Neuroprotective effects of Kukoamine A against cerebral ischemia via antioxidant and inactivation of apoptosis pathway

Accepted Manuscript Neuroprotective effects of Kukoamine A against cerebral ischemia via antioxidant and inactivation of apoptosis pathway Jia Liu, Xiaowen Jiang, Qiao Zhang, Sen Lin, Jun Zhu, Yajun Zhang, Jiabao Du, Xiaolong Hu, Weihong Meng, Qingchun Zhao PII:

S0197-0186(16)30187-5

DOI:

10.1016/j.neuint.2016.12.024

Reference:

NCI 3982

To appear in:

Neurochemistry International

Received Date: 28 June 2016 Revised Date:

15 December 2016

Accepted Date: 21 December 2016

Please cite this article as: Liu, J., Jiang, X., Zhang, Q., Lin, S., Zhu, J., Zhang, Y., Du, J., Hu, X., Meng, W., Zhao, Q., Neuroprotective effects of Kukoamine A against cerebral ischemia via antioxidant and inactivation of apoptosis pathway, Neurochemistry International (2017), doi: 10.1016/ j.neuint.2016.12.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Neuroprotective effects of Kukoamine A against cerebral ischemia via antioxidant and inactivation of apoptosis pathway Jia Liu1, 2, Xiaowen Jiang3, Qiao Zhang2, Sen Lin4, Jun Zhu2, Yajun Zhang2, Jiabao Du2, Xiaolong Hu3, Weihong Meng1 and Qingchun Zhao1

1

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Affiliation Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840,

China

Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China

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Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016,

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2

China

Department of Radiology, General Hospital of Shenyang Military Area Command, Shenyang 110840,

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4

China

Correspondence

Prof. Dr. Qingchun Zhao, General Hospital of Shenyang Military Area Command, Department of

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Pharmaceutical, 83 Wenhua Road, Shenyang, People’s republic of China 110840.

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Tel/Fax: +86-024-28856205 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

Kukoamine A (KuA) is a bioactive compound, which is known for a hypotensive effect. Recent studies have shown that KuA has anti-oxidative effect and anti-apoptosis stress in vitro. However, its

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neuroprotective effect in rats with cerebral ischemia is still unclear. In the study, we investigated whether KuA could attenuate cerebral ischemia induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Results revealed that KuA could significantly reduce infarct volume both pre-treatment and post-treatment, and increase corresponding Garcia neurological scores. Acute KuA

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postconditioning not only significantly reduced cerebral infarct volume, brain water content and improved neurological deficit scores, but also decreased the number of TUNEL-positive cells.

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Moreover, it markedly increased the activities of Cu/Zn-SOD and Mn-SOD, reduced levels of MDA and H2O2. Increased expressions of caspase-3, cytochrome c and the ratio of Bax/Bcl-2 were significantly alleviated with KuA treatment. These findings demonstrated that KuA was able to protect the brain against injury induced by pMCAO via mitochondria mediated apoptosis signaling pathway.

Kukoamine A cerebral ischemia

antioxidant

mitochondrial apoptosis

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Key words

Abbreviations: ADC, Apparent diffusion coefficient; CCA, right common carotid artery; DAPI,

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4',6-diamidino-2-phenylindole; DWI, Diffusion weighted imaging; ECA, external carotid artery; ICA, internal carotid artery; KuA, Kukoamine A; NMDA, N-methyl-D-aspartate; PFA, paraformaldehyde;

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pMCAO, permanent middle cerebral artery occlusion; rADC, value of relative ADC; ROI, region of interest; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling

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ACCEPTED MANUSCRIPT 1. Introduction Ischemic stroke is one of the major causes of morbidity and long-term disability worldwide in the past several decades (Green and Shuaib, 2006; Donnan et al., 2008). There were increasing evidences that oxidative stress could lead to ischemic cell death and the potential molecular mechanism involves

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the massive formation of reactive oxygen species (ROS) via mitochondrial apoptosis pathway (Nakka et al., 2008). Besides, there were complicated connections among the multiple mechanisms of cerebral ischemia. Therefore, agents directed at the multiple mechanisms would offer effective protection against ischemic stroke.

