Oxymatrine attenuated hypoxic-ischemic brain damage in neonatal rats via improving antioxidant enzyme activities and inhibiting cell death

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Oxymatrine attenuated hypoxic-ischemic brain damage in neonatal rats via improving antioxidant enzyme activities and inhibiting cell death Peng Zhao a, Ru Zhou a, Hai-Ning Li b, Wan-Xia Yao a, Hai-Qi Qiao a, Shu-Jing Wang c, Yang Niu d, Tao Sun e, Yu-Xiang Li f, Jian-Qiang Yu a,g,* a

Department of Pharmacology, Ningxia Medical University, Yinchuan 750004, China Department of Neurology, General Hospital of Ningxia Medical University, Yinchuan 750004, China c Medical Sci-tech Research Center, Ningxia Medical University, Yinchuan 750004, China d Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan 750004, China e Ningxia Key Laboratory of Craniocerebral Diseases of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan 750004, China f College of Nursing, Ningxia Medical University, Yinchuan 750004, China g Ningxia Hui Medicine Modern Engineering Research Center and Collaborative Innovation Center, Ningxia Medical University, Yinchuan 750004, China b

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Article history: Received 30 March 2015 Received in revised form 11 June 2015 Accepted 12 June 2015 Available online Keywords: Oxymatrine Neuroprotection Hypoxic-ischemia Neonatal Cell death Antioxidant enzyme

A B S T R A C T

Oxymatrine (OMT), an active constituent of Chinese herb Sophora flavescens Ait, has been proved to possess anti-tumor, anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Previous study has demonstrated that OMT had protective roles on multiple in vitro and in vivo brain injury models including regulation of apoptosis-related proteins caspase-3, Bax and Bcl-2. In this study, we investigated whether this protective effect could apply to neonatal hypoxic-ischemic brain damage. Seven-day-old Sprague–Dawley rats were treated with the left carotid artery ligation followed by exposure to 8% oxygen (balanced with nitrogen) for 2.5 h at 37 °C. In sham group rats, neither ligation nor hypoxia was performed. After two successive days intraperitoneal injection with OMT (30, 60 and 120 mg/kg), Nimodipine (1 mg/kg), and saline, brain infarct volume was estimated, histomorphology changes were performed by hematoxylin– eosin (HE) staining as well as electron microscopy. In addition, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (T-AOC), as well as production of malondialdehyde (MDA) were assayed in ipsilateral hemisphere homogenates to evaluate the redox status after hypoxic-ischemic. Expression of apoptosis-related proteins Caspase-3, Bax and Bcl-2 in brain were analyzed by western-blot analysis and immunofluorescence. Administration of OMT significantly decreased brain infarct volume and the percentage of injured cells, and ameliorated histopathology and morphological injury as well. Furthermore, OMT obviously increased the activities of SOD, GSH-Px, CAT and T-AOC, and decreased MDA content. Western-blot analysis showed a marked decrease in Caspase-3 expression and increase in the ratio of Bcl-2/Bax after OMT (120 mg/kg) posttreatment as compared with hypoxic-ischemic group. These results suggest that OMT exerts a neuroprotective effect against hypoxic-ischemic brain damage in neonatal rats, which is likely to be mediated through increasing anti-oxidant enzyme activities and inhibiting cell death. © 2015 Published by Elsevier Ltd.

1. Introduction Neonatal hypoxic-ischemic brain damage (HIBD), resulting from perinatal asphyxia, is one of the most common causes of severe

Peng Zhao and Ru Zhou have contributed equally to this study. * Corresponding author. Department of Pharmacology, Ningxia Medical University and Ningxia Hui Medicine Modern Engineering Research Center and Collaborative Innovation Center, Yinchuan, Ningxia 750004, China. Tel.: +86 951 4081046; fax: +86 951 6880693. E-mail address: [email protected] (J. Yu).

neurological handicap in newborns (Charriaut-Marlangue et al., 2014; Koonrungsesomboon et al., 2014), which is associated with poor prognosis, a high case fatality rate and complex pathogenesis (Hagberg et al., 2009; Xiao et al., 2015). The prevalence of neonatal HIBD occurs in 1–2‰ of term or near-term infants, among them about 20% die and up to 40% of the survivors suffer devastating disabilities such as cerebral palsy, mental retardation and epilepsy (Pan et al., 2012), which seriously affects the quality of life of the newborn. Unfortunately, no definitive therapeutic option is available for HIBD nowadays except that several studies indicated the potential benefits of hypothermia in some mild or moderate case within 6 hours after birth (Gonzalez-Rodriguez et al., 2014). Thus, there is an urgent,

http://dx.doi.org/10.1016/j.neuint.2015.06.008 0197-0186/© 2015 Published by Elsevier Ltd.

