < 0.01) compared with the sham group. Expression of Bcl-2

each region of hippocampus in all groups. The ratio of the ROIs

immunoreactivity of the model group was signiﬁcantly lower

in model group compared with the sham group, and both ASG IV

than that of the ASG IV group (P < 0.05), the ASG IV–TMPZ group

group and ASG IV–TMPZ group were all increased. Furthermore

(P < 0.01), and nimodipine group (P < 0.01). In the ASG IV- TMPZ

the effect of ASG IV–TMPZ was better than ASG IV in the recovery

group (P < 0.01) and nimodipine group (P < 0.01), the Bcl-2 level was

of glucose metabolism. Obvious metabolic asymmetry in the right

showed to be higher than ASG IV group (Table 6). As shown in Fig. 3,

× and left hemisphere of rat after the operation was observed by 7 d

Bcl-2 immunostained tissue (magniﬁcation, 200 ) changes in pro-

after cerebral IR, the hypometabolic region was enlarged distinctly

tein expression of Bcl-2 in all groups after cerebral IR damage were

in the three treatment groups, among which was the smallest in

observed by the immunohistochemical method. 18

the ASG IV–TMPZ group, as shown in Fig. 1. The data of F-FDG

micro-PET images were consistent with the triphenyltetrazolium

4. Discussion chloride evaluation (Fig. 2). However, by 1 h and 6 h after MCAO,

the mild decrease of metabolic activity of glucose was observed in

Cerebral I/R injury, which results in cerebral infarction, is the the right brain (data not shown), whereas a signiﬁcant increase was

main cause for the aggravation of brain dysfunction and cell death also evident on the border of the ischemic area (Read et al., 1998;

(Schaller and Graf, 2004). The early moments of reperfusion are Sarrafzadeh et al., 2010).

important in the pathogenesis at excessive formation of free radi- Generation of excessive reactive oxygen species (ROS) dur-

cals, cascade reactions, overload of calcium inside neurocytes, and ing reperfusion plays a major role in brain injury. Because of the

cytotoxic substance of excitatory amino acids are the main reasons brain’s low activities of antioxidative enzymes, it is very vulnera-

contributing to cerebral IR injury. As the main cause for the aggrava- ble to ROS induced by cerebral IR injury, which causes oxidative

tion of cerebral injury, acute inﬂammatory cascade reactions draw damage to brain lipids, proteins, and DNA, leading to brain dys-

speciﬁc attention (Danton and Dietrich, 2003; Huang et al., 2006). function and cell death. Experiment indicated that in cerebral

MCAO, a reliable, less invasive stroke model of temporary regional IR, SOD activity was remarkably decreased and some antioxi-

ischemia has been prompted by the extensive neurochemical data dants up regulated SOD gene expression in rats after I/R injury

already available in rats, so as to reduce the rising cost of exper- (Shahed et al., 2001). The content of MDA, one of the most sen-

iments with larger animals, and the limitations of other rodent sitive indicators of lipid peroxidation (Gutteridge, 1995), increased

models of focal cerebral IR. Because cerebral cortex and hippocam- remarkably. Besides superoxide, recent studies demonstrated that

pus damage are the main event of brain ischemia in MCAO, the iNOS increased in cerebral IR. It has demonstrated that inhibition of

neurons in hippocampus should be a target for this protection (Xing apoptosis formation (active Caspase-3) and inﬂammatory response

et al., 2008). Neuroprotective effects of ASG IV–TMPZ were stud- (iNOS), resulting in a reduction in the infarct volume in ischemia-

ied in the MCAO model in rats for the ﬁrst time. The neurological reperfusion brain injury (Chang et al., 2007). The results of MDA,

score was used to evaluate the neuroprotective effects (Garcia et al., iNOS level, and SOD activity were consistent with the reduction of

1995). The neurological score was improved by treatment with ASG cerebral infarction as well as neurological recovery (Vaughan and

IV and ASG IV–TMPZ, which indicated the protective potential of Delanty, 1999). In this experiment, we also found that free radical

ASG IV and ASG IV–TMPZ in focal cerebral ischemia. In the ASG IV production may be an important factor to represent an impor-

and ASG IV–TMPZ groups, moderate focal deﬁcits were observed tant modulator after longer periods of cerebral IR. The effects of

ASG IV and ASG IV–TMPZ on brain MDA content, SOD activity, and

iNOS levels in MCAO–reperfusion rats are shown in Table 3. After

Table 6

the experiment, we found that the levels of MDA and iNOS in the

The expression of Bcl-2 immunoreactive (numbers of positive cells/ﬁeld of vision).

brain tissue in the model group were remarkably increased and

Group n Bcl-2 protein SOD activity was remarkably decreased. In the ASG IV and ASG

IV–TMPZ groups, the levels of iNOS were reduced and the level

Sham operation group 50 34.92 ± 2.93

Model group 50 11.36 ± 2.44 of SOD was increased compared with the model group. These data

Astragaloside IV group 50 12.94 3.95 suggested that the antioxidant property of ASG IV–TMPZ could play

***

Astragaloside IV–tetramethylpyrazine 50 19.70 ± 3.49

an important role in protecting the neurons, possibly by increasing

Nimodipine group 50 21.22 ± 3.94

the endogenous defensive capacity of the brain to combat oxidative

Values are means ± SD.

stress induced by cerebral I/R.

