• 1 •Chin J Integr Med

Ischemic stroke is the third most common cause of death and the leading cause of disability in adults.(1) Cellular toxicity of thrombin has been reported to be extremely associated with the nerve injury secondary to cerebral ischemia as thrombin could interact with the protease-activated receptor-1 (PAR-1) in brain tissues. Direct thrombin inhibitor such as argatroban could attenuate the neural injury after cerebral ischemia through binding directly to the active site of thrombin.(2) However, its disadvantages including higher price and postoperative hemorrhage have limited its further application in clinical practice. According to the Chinese medicine (CM) theory, toxic heat was the leading cause for acute stage of stroke.(3) Moreover, a commercial CM functioned in clearing away the heat evil and expelling superficial evils showed neuroprotective effects in rats with cerebral ischemia.(4)

Baicalin, the major flavonoids isolated from the dry roots of Scutellaria baicalensis Georgi, has been frequently applied in the traditional herbal medicine for the treatment of various inflammatory diseases. Previous reports mentioned baicalin has multiple

biological activities including antiviral,(5) antioxidant,(6) antiapoptosis,(7) and antitumor activities.(8) Recent studies revealed that baicalin possessed neuroprotective effects in the ischemic cerebral injury,(9) which raised the possibility of using baicalin as a potential agent for stroke therapy.

Protease-activated receptors (PARs) belong to a

ORIGINAL ARTICLEBaicalin Attenuates Focal Cerebral Ischemic Reperfusion

Injury by Inhibition of Protease-Activated Receptor-1 and Apoptosis

ZHOU Qing-bo (周庆博)1, DUAN Cheng-zhu (段成竹)2, JIA Qing (贾 青)3, LIU Ping (刘 萍)4, and LI Lu-yang (李鲁杨)5

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2013Supported by National Natural Science Foundation of China (No. 81072916), Shandong Science and Technique Foundation (No. 2005GG3202062), Shandong Tradit ional Chinese Medicine Administration Fund Program (No.2009-160) and Free Exploration Program of Shandong University (No.2009TS009)1. Department of Neurology, The Second Hospital, Shandong University, Jinan (250033), China; 2. Shandong University School of Medicine, Jinan (250012), China; 3. Department of Experimental Pathology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (250012), China; 4. China Institute of Pharmacology, College of Medicine, Shandong University, Jinan (250012), China; 5. Department of Gerontology, Qilu Hospital, Shandong University, Jinan (250012), ChinaCorrespondence to: Dr. LI Lu-yang, Tel: 86-531-85875207, Fax: 86-531-8896254, E-mail:lianxiang1210@163.comDOI: 10.1007/s11655-013-1441-7

ABSTRACT Objective: To investigate the neuro-protective effects of baicalin in Wistar rats with focal cerebral ischemic reperfusion injury. Methods: Ninety adult male Wistar rats weighing 320–350 g were randomly divided into the following groups (n=5): (a) sham control group; (b) vehicle group, subjected to middle cerebral artery occlusion and received vehicle intraperitoneally; (c-e) baicalin groups, which were subjected to the middle cerebral artery occlusion and treated with baicalin 25, 50 and 100 mg/kg, respectively. The neurological scores were determined at postoperative 1, 3 and 7 d after the treatment. The expression of protease-activated receptor-1 (PAR-1), PAR-1 mRNA and Caspase-3 were determined using Western blot, reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry, respectively. Results: Signifi cant decrease was noted in the neurological score in the baicalin group compared with that of the vehicle group (P<0.01). Additionally, down-regulation of PAR-1 mRNA, PAR-1 and Caspase-3 was observed in the baicalin groups compared with those obtained from the vehicle group (P<0.01). Compared with the low-dose baicalin group (25 mg/kg), remarkable decrease was noted in neurological score, and the expression of PAR-1 mRNA, PAR-1 as well as Caspase-3 in the high-dose group (P<0.05). Conclusion: Baicalin showed neuro-protective effects in focal cerebral ischemic reperfusion injury through inhibiting the expression of PAR-1 and apoptosis. KEYWORDS baicalin, cerebral ischemia-reperfusion, protease-activated receptor-1, Caspase-3, neuroprotection