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Kukoamine A (KuA) is a major bioactive component extracted from the root barks of Lycium chinense (L. chinense) Miller, which has been proved to possess series of pharmacological effects, such

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as anti-hypertension, anti-analgesic, anti-inflammatory, antisepsis and enhancing autoimmune (Funayama et al., 1980; Yamahara et al., 1980; Zheng et al., 2011). Our previous study has confirmed that KuA could significantly attenuate H2O2-induced SH-SY5Y cell apoptosis via anti-oxidative stress and inactivation of the apoptosis pathway (Hu et al., 2015). Besides, Li et al. has also indicated KuA could effectively prevent H2O2-induced toxicity in primary cerebellar granule neurons through

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anti-apoptotic progress to antagonize the oxidative stress (Li et al., 2015). However, the protection of KuA in acute ischemic cerebral infarction was unreported. Based on the neuroprotection of KuA in vitro, the purpose of this study was to determine whether

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KuA has neuroprotective effects against permanent middle cerebral artery occlusion (pMCAO) injury in rats by determining infarct volume, neurological function, brain water content and cellular apoptosis.

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In addition, we also examined the neuroprotective effects of KuA related to anti-oxidative stress and mitochondrial apoptosis pathway by determining the activities of Cu/Zn-SOD, Mn-SOD, the levels of MDA and H2O2 as well as the expressions of Bax, Bcl-2, caspase-3 and cytochrome c.

2. Materials and Methods 2.1 Animals

Male Sprague-Dawley rats, weighing 250-270 g (SPF grade, Certificate No: SCXK 20100001), were purchased from Liaoning Chang Sheng Biotechnology Co., Ltd. Animals were maintained on a 12 h dark-light cycle in a 25

temperature-controlled room with free access to water and food. All

experiments and procedures were conducted in accordance with the Regulations of Experimental 3

ACCEPTED MANUSCRIPT Animal Administration issued by the State Committee of Science and Technology of China. This study was approved by the Animal Experiment Committee of General Hospital of Shenyang Military Area Command (approval number 2014100102; approval date July 8, 2014). 2.2 Surgical procedures

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The operation was carried out as described (Longa et al., 1989) with some modifications. In brief, rats were fasted overnight with free access to water. Rats were anaesthetized with chloral hydrate (300 mg/kg, intraperitoneal injection). The right common carotid artery (CCA) was exposed through a midline incision in the neck region. The neck muscles were separated to expose the external carotid

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artery (ECA) and internal carotid artery (ICA). After ligating the ECA, a thread with tip rounded (Beijing Cinontech Co. Ltd) was inserted into the stump of the CCA and advanced into the ICA

temperature was maintained at 37

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approximately 19-20 mm from the bifurcation to occlude the origin of middle cerebral artery. Body with a heat pad. Rats in sham-operated underwent the same surgery

except for thread insertion. Animals were allowed to cages after surgery. 2.3 Drug Preparation and Administration

KuA (purity ≧98%, Liaoning University, Shenyang, China) was dissolved in normal saline. All

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related reagents were of analytical or pharmaceutical grade. KuA was dissolved in normal saline and administered by the intravenous (i.v.) route. All rats were randomly divided into 5 groups (20 rats in each group): a sham-operated group (normal saline), a vehicle group (rats suffered from pMCAO and

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given normal saline), KuA groups (rats suffered from pMCAO and given KuA at concentrations of 5, 10, 20 mg/kg). To determine the therapeutic window of KuA administration, rats were treated with