P < 0.01; signiﬁcantly lower compared with values of sham operation group.

** Caspase-3 is a major cell death effector protease in the adult and

P < 0.05; signiﬁcantly higher compared with values of model group.

*** neonatal nervous system (Le et al., 2002), and initiate the cleavage

P < 0.01; signiﬁcantly higher compared with values of model group and astraga-

loside IV group. of key cellular substrates (Namura et al., 1998; Zhou et al., 2003).

P < 0.01; signiﬁcantly higher compared with values of model group. In our study, we found that the level of Caspase-3 after ischemia

J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72 71

was increased, connecting with the worsening of infarct severity, Chang, Y., Hsiao, G., Chen, S.H., Chen, Y.C., Lin, J.H., Lin, K.H., Chou, D.S., Sheu, J.R., 2007.

Tetramethylpyrazine suppresses HIF-1alpha, TNF-alpha, and activated caspase-

which means that the role of Caspase-3 was propagating tissue

3 expression in middle cerebral artery occlusion-induced brain ischemia in rats.

damage. After cerebral I/R, the expression of Caspase-3 mRNA in

Acta Pharmacologica Sinica 28, 327–333.

the model group compared with the sham operation group. The Cherry, S.R., Gambhir, S.S., 2001. Use of positron emission tomography in animal

research. ILAR Journal 42, 219–232.

Caspase-3-mRNA expression of the ASG IV, ASG IV–TMPZ, and

Chugani, H.T., Hovda, D.A., Villablanca, J.R., Phelps, M.E., Xu, W.F., 1991. Metabolic

nimodipine groups was decreased signiﬁcantly compared with the

maturation of the brain: a study of local cerebral glucose utilization in the devel-

model group. ASG IV–TMPZ played an important role in prevent- oping cat. Journal of Cerebral Blood Flow and Metabolism 11, 35–47.

Danton, G.H., Dietrich, W.D., 2003. Inﬂammatory mechanisms after ischemia

ing expression of the Caspase 3 mRNA. The bcl-2 family members

and stroke. Journal of Neuropathology and Experimental Neurology 62,

are major regulators of the apoptotic process (Ivins et al., 1998;

127–136.

Hata et al., 1999; Solaroglu et al., 2006). In our experiment, we Gao, F., Wang, S., Guo, Y., Wang, J., Lou, M., Wu, J., Ding, M., Tian, M., Zhang, H., 2010.

investigated the immunoreactivity expression to study the protec- Protective effects of repetitive transcranial magnetic stimulation in a rat model

of transient cerebral ischaemia: a microPET study. European Journal of Nuclear

tion by ASG IV–TMPZ of cortical neurons from cerebral IR injury.

Medicine and Molecular Imaging 37, 954–961.

Bcl-2 immunoreactivity of neural cells was quantiﬁed by using

Garcia, J.H., Wagner, S., Liu, K.F., Hu, X.J., 1995. Neurological deﬁcit and extent of neu-

light microscopy and a computerized image analysis system. Our ronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical

validation. Stroke 26, 627–634, discussion 635.

results showed that a signiﬁcant increase in the number of Bcl-2

Ginsberg, M.D., 2001. Role of free radical reactions in ischemic brain injury. Drug

immunoreactivities in neural cells was found in the cortex in the

News & Perspectives 14, 81–88.

ASG IV–TMPZ group among all three treatment groups, as shown in Gutteridge, J.M., 1995. Lipid peroxidation and antioxidants as biomarkers of tissue

damage. Clinical Chemistry 41, 1819–1828.

Table 5, which indicated that ASG IV–TMPZ has obvious protective

Han, B.H., Xu, D., Choi, J., Han, Y., Xanthoudakis, S., Roy, S., Tam, J., Vaillancourt, J.,

effect on nerve cells.