• 2 • Chin J Integr Med

superfamily of seven-transmembrane domain G-protein-coupled receptors. To date, four subtypes of PAR members (PAR-1, 2, 3 and 4) are identified, among which PAR-1, PAR-3 and PAR-4 are called thrombin receptors as they can be activated by thrombin.(10) Thrombin and its PARs, have been reported to modulate ischemic, hemorrhagic, and traumatic brain injuries.(11) Studies involved thrombin and PARs in the nervous system indicated these receptors played a pivotal role in maintaining a delicate balance between neuro-protection and neuro-degeneration during infl ammation, injury, and disease conditions.(12,13) Additionally, activation of PAR-1 by thrombin or plasmin initiates a series of cellular events in brain including neurite outgrowth inhibition, astrocytic proliferation, microglia activation, and apoptosis.(14-16) Some studies performed in null mice indicated that PAR1 increased infarct volume and caused neuronal damage after transient focal cerebral ischemia and combined cerebral hypoxia/ischemia.(17-19) All these lead us to investigate the effects of baicalin on the expression of PAR-1 in rats with focal cerebral ischemic reperfusion injury.

METHODSDrug Preparation

Baicalin, 7-D-glucuronic acid-5, 6-dihydroxy fl avone, was purchased from Xieli Pharmaceutical Co. Ltd., (Chinese Drug Approval Number: H20053191, Chengdu, China). The purity of baicalin as measured by high performance liquid chromatography (HPLC) was 98.1%. Baicalin was dissolved in saline for intravenously injection.

Animals GroupingAdult male Wistar rats weighing 320–350 g

were provided by the Laboratory Animal Center of Shandong University (Jinan, China). The animals were housed under controlled temperatures and a 12 h/12 h light/dark cycle with food and water. All the experiments were performed according to the Principles of Laboratory Animal Care (NIH Publication No. 86-23, revised 1985) and the regulation of the Committee on the Use and Care of Animals of Fudan University (Shanghai, China), and approved by the Ethics Committee of the 2nd Hospital of Shandong University (Jinan, China). The animals were randomly divided into the following groups (n=5): (a) sham control group, which was subjected to sham operation and received vehicle intraperitoneally (physiological saline 0.1 mL/100 g); (b) vehicle group, which

underwent the occlusion of middle cerebral artery and received vehicle intraperitoneally (physiological saline 0.1 mL/100 g); (c-e) baicalin groups, which were subjected to the occlusion of middle cerebral artery and treated with low dose (25 mg/kg), moderate dose (50 mg/kg) and high dose (100 mg/kg) of baicalin respectively. The animals were sacrificed by decapitation under deep ether anaesthesia 1, 3 and 7 d after cerebral ischemia.

Model EstablishmentThe animals were subjected to middle cerebral

artery occlusion (MCAO) described by Koizumi, et al.(20) In brief, the rats were anesthetized with 10% chloral hydrate (360 mg/100 g, via intraperitoneal injection). The right common carotid artery (CCA) was exposed and clipped with artery clamp. The external carotid artery (ECA) was isolated and ligatured. A nylon suture (with a diameter of 0.22–0.30 mm) with a blunted tip was introduced through a small incision in internal carotid artery and then into the proximate end of the anterior cerebral artery. Ischemia reperfusion injury was performed by withdrawal of the inserted nylon suture after 3 h of occlusion. The animals were returned to cage with free access to water and food.

Neurological Examination

The neurological examination was conducted blindly 24, 48 and 72 h after reperfusion according to the previous report.(21) Neurological tests were scored on a five-point scale. 0: no neurological deficit; 1: failure to extend left forepaw fully on lifting whole body by tail; 2: circling to left; 3: falling to left; 4: unable to walk spontaneously and had depressed levels of consciousness.

