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Phytomedicine 18 (2011) 811– 818

Contents lists available at ScienceDirect

Phytomedicine

j ourna l ho mepage: www.elsev ier .de /phymed

rotective effects of alkaloid extract from Leonurus heterophyllus on cerebralschemia reperfusion injury by middle cerebral ischemic injury (MCAO) in rats

ao Lianga, Ping Liua,b, Yunshan Wanga, Shuliang Songa, Aiguo Ji a,∗

SDU-Weihai International Biotechnology R&D Center, Shandong University at Weihai, 180 Wenhua Xilu, Weihai 264209, PR ChinaNordic Bioscience (Beijing) Ltd., Beijing 102206, PR China

r t i c l e i n f o

eywords:eonurus heterophyllus (Yimucao)schemiaeperfusionlkaloidseuroprotection

a b s t r a c t

The neuronal damage following cerebral ischemia is a serious risk to stroke patients. The aim of this studywas to investigate the neuroprotective effects of alkaloid extract from Leonurus heterophyllus (LHAE) oncerebral ischemic injury. After 24 h of reperfusion following ischemia for 2 h induced by middle cerebralartery occlusion (MCAO), some rats were intraperitoneally administered different doses of LHAE (3.6,7.2, 14.4 mg/kg, respectively). Neurological examination was measured in all animals. Infarct volume,myeloperoxidase (MPO) activity, levels of nitrate/nitrite metabolite (NO) and apoptosis ratio of nervefiber in brain were determined. The results showed that LHAE at 7.2 mg/kg or 14.4 mg/kg exerted signif-icantly decreasing neurological deficit scores and reducing the infarct volume on rats with focal cerebral

ischemic injury (p < 0.05). At those dose, the MPO content were significantly decreased in ischemic brainas compared with model group (p < 0.05). LHAE at 14.4 mg/kg significantly decreased the NO level com-pared with the model group (p < 0.05). In addition, LHAE significantly decreased the apoptosis ratio ofnerve fiber compared with the model group (p < 0.05). This study suggests that LHAE may be used fortreatment of ischemic stroke as a neuroprotective agent. Further studies are warranted to assess the

E in p

efficacy and safety of LHA

ntroduction

Stroke is the third most common cause of death world-ide and the leading cause of disability in adult (Wong et al.

007). Prevention and effective treatment of stroke is of outmostmportance. A number of medications that help prevent stroken high-risk patients, are under investigation (Hohnloser et al.008). However, there is currently no treatment that has beenonclusively proven by controlled clinical trials to be beneficial,xcept for thrombolytic treatment with recombinant tissue plas-inogen activator for highly selected patients (Alexandrov et al.

004; National Institute of Neurological Disorders and Stroke r-PA Stroke Study Group 1995). Nonetheless, ischemia/reperfusionfter thrombolysis therapy is a big problem, which restrained clin-cal application of thrombolytic drugs (Warach and Latour 2004).p to now, there is yet no valid therapic drug for the treatmentf ischemia/reperfusion injury, especially for the therapy of neural

amage.

Over the last few years, intense interest has focused on thepplication of traditional Chinese herbal medicines (CHM) in thereatment of ischemic stroke since many CHMs are reported to

∗ Corresponding author. Tel.: +86 138 6316 8701; fax: +86 631 568 8049.E-mail address: jiaiguo@sdu.edu.cn (A. Ji).

944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.phymed.2011.01.020

atients.© 2011 Elsevier GmbH. All rights reserved.

possess cardioprotective or neuroprotective compounds (Hua et al.2008; Tan et al. 2008; Zhang et al. 2006). In fact, there are over onehundred traditional Chinese patent medicine (TCPM) commonlyused for ischemic stroke in China (Wu et al. 2007). These TCPMhave been used in clinical practice over 60 years and approved bythe Chinese State Food and Drug Administration for the treatmentof stroke. They are listed in the Chinese National Essential Drug of2004. Many researches have suggested that some of these drugsare therapeutic for ischemia/reperfusion injury (He et al. 2005).