Colucci, J., Siman, R., Giroux, A., Robertson, G.S., Zamboni, R., Nicholson, D.W.,

Rats with large infarct volume showed a lower SOD and Bcl-2, Holtzman, D.M., 2002. Selective, reversible caspase-3 inhibitor is neuroprotec-

tive and reveals distinct pathways of cell death after neonatal hypoxic–ischemic

higher MDA, iNOS, Caspase-3, and lower glucose metabolism com-

brain injury. Journal of Biological Chemistry 277, 30128–30136.

pared with that of small infarcts (Zhang et al., 2003; Moro et al.,

Hata, R., Gillardon, F., Michaelidis, T.M., Hossmann, K.A., 1999. Targeted disruption of

2005). Treatment drugs improving energy metabolism, scavenging the bcl-2 gene in mice exacerbates focal ischemic brain injury. Metabolic Brain

Disease 14, 117–124.

ROS and inhibiting apoptosis of cerebral IR injury, among which

Ho, W.K., Wen, H.L., Lee, C.M., 1989. Tetramethylpyrazine for treatment of experi-

ASG IV–TMPZ treatment showed the most signiﬁcant protective

mentally induced stroke in Mongolian gerbils. Stroke 20, 96–99.

effect. However, it is believed that the technology of neural stem Huang, J., Upadhyay, U.M., Tamargo, R.J., 2006. Inﬂammation in stroke and focal

cell transplantation and repetitive transcranial magnetic stimula- cerebral ischemia. Surgical Neurology 66, 232–245.

Ivins, K.J., Bui, E.T., Cotman, C.W., 1998. Beta-amyloid induces local neurite degen-

tion combined with ASG IV may have amazing protective effect to

eration in cultured hippocampal neurons: evidence for neuritic apoptosis.

cerebral I/R injury in near future (Zhang et al., 2008; Gao et al.,

Neurobiology of Disease 5, 365–378.

2010). Le, D.A., Wu, Y., Huang, Z., Matsushita, K., Plesnila, N., Augustinack, J.C., Hyman, B.T.,

Yuan, J., Kuida, K., Flavell, R.A., Moskowitz, M.A., 2002. Caspase activation and

neuroprotection in caspase-3-deﬁcient mice after in vivo cerebral ischemia and

5. Conclusion in vitro oxygen glucose deprivation. Proceedings of the National Academy of

Sciences of the United States of America 99, 15188–15193.

Liu, K.Z., Li, J.B., Lu, H.L., Wen, J.K., Han, M., 2004. Effects of Astragalus and saponins of

The synergistic protective effect of ASG IV–TMPZ is related to

Panax notoginseng on MMP-9 in patients with type 2 diabetic macroangiopathy.

improve glucose metabolism and protect hippocampus neurons, Zhongguo Zhong Yao Za Zhi 29, 264–266.

Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cere-

followed by increase the activity of SOD, the expression of Bcl-2 pro-

bral artery occlusion without craniectomy in rats. Stroke 20, 84–91.

tein, mediate a portion of the inhibition of the reduction of oxidative

Lubec, B., Chiappe-Gutierrez, M., Hoeger, H., Kitzmueller, E., Lubec, G., 2000. Glu-

stress, and anti-apoptosis, followed by inhibition of apoptosis for- cose transporters, hexokinase, and phosphofructokinase in brain of rats with

perinatal asphyxia. Pediatric Research 47, 84–88.

mation and inﬂammatory response, resulting in a reduction in

18 Luo, Y., Qin, Z., Hong, Z., Zhang, X., Ding, D., Fu, J.H., Zhang, W.D., Chen, J., 2004.

the infarct volume in ischemia-reperfusion brain injury. F-FDG

Astragaloside IV protects against ischemic brain injury in a murine model of

micro-PET could be used to monitor the therapeutic response of transient focal ischemia. Neuroscience Letters 363, 218–223.

ASG IV–TMPZ or ASG IV. Thus, ASG IV–TMPZ treatment may repre- Moro, M.A., Almeida, A., Bolanos, J.P., Lizasoain, I., 2005. Mitochondrial respiratory

chain and free radical generation in stroke. Free Radical Biology and Medicine

sent an ideal approach to lowering the risk of or improving function

39, 1291–1304.

in cerebral IR injury.

Namura, S., Zhu, J., Fink, K., Endres, M., Srinivasan, A., Tomaselli, K.J., Yuan,

J., Moskowitz, M.A., 1998. Activation and cleavage of caspase-3 in apopto-

sis induced by experimental cerebral ischemia. Journal of Neuroscience 18,

Conﬂict of interest 3659–3668.

Qu, Y.Z., Li, M., Zhao, Y.L., Zhao, Z.W., Wei, X.Y., Liu, J.P., Gao, L., Gao, G.D., 2009.

Astragaloside IV attenuates cerebral ischemia-reperfusion-induced increase in

The authors declare that there are no conﬂicts of interest.

permeability of the blood–brain barrier in rats. European Journal of Pharmacol-

ogy 606, 137–141.

