Journal of Ethnopharmacology 155 (2014) 1053–1060
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Huangqi Honghua combination and its main components ameliorate cerebral infarction with Qi deficiency and blood stasis syndrome by antioxidant action in rats Jinyi Cao a,1, Zhengyu Chen b,1, Yanrong Zhu a,1, Yuwen Li a, Chao Guo a, Kai Gao a, Lei Chen a, Xiaopeng Shi a, Xiaofang Zhang a, Zhifu Yang a,n, Aidong Wen a,nn a b
Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Shaanxi, Xi'an 710032, PR China Health Department of General Logistics Department, CPLA, Beijing 010842, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 December 2013 Received in revised form 22 May 2014 Accepted 31 May 2014 Available online 21 June 2014
Ethnopharmacological relevance: Combination of Radix Astragali (Huangqi) and Carthamus tinctorius L. (Honghua) has been extensively used as traditional herb medicine in China for the treatment of stroke and myocardial ischemia diseases with Qi deficiency and blood stasis (QDBS) syndrome. Aim: To investigate the effect of Huangqi Honghua combination (HH) and its main components astragaloside IV (AS-IV) and Hydroxysafflor yellow A (HSYA) on cerebral ischemia-reperfusion (IR) with QDBS in rat model. Materials and methods: Male rats were randomly divided into the following six groups: sham group, QDBS þI/R model group and treatment group including AS-IV, HSYA, AS-IV þHSYA and HH. The whole blood viscosity (WBV), plasma viscosity (PV), neurological examination, infarct volume, histopathology changes and some oxidative stress markers were assessed after 24 h of reperfusion. Results: HH and its main components AS-IV þ HSYA could significantly decrease WBV, PV, and also significantly ameliorate neurological examination and infarct volume after 24 h of reperfusion. They also significantly increased expression of Nuclear factor erythroid 2-related factor 2 (Nrf2), activities of antioxidants, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px), led to decrease levels of malondialdehyde (MDA) and reactive oxygen species (ROS). Conclusion: AS-IV and HSYA are responsible for the main curative effects of HH. The study may provide scientific information to further understanding the mechanism(s) of HH and its main components in removing blood stasis and ameliorating cerebral infarction. Additionally, AS-IV and HSYA appear to have synergistic effects on neuroprotection. & 2014 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Astragaloside IV (PubChem CID: 13943297) Hydroxysafflor yellow A (PubChem CID: 6443665) Keywords: Radix Astragali Carthamus tinctorius L. Astragaloside IV Hydroxysafflor yellow A Cerebral infarction Qi deficiency and blood stasis syndrome
1. Introduction Stroke is one of the leading cause of morbidity and mortality in the both developing and developed countries (Truelsen et al., 2005; Liu et al., 2007a, 2007b) and is a major challenge to public health due to its high incidence and life-threatening nature (Rossi et al., 2007). Nearly 80% of stroke is ischemic (Donnan et al., 2008), a consequence of a transient or permanent reduction in cerebral blood flow that is restricted to the territory of a major brain artery (Mahajan et al., 2004). In Traditional Chinese Medicine (TCM)
n
Corresponding author. Tel./fax: þ 86 29 84775471. Corresponding author. Tel./fax: þ86 29 84773636. E-mail addresses:
[email protected] (Z. Yang),
[email protected] (A. Wen). 1 These authors contributed equally to this work. nn
http://dx.doi.org/10.1016/j.jep.2014.05.061 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
theory, Qi deficiency and blood stasis (QDBS) syndrome is at the core of ischemic stroke. QDBS is one of the common syndromes, characterized by short breath, pale complexion, tiredness, dark tongue and deep and thin pulse, etc., that often appear in the course of diseases such as cerebrovascular and cardiovascular diseases (Liu et al., 2007a, 2007b; Miao et al., 2008). Radix Astragali (Huangqi), the dry root of Astragalus membranceus (Fisch) Bge, has been routinely used in China for patients with stroke or chronic debilitating diseases, because it can reinforce Qi, strengthen superficial resistance and promote the discharge of pus and the growth of new tissue (Lin et al., 2000; Guo et al., 2012). Carthamus tinctorius L. (Honghua), a dried flower, is popularly used to promote blood circulation to remove blood stasis and alleviate pain (Li et al., 2009; Han et al., 2013). Huangqi Honghua combination (HH) widely used in clinical practice for treating cerebrovascular and cardiovascular diseases with QDBS, such as