Leonurus heterophyllus (LH), also known as Yimucao, is a tra-ditional Chinese medicine for the treatment of menstruation andchild delivery in gynaecology (Ruan et al. 2003). In the past 30years, LH-based pharmaceutical preparations have also been usedfor the treatment of blood hyperviscosity and myocardial ischemiain China. In addition, it has been reported that LH possessed antiox-idant activity, and the mechanisms involved of scavenging freeradicals and inhibiting the formation of reactive oxygen species(ROS) (Sun et al. 2005).

ROS are excessively produced during reperfusion followingcerebral ischemia (Piantadosi and Zhang 1996). These radicals ini-

tiate a radical chain reaction or signaling pathways that involvemitochondria and lead to cell death (Christophe and Nicolas 2006).Some natural antioxidants like Vitamin E, Vitamin C and uric acidhave been shown to be neuroprotective in animal models or inclinical studies (Gilgun-Sherki et al. 2002). These results suggest

812 H. Liang et al. / Phytomedicine 18 (2011) 811– 818

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0.3 mg/mL with saline for different treatment groups. The LHAEwere administrated by intravenous injection 1 h after reperfusionfor 0.3 mL (1.2 mg/mL, 0.6 mg/mL and 0.3 mg/mL). The sham groupand the MCAO model group were given blank saline of the samevolume in the same manner.

Fig. 1. A representative chromat

hat reducing excessive ROS can ameliorate oxidant injury afterschemia/reperfusion.

LH extracts possess strong anti-oxidant and anti-inflammatoryctivities, which involve of scavenging free radicals and inhibitinghe formation of ROS (Liu et al. 2001). The pharmacological effects ofH extracts include anti-platelet aggregation, inhibitory action onulsating myocardial cells in vitro and vasorelaxative effect (Changnd Li 1986; Xia 1983; Chen and Kwan 2001). However, the effectf LH extracts on cerebral stroke has not been evaluated. The majorharmacologically active constituents of LH extracts are alkaloids,uch as stachydrine, leonurine, leonurinine and betonicine. The goalf our study aimed to develop a rat ischemia model to examinehether alkaloid extract from LH (LHAE) possessed neuroprotec-

ive effects and possible mechanism of LHAE on ischemia-inducedeuronal death.

aterials and methods

aterials

The LHAE was a mixture of alkaloids, and prepared by Kairuinnovative Pharmaceutical Technology Co., Ltd. (Beijing, China).n their preparation, the herb was extracted with water and thextract was concentrated. After precipitated with alcohol, pH of theerbal extract was adjusted to 2–2.5, and strong cation exchangeSCX) resin was employed for further purification. Eluted withodium chloride solution, the eluent was concentrated, desaltednd dried for the following experiments. Chromatographic fin-erprint was employed as quality control for different batches.hromatography was performed using a 4.6 × 250 mm Waterspherisorb 5 �m SCX column (Waters, USA) at ambient temper-ture. The mobile phase was 15 mmol/L KH2PO4 (contain 0.04%riethylamine and 0.15% phosphoric acid). The LHAE was added to

obile phase to get a final concentration of 0.4 mg/mL, and injec-ion volume was 10 �L. The analysis was carried out in an isocratic

ode with a flow rate of 1 mL/min and detection at 192 nm. Eighteaks were indicated, the area of uncommon peaks was no morehan 5% of total peak areas on the base of stachydrine hydrochlorideNational Institute for the Control of Pharmaceutical and Biological

roducts, China; purity �99.2%) as standard sample (Figs. 1 and 2).

The protocols were approved by the Committee for Animalesearch at Shandong University. The animal experiments wereerformed in accordance with the Care Standard of the Labora-ory Animal [China Ministry of Health Publication, 1998]. Adult

of LHAE. Peak 5 is stachydrine.

male Sprague–Dawley rats (250 ± 50 g, Shandong University Ani-mal Service) were maintained under natural illumination with freeaccess to food and water, and were randomly divided into 5 groups:sham control group, ischemia model group, low dose LHAE group(3.6 mg/kg), medium dose LHAE group (7.2 mg/kg) and high doseLHAE group (14.4 mg/kg).

