Journal of Ethnopharmacology 151 (2014) 660–666

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Therapeutic time window and underlying therapeutic mechanism of breviscapine injection against cerebral ischemia/reperfusion injury in rats Chao Guo a,1, Yanrong Zhu a,1, Yan Weng a,1, Shiquan Wang b, Yue Guan a, Guo Wei a, Ying Yin a, Miaomaio Xi a,n, Aidong Wen a,nn a b

Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, PR China Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 7 August 2013 Received in revised form 12 November 2013 Accepted 13 November 2013 Available online 26 November 2013

Ethnopharmacological relevance: Breviscapine injection is a Chinese herbal medicine standardized product extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been widely used for treating cardiovascular and cerebrovascular diseases. However, the therapeutic time window and the action mechanism of breviscapine are still unclear. The present study was designed to investigate the therapeutic time window and underlying therapeutic mechanism of breviscapine injection against cerebral ischemic/reperfusion injury. Materials and methods: Sprague–Dawley rats were subjected to middle cerebral artery occlusion for 2 h followed by 24 h of reperfusion. Experiment part 1 was used to investigate the therapeutic time window of breviscapine. Rats were injected intravenously with 50 mg/kg breviscapine at different time-points of reperfusion. After 24 h of reperfusion, neurologic score, infarct volume, brain water content and serum level of neuron specific enolase (NSE) were measured in a masked fashion. Part 2 was used to explore the therapeutic mechanism of breviscapine. 4-Hydroxy-2-nonenal (4-HNE), 8-hydroxyl-2′- deoxyguanosine (8-OHdG) and the antioxidant capacity of ischemia cortex were measured by ELISA and ferric-reducing antioxidant power (FRAP) assay, respectively. Immunofluorescence and western blot analysis were used to analyze the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Results: Part 1: breviscapine injection significantly ameliorated neurologic deficit, reduced infarct volume and water content, and suppressed the levels of NSE in a time-dependent manner. Part 2: breviscapine inhibited the increased levels of 4-HNE and 8-OHdG, and enhanced the antioxidant capacity of cortex tissue. Moreover, breviscapine obviously raised the expression of Nrf2 and HO-1 proteins after 24 h of reperfusion. Conclusion: The therapeutic time window of breviscapine injection for cerebral ischemia/reperfusion injury seemed to be within 5 h after reperfusion. By up-regulating the expression of Nrf2/HO-1 pathway might be involved in the therapeutic mechanism of breviscapine injection. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Breviscapine I/R Therapeutic time window Nrf2 HO-1 Scutellarin-7-O-glucuronide (Pubchem CID: 185617) Apigenin-7-O-glucuronide (Pubchem CID: 5319484)

1. Introduction Stroke is the second most common cause of death worldwide and the leading cause of disability in adults (Zhou et al., 2008). Application of traditional Chinese medicine for stroke treatment is an effective way (Wu et al., 2007; He et al., 2012; Ning et al., 2012). Erigeron breviscapus (vant.) Hand.-Mazz (specimen deposited in museum of medicinal plants from Yunnan province of China) has a long history of medicinal use in Chinese medicine. It possesses

n

Corresponding author. Tel.: þ 86 29 84775475. Corresponding author. Tel./fax: þ 86 29 84773636. E-mail addresses: handsomfi[email protected] (M. Xi), [email protected] (A. Wen). 1 These authors contributed equally to the manuscript. nn

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.11.026

anti-platelet, anti-thrombus action, decreases plasma fibrin content and promotes fibrinolytic activity (Wang et al., 2010). Erigeron breviscapus is generally used for treating a wide variety of cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, angina pectoris and paralysis caused by cerebral infarction (Domoki et al., 2009). Breviscapine injection, produced by Shineway Pharmaceutical Co., Ltd of China with country medicine accurate character Z13020778, is a Chinese herbal medicine standardized product extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been used in the treatment of disorders in blood supply to the heart and brain, as well as in ischemic disease in China. Previous studies had demonstrated that breviscapine can induce the neuroprotective effect by improving neurological function and decreasing infarct size (Yiming et al., 2008; Guo et al., 2011).