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Buyang Huanwu decoction, a well-known TCM formula (Cai et al., 2007; Zhang et al., 2010). In clinical, Huangqi Injection (HQI) and Honghua Injection (HHI), which are the water extract of Huangqi and Honghua, respectively, both are the most popular TCM injections in China. Moreover, the combination of HQI and HHI is widely used and the protection for cerebral infarction with QDBS model is remarkable (Liang et al., 2007;Hu et al., 2008a, 2008b; Lai et al., 2008). Recent research works show that, astragaloside IV(AS-IV), one of the major and active components of Huangqi, has the anti-infarction effect by its antioxidant properties and attenuates permeability of the blood-brain barrier (Luo et al., 2004; Qu et al., 2009). It has been reported that Hydroxysafflor yellow A (HSYA) could protect rat brains against ischemia-reperfusion injury by antioxidant action (Wei, et al., 2005; Yang, et al., 2010; Chen, et al., 2013). But the further biochemical mechanism(s) of AS-IV and HYSA in treating cerebral infarction with QDBS is still unreported. In addition, as the main components of Huangqi and Honghua, their synergism on cerebral infarction with QDBS is little known, either. The widely used modeling method of QDBS is exhaustive swimming exercising (Li, 1991), and the routine cerebral infarction modeling method is middle cerebral artery occlusion (MCAO) (Guo et al., 2012; Raza et al., 2013). In this study, the animals are used in the QDBS model firstly, then operated the MCAO; finally, the animals would become the cerebral infarction with QDBS model. This model is more similar to the stroke patients syndrome in clinical (Liu et al., 2007a; Zhang et al., 2008) and reflects the cerebral protection of the subject drugs more accurately. Oxidative stress is a major contributor in the pathogenesis of I/ R injury (Janardhan and Qureshi, 2004) and one of the main causes of tissue damage following ischemic injury in the brain (Chen et al., 2011). Antioxidant enzymes are the primary means by which neuronal cells protect themselves from toxic reactive oxygen species (ROS).The induction of such enzymes is governed by the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2). Nrf2 can promote the transcription of cytoprotective genes in response to oxidative and electrophilic stresses, and is becoming a promising therapeutic target for neuroprotection (Jing et al., 2013.) In this paper, the effects of HH and their main components AS-IV, HSYA on cerebral infarction with QDBS were studied and their synergistic effect was assessed. The aim of this study is to provide scientific information to further understand the mechanisms of HH and their main components in neuroprotection, exploring the effective basic material of HH, and promoting the modernization of Traditional Chinese Medicine.
2. Materials and methods 2.1. Materials Adult male Sprague-Dawley rats weighing 280 720 g were supplied by the Experimental Animal Center of the Fourth Military Medical University. All experimental procedures were carried out according to protocols approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University (Xi'an, Shaanxi, China) and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Huangqi Injection (HQI) from Chiatai Qingchunbao Pharmaceutical Co., Ltd. (China), Honghua Injection (HHI) was purchased from Ya'an Sanjiu Pharmaceutical Co., Ltd. (China). Standard substances of astragaloside IV (Lot number: 110781-200613) and Hydroxysafflor yellow A(Lot number: 111637-201106) were obtained from the Chinese National Institute for the Control of