A reperfusion rat model

Animals were anaesthetized with chloral hydrate (300 mg/kg),and underwent middle cerebral artery occlusion (MCAO) using anintraluminal suture technique as described in detail previously(Aspey et al. 1997; Longa et al. 1989). After the MCAO for 2 h, theoperator carefully removed the suture to restore blood flow andthen sutured the skin and allowed the rat to wake up. The Sham-operated animals underwent the same surgical procedure, expectfor arterial occlusion. After reperfusion for 24 h, rats were decapi-tated under anesthesia with chloral hydrate (300 mg/kg), and thebrains were removed and frozen in liquid nitrogen.

Administration

The injection solution (1.2 mg/mL) was diluted to 0.6 mg/mL and

Fig. 2. Chemical structure of stachydrine.

H. Liang et al. / Phytomedicine 18 (2011) 811– 818 813

Table 1Effects of LHAE on neurological scores, MPO activity, and NO level in the brain of rats after reperfusion for 24 h (n = 10).

Group Dose (mg/kg) Neurological evaluation MPO activity (U/g wet tissue) Nitrite/nitrate level (�mol/g protein)

Sham Saline 0.1 ± 0.32** 0.042 ± 0.024** 0.34 ± 0.053**

Model Saline 1.9 ± 0.32 0.22 ± 0.054 0.52 ± 0.030Low dose 3.6 1.6 ± 0.52 0.15 ± 0.05 0.51 ± 0.031Medium dose 7.2 1.4 ± 0.52* 0.12 ± 0.059* 0.48 ± 0.023

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High dose 14.4 0.6 ± 0.52**

* p < 0.05 represents a significant difference between model control group and ot** p < 0.01 represents a significant difference between model control group and ot

eurological examination

A neurological examination was blindly performed by a singlexaminer just before the animals were killed. Performance wascored using a four-point scale (Longa et al. 1989) (0 = no neurolog-cal deficit, 1 = failure to extend the left forepaw fully, 2 = circlingo the left, 3 = falling to the left, 4 = no spontaneous walking with aepressed level of consciousness).

ssessment of cerebral infarct volume

Animals were killed by decapitation 24 h after the reperfusionnd the brain was kept at −20 ◦C for 40 min. Frozen brain was slicednto uniform coronal sections of approximately 2 mm thicknessach. The slices were incubated in 0.5% 2,3,5-triphenyltetrazoliumhloride (TTC) in 0.2 mol/L phosphate buffer (pH 7.4) at 37 ◦C for0 min. Unstained areas were defined as ischemia lesions (Bedersont al. 1986). After 24 h of postfixation in 10% paraformaldehydePFA) solution at 4 ◦C, the slices were photographed on each side.he areas of infarct and both hemisphere for each slice were mea-ured using an image analysis system (JM VIS-TEC, Suzhou, China).he total infarct volume was determined by measuring the infarctrea in each slice section and integrating the values of all slices.nfarct volumes were expressed as percentage of the contralateralemisphere volume (Block et al. 1993).

istological analysis

To observe the ischemic damaged cells, the histological analysisf coronal sections was carried out. Animals were killed by decap-tation and brains were removed and fixed in 4% PFA solution (in.1 mol/L phosphate buffer), overnight for histological analysis. Ratrains were rapidly and carefully removed 24 h after the occlusion,nd then placed in 10% buffered PFA for 24 h. Fixed brains were cutoronally into blocks and then embedded in 10% PFA solution. Then

�m thick coronal sections were obtained at 0.5 mm intervals andlaced on adhesive glass for hematoxylin and eosin (HE) stainingIsayama et al. 1991). The damaged area was delineated accordingo a stereotaxic atlas.

easurement of myeloperoxidase (MPO) activity

To determine MPO activity in ischemic brain, the cortex wasomogenized in 0.86% cold saline after blotted and weighed. The0% homogenate was used to determine MPO activity at 460 nm ashe methods provided by the assay kit (JianChengShengWu, Nan-ing, China). The activity was expressed as U/g wet tissue.

easurement of NO metabolite in the brain

Tissue samples were disposed as described above and the pro-uction of NO (the sum of nitrite and nitrate) was determined using

nitrate reductase method. The measurements were performed asescribed by the assay kits (JianChengShengWu, NanJing, China).he content was expressed as �mol/mg protein.