Preparation of Brain TissuesAfter neurological evaluation, the animals were

sacrifi ced to fetch the whole brain. The brain tissues were washed with physiological saline at 4 ℃. The right hemisphere was obtained after removal of rhinencephalon, cerebellum and brain stem. The samples were stored at –70 ℃.

Reverse Transcription Polymerase Chain Reaction Analysis for PAR-1 mRNA

RNA was extracted using Trizol reagent and subject to measurement of PAR-1 mRNA abundance using AnAMV one-step Reverse Transcription Polymerase Chain Reaction (RT-PCR) amplification

• 3 •Chin J Integr Med

kit according to manufacturer's instructions. Briefly, the first-strand cDNA was synthesized by reverse transcription using a random primer. The RT product was subsequently amplified by PCR using a Perkin-Elmer 9600 thermal cycler. The sequences of the sense and anti-sense primers used in the PCR reaction were: 5'-ACT ATT TCT CCG CC TTC TCC GCC AT-3' and 5'-TCA CGC AGA CGC AGA GGA GGA GGT AAG C-3'. β-actin was amplified under the same reaction conditions to serve as an internal control. Amplifications were performed under the following conditions: denaturation 180 s at 94 ℃, 30 cycles of 30 s at 94 ℃, 30 s at 64 ℃, 60 s at 72 ℃, terminal elongation 8 min at 72 ℃. The PCR products were resolved by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining. PCR results were analyzed using a Gel Documentation System (Bio-Rad Laboratories Inc., USA). The semi-quantitative analysis of PAR-1 mRNA abundance was performed using the TotalLab TL120 Image software (TotalLab Life Science Analysis Essentials, Greensboro, NC, USA).

Western Blotting Analysis for PAR-1 Expression Western blott ing analysis was performed

as previously described. Briefly, the brain tissues were homogenized in radio immumoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors. Proteins were separated by electrophoresis on a 10 % SDS-PAGE gel and transferred to a Hybond-P PVDF membrane. Subsequently, the membrane was blocked in 5% nonfat milk and incubated with a PAR-1 primary antibody (1:1000 dilution) overnight at 4 ℃, followed by incubation with the peroxidase-conjugated rabbit anti-goat secondary antibody (1:1000 dilution) for 1 h at room temperature. After washing with PBS, the bound primary antibody was visualized with the Enhanced Chemiluminescence System from Amersham (Piscataway, NJ, USA) and exposed to fi lm. The same membrane was probed for β-actin for loading control.

The relative density of PAR to β-actin was analyzed with the TotalLab TL120 Image software.

Immunohistochemistry of Caspase-3 Sections of paraffin-embedded tissues were

stained with the following antibodies: Primary antibody, rabbit anti-mouse Caspase-3 polyclonal antibody (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China); Secondary antibody, horseradish peroxidase (HRP) conjugated with goat anti-rabbit IgG (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China). The sections were developed with DAB (3,3'-diaminobenzidine tetrahydrochloride) substrate and counter stained with Mayers Haematoxylin. The images were analyzed with Leica QWin V3 system (Leica Microsystems Ltd., Germany).

Statistical Analysis The statistics analyzes were performed by

SPSS/Win13.0 software. All data were presented as means ± standard deviation, and analyzed with one-way analysis of variance (ANOVA) and Student's t-test. P<0.05 was considered statistically signifi cant.

RESULTSEffects of Baicalin on Neurological Scores

In this study, the effects of baicalin on neurological scores were determined after ischemia reperfusion injury was induced. As illustrated in Table 1, no signs of neurological defi cit in sham animals were observed. Significant increase of neurological deficit score was noted in the vehicle group (P<0.01). With regards to the baicalin groups, remarkable decrease was noted in the neurological defi cit scores on postoperative day 1, 3 and 7 after ischemia reperfusion injury compared with the vehicle group (P<0.05). On postoperative day 3 and 7 after the baicalin administration, the neurological scores obtained in the high dose group (100 mg/kg) showed a significant difference compared with those obtained in the low dose group (25 mg/kg) (P<0.05, Table 1).