Pharmaceutical and Biological Products. The chemical structure is shown in Fig. 1. 2.2. Quantitative analysis of marker components 2.2.1. Content of astragaloside IV in Huangqi Injection (HQI) Analyses were performed on a Shimadzu Instruments (Kyoto, Japan) liquid chromatographic system which was composed of a LC-10Avp binary pump, an evaporative light-scattering detector and a computer system for data acquisition (LC-Solution). The analytical column employed was an Agilent Zorbax Extend C18 column (250 mm 4.6 mm, 5 mm, Agilent Corporation) and acetonitrile water (36:64) as mobile phase, at a flow rate of 1.0 mL/min. The injection volume was 10 mL. The detector temperature was 40 1C, and the pressure was 0.35 MPa. 2.2.2. Content of Hydroxysafflor yellow A in Honghua Injection (HHI) Analyses were performed on an Agilent 1100 series system (Agilent Corporation, USA) with a diode-array detector. The analytical column employed was an Agilent Zorbax Extend C18 column (250 mm 4.6 mm, 5 mm, Agilent Corporation). The mobile phase was composed of acetonitrile (A) and 0.02 M NaH2PO4 (adjusted to pH 3.5 with ortho-phosphoric acid (B)). Gradient elution was employed and the gradient program was set as follows: initial 0 min at 10% solvent A; 0–16 min, linear increased from 10% to 22% solvent A. The system was balanced for 5 min by the initial mobile phase (A:B ¼10:90) after detecting each sample. The flow rate was set at 0.8 mL/min and the injection volume was 10 mL. A detection wavelength was set at 403 nm. 2.3. Animal model The rats were divided into six groups randomly: (1) sham group, (2) QDBS þ I/R model group, (3) AS-IV group, (4) HSYA group, (5) AS-IV þ HSYA group, and (6) HH group. The injection volume was 3 mL/kg, throughout. The dosage of HQI and HHI in clinical is 0.3 ml/kg (20 ml/60 kg) for both (Liang et al., 2007; Hu et al., 2008a, 2008b). The most effective dosage in QDBS þI/R rats, confirmed through preliminary experiments, was 3 ml/kg. Rats in AS-IV group, HSYA group, AS-IVþ HSYA group and HH group were treated with AS-IV (equal to the contents of HQI), HSYA ( equal to the contents of HHI), AS-IV þHSYA (equal to the contents of HQI and HHI) and HQI þHHI via tail intravenous injection, respectively, other groups were treated with normal saline once a day. 40 min after administration, except the sham group, rats in other groups were treated with exhaustive swimming exercising once a day so that they were in a chronic state with QDBS (Li, 1991). On the 22nd day, the middle cerebral artery occlusion (MCAO) was performed using a traluminal filament model (Longa et al., 1989) as described previously (Khan et al., 2009). The animals were anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg) and placed in dorsal recumbency. A 3-0 monofilament nylon suture (Ethicon, Inc., Osaka, Japan) was introduced from the external carotid artery (ECA) to the right internal carotid artery (ICA) to occlude the origin of the right middle cerebral artery. 2 h after the induction of ischemia, the suture was slowly withdrawn. At the same time, the rats were administrated with AS-IV, HSYA, AS-IV þHSYA and HQI þHHI via tail intravenous injection and other groups were treated with normal saline. The neck incision was closed. Sham group rats went the same surgical procedures except the monofilament insertion. Body temperature was maintained at 37 1C with a heating light during surgery. Thereafter the rats were returned to their cages and given free access to food and water.
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Fig. 1. Chemical structure of astragaloside IV (A, AS-IV, molecular weight¼ 784) and Hydroxysafflor yellow A (B, HSYA, molecular weight¼ 612).
2.4. Neurological examination Neurological tests were analyzed in a blinded fashion with regard to treatments before the rats were killed. Neurological function was evaluated using a 0–5-point scale neurological score: 0 ¼no neurological dysfunction; 1 ¼failure to extend left forelimb fully when lifted by tail; 2 ¼circling to the contralateral side; 3 ¼falling to the left; 4¼ no spontaneous walk or in a comatose state; 5 ¼death. The scoring was based on the method of Masuo et al. (1997). 2.5. Blood collection Rats were anesthetized with chloral hydrate (400 mg/kg) by intraperitoneal injection after the neurological tests, and blood was drawn from the abdominal aorta to determine hemorheological variables. Blood was collected into dry vacuum tubes with heparin lithium for whole blood viscosity (WBV). Then plasma was separated from blood by centrifugation at 3000 rpm for 10 min and detected for plasma viscosity (PV). All experiments were completed within 3 h after blood collection. 2.6. Viscosity determination A total of 400 mL blood or plasma was used to determine the viscosity with a cone-plate viscometer (Model ZL9000, Zonci, Co., China) at different shear rates maintained at 37 1C. WBV was measured with shear rates' varying from 1 to 200/s.