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KuA (10 mg/kg) in pre-occlusion and 0 h, 0.5 h, 2 h, 4 h post-occlusion. A single KuA (5, 10, or 20 mg/kg) was administered i.v. at 0 h after pMCAO for the rest of experiments. These tests were conducted at 24 h after occlusion, including infarct volume, TUNEL-positive cells, the expressions of proteins, as well as the activities of Mn-SOD, Cu/Zn-SOD, the levels of MDA, H2O2, except for the assessment of cerebral edema which was determined at 8 h after occlusion. 2.4 Neurological deficit The neurological deficit score of each animal was measured. Neurological functions were evaluated using an 18-point sensory motor assessment modified Garcia score (Garcia et al., 1995), which includes six tests: spontaneous activity (in cage for 5 min, 0-3 scores); symmetry of movements (four limbs, 0-3 scores); symmetry of forelimbs (outstretching while held by tail, 0-3 scores); climbing 4

ACCEPTED MANUSCRIPT wall of wire cage (1-3 scores); reaction to touch on either side of trunk (1-3 scores) and response to vibrissae touch (1-3 scores). 2.5 Infarct size assessment After neurological examination, rats were deeply anaesthetized with chloral hydrate for

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determination infarct volume using the 2,3,5-triphenyltetrazolium chloride (TTC). Firstly, rats were perfused with normal saline and their heads were isolated by decapitation. Then the cerebellum and rest of the brain were removed, only brains were immediately transferred to a -20

freezer. Frozen brains

were sliced into uniform coronal sections and immersed in 1% TTC (Solarbio) at 37

for 30 min and

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finally fixed in 4% paraformaldehyde (PFA) for the photograph. To compensate for the effect of cerebral edema, the corrected infarct volume was calculated as previously described (Lin et al., 1993):

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corrected infarct area = left hemisphere area - (right hemisphere - area infarct area). 2.6 Brain water content

Diffusion weighted imaging (DWI) was used to examine cerebral edema, as it is sensitive to early water distribution change in brain tissue. Besides, the method of DWI is non-invasive test which can minimize the number of animals used in the experiment. The result of a reduction in the apparent

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diffusion coefficient (ADC) of water during acute ischemia presents the hyperintensity and abnormal blue signal on DWI. All rats were examined at 8 h after pMCAO using Signa HD 3.0 T magnetic resonance imaging scanners (General Electric Company) with a wrist radio frequency coil (Xu et al.,

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2014). Rats were anaesthetized with chloral hydrate and the heads were put in the middle of the coil in the prone position. The main scanning parameters were as follows: TR = 2775 ms, TE = 100 ms, Slice

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thickness = 2.9 mm, Matrix = 96 × 96, FOV = 8 cm × 0.80 cm, b = 800 s/mm2. The ADC values of the region of interest (ROI) on each of the lesion-containing slices and contralateral slices were calculated using the corresponding outline workstation. The rADC was evaluated as a ratio of the ADC in ischemic region to the contralateral hemisphere. 2.7 Cell apoptosis assay

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) analysis was used to examine apoptosis cells via measuring fracture of DNA fragmentation. Rats were deeply anesthetized and perfused with normal saline followed by 4% PFA. Brains were removed and fixed in 4% PFA solution about 30 days. After being dehydrated in alcohol, the brains were embedded in paraffin and cut into 3µm sections. Sections were prepared for TUNEL and DAPI assay. DAPI was 5

ACCEPTED MANUSCRIPT used to mark the nuclei of all cells. TUNEL assay was performed by using the cell death detection kit (Beyotime, C1088) according to the manufacturer’s instructions. After TUNEL assay, sections were dyed with DAPI staining fluid for 5 min and subsequently washed with PBS. Then, the TUNEL-positive cells in the peri-infarct region were viewed under the fluorescence microscopy (IX71,

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OLYMPUS) with magnification (×100) by the observer who was blinded to the group assignments. Data obtained in every field were added together to make the final count for each slice. The results were expressed as a ratio of TUNEL-positive cells to the DAPI-positive cells. 2.8 Protein carbonyl assay