Table 1. Neurologocal Scores of Each Group

Group n Dosage (mg/kg)Neurological scores

1 d 3 d 7 d

Sham 18 – 0 0 0

Vehicle 18 – 2.76±0.48 2.45±0.38 1.92±0.36

Baicalin-25 18 25 2.53±0.42△ 1.99±0.35△ 1.00±0.32△

Baicalin-50 18 50 2.47±0.50△ 1.81±0.36△ 0.60±0.22△

Baicalin-100 18 100 2.38±0.52△▲ 1.40±0.33△▲ 0.40±0.13△▲

Notes: P<0.01, compared with the sham group; △P<0.05, compared with the vehicle group; ▲P<0.05, compared with the low-dose group

• 4 • Chin J Integr Med

was noted in the baicalin groups (P<0.05). Additionally, high-dose administration of baicalin (100 mg/kg) caused a dramatic decrease of the PAR-1 mRNA on postoperative day 3 and 7 compared with the low dose group (25 mg/kg).

The Expression of PAR-1 Protein after Baicalin Administration

Western blot analysis was conducted to evaluate the effect of baicalin on the protein expression of PAR-1. As compared with the sham control, the protein expression of PAR-1 in the vehicle group showed significant increase on postoperative 1 d (0.20±0.019 vs 0.96±0.047, P<0.01), 3 d (0.20±0.02 vs 1.49±0.03, P<0.01) and 7 d (0.20±0.02 vs 0.92±0.05, P<0.01), respectively. In addition, administration of baicalin caused remarkable decrease in PAR-1 compared with the vehicle group (P<0.01). Moreover, dramatic decrease of the PAR-1 expression was observed at 3 d and 7 d after administration of high dose of baicalin compared with the low dose group (Figure 2).

C a s p a s e - 3 E x p r e s s i o n a f t e r B a i c a l i n Administration

To evaluate the effects of baicalin on cellular apoptosis, the expression of Caspase-3 was determined using immunohistochemical staining. Compared with the sham control, the protein expression of Caspase-3 in the vehicle group increased significantly on postoperative 1 d (24.20±3.84 vs 3.15±1.10, P<0.01), 3 d (29.54±3.34 vs 3.11±1.05, P<0.01) and 7 d (17.65±2.33 vs 3.13±1.10, P<0.01) after reperfusion injury, respectively. Additionally, administration of baicalin caused a remarkable decrease in PAR-1 compared with the vehicle group (P<0.01). Moreover, high dose administration of baicalin caused a dramatic decrease of the PAR-1 on postoperative day 3 and 7 compared with the low dose group (Figure 3).

DISCUSSION

Cerebral ischemia-reperfusion injury is a complicated pathological process, which caused great threats to the lives of human beings. To date, the mechanism of ischemic cerebrovascular disease has not been well defined. Previous study indicated that neurotoxicity of thrombin played an important role in the pathogenesis of acute ischemic stroke as thrombin activity was closely related with stroke severity.(22) Moreover, enhanced expression of thrombin was detected in patients with ischemic cerebrovascular

Figure 1. Baicalin Suppressed PAR-1 mRNA Expression Following Focal Cerebral

Ischemic Reperfusion Injury Notes: A: PAR-1 mRNA expression of each group. B:

Semi-quantitative analysis of PAR-1 mRNA expression. The quantitation represents the average relative ratio of PAR-1 mRNA to beta-actin per animal (n=18 for each group). P<0.01, compared with the sham group; △P<0.05, compared with the vehicle group; ▲P<0.05, compared with the low-dose baicalin group

The Expression of PAR-1 mRNA after Baicalin Administration

The expression of PAR-1 mRNA was analyzed using RT-PCR. The results indicated the length of amplified fragments for PAR-1 was 273 bp. Figure 1 revealed signifi cant increase of PAR-1 mRNA on postoperative day 1, 3 and 7 after reperfusion injury compared with the sham control. Compared with the vehicle group, significant decrease of PAR-1 mRNA

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• 5 •Chin J Integr Med

disease (ICD) and those with high risks of ICD.(23) Therefore, the toxic effect of thrombin on the ICD has been considered as a new hot. Hua, et al(24) reported brain thrombin activity and the expression of prothrombin-mRNA were elevated in adult male Sprague-Dawley rats after permanent middle cerebral artery occlusion. In addition, cerebral edema together with infl ammatory injury and cell death were induced due to high level of thrombin.