area {1 [ (ipsilateral hemisphere area contralateral hemisphere area)/contralateral hemisphere ] }. 2.8. Histological studies After 24 h of reperfusion the rats were killed and then perfused with physiological saline solution at 4 1C, followed by freshly prepared 4% (v/v) paraformaldehyde in 0.1 M phosphate buffered saline (PBS) buffer (pH 7.4). The brain was removed and fixed in 4% (w/v) paraformaldehyde for 24 h. Then the brain block was embedded in paraffin and cut into 5 mm coronal sections. Then the sections were stained with hematoxylin–eosin (H–E) using standard methods. 2.9. Tissue preparation After the neurological tests, the rats were killed and their brains were taken out to dissect the ipsilateral hemisphere to give 5% (w/v) homogenate (10 mM Tris–HCl, pH 7.4 having 10 ml/ml protease inhibitors: 5 mM leupeptin, 1.5 mM aprotinin, 2 mM phenylethylsulfonylfluoride (PMSF), 3 mM peptastatin A, 0.1 mM EGTA, 1 mM benzamidine and 0.04% butylated hydroxytoluene) and centrifuged at 1000 rpm for 5 min at 4 1C to separate the debris. This supernatant 1 (S1) was used for the measurement of mitochondria-generated reactive oxygen species (ROS) and the remaining was recentrifuged at 10,500g for 15 min at 4 1C. The post mitochondrial supernatant (PMS) was used for the estimation of antioxidant enzyme SOD and GSH.
2.7. Infarct volume measurement
2.10. Measurement of mitochondria-generated reactive oxygen species
The brains were dissected out after the blood drawn and kept at 20 1C for 10 min. Coronal sections (2 mm) of the frozen brains were cut with the sharp blades and stained with 1% 2,3,5triphenyltetrazolium chloride (TTC) at 37 1C for 10 min. Viable tissues stained deep red while the infarcts remain unstained. The TTC-stained sections were photographed with a digital camera and the infarct volume of each section was calculated using an image analysis system (Adobe Photoshop 9.0, Adobe Systems Incorporated, San Jose, CA). To compensate for the effect of brain edema, corrected infarct volumes were calculated as previously described using the following equation (Belayev et al., 2003). Infarct volumes were expressed as percentages of contralateral hemispheric volumes. Corrected infarct area ¼measured infarct
Mitochondria are major generators of reactive oxygen species (ROS) in cells and tissues (Moro et al., 2005). Mitochondria in brain tissues were isolated according to a previously described method (Sims and Anderson, 2008). ROS were measured by using the indicator MitoSOX Red (Molecular Probes), according to manufacturer's instructions. Briefly, cells were washed with warm Hank's Buffered Salt Solution (HBSS) containing calcium and magnesium, and then incubated with 5 μM MitoSOX reagent working solution for 10 min at 37 1C. After removing MitoSOX Red, cells were washed with warm HBSS, and fluorescence was read at 510 nm for excitation and 580 nm for emission with a fluorescence plate reader. ROS level was expressed as percentage in fluorescence relative to model group.
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2.11. Measurement of antioxidant enzyme activity and malondialdehyde content Both the measurements of antioxidant enzyme activities and malondialdehyde (MDA) content in tissue homogenate were performed according to the technical manual of the detection kit (Jianchen Biological Institutes, China) (Dong et al., 2002). MDA content was measured by using the thiobarbituric acid reactive substances assay and expressed as nmol/mg protein. Super-oxide dismutase (SOD) activity was measured following the reduction of nitrite by a xanthine–xanthine oxidase system. One unit of SOD is defined as the amount that shows 50% inhibition. The SOD activity was expressed as U/mg protein. Catalase (CAT) activity was assayed by measuring absorbance at 240 nm using an ultraviolet light spectrophotometer (Beckman, USA) and was expressed as U/g protein. The definition of its activity was based on the hydrogen peroxide decomposition rate at 240 nm in the reactive mixture, of which the absorbance was between 0.5 and 0.55. Glutathioneperoxidase (GSH-Px) activity was measured by measuring the absorbance at 412 nm and was expressed as U/mg protein. One unit of GSH-Px activity was defined as the GSH-Px in1 g protein that led to the decrease of 1 mmol/L GSH in the reactive system per minute. 2.12. Western Blotting analysis Proteins were extracted from cerebral cells and fractionated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat dried milk and incubated overnight with primary antibodies (Nrf2, Cell Signaling, USA) at 4 1C. The primary antibody was used at a dilution of 1:1000. Subsequently, membranes were incubated with secondary antibody at a 1:5000 dilution at 37 1C for 30 min. The
blots were visualized with ECL-Plus reagent (Santa Cruz, USA) and analyzed with Quantity One System image analysis software (BioRad, USA).