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The brains of rats were quickly removed by decapitation under anesthesia at 24 h after pMCAO. The right brain parts were quickly homogenized (1:3 v/w) in cold lysis buffer. Lysis buffer commercial

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kits used in our study was purchased from Beyotime (P0013B). The protease inhibitors in the lysis buffer contain 4mM sodium pyrophosphate, 3mM β-glycerophosphate, 1mM EDTA, 5mM Na3VO4, 0.02mM leupeptin, 1mM AEBSF, 0.01mM E-64. The homogenate was centrifuged at 15,000×g for 10 min at 4 , the supernatant was the protein used for the analysis of oxidative stress markers and the expressions of caspase-3. The mitochondria were used to detect the expressions of bax and bcl-2 by the

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commercial mitochondrial protein assay kit (Beyotime, C3606). The protein in cytoplasm was utilized to examine the expression of cytochrome c. Protein carbonyl assay was conducted by a BCA protein assay kit (Beyotime).

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2.9 Oxidative stress markers

To investigate the effect of KuA on pMCAO-induced oxidative stress, we evaluated the activities

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of Mn-SOD, Cu/Zn-SOD as well as the levels of malondialdehyde (MDA), hydrogen peroxide (H2O2) in the brain. These markers were detected using commercial assay kits which were purchased from Beyotime Biotechnology, Jiansu, China. Our procedures entirely were carried out in accordance with the instructions of kits.

The method of SOD was based on reaction of WST-8 formazan dye and SOD. Briefly, the protein

was reacted with Cu/Zn-SOD inhibitors. Then the WST-8/enzyme working fluid was mixed with these proteins. Finally, mixtures were reacted with specific start working liquid at 37

for 30 min. The

absorbance was determined at 450 nm. Refer to the following formula to calculate the activity of the Mn-SOD. Mn-SOD enzyme activity units = inhibition percentage / (1- inhibition percentage) units. The total SOD enzyme activity could also be determined by the total SOD kit and the operation procedure 6

ACCEPTED MANUSCRIPT was according to the Mn-SOD except for addition of Cu/Zn-SOD inhibitors. The Cu/Zn-SOD enzyme activity was subtracting Mn-SOD activity units from the total SOD. The kit of MDA was based on the absorbance of the MDA-TBA. The products of other oxidation analogues and TBA have little absorbance than MDA-TBA at 535nm. The MDA-TBA has max

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absorbance at 535nm. The absorbance at 535nm was only determined. The measurement of MDA was similar to the experiment on SOD. However, the working fluid and reaction start working liquid were specific, which were only applied to the analysis of MDA.

The H2O2 kit was based on that H2O2 could oxidize Fe2+ to Fe3+, and then Fe3+ reacted with

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xylenol orange. The colorimetric reaction could be further detected by a spectrometer. The standard of H2O2 should be recalibrated before the reaction. Firstly, sample was added in 96-well plates. Then the

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particular detection reagent was added into the 96-well plates. Finally, the mixture was reacted at 37 for 30 min. The absorbance was determined at 560 nm. The standard curve was used to calculate the concentration of H2O2 in the sample. 2.10 Western-blot analysis

Western-blot analysis was performed to determine protein related to mitochondrial apoptosis.

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Briefly, proteins were separated by electrophoretic separation on SDS polyacrylamide gel. After transferring to PVDF membranes, phosphorylated proteins were incubated with 5% BSA in TBST, whereas non-phosphorylated proteins incubated with 5% nonfat milk. All proteins were reacted

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overnight with primary antibodies Bax, Bcl-2, caspase-3, cytochrome c (1:500, Beijing Zhongshan Golden Bridge Biotechnology Co Ltd.). Proteins were detected by the enhanced chemiluminescence

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method and β-actin was used as an internal control. 2.11 Statistical analysis

Data was analyzed by GraphPad Prism 5 software and the results were represented as the mean ±

SD. Statistical significance was analyzed with one-way analysis of variance followed by a Tukey post-test. Difference with a p value less than 0.05 was considered statistically significant.