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However, the therapeutic time window and mechanism are still unclear. Recent evidence has suggested that oxidative stress is an important pathological mechanism in cerebral ischemia injury (Janardhan and Qureshi, 2004). Reactive oxygen species (ROS) have been found to be over-produced in cerebral ischemia/reperfusion (I/R), which can lead to oxidative stress (Ren et al., 2011). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of anti-oxidative defense responses, which enhances the transcription of an expansive set of anti-oxidant enzymes and phase II detoxification enzymes, such as heme oxygenase-1 (HO-1), glutathione S-transferases (GSTs) and NAD(P)H quinone oxidoreductase (NQO1) (Shah et al., 2007; Liu et al., 2004). HO-1 is a ubiquitous and redoxsensitive inducible stress protein, which can exert potent indirect anti-oxidative function by degrading heme to CO, iron and biliverdin (Motterlini et al., 2002). A number of studies have proved that increasing the expression of Nrf2 and HO-1 can attenuate oxidative injury induced by brain ischemia (Li et al., 2013; Chen et al., 2012). Therefore, the present study was designed to investigate the therapeutic time window of breviscapine injection by using a rat model and explore whether the therapeutic benefit of breviscapine was associated with the activities of Nrf2, HO-1.

2. Materials and methods 2.1. Preparation and quality control of breviscapine injection Around 4 g pure powder of breviscapine was dissolved into 700 ml 0.1% edetate disodium. Then a small amount of sodium bicarbonate was added to obtain a physiological solution with pH value 7.0–7.5. Subsequently, water was added to the mixed solution for injection to 1000 ml and then the pyrogens were removed by treating with activated charcoal. Finally, the solution was filtered, sterilized and encapsulated into ampoules (Ministry of Public Health, 1998). The main active components of breviscapine are two flavonoids, including the main scutellarin-7-O-glucuronide and a small amount of apigenin-7-O-glucuronide (Fig. 1). In accordance with the corresponding quality control standard, breviscapine injection should contain not less than 95.0% and not more than 105.0% of the labeled amount of scutellarin-7-O-glucuronide (molecular formula: C21H18O12) when determined by ultraviolet spectrophotometry under 335 nm (Ministry of Public Health, 1998).

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2.3. Experimental design Part 1: To investigate the therapeutic time window of breviscapine injection, rats were divided randomly into 3 large groups: sham (n ¼8), control (n¼ 8) and breviscapine groups (n ¼8 in each subgroup). At 0, 1–3, 5 and 7 h after starting reperfusion, rats in the breviscapine group were injected intravenously with 50 mg/kg breviscapine injection. The dosage of 50 mg/kg of breviscapine in this study was selected based on a previous study (Yiming et al., 2008). Sham and control groups were injected with equi-volume saline. After 24 h of reperfusion, neurologic score, infarct volume and brain water content, the level of neuron specific enolase (NSE) was evaluated in a masked fashion. Part 2: To explore the underlying therapeutic mechanism of breviscapine injection, rats were randomly divided into 3 groups (n ¼6 in each group): sham, control and breviscapine groups. At the onset of reperfusion, rats were randomized to receive either saline or breviscapine (50 mg/kg) by intravenous injection. Some oxidative stress markers were measured by ELISA and ferricreducing antioxidant power (FRAP) assay. Immunofluorescence staining and western blot analysis were performed after 24 h of reperfusion. 2.4. Middle cerebral artery occlusion model Middle cerebral artery occlusion (MCAO) was induced using an intraluminal monofilament as previously described (Guo et al., 2012). Rats were anesthetized with sodium pentobarbital (40– 45 mg/kg IP). The right common carotid artery (CCA), the right external carotid artery (ECA) and the internal carotid artery (ICA) were exposed and carefully isolated. A 3-0 monofilament nylon suture (Ethicon, Inc., Osaka, Japan) with a rounded tip was inserted from the lumen of the ECA to that of the right ICA to occlude the origin of the middle cerebral artery (MCA). After 120 min of MCAO, the suture was carefully removed to restore blood flow. The neck incision was closed and rats were allowed to recover. Shamoperated rats underwent the same surgical procedure, but the MCA was not occluded. To monitor occlusion and reperfusion, regional cerebral blood flow (rCBF) was monitored by laser Doppler flowmetry (PeriFluxsystem 5000; Perimed AB, Stockholm, Sweden) positioned at 1 mm posterior and 5 mm lateral to bregma. During the surgical procedure, the body temperature was maintained at 37 1C with a heating pad.