2.13. Statistical analysis The results, except for neurologic scores, were expressed as mean 7standard deviation (S.D.) and analyzed with one-way analysis of variance (ANOVA) based on Student's two-tailed unpaired t-test or Dunnett's multiple comparisons test. Neurologic scores were expressed as median (range) and were compared using a nonparametric method (Kruskal–Wallistest) followed by the Mann–Whitney U statistic with Bonferronicorrection. P-value less than 0.05 presented statistical significance.
Table 1 Effects on WBV (mPa s) at various shear rats (n¼ 8 in each group). Group
1/s
5/s
30/s
200/s
Sham QDBSþ I/R AS-IV HSYA AS-IV þHSYA HH
26.317 2.56 34.41## 72.53 30.53 7 1.14 29.007 1.67 27.80* 7 2.12 20.46** 7 2.34
11.517 0.95 13.62# 7 0.96 12.167 0.54 11.667 0.82 11.93* 7 0.46 8.64** 7 0.68
6.11 7 0.36 7.50## 70.57 6.86 7 0.27 6.36* 7 0.22 5.83** 7 0.42 4.76** 7 0.21
4.34 7 0.31 5.41## 7 0.43 4.75 7 0.21 4.54* 70.29 4.68* 70.12 3.47** 7 0.08
Sham¼ sham group; QDBSþ I/R ¼QDBSþ I/R model group; AS-IV ¼AS-IV group; HSYA ¼ HSYA group; AS-IVþ HSYA ¼AS-IV þHSYA group; HH¼ HQI þ HHI group. Data were represented as mean7 S.D. n¼ 8. ## #
Po 0.01 vs. sham group. Po 0.05 vs. sham group. P o 0.01 vs. QDBSþI/R model group. P o0.05 vs. QDBSþ I/R model group.
nn n
Fig. 2. HPLC chromatogram of HQI (A), HHI (C), standard (B, D): AS-IV (1), HSYA (2).
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3. Results 3.1. HPLC analysis The main components contents of HQI and HHI were measured by an HPLC method as described in materials and methods. The value of AS-IV and HSYA given was the equivalent amount of the components presented in HQI and HHI used for animal treatment. The content of AS-IV in HQI was 0.15 mg/ml, and the content of HSYA in HHI was 0.24 mg/ml. The quantitatively results are show in Fig. 2. 3.2. Effects on WBV
Fig. 3. Effects on PV. ♯♯P o0.01 vs. sham group, nPo 0.05 vs. QDBSþ I/R model group, nnPo 0.01 vs. QDBSþ I/R model group.
The effects on WBV and PV are shown in Table 1. In the QDBS þ I/R model group, WBV significantly increased at all shear rates in the blood stasis. After administration, WBV at high shear rate significantly decreased except for AS-IV group and significantly decreased at low shear rate in HH as well as AS-IVþ HSYA groups. 3.3. Effects on PV The Effects on PV are shown in Fig. 3. In the QDBS þI/R model group, PV significantly increased, while in HH and AS-IVþ HSYA groups, PV significantly decreased compared with the QDBS þI/R model group. 3.4. Neurological deficit evaluation Neurological scores assessed at 24 h after reperfusion are shown in Fig. 4. The scores in HH and AS-IVþ HSYA groups were significantly better than those in the QDBS þI/R group (P o0.05). 3.5. Infarction volume assessment
Fig. 4. Neurological scores after MCAO in each group (n¼ 8). Neurological scores were expressed as median (range); nPo 0.05, nnP o 0.01 compared with QDBS þ I/R model group,♯♯Po 0.01 compared with sham group.
The representative photographs of coronal sections from each group are shown in Fig. 5A. All treatment groups significantly decreased the infarct volume compared with the QDBS þ I/R model group (Fig. 5B). The group with the best protection effect was the HH group, as well as the AS-IV þHSYA group.
Fig. 5. Infarct size in each group. (A) TTC staining of the cerebral infarct in the rat brain at 24 h after reperfusion. (B) Statistical analysis of the percentage of infarct volume was assessed for each group. All data were expressed as mean 7 S.D. ( n¼ 8 ); nP o 0.05, nnPo 0.01 compared with QDBS þI/R model group, ♯♯P o0.01 compared with sham group.