3. Results 3.1 KuA protects rats against ischemic brain injury To determine the dose-dependent protection effect of KuA, the prominent cerebral infarct size and lowest neurological deficit scores in the vehicle group were observed (p < 0.01 vs. sham group). The 7

ACCEPTED MANUSCRIPT reductions in infarct size were observed at KuA-treated groups (p < 0.01 vs. vehicle group, Fig. 1A-B). Furthermore, neurological scores developed by Garcia et al (Garcia et al., 1995) were significantly higher in the groups with KuA treatment (p < 0.01 vs. vehicle group, Fig. 1C). There was no significant difference between 10 mg/kg and 20 mg/kg group in infarct size as well as neurological scores.

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Therefore, the lower dosage (10 mg/kg) was chosen to explore the therapeutic window. Rats were treated with KuA (10 mg/kg) at pre-ischemia and 0 h, 0.5 h, 2 h, 4 h post-ischemia in the test of therapeutic window. Obvious reductions in stroke volume were observed in both pre-treatment and post-treatment groups (Fig. 2A). Consistently, KuA-treated groups also increased the

3.2 KuA could reduce brain water content after pMCAO

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neurological scores except for at the time of 2 h (p >0.05 vs. vehicle group, Fig. 2B).

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As shown in Fig. 3A, the sham group did not emerge large hyperintense area. However, in the vehicle group, large hyperintense area and lower rADC value were observed, which suggested obvious brain edema (p < 0.01 vs. sham group). However, the rADC values were markedly increased (p < 0.05, p < 0.05, p < 0.01 vs. vehicle group, Fig. 3B) and large hyperintense area decreased in KuA treated group, indicating that brain water content was relieved significantly.

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3.3 KuA could decrease the cell apoptosis

TUNEL analysis was used to detect apoptosis cells. In the sham group, it was hard to see TUNEL-positive cells in Fig. 4. But there were significant TUNEL-positive cells in the vehicle group

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(p < 0.01 vs. sham group). Besides, the increase can be attenuated by KuA treatment (p < 0.01, p < 0.01, p < 0.01 vs. vehicle group).

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3.4 KuA prevented the oxidative stress To illustrate the effect of KuA on oxidative stress induced by pMCAO, the activities of Mn-SOD,

Cu/Zn-SOD and the levels of MDA, H2O2 were determined in brain. As shown in Fig. 5A-B, the activities of Mn-SOD and Cu/Zn-SOD were significantly decreased in vehicle groups compared with the sham group. Pre-treatment with KuA obviously restored their activities in dose-dependent manners. In the vehicle group, rats had remarkable augmentations of the levels of MDA and H2O2. However, the pre-treatment with KuA could efficiently decrease the levels of MDA and H2O2. And there was no significant effect on the H2O2 content at the dose of 5 mg/kg. 3.5 KuA could inhibit the expressions of related proteins To identify the underlying mechanism of the protective role of KuA, we measured associated 8

ACCEPTED MANUSCRIPT proteins. As shown in Fig. 6, there were significant increases in the ratio of Bax/Bcl-2, caspase-3 and cytochrome c in the vehicle group compared with the sham-operated group. But KuA significantly reversed the increases in the ratio of Bax/Bcl-2, the expressions of caspase-3 and cytochrome c.

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4. Discussion Infarct region is a typical characteristic in ischemic stroke patients. The present study identified that KuA could reduce the infarct volume and improve neurological dysfunction both in pre-occlusion and post-occlusion, indicating that KuA might be benifit to the people who were higher risk of cerebral

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infarction regardless of before or within the beginning of a few hours after the onset of the stroke.