0.094 ± 0.065** 0.43 ± 0.044**

oups.oups.

The apoptosis ratio of nerve fiber in the brains mensurate

The ischemic cortex was crumbed by a scissor and was madeto single cell suspension by mechanical process (Campanella et al.2002). RNaseA (1 mg/mL, Sigma, USA) was added. The samples wereincubated for 30 min at 37 ◦C, dyed by Annexin V-FITC & PI Apo-ptosis Kit (Biouniquer, USA) and then detected at 488 nm by a flowcytometry (BD FACScan, USA).

Statistical analysis

Data were expressed as mean ± standard deviation (S.D.). Statis-tical significance was determined by one-way analysis of variancefollowed by Dunnett’s test. All the statistics analyses were per-formed by soft-ware SPSS for Windows. A p < 0.05 was consideredstatistically significant.

Results

Neurological evaluation

Twenty-four hours after the onset of MCAO, neurological deficitscores in MCAO rats were significantly higher (p < 0.01) than thoseof the control rats (Table 1), indicating that the MCAO model hadeffectively established cerebral ischemia. As compared to the modelcontrol group, after the treatments with 7.2 mg/kg and 14.4 mg/kgLHAE, the neurological deficit scores significantly decreased.

Infarction volume assessment

Fig. 3a shows typical images of the coronal sections from eachgroup. Treatments with LHAE at all doses significantly reducedthe infarct volume and showed dose-dependent reductions com-pared with the control group. Infarct volumes after treatmentswith 3.6, 7.2 and 14.4 mg/kg LHAE were reduced approximately by31.5%, 68.0%, and 76.4%, respectively (Fig. 3b), versus the controlgroup (control, 30.2 ± 0.64%; 3.6 mg/kg, 20.7 ± 0.30%; 7.2 mg/kg,9.68 ± 0.31%; 14.4 mg/kg, 7.14 ± 0.50%). The protective effects ofLHAE at 14.4 mg/kg were the best of all groups.

Histology analysis

As shown in Fig. 4e, most neurons in the damaged area appearedshrunken with eosinophilic cytoplasm and triangulated pyknoticnuclei at 24 h after reperfusion. Edema of the neuropile wasobserved in the ischemic zone, furthermore there were no neuronswith normal morphology in the deep-ischemic zone. Coagulativenecrosis and cavitation with edema of the neuropile and degrada-tion of cells were detected in the damaged area. The infarct corewas surrounded with ischemic injured neurons (penumbra). Com-

pared with rats in the MCAO control group, the cerebral cortexof rats in the sham-control group (Fig. 4a) appeared intact, theneurons of those were arranged orderly and were normal mor-phology with intact structure, and had abundant cytoplasm andclear nucleolus. There was no edema in the intracellular space, nor

814 H. Liang et al. / Phytomedicine 18 (2011) 811– 818

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ig. 3. Effect of LHAE on the infarct volume of brains in rats. (a) Images of typical cn each group. Values are expressed as mean ± S.D. (n = 10). *p < 0.05, **p < 0.01 com

nflammatory cell infiltration or infarct focus occurred. After treat-ent with LHAE, the central region of infarct focus in the ischemic

rain tissue were markedly shrunken. The pathological changesnvolving cell swelling, eosinophilic cytoplasm and pyknotic nucleiignificantly decreased (Fig. 4c and d).

he LHAE treatment decreased ischemia-inducedyeloperoxidase activity

The accumulation of neutrophils in the ischemic area was inves-igated by measuring MPO activity in the brain tissue. As shown inable 1 and Fig. 5, MPO activity was significantly higher (p < 0.01) inhe brain tissue of model control rats as compared to the values inhe sham-operated group. Treatment with 3.6 mg/kg LHAE showed

tendency to decrease the ischemia-induced nitrite/nitrate levels.t the doses of 7.2 mg/kg or 14.4 mg/kg, MPO activity was signif-

cantly decreased as compared to the model control (p < 0.05 or.01).

evels of NO metabolite

In comparison with the sham control animals, the nitrite/nitrateevels in the brain tissue of the model control rats showedn markedly increase. Treatment with 7.2 mg/kg LHAE led to aecrease of the ischemia-induced nitrite/nitrate levels, and the

l sections of each group; (b) the columnar diagram for the infarct volume of brains to the model control group.

decrease at the dose of 14.4 mg/kg was significant as compared tothe model control (Table 1 and Fig. 5).