Acknowledgments Read, S.J., Hirano, T., Abbott, D.F., Sachinidis, J.I., Tochon-Danguy, H.J., Chan, J.G., Egan,

G.F., Scott, A.M., Bladin, C.F., McKay, W.J., Donnan, G.A., 1998. Identifying hypoxic

tissue after acute ischemic stroke using PET and 18F-ﬂuoromisonidazole. Neu-

This study was supported by the National Science Foundation rology 51, 1617–1621.

of China (NSFC) (Nos. 30873430, 30973933, 81173647), Natural Rohren, E.M., Turkington, T.G., Coleman, R.E., 2004. Clinical applications of PET in

oncology. Radiology 231, 305–332.

Science Foundation of Zhejiang Province (Z2101201) and Zhejiang

Sarrafzadeh, A.S., Nagel, A., Czabanka, M., Denecke, T., Vajkoczy, P., Plotkin, M.,

Provincial Program for the Cultivation of High-level Innovative

2010. Imaging of hypoxic-ischemic penumbra with (18)F-ﬂuoromisonidazole

Health Talent Fellowship. PET/CT and measurement of related cerebral metabolism in aneurysmal sub-

arachnoid hemorrhage. Journal of Cerebral Blood Flow and Metabolism 30,

36–45.

References Schaller, B., Graf, R., 2004. Cerebral ischemia and reperfusion: the pathophysio-

logic concept as a basis for clinical therapy. Journal of Cerebral Blood Flow and

Metabolism 24, 351–371.

Balch, P.A., 2006. Prescription for Nutritional Healing, 4th ed. Avery, New York.

Shahed, A.R., Jones, E., Shoskes, D., 2001. Quercetin and curcumin up-regulate

Bederson, J.B., Pitts, L.H., Germano, S.M., Nishimura, M.C., Davis, R.L., Bartkowski,

antioxidant gene expression in rat kidney after ureteral obstruction or

H.M., 1986. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for

ischemia/reperfusion injury. Transplantation Proceedings 33, 2988.

detection and quantiﬁcation of experimental cerebral infarction in rats. Stroke

Shao, B.M., Xu, W., Dai, H., Tu, P., Li, Z., Gao, X.M., 2004. A study on the immune

17, 1304–1308.

receptors for polysaccharides from the roots of Astragalus membranaceus, a

Belayev, L., Alonso, O.F., Busto, R., Zhao, W., Ginsberg, M.D., 1996. Middle cerebral

Chinese medicinal herb. Biochemical and Biophysical Research Communications

artery occlusion in the rat by intraluminal suture. Neurological and pathological

320, 1103–1111.

evaluation of an improved model. Stroke 27, 1616–1622, discussion 1623.

72 J. Yang et al. / Journal of Ethnopharmacology 140 (2012) 64–72

Solaroglu, I., Tsubokawa, T., Cahill, J., Zhang, J.H., 2006. Anti-apoptotic effect of model of traumatic brain injury. European Journal of Nuclear Medicine and

granulocyte-colony stimulating factor after focal cerebral ischemia in the rat. Molecular Imaging 35, 1699–1708.

Neuroscience 143, 965–974. Zhang, W.J., Hufnagl, P., Binder, B.R., Wojta, J., 2003. Antiinﬂammatory

Vaughan, C.J., Delanty, N., 1999. Neuroprotective properties of statins in cerebral activity of astragaloside IV is mediated by inhibition of NF-kappaB activa-

ischemia and stroke. Stroke 30, 1969–1973. tion and adhesion molecule expression. Thrombosis and Haemostasis 90,

Wan, H., Zhu, H., Tian, M., Hu, X., Yang, J., Zhao, C., Zhang, H., 2008. Protective effect 904–914.

of chuanxiongzine-puerarin in a rat model of transient middle cerebral artery Zhao, C., Chen, Z., Ye, X., Zhang, Y., Zhan, H., Aburano, T., Tian, M., Zhang, H.,

occlusion-induced focal cerebral ischemia. Nuclear Medicine Communications 2009. Imaging a pancreatic carcinoma xenograft model with 11C-acetate:

29, 1113–1122. a comparison study with 18F-FDG. Nuclear Medicine Communications 30,

Xing, B., Chen, H., Zhang, M., Zhao, D., Jiang, R., Liu, X., Zhang, S., 2008. Ischemic post- 971–977.

conditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury Zhou, H., Ma, Y., Zhou, Y., Liu, Z., Wang, K., Chen, G., 2003. Effects of mag-

in the rat. Stroke 39, 2362–2369. nesium sulfate on neuron apoptosis and expression of caspase-3, bax and

Zhang, H., Zheng, X., Yang, X., Fang, S., Shen, G., Zhao, C., Tian, M., 2008. 11C- bcl-2 after cerebral ischemia-reperfusion injury. Chinese Medical Journal 116,

NMSP/18F-FDG microPET to monitor neural stem cell transplantation in a rat 1532–1534.