2.2. Animals 2.5. Neurologic deficits score Adult male Sprague–Dawley rats (220–240 g) were obtained from the Experimental Animal Center of the Fourth Military Medical University. All animals were cared for according to National Institutes of Health Guide for the Care and Use of Laboratory Animals. In addition, the study was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University (Xi'an, China).

After 24 h of reperfusion, neurological deficits were evaluated by a blinded observer with a 5-point-scale scoring system as described previously (Liang et al., 2011). 0 ¼ no deficit; 1 ¼failure to extend left forepaw fully; 2 ¼circling to the left; 3¼ falling to the left; 4 ¼no spontaneous walking with a depressed level of consciousness.

Fig. 1. The chemical constituent of breviscapine. (A) scutellarin-7-O-glucuronide. (B) apigenin-7-O-glucuronide.

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2.6. Measurement of infarct volume and brain water content After neurological evaluation, rats were decapitated and brains were removed for infarct volume measurement. The brains were sliced into uniform coronal sections of 2 mm thickness each. Slices were stained using 1% 2, 3, 5-triphenyltetrazolium chloride (TTC) at 37 1C for 10 min and fixed in 10% buffered formaldehyde solution. For analysis, sections were photographed with a digital camera and the infarct areas of each section were determined with a computerized image analysis system. To compensate for the effect of brain edema, corrected infarct volumes were calculated as described in a previous equation (Belayev et al., 2003). Infarct volume was expressed as percentages of contralateral hemispheric volume. After brains were used for measurement of infarct volume as described above, all of the samples including infarct section and non-infarct sections were weighed immediately to obtain the wet weight. Then these samples were dried in a desiccating oven at 110 1C for 24 h and weighed again to obtain the dry weight. Brain water content was calculated as follows: brain water content (%)¼ (wet weight dry weight)/wet weight  100%.

with a cover glass. The stained sections were examined under a fluorescence microscope (Olympus, Tokyo, Japan). 2.11. Western blot analysis After 24 h of reperfusion, protein extractions for Nrf2 and HO-1 in the ischemic cortex were obtained using a total protein extraction kit (Vazyme, Piscataway, USA) following the manufacturer's protocols. The protein concentrations were then determined using the BCA protein assay kit (Vazyme, Piscataway, USA). Equal amounts of protein samples were loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then electrotransferred to a nitrocellulose membrane. The membrane was blocked with 10% (w/v) nonfat dry milk and 0.5% (v/v) Tween20 in Tris-buffered saline and incubated with anti-Nrf2 (1/500, ab31163, Abcam), anti-HO-1 (1/400, ab13243, Abcam) and anti-βactin (1/1000, Bioss Inc., China) overnight at 4 1C. Membranes were then incubated with HRP-conjugated secondary antibody for 2 h at 37 1C and developed with an enhanced chemiluminescence system. Densitometric analysis of the protein bands was performed with image analysis software. The values were normalized to the β-actin content and expressed as relative intensity.

2.7. Measurement of NSE in serum 2.12. Statistical analysis The blood samples were collected before the rats were decapitated for infarct volume measurement. The sample was centrifuged at 1000 rpm for 20 min at 4 1C and the supernatant was removed to a tube for NSE detection. The levels of NSE and indices of neuronal injury were quantified using an enzyme-linked ELISA kit according to the manufacturer's instructions (Uscn Life Science Inc., Wuhan, China). Serum level of NSE was expressed as ng/ml. 2.8. Measurement of 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxyl2′-deoxyguano- sine (8-OHdG)

All data, except for neurologic score, were expressed as mean 7 standard deviation (S.D.) and were compared using a one-way analysis of variance (ANOVA) followed by Tukey's multiplecomparison test. Neurologic scores were expressed as median (range) and were compared using a nonparametric method (Kruskal– Wallis test) followed by the Mann–Whitney U statistic with Bonferroni correction. A value of P o0.05 was considered as statistically significant.