Effect of LHAE on the apoptosis ratio of nerve fiber

In the earlier stages of apoptosis, phosphatidylserine (PS) inthe plasma membrane inner leaflet appears in the outer leaflet.Annexin V is a 35–36 kDa Ca2+ dependent phospholipid-bindingprotein that has a high affinity for PS. So annexin V-FITC staining canidentify apoptosis at an earlier stage. For impermeable to cell mem-branes, only necrotic cells or apoptotic cells undergoing secondarynecrosis will be dyed by propidium iodide (PI).

Fig. 6 shows the dot plots of the samples labeled with annexin V-FITC and PI and analyzed by flow cytometry; X axis shows annexinV log fluorescence on FL1-H and Y axis shows PI log fluorescenceon FL2-H. Cells in left lower region represent living cells, cells inright lower early apoptotic cells, cells in right upper late apoptoticcells and cells in left upper those with a damaged membrane only.

As shown in Fig. 6 and Table 2, the early apoptotic ratio of modelcontrol cells was significantly higher (p < 0.01) as compared to thoseof the sham-operated group and all LHAE treatment groups. Butthe ratio of cells in late apoptotic was no significant difference insham-operated group and all LHAE treatment groups.

H. Liang et al. / Phytomedicine 18 (2011) 811– 818 815

Fig. 4. Histochemical staining of coronal sections of rats brain. (a) Normal neurons (10 × 10); (b) neurons of MCAO rats treated with LHAE at low dose (10 × 10); (c) neuronsof MCAO rats treated with LHAE at medium dose (10 × 10); (d) neurons of MCAO rats treated with LHAE at high dose (10 × 10); (e) neurons of MCAO model rats (10 × 10).N th intai d pyka in thel

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ormal neurons were arranged orderly and the neurons had normal morphology win the damaged area appeared shrunken with eosinophilic cytoplasm and triangulatend cavitation (black arrows) with edema of the neuropile and degradation of cellsegend, the reader is referred to the web version of the article.)

iscussion

The main aim of the present study was to examine whetherreatments with alkaloid extract from Leonurus heterophyllus

able 2he apoptosis ratio of brain cells (%) (n = 10). Asterisks represent a significant difference b

Group Dose (mg/kg) Ratios of early apo

Sham Saline 14.1 ± 0.31**

Model Saline 39.0 ± 0.80

Low dose 3.6 28.3 ± 0.20**

Medium dose 7.2 21.4 ± 0.35**

High dose 14.4 17.8 ± 0.37**

ct structure, abundant cytoplasm and clear nucleolus (arrowheads). Some neuronsnotic nuclei (red arrows). There were extensive coagulative necrosis (green arrows)

region of infarct focus. (For interpretation of the references to color in this figure

have neuroprotection in rats undergoing transient focal cerebralischemia followed by reperfusion.

The mechanism of ischemia/reperfusion injury is still unclear. Sofar, it is believed that the mechanism consists of free radical injury,

etween model control group and other groups (**p < 0.01).

ptotic cells (%) Ratios of late apoptotic and necrotic cells (%)

0.033 ± 0.012**

0.17 ± 0.0150.088 ± 0.023**

0.067 ± 0.029**

0.07 ± 0.018**

816 H. Liang et al. / Phytomedicin

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the early apoptosis rate of brain cells, the model group is higherthan control group and LHAE-treated groups significantly (Fig. 6

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ig. 5. MPO activity and NO level in the brain tissue after 24 h ischemia from eachroup (n = 10). Values are mean ± S.D. *p < 0.05, **p < 0.01 compared to the modelontrol group.

alcium overload, inflammatory response, and so on (Bright andochly-Rosen 2005). The antioxidant activity of LH extraction has

een reported (Sun et al. 2005). Considering the anti-inflammatoryffect of alkaloid extract from LH (Li and Cai 2002), we consideredhe protective effects on ischemia/reperfusion may be correlatedith anti-inflammation.