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unmet need to develop effective intervention strategies that prevent and reverse HIBD, promote the repair after injury, and improve the prognosis, which are major subjects in the field of perinatal medical and nerve medicine, seeking effective neuroprotective agents has been a research focus. The exact pathogenesis responsible for neonatal HIBD is still inconclusive, although there is increasing evidence suggesting that oxidative stress and apoptosis are significant contributing factors to the pathogenic process (Taylor et al., 1999). Excessive reactive oxygen species (ROS) and subsequent oxidative stress play harmful roles during the process of delayed neuronal death after hypoxicischemic, which directly provoke damage to nucleic acids, lipid, and protein, leading to membrane damage, cell death and brain dysfunction (Chan, 2001; Kontos, 2001). Endogenous antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) can scavenge overproduction of oxidants, prevent the deleterious ROS generation (Chen et al., 2011). Apoptosis is actively executed by several members of the caspase family including caspase-3, which is involved in the final execution phase of apoptosis (Han et al., 2014; Liu et al., 2013). One mechanism pertaining to the death of immature neurons is the accumulation of Bax, which is one of the pro-apoptotic Bcl-2 family members. The balance between anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax plays a critical role in regulating cell death (Tsujimoto, 2002; Yang et al., 2015). Therefore, anti-apoptotic therapies via inhibiting caspase-3 expression and regulating by the balance of Bcl2/Bax have been proposed to be useful in attenuating neonatal HIBD. Over the past decades, Chinese medicines have received more attention and are being increasingly investigated due to the advantage in terms of abundant resources, multi-targeted efficacy, few side effects, as well as lower cost (Xu et al., 2014). Oxymatrine (OMT), a quinolizidine alkaloid extracted from traditional Chinese herb Sophora flavescens Ait, has a tetracyclic quinolizine structure (Dong et al., 2013), it has been proved to possess extensive pharmacological activities, including anti-inflammatory (Wang and Jia, 2014), antiviral, hepatoprotective (Wen et al., 2014), anti-tumor (Liu et al., 2014; Ying et al., 2015), immune-modulating, anti-oxidant (Guo et al., 2014; Wang et al., 2015) and anti-apoptotic (Hong-Li et al., 2008; Jiang et al., 2005; Wang and Jia, 2014; Xiao et al., 2014), etc. In recent years, OMT has been demonstrated to possess neuroprotective activity in various kinds of in vitro and in vivo brain injury models. The molecular pathways by which OMT mediates its protective effects remain unclear but seem to involve both inhibition of phosphor-p38MAPK and decreased Toll-like receptor 4/NF-KB subunit p65 protein expression (Cui et al., 2011; Dong et al., 2011, 2013). Other than the molecular pathways mentioned earlier, evidence implicates pathways inhibiting cell death via regulation of Bcl-2 and Bax expression (Zhang et al., 2013; Zhou et al., 2014). However, no information is available on possible effects of OMT in neonatal brain injury induced by hypoxia-ischemia. Therefore, it was speculated that OMT might exert a protective effect on neonatal HIBD. To test this hypothesis, the present experiment was designed to investigate the potential neuroprotective effects of postinsult administration of OMT on neonatal HIBD using a neonatal rat model of hypoxic-ischemic as well as to further identify its underlying mechanisms. 2. Materials and methods 2.1. Experiment animals Female Sprague–Dawley rats with 7-day-old neonates were obtained from the Experimental Animal Center of Ningxia Medical University (Certificate number was SYXK Ningxia 20050001). The animal house temperature was controlled at 22–24 °C under a 12 h light and dark cycles and animals had access to food and water ad

libitum. The experimental protocol was duly approved by the Institutional Animal Care Ethics Committee of Ningxia Medical University, Yinchuan City, Ningxia. This study complied with the internationally accredited guidelines and ethical regulations on animal research. During the whole study, all researchers adhered to the updated STAIR recommendations (Fisher et al., 2009). All surgery was performed under diethyl ether anesthesia, and all efforts were made to minimize suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. 2.2. Drug administration OMT, white or almost white crystalline powder with purity ≥98.0%, was purchased from Ningxia Institute of Material Medical (Yinchuan, China) and dissolved in normal saline before use. Nimodipine injection (Nim) (0.2 mg/ml) was obtained from the German Bayer Company (Leverkusen, Germany). Both compounds were injected intraperitoneally (i.p.) in an application volume of 0.1 ml/10 g body weight and administered 15 min prior to testing. Pups of mixed sex from different litters were randomly divided into the following groups (n = 42, for each group): (1) (2) (3) (4) (5) (6)

Sham (sham surgery) with NS group; HI (cerebral hypoxic-ischemic) with NS group; HI (cerebral hypoxic-ischemic) with OMT (30 mg/kg) group; HI (cerebral hypoxic-ischemic) with OMT (60 mg/kg) group; HI (cerebral hypoxic-ischemic) with OMT (120 mg/kg) group; HI (cerebral hypoxic-ischemic) with Nimodipine (1 mg/kg) group.