The contents of 4-HNE and 8-OHdG were measured in ischemia cortex using specific ELISA kits according to the manufacturers' instructions (Cell Biolabs, San Diego, USA).

3. Results

2.9. Ferric-reducing antioxidant power (FRAP) assay

The rCBF was immediately reduced to o20% of the preischemic baseline after MCAO and remained constant during the ischemic period in all rats. After the suture was removed, the rCBF returned to the pre-ischemic value within 2 min. Monitoring of rCBF demonstrated that MCAO was successful (Fig. 2).

The antioxidant capacity of cortex tissue was determined by FRAP according to the manufacturers' instructions (Beyotime, Jiangsu, China). Briefly, the FRAP reagent was prepared from acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl and 20 mM FeCl3  6H2O in proportions of 10:1:1 (v/v), respectively. Around 50 ml of tissue homogenate was added to 1.5 ml freshly prepared and pre-warmed FRAP reagent in a tube and incubated at 37 1C for 10 min. The absorbance of the reaction mixture was then recorded at 593 nm. Standard solutions of Fe II in the range of 100– 1000 mM were prepared from ferrous sulfate (FeSO4  7H2O) in distilled water. The data was expressed as mM ferric ions reduced to ferrous form per liter.

3.1. Regional cerebral blood flow

3.2. Neurologic score As shown in Fig. 3, neurologic scores were examined at 24 h after reperfusion in all groups. The sham group did not have any neurological deficits, whereas severe neurological deficits were

2.10. Immunofluorescence staining The frozen coronal brain sections that were harvested after 24 h of reperfusion were prepared as described previously (Kao et al., 2013). The 10 mm sections were first incubated with 3% (v/v) normal goat serum for 1 h at room temperature. Sections were incubated at 4 1C overnight with anti-Nrf2 (1/100, abcam31163, Abcam) and anti-NeuN antibody (1/200, monoclonal clone A60, Chemicon). Sections were then incubated with Alexa Fluors 488 anti-rabbit IgG or Alexa Fluors 594 anti-mouse IgG secondary antibodies for 2 h. Then, the sections were incubated with DAPI for nuclear counterstaining. Finally, the sections were coverslipped

Fig. 2. Regional cerebral blood flow (rCBF) was monitored 5 min before MCAO, at 5, 60 and 115 min during MCAO and 5 min after MCAO. The rCBF monitoring demonstrated whether the MCAO model was successful.

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observed in the control group. Neurological deficits were significantly improved by breviscapine treatment at 0–5 h after reperfusion. 3.3. Infarction volume assessment As shown in Fig. 4, there was no detectable infarction in the sham group, but a large infarct volume was observed in the control group. Compared with the control group, infarct volume was markedly reduced by breviscapine treatment at 0–5 h after reperfusion. 3.4. Brain water content As shown in Fig. 5, treatment with breviscapine at 0–3 h after reperfusion showed a significant decline in the brain water content compared with the control group. Brain water content was also reduced by breviscapine treatment at 5 h after reperfusion, but it did not reach a significant level.

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3.6. Breviscapine attenuated oxidative damage As shown in Fig. 7, the protein concentrations of 4-HNE and 8OHdG were very low in the cortex of sham-operated rats. After I/R injury, 4-HNE and 8-OHdG protein levels in the ischemia cortex of the control group were markedly increased compared with the sham group. Treatment with breviscapine significantly reduced the concentrations of 4-HNE and 8-OHdG (Fig. 7A and B).

3.7. Breviscapine increased the FRAP value As shown in Fig. 8, after 24 h of reperfusion, the antioxidant power (FRAP value) of ischemic cortex in the control group had a significant reduction when compared with the sham group.

3.5. The level of NSE As shown in Fig. 6, serum levels of NSE were significantly elevated in the control group, whereas treatment with breviscapine at 0–5 h after reperfusion markedly suppressed the elevation. Treatment with breviscapine at 7 h after reperfusion had nearly no effect on the elevation of NSE. Fig. 5. The brain water content in each group was measured and the data were shown as mean 7 S.D., n ¼8; ♯p o 0.05 compared with the control group.