The MCAO model is by far the most commonly used strokeodel, since the pathophysiology presented in this model closelyatches that in stroke patients, many of those patients suffered

rom focal cerebral arterial thrombosis (Longa et al. 1989).Neurological test was a commom method to estimate the suc-

ess rate of MCAO modeling and the ischemia degree of theodeling. In our study, the neurological test and histological anal-

sis showed the curative efficacy of LHAE on ischemia injury.he anti-apoptotic properties of LHAE were proven in brain cells.n the present study, LHAE with all examined doses decreased

PO activity, which suggested the anti-inflammation of LH. Ouresults showed a great increase in nitrite/nitrate level 24 h after theschemia/reperfusion, which showed a great increase in NO levelfter ischemic/reperfusion.

ig. 6. Flow cytometric analysis of apoptosis in rat brain cells. (a) Dot plot of sham; (b) dlot of high dose.

e 18 (2011) 811– 818

In rats, occlusion of the middle cerebral artery resultedin significant neurological deficit, which was evident fromthe neurological score (Devries et al. 2001). Therefore, weemployed a set of most commonly used neurological tests toassess the changed functions following focal cerebral ischemiain rats. According to our observations, 14.4 mg/kg LHAE sig-nificantly decreased neurological deficit scores (Table 1).These changes were also reflected in the infarct volume,which is the most vigorous index of ischemic brain damage(Fig. 3).

The histological analysis supported the functional evidencefor the neuroprotection by LHAE. At the doses of 7.2 mg/kg or14.4 mg/kg, LHAE attenuated the ischemia-induced ill effect sig-nificantly (Fig. 4c and d). At 24 h after reperfusion, most neuronsin the damaged area appeared shrunken with eosinophilic cyto-plasm and triangulated pyknotic nuclei. Edema of the neuropilewas observed in the ischemic zone, while there were no neuronswith normal morphology in the deep-ischemic zone (Fig. 4e). Aftertreatment with 7.2 mg/kg or 14.4 mg/kg LHAE, the central region ofinfarct focus in the ischemic brain tissue had potency to shrunken.The pathological phenomena involving cell swelling, eosinophiliccytoplasm and pyknotic nuclei were decreased significantly.

The reliability of our animal model was confirmed in thisstudy. Using this model, we showed the effect of LHAE onischemia/reperfusion injury from behavior and histological aspects.Then we carried on research from cellular aspect. It has been sug-gested that an alkaloid extracted from LH may have anti-oxidantand anti-apoptotic effects in hypoxic neonatal rat cardiomyocytesand infarcted rat heart (Liu et al. 2009). By means of Annexin V/PIdouble-staining method, we read early apoptosis rate and late apo-ptosis/necrosis rate of rat brain cells from flow cytometry. As to

and Table 2). Moreover, the descending trend of the three LHAE-treated groups is dose-depended. What is interesting, contrary toour expectation, although the late apoptosis of the model group

ot plot of model; (c) dot plot of low dose; (d) dot plot of medium dose; and (e) dot

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ad significant difference with control group, there is no remark-ble difference between model group and LHAE-treated groups,nd also no obvious intraclass difference in the three LHAE-treatedroups. Therefore we believed that, LHAE intervenes in the earlypoptosis process of ischemic/reperfusion injury, preventing therogrammed cell death, and protecting effects on brain cells. As toells in late apoptosis or necrosis, LHAE turned helpless.

In this study, we conducted a set of further research on LHAEor ischemic/reperfusion brain tissue in the biochemical level. Ouresults showed a great increase in MPO activity 24 h after theschemic/reperfusion (Fig. 5 and Table 1). LHAE at medium and highoses decreased MPO activity, suggesting the anti-inflammation ofH. Our results showed a great increase in nitrite/nitrate level 24 hfter the ischemia/reperfusion, and showed a great decrease in NOevel after LHAE-treated with high dose.