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Fig. 6. Hematoxylin–eosin stains of coronal sections of brain after 24 h reperfusion. (A) Sham group; (B) QDBS þ I/R model group; (C) AS-IV group; (D) HSYA group; (E) AS-IV þHSYA group; (F) HH group.
3.6. Histopathology analysis Fig. 6 shows the histopathological results from each group. In the sham group, no histopathological abnormalities were observed, and the neurons of those were arranged orderly and had abundant cytoplasm and clear nucleolus (Fig. 6A). On the contrary, in the QDBS þI/R model group, most neurons in the damage area appeared shrunken with eosinophilic cytoplasm and triangulated pyknotic nuclei at 24 h after reperfusion. Edema of the neuropile was observed in the ischemic zone, furthermore there were no neurons with normal morphology in the deepischemic zone. The infarct core was surrounded with ischemic injured neurons (penumbra) (Fig. 6B). After treatment, the central regions of infarct core in the ischemic brain tissue were significantly shrunken. The pathological changes involving cell swelling, eosinophilic cytoplasm and pyknotic nuclei significantly decreased (Fig. 6C F).
Fig. 7. Mitochondria-generated reactive oxygen species level determined after 24 h reperfusion. All data were expressed as mean 7 S.D. ( n¼ 8 ); ♯♯Po 0.01 compared with sham group, nnP o0.01 compared with QDBS þI/R model group.
3.7. Effect on mitochondria-generated reactive oxygen species ROS production in the mitochondria of the QDBS þI/R model group significantly increased compared with the sham group as shown in Fig. 7. In the HH group and AS-IV þHSYA group, ROS were markedly decreased (P o0.01) compared with the QDBS þI/R model group. 3.8. Effect on antioxidant enzyme activities and MDA content After 2 h of ischemia followed by 24 h reperfusion, the activities of SOD, catalase and GSH-Px were significantly decreased in the QDBS þI/R model group compared with the sham group as shown in Table 2. Nevertheless, after treatment with HH, it showed a markedly increase compared with the QDBS þI/R model group (P o0.01). MDA was used as a maker of lipid peroxidation. After 24 h reperfusion, the MDA contents of the AS-IV þHSYA and HH groups were obviously decreased compared with the QDBS þI/R model group (P o0.01).
3.9. Western blotting analysis We examined the expression level of Nrf2 to explore the mechanisms responsible for the beneficial effects of HH. The level of Nrf2 plays an important role in oxidative damage. As shown in Fig. 8, administration of AS-IV þHSYA or HH, individually, significantly increased the expression of Nrf2 compared with the QDBS þI/R model group.
4. Discussion and conclusion In the TCM theory, Qi and blood dysfunction is one major pathogenesis of cerebral diseases. The treatment of cerebral ischemia with QDBS by combination of Qi-tonifying drug (e.g. Huangqi) and drug for invigorating blood circulation and eliminating stasis (e.g. Honghua) has shown satisfactory efficacy in
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Table 2 Effects on the antioxidant activities and MDA content of brain tissue after ischemia/reperfusion injury in rats (n¼ 8 in each group). Group
SOD(m/mg.prot)
CAT(m/g.prot)
GSH-px(vital unit)
MDA(nmol/mg.prot)
Sham QDBS þI/R AS-IV HSYA AS-IV þ HSYA HH
204.60 728.03 80.46## 7 28.53 128.53 712.14 122.00 710.67 155.80* 7 20.25 200.46** 7 31.34
42.357 8.05 13.59## 7 1.96 20.93 7 8.48 19.167 6.54 29.36 7 6.72 38.64** 7 6.08
66.787 9.30 17.10## 7 3.57 26.36 7 9.25 24.977 8.22 38.83* 74.42 54.76** 7 5.71
11.52 73.21 45.33## 7 9.43 34.75 76.31 33.7875.12 20.54** 73.39 14.47** 75.08
Data were shown as mean7 S.D. n¼ 8. ##
P o0.01 vs. sham group. Po 0.05 vs. QDBS þI/R model group. nn Po 0.01 vs. QDBSþ I/R model group. n
Fig. 8. Effects on the levels of Nrf2. nPo 0.05 vs. QDBSþ I/R model group, nnPo 0.01 vs. QDBS þI/R model group.