Fig. 3. Neurologic scores were recorded in each group at 24 h after reperfusion and were presented as median (n ¼8);np o 0.05, ♯p o 0.01 compared with the control group.

Fig. 6. The levels of serum NSE in each group were estimated at 24 h after reperfusion. All the data were shown as mean 7S.D., n ¼8; ♯p o0.01 compared with the control group.

Fig. 4. Infarct volume in each group at 24 h after reperfusion. (A) TTC staining of the cerebral infarct in the sham, control and treatment with breviscapine at different time. (B) Statistical analysis of the percentage of infarct volume was determined for each group. All data were expressed as mean7 S.D. (n¼ 8); np o 0.05, ♯p o 0.01 compared with the control group.

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Fig. 7. The oxidative stress markers of 4-HNE and 8-OHdG in the injured cortex were assessed by ELISA after 24 h of reperfusion. All the data were shown as mean 7 S.D., n¼ 6; np o 0.05 compared with the control group.

Fig. 8. The FRAP values in each group were detected at 24 h of reperfusion. All the data were shown as mean 7 S.D., n¼ 6; np o0.05 compared with the control group.

Treatment with breviscapine significantly increased the FRAP value of the ischemic cortex. 3.8. Breviscapine enhanced Nrf2 expression As shown in Fig. 9, the expression of Nrf2 was defined by their specific immunoreactivities together with DAPI positive staining. The Nrf2 immuno-fluorescence was very faintly visualized in the sham group. At 24 h after reperfusion, the expressions of Nrf2 in the control group were increased in cytoplasm and nucleus. Breviscapine treatment further enhanced the expression of Nrf2 after 24 h of reperfusion. 3.9. Breviscapine increased the expression levels of Nrf2 and HO-1 Protein levels of Nrf2 and HO-1 in the cortex from the control group were up-regulated at 24 h of reperfusion when compared with the sham group. Breviscapine treatment significantly increased the levels of Nrf2 and HO-1 when compared with the control group (Fig. 10).

4. Discussion and conclusion In the current study, we demonstrated that breviscapine injection markedly ameliorated neurologic deficits, reduced infarct volume and water content and suppressed the levels of NSE induced by cerebral I/R injury. The optimal neuroprotective effects of breviscapine were 0–3 h after reperfusion, with declining effects at 5 h and almost no effects at 7 h. Thus, the therapeutic time window of breviscapine injection for I/R injury was considered to be within 5 h after reperfusion. Furthermore, immunofluorescence

studies and western blotting analysis revealed that breviscapine obviously increased the expression of Nrf2 and HO-1 proteins after MCAO. The results suggested that the up-regulation of Nrf2/HO-1 pathway could be one of the key mechanisms of neuroprotection after treatment with breviscapine injection. Neurologic score, infarct volume and brain water content are the specific markers for evaluating brain injury. NSE is a glycolytic enzyme concentrated in the cytoplasm of neurons and released in the setting of cell death (Gonzalez-Garcia et al., 2012). Studies suggested that the serum level of NSE was also a sensitive biomarker for estimating brain injury induced by stroke (Celtik et al., 2004). Therefore, it has been studied in this paper. The results showed that breviscapine could ameliorate neurologic deficits, reduce infarct volume and suppress the levels of NSE in a time-dependent manner. Erigeron breviscapus (Vant.) Hand.-Mazz is the endemic medicinal plant resource in Yunnan Province of China. According to records of Chinese ancient book “Dian Nan Ben Cao”, it can promote blood circulation, remove blood stasis and dredge the meridian passage. Recent studies showed that breviscapine was the active ingredient of Erigeron breviscapus for treating cardiovascular and cerebrovascular diseases (Wei et al., 2012). Breviscapine has been prepared into some Chinese patent medicines including injection and used generally in clinical stroke treatment. However, because the therapeutic time window of breviscapine injection is unclear, it often leads to irrational drug use. Our studies showed that the therapeutic time window of breviscapine injection seemed to be within 5 h after reperfusion through the evaluation markers mentioned above. This result suggested that the earlier administration of breviscapine injection for I/R injury might produce a better outcome. There are complex mechanisms that cause progressive brain damage during cerebral ischemia and reperfusion. Among the proposed mechanisms, oxidative stress generated by ROS is considered to play a vital role in cerebral I/R injury (CandelarioJalil, 2009). During cerebral I/R, excessive production of ROS can cause oxidative damage to brain lipids, proteins and DNA, leading to brain dysfunction and cell death (Crack and Taylor, 2005). 4-HNE is one of the reaction products of lipid hydroperoxide breakdown occurring in response to oxidative stress (Cindric et al., 2013). It rapidly modifies proteins on several amino acids residues, leading to the loss of protein functions (Pedraza-Chaverri et al., 2004). 8-OHdG, another marker of oxidative stress to DNA, is produced when nucleic acid is exposed for oxidative stress (Hong et al., 2010). As two important markers of oxidative stress, our study showed that treatment with breviscapine inhibited injury-induced elevation of 4-HNE and 8-OHdG in brain tissue. The results indicated that breviscapine could attenuate oxidative stress in cerebral ischemia/reperfusion injury.