It has been reported that inflammation plays an important rolen the pathogenesis of central nervous system injury (Barone et al.992; Frijns and Kappelle 2002; Huang et al. 2006). NeutrophilsPMNs) accumulation is one of the initial events of the injury,hich triggers the release of oxygen free radicals, arachidonic acidetabolites and lysosomal proteases; these factors worsen sub-

equent tissue injury. Experimental studies in rat models of focalschemic stroke suggested that polymorphonuclear leukocytes play

role in the development of secondary injury after acute ischemicnfarction (Hwang et al. 2006; Lin et al. 2000). MPO is a reductaserimarily existing in neutrophil, and traditionally described as aotential marker for neutrophil infiltration within damaged brainissue (Barone et al. 1992). In humans, it has also been shown thatn elevated leukocyte count in the acute stage is associated with aelatively poor clinical outcome (Jean et al. 1998).

Previous studies suggested that neutrophil infiltration occurredt 1 day after cerebral ischemia, and then was replaced byacrophages phagocytosis by day 3 (Becker et al. 2001; Lin et al.

000). In our study, MPO activity in all LHAE groups decreasedFig. 5 and Table 1); this indicates the potent anti-inflammatoryffect of LHAE on focal cerebral ischemia. The dose comparison sug-ested that the anti-inflammatory of LHAE is dose dependent. Theigh dose (i.e. 14.4 mg/kg) brought greater damage decreased.

A number of studies suggested that inhibition of neutrophil infil-ration led to a reduction in infarct volumes, and neutrophils mayontribute to ischemic injury due to stroke (Galvão et al. 2005;arcia et al. 1994). Similarly, in the present study that striatalnd hippocampal neurons became markedly shrunken and vac-olizated in the ischemia rats (Fig. 4e). These changes were reversedy LHAE treatment (Fig. 4c and d), indicating that neutrophil infil-ration may associate with infarct formation.

In rats, the content of mitochondria NO rapidly increasedn ischemic cerebral cortex after focal cerebral ischemia, andncreased furthermore after reperfusion (Kader et al. 1993; Suzukit al. 2002). The final products of NO in vivo are nitrite and nitrate,hus the sum of both species was employed as an index of totalO production in the present study. The authors suggested that

he nitrite levels were increased after MCAO, peaking at 5–10 minfter arterial occlusion and returning to baseline by 60 min (Kadert al. 1993). However, another study suggested that dialyzed NOetabolites from the jugular vein increased immediately after

at middle cerebral artery occlusion, reaching a maximum at6 h (Suzuki et al. 2002). Consistent with the latter study, ouresults showed a great increase in nitrite/nitrate level 24 h afterhe ischemia/reperfusion (Fig. 5 and Table 1), which showed areat increase in NO level after ischemia/reperfusion. Although

ot significant, there was a tendency in our study for decreaseditrite/nitrate concentrations in the ischemic group which werereated with LHAE (3.6 mg/kg and 7.2 mg/kg), as compared with theschemia model group. The nitrite/nitrate concentrations decreasedignificantly at the dose of 14.4 mg/kg (Fig. 5 and Table 1). There-

e 18 (2011) 811– 818 817

fore, we proposed that the neuroprotective effect of LHAE was alsorelated to endothelium-derived NO.

The role of NO in the progression of ischemic damage has beenextensively studied in transient focal cerebral ischemia (Dejamet al. 2007). The overproduction of NO after reperfusion maydirectly influence the activity of enzymes involved, or reactswith superoxide to yield the peroxynitrite anion (ONOO–), whichdecomposes to highly toxic hydroxyl and nitrogen dioxide radi-cals. These radicals lead to the loss of protein, nucleic acids andmembrane lipids, inhibit the enzymes involved in mitochondrialelectron transport, and eventually inhibit the respiration of mito-chondria and cause cell deep-injury (Beckman et al. 1990; Formanet al. 1998; Paradies et al. 2002; Schmidley 1990). It has beenreported that LH is able to scavenge superoxide (Liu et al. 2001).Therefore, the NO removal by LH may contribute to inhibiting thereaction of NO with superoxide (which yields peroxynitrite anion).

In summary, our study first demonstrated that LHAE plays a pro-tective role in focal cerebral ischemia via multifaceted and multiplemechanisms. Further investigation is required to determine howthe different mechanisms interact with each other.

Acknowledgement

We would like to thank the manufacturer (Kairui InnovativePharmaceutical Technology Co., Ltd.) for providing free samples ofLHAE.

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