The permeability of the blood brain barrier was destroyed for the impaired capillaries when the

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cerebral ischemia appeared, which could no longer hold intravascular elements, such as Na+, water and serum proteins. These substances in the intracranial space entered into the extracellular space of the brain and caused brain swelling subsequently (Simard et al., 2007). The intracranial pressure significantly increased, which could lead to serious complications or cerebral hernia. Our results showed that KuA could remarkably ameliorate the brain swelling to protect brain from the damage of

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cerebral ischemia.

Mitochondrial apoptosis and oxidative stress play important roles in the development of the pathological process of ischemic brain injury (Broughton et al., 2009). Thus, novel agents targeting

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apoptosis inhibition and/or antioxidation might be potential in neuroprotective drugs in cerebral ischemic stroke. Many substances have been confirmed that they can reduce the injury induced by

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pMCAO via antioxidation and/or inhibiting the mitochondrial mediated apoptosis signaling pathway, such as Liraglutide (Briyal et al., 2014), Isoflurane (Li and Zuo, 2009), Ginsenoside Rd (Ye et al., 2011), Formononetin (Liang et al., 2014). In the ischemic stroke, overproduction of free radicals, contain one or more unpaired valence

electrons, which couldn’t contribute to intramolecular bonding under normal conditions. Free radicals are highly active with other molecules and lead to oxidation of those molecules (Droge., 2002). Superoxide anions are formed when oxygen acquires an additional electron in mitochondria. SOD could detoxify superoxide anions to H2O2, which is subsequential converted to H2O by catalase or glutathione peroxidase (Chan., 2001). Result demonstrated that KuA could decrease the amount of H2O2. The specific mechenisms of the falling H2O2 level need to be clarified by more researches. SOD 9

ACCEPTED MANUSCRIPT as the endogenous antioxidants could control the levels of ROS (Sugawara et al., 2002). The SOD is believed to be the antioxidase comprised of Cu/Zn-SOD and Mn-SOD. The Mn-SOD in mitochondria is usually inducible by stimuli. The present study demonstrated that KuA could elevate the activities of Mn-SOD and Cu/Zn-SOD. Besides, the activity of Mn-SOD in mitochondria was more reactive than

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Cu/Zn-SOD in cytoplasm, which indicated that the ability of ameliorating oxidative damage was closely associated with mitochondria. As the lipid oxidation end-product, MDA could make macromolecules cross-linked, which leads to cytotoxicity. Result confirmed that the level of MDA could be decreased by KuA. Collectively, results obtained in the present research confirmed the

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neuroprotective efficacy of KuA via ameliorating oxidative damage in vivo and confirm our previous findings in vitro.

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Mitochondria are considered to affect neuron apoptosis primarily via releasing of factors associated with apoptosis into the cytoplasm, including the proapoptotic protein Bax and antiapoptosis protein Bcl-2 (Mehta al., 2007). After the opening of the mitochondrial transition pores, cytochrome c which is released from the intermembrane space into the cytosol activates the caspase-dependent mitochondrial pathway (Love et al., 2003; Susan et al., 2007). The release of cytochrome c could

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activate the procaspase-9. The clustering of procaspase-9 leads to caspase-9 activation which is presumably an initiator of the cytochrome c-dependent caspase cascade. Then the caspase-3 was activated. It could cleave nDNA repair enzymes, which lead to nuclear DNA damage without repair

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and finally cause apoptosis (Sims et al., 2002; Sugawara et al., 2004; Broughton et al., 2009). The results of western blot showed that the ratio of Bax/Bcl-2, expressions of caspase-3 and cytochrome c

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were reversed by KuA, which indicated that the neuroprotective effect of KuA may be involved in mitochondrial apoptosis.