clinical practice in China (Liang et al., 2007; Ma et al., 2013). In the present study, our results indicated that HH treatment could significantly ameliorate cerebral ischemia and up-regulate Nrf2 to coordinately increase expression of several anti-oxidative enzymes such as SOD and CAT, which play important role in combating oxidative stress. Furthermore, AS-IV and HSYA are mainly responsible for curative effects of HH and play significant roles. Sometimes two main components show synergistic effects. WBV is one of the measurement indexes of hemorheology, the higher the viscosity, the smaller the liquidity, the more easily to form tissue ischemia (Liu et al., 2012). PV plays an important role in WBV. In the present study, our results showed that the level of WBV and PV increasing significantly in the QDBS þ I/R model compared with the control group and HH treatment could significantly decrease WBV at all shear rates and PV. HSYA and AS-IV þHSYA treatments could decrease WBV at high shear rate and PV, while AS-IV had no effect on PV and WBV, which suggested that HSYA may contribute to the effect of HH in decreasing WBV and PV. The above results showed that AS-IV and HSYA had no synergistic effect in decreasing WBV and PV. In the current study, the results of infarct size and histological damage demonstrated that HH or AS-IV þHSYA treatment could significantly decreased the infarct volume and shrink the central region of infarct focus in the ischemic brain tissue. However, treatment with AS-IV or HSYA alone had worse cerebral protective. The results showed that AS-IV and HSYA had a synergetic effect on cerebral protection.
Mitochondria are the main sites of ROS generation, and they function in several defense mechanisms under normal conditions (Li et al., 2012). ROS can lead to the free radical attack of membrane phospholipids and loss of mitochondria membrane potential (Li et al., 2006). During exhaustive swimming exercising and I/R injury, high concentrations of ROS cannot be efficiently removed by the endogenous antioxidant systems, thus the accumulation of ROS resulted in oxidative damage to cellular membrane, DNA and protein. In our results, treatment with HH and AS-IVþ HSYA markedly decreased ROS level compared with the QDBS þI/R model group. What is more, treatment with AS-IV or HSYA alone could not decrease ROS. The results demonstrated that AS-IV and HSYA had a synergetic effect on decreasing ROS. Nrf2 is a transcription factor that regulates an expansive set of antioxidant-related genes, which acts in synergy to remove ROS through sequential enzymatic reactions (Qin et al., 2009). Among the spectrum of anti-oxidant genes, expression of those encoding catalase, SOD, glutathione reductase, GSH-Px is all controlled by Nrf2 (Dai et al., 2012). In our findings, Nrf2 expression was significantly increased by treatment with AS-IVþHSYA and HH, followed by increasing the activities of SOD, catalase and GSH-Px, decomposing O2 and H2O2 before their interaction to form the more harmful hydroxyl (OH þ ) radical (El-Missiry et al., 2001). MDA, a crucial product and one of the most sensitive indicators of lipid peroxidation, also indirectly reflects the production of intracellular ROS (Gutteridge, 1995; Qin et al., 2009). The content of MDA increased remarkably in rats after I/R injury, and AS-IVþHSYA and HH treatments significantly reduced the level of MDA. These data suggested that AS-IVþHSYA could play an important role in the antioxidant property of HH, possibly increasing the endogenous defensive capacity of the brain to combat oxidative stress induced by cerebral I/R (Yang et al., 2012). The synergistic protective effect of AS-IV and HSYA is related to ameliorate cerebral infarction followed by increasing the activity of SOD, catalase and GSH-Px, decreasing MDA and ROS, upregulating the expression of Nrf2. While, there is no synergistic protective effect on activating blood to dissipate blood stasis. Thus AS-IV and HSYA are mainly responsible for curative effects of HH and play important roles in neuroprotection, the effect of HH in promoting blood circulation may be associated with other components from Huangqi or Honghua besides HSYA.
Acknowledgment This work was supported by National Natural Science Foundation of China (Nos. 81173514 and 81373947), Xijing Research Booting Program (No. XJZT10D01) and Excellent Civil Service Training Fund of Forth Military Medical University (No. 4138C4IDK6). We thank our colleagues for their constructive advices on our experiments.
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