Currently, four subtypes of PAR members (PAR-1, 2, 3 and 4) are identifi ed, among which PAR-1

has been reported to be commonly distributed in the cerebral cortex, striatum, hypothalamus, hippocampus and cerebellum.(25) Another study performed in adult male Sprague-Dawley rats indicated thrombin involved in the cerebral ischemic reperfusion injury through activating PAR-1.(26) Junge, et al(17) reported that mice lacking PAR-1 showed a 3.1-fold reduction in infarct volume after transient focal cerebral ischemia. Additionally, intracerebroventricular injection of PAR-1 antagnoist could reduce the infarct volume by 2.7-fold.

1 d 3 d 7 d

Figure 3. Baicalin Suppressed Cell Apoptosis Following Focal Cerebral Ischemic

Reperfusion InjuryNotes: A: Caspase-3 positive cells analysis of each group.

B: Semi-quantitative analysis of Caspase-3 positive cells. The quantitation represents the average number of caspase-3 positive cells per animal (n=18 for each group). P<0.01, compared with the sham group; △P<0.05, compared with the vehicle group; ▲P<0.05, compared with the low-dose baicalin group

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Figure 2. Baicalin Suppressed PAR-1 Protein Expression Following Focal Cerebral

Ischemic Reperfusion Injury Notes: A: PAR-1 protein expression of each group. B: Semi-

quantitative analysis of PAR-1 protein expression. The quantitation represents the average relative ratio of PAR-1 protein to beta-actin per animal (n=18 for each group). P<0.01, compared with the sham group; △P<0.05, compared with the vehicle group; ▲P<0.05, compared with the low-dose baicalin group

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• 6 • Chin J Integr Med

As is known to all, Caspases are a family of cystein-dependent proteases with a critical role in the initiation and execution of cell apoptosis. To date, Caspase-3 is considered as an executioner of apoptosis. In vivo study revealed that after thrombin induction, Caspase-3 positive cells appeared in the experimental group (but not in the saline control brains) and were present for more than 2 weeks after neural injury, peaking at 3 days.(27) Another study revealed that thrombin could induce apoptosis in primary cortical neurons, which demonstrated that thrombin played a pivotal role in the apoptosis of cortical neurons.(28) PAR-1 activation induced by thrombin could lead to cellular response triggered by N-methyl-D-aspartate (NMDA). Additionally, thrombin could result in increased levels of free intracellular Ca2+ in primary cultured hippocampal neurons significantly in a dose-response manner, which can lead to apoptosis finally.(29) In our study, significant decreases were observed in the expression of PAR-1 mRNA, PAR-1, Caspase-3, as well as the neurological scores after cerebral ischemic reperfusion injury, which all reached the peak level on day 3, and decreased on day 7. After baicalin administration, significant decreases were noted in the expression of PAR-1 mRNA, PAR-1 and Caspase-3, which demonstrated that baicalin might attenuate focal cerebral ischemic reperfusion injury through inhibition of PAR-1 and its apoptosis.

As thrombin played an important role in the neural injury secondary to the cerebral hemorrhage, anti-thrombin therapy may be attractive in the clinical practices in the near future. Our results indicated baicalin could inhibit the expression of PAR-1 mRNA, PAR-1 protein and Caspase-3 in rats with focal cerebral ischemic reperfusion injury. Also, our study demonstrated that baicalin possessed neuro-protective effects by attenuating the neurologic impairments. We speculated that the neuro-protective effects of baicalin might be associated with the inhibition of PAR-1 expression and the attenuation of cell apoptosis as well as cerebral edema.

Confl ict of InterestThe authors claimed no competing interests.

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(Received July 7, 2012)Edited by CHEN Yi-yu