5. Conclusions

In conclusion, it was the first time to find that KuA treatment could reduce cerebral infarct volume,

brain edema, apoptosis cells, and increase the neurological deficit scores. Besides, KuA could reduce the infarct volume both in pre-occlusion and post-occlusion. The potential mechanisms may be involved in reducing brain swelling, modulating oxidative status and inactivation mitochondrial apoptosis pathway. Considering above results, KuA may be a promising neuroprotectant for preventing or treating ischemic stroke. 10

ACCEPTED MANUSCRIPT Conflicts of Interest The authors have not any conflict of interest to declare. Acknowledgments This work was supported by the National Science and Technology Major Project, China. (Project

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number: 2014ZX09J14101-05C).

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ACCEPTED MANUSCRIPT Legends for Figures Fig. 1 Dose-response study of the neuroprotection afforded by KuA (5, 10, 20 mg/kg) at post-occlusion in rats at 24 h after pMCAO. (A) TTC was used to visualize the infarct area. Non-ischemic region is red and the infarct region is white. (B) Column chart showed corresponding the percentages of corrected infarct area in right brain to the corresponding left brain. (C) Column chart showed corresponding the neurological scores of rats developed by Garcia et al. Values were shown as means ±

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SD (n = 6). ##p < 0.01 versus the sham group. *p < 0.05 and **p < 0.01 versus the vehicle group.

Fig. 2 Therapeutic window study of the neuroprotection of KuA (10 mg/kg) in adult rats subjected to pMCAO. (A) Rats were treated with KuA at pre-occlusion and respectively 0 h, 0.5 h, 2 h, 4 h post occlusion. (B) Column chart showed the corresponding neurological score of rats developed by Garcia 6). *p < 0.05 and **p < 0.01 versus the vehicle group.

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et al. “Pre” represents that KuA were given before occlusion. Values were shown as means ± SD (n =

Fig. 3 DWI was used to detemine the effects of KuA (5, 10, 20 mg/kg) at post-occlusion on brain

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edema at 8 h after pMCAO. (A) DWI revealed signal in right hemisphere in rats. (B) Column chart showed the values of corresponding rADC. Values were shown as means ± SD (n = 6).

##

p < 0.01

versus the sham group. *p < 0.05 and **p < 0.01 versus the vehicle group.

Fig. 4 TUNEL assay was used to detemine the effects of KuA (5, 10, 20 mg/kg) at post-occlusion on apoptosic cells at 24 h after pMCAO. (A) TUNEL detected apoptosic cells (green) in brain. While DAPI determined all cells (blue). (B) Column chart showed the percentages of TUNEL-positive cells

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to the total cell numbers. Values were shown as means ± SD (n = 6). ##p < 0.01 versus the sham group. *p < 0.05 and **p < 0.01 versus the vehicle group.

Fig. 5 The effects of KuA (5, 10, 20 mg/kg) at post-occlusion on oxidative stress markers at 24 h after pMCAO. (A) The effect of KuA on the activity of Mn-SOD. (B) The effect of KuA on the activity of

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Cu/Zn-SOD. (C) The effect of KuA on the level of MDA. (D) The effect of KuA on the level of H2O2. The Data was shown as means ± SD (n = 6). ##p < 0.01 versus the sham group. *p < 0.05 and **p <

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0.01 versus the vehicle group.

Fig. 6 Representative western blot showed effect of KuA (5, 10, 20 mg/kg) at post-occlusion on protein expressions at 24 h after pMCAO. (A) The effects of KuA on the expressions of Bax and Bcl-2. (B) The effects of KuA on the expressions of caspase-3 as well as cytochrome c (cytosol). (a-b) Column charts showed the densitometric analysis of the ratio of the densities of corresponding protein to β-actin band. The Values were shown as means ± SD (n = 6). ##p < 0.01 versus the sham group. *p < 0.05 and **p < 0.01 versus the vehicle group.

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KuA could reduce the infarct volume both in pre-occlusion and post-occlusion. KuA could attenuate brain swelling and the number of apoptotic cells in the ischemic

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brain. The potential mechanisms may be involved in modulating oxidative status and

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inactivation mitochondrial apoptosis pathway.