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Fig. 9. Representative immunofluorescence staining for Nrf2 (green), NeuN (red) and DAPI-nuclear (blue) staining in the cortex of each group at 24 h of reperfusion. The merged image showed co-localization, depicting the presence of Nrf2 in the nucleus. N ¼6/each group. Scale bar: 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 10. Protein levels of Nrf2 and HO-1 in the cortex of sham, control and breviscapine treatment groups were evaluated by western blot analysis. Representative bands and corresponding β-actin bands and analysis of Nrf2 and HO-1 protein levels were shown for each group. Data were presented as mean7 S.D, n¼ 6. nP o0.05, compared with the control group.

In order to further clarify the neuroprotective mechanisms of breviscapine, Nrf2, a master regulator of antioxidative defense responses, is investigated in this paper. Nrf2 is a basic leucine zipper transcription factor that regulates the expression of numerous ROS detoxifying and antioxidant genes (Kobayashi and Yamamoto, 2005). Under normal conditions, Nrf2 interacts with Kelch-like ECH-associated protein 1 (Keap l) to form the Keap-1– Nrf2 complex and limits Nrf2-mediated gene expression. Once stimulated, Nrf2 dissociates from its Keap1, and is translocated

into the nucleus where it binds to an antioxidant responsive element (ARE) and induces the expression of ARE-dependent genes, including HO-1 (Lee et al., 2011; Ungvari et al., 2010). HO1, along with other phase II enzymes, serves as a defense system against oxidative stress (Satoh et al., 2006). In recent research, sufficient evidences have suggested that increasing the activity of Nrf2 pathways and gene targets could exert highly neuroprotective effects against oxidative, metabolic and excitotoxic insults relevant to stroke both in cell culture and in animal models (Lee et al., 2003; Satoh et al., 2008). Previous studies had proved that Nrf2 and HO-1 were up-regulated beginning at 3 h and peaking at 24 h after MCAO (Yang et al., 2009). Therefore activation Nrf2 pathway may gain great benefits at an early stage of ischemia. By immunofluorescence staining and western blot analysis, our results showed that treatment with breviscapine significantly increased the expression of Nrf2 and HO-1 in the ischemic cortex at 24 h after MCAO. The results suggested that the protective effect of breviscapine might be associated with the up-regulation expression of Nrf2 and HO-1. Some limitations of this study should be acknowledged. First, further study is needed to further demonstrate the involvement of Nrf2 in the neuroprotective effects of breviscapine by the model of Nrf2-deficient mice. Second, the therapeutic time window of breviscapine injection for I/R injury was found only by animal observation, and did not involve human subjects. Thus, it may need further clinical trials to support the finding. In conclusion, our results in this study suggested that the therapeutic time window of breviscapine injection for I/R injury seemed to be within 5 h after reperfusion. Moreover, the neuroprotective mechanism of breviscapine injection against cerebral I/R injury may be mediated at least in part by the Nrf2/HO-1 pathway. These results will provide a better understanding of the action mechanism by which breviscapine can be beneficial and might help promote the design of improved neuroprotective agents for use in stroke.

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reperfusion injury in rats.

Breviscapine injection is a Chinese herbal medicine standardized product extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been widely us...
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