OMT and Nimodipine were given by intraperitoneal injection every 12 h for two consecutive days after HI. Sham and HI groups were treated with physiological saline under the same conditions. 2.3. Hypoxic-ischemic brain injury model Hypoxia-ischemia was induced as previously described with slight modifications (Vannucci and Vannucci, 2005) except for mice in sham group. Briefly, 7-day-old Sprague–Dawley rats of both sexes were anesthetized with ether inhalation, a small incision was made in the left side of the neck where the left common carotid artery was exposed, isolated, and cut between double ligatures with 6-0 silk surgical suture. The incision was sutured. Each surgery was completed within 5 min. The neonates were sent back to their cages with their mothers for 1.5 h. After recovery, pups were treated with 8% oxygen (balanced with nitrogen) at 4 l/min for 2 h. A constant temperature of 37 °C was maintained throughout all the procedures. After hypoxic exposure, all surviving pups were returned to their cages with their mothers until they were sacrificed. Animals in sham group were randomly chosen from the same litters of hypoxiaischemia rats and were given anesthesia but not subjected to hypoxia-ischemia. 2.4. Determination of infarct volume Pups (n = 6, for each group) were anesthetized and euthanized at 48 h after the HI treatment. Rat brains were removed and sectioned coronally into five 2 mm slices and stained in 2% solution of 2,3,5,-triphenyl tetrazolium chloride (TTC) (Sigma, USA) for 30 min at 37 °C followed by overnight fixing in 4% formaldehyde solution. The TTC stained sections were photographed with a digital camera and the infarct volumes were calculated with microscope imageanalysis software (Image-Pro plus, USA) by an investigator blinded to the treatment groups. To compensate for the effect of brain edema, the exact infarct volumes were calculated as the following equation: percentage of corrected infarct volume = (normal hemisphere

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volume − non-infarct volume of infarct side)/normal hemispheric volume × 100%. 2.5. Examination of histopathology Forty-eight hours after HI, neonatal rats (n = 6, for each group) were anesthetized with ether inhalation, and perfused with physiological saline via an aortic root catheter until the liver appeared white, followed by 4% paraformaldehyde solution that had been cooled to 4 °C. The brains were removed and post-fixed in 4% formaldehyde solution overnight at 4 °C. Afterwards, brains were processed through graded alcohols and xylene. After detaching the hindbrain, the forebrain was cut coronally with a brain matrix into four equally spaced slices each with a 2 mm interval and embedded in paraffin blocks. Brain sections obtained 1.5 mm behind the bregma in the coronal plane were cut into 5 μm sections using a vibratome (Leica, Solms, Germany) and mounted on glass slides. Section were deparaffinized in xylene and rehydrated in gradient ethanol from 100% to 70%. Finally, stained with hematoxylin and eosin (HE staining). Serial sections corresponding to the plate 35 of the rat brain atlas were selected for analysis. The degree of brain damage in the cerebral cortex and the hippocampus CA3 was scored on a 6-point scale as follows: 0 = normal, 1 = few neurons damaged (1–5%), 2 = several neurons damaged (6–25%), 3 = moderate neurons damaged (26–50%), 4 = greater than half of neurons damaged (51– 75%), 5 = majority of neurons damaged (>75%). Evaluation was performed in a blind manner by a neuropathologist. The images were captured using a computer assisted image analyzer system consisting of a microscope (Olympus BX-51, Tokyo, Japan) magnification at ×400 and photographed. Six randomly chosen microscopic fields in each section were analyzed. 2.6. Morphological evaluation by electron microscopy After 48 h of HI injury, 1 mm × 1 mm × 1 mm of hippocampus tissue in ischemic hemispheres (n = 6, for each group) was gathered, fixed for 2 h at 4 °C with 2.5% glutaraldehyde then purged with PBS and soaked in 2% osmium tetroxide. And then, dehydrated, embedded in epon. Ultrathin (60 nm) sections were cut with a diamond knife and put onto colloid coated copper grids, double-stained with 0.4% uranyl acetate and 2% lead acetate. Finally, morphological changes of neurons were observed and determined at each crosssectional level by a morphologist, with no prior knowledge of the experimental group, using a transmission electron microscope (H7650, Hitachi, Japan). 2.7. Determination of oxidative stress indicators 2.7.1. Assay of the activities of SOD, GSH-PX, CAT and T-AOC capability Brain injury 48 h after HI, neonatal rats (n = 6, for each group) were decapitated, and the ischemic hemispheres were collected to homogenized with 0.9% saline using glass homogenate for 15 min to prepare a 10% (w/v) homogenate. The homogenate was centrifuged at 2500 rpm and 4 °C for 10 min. Afterwards the supernatant was employed for this test. Tissue protein concentration, the activities of SOD, GSH-PX, CAT and T-AOC were estimated by a single investigator blinded to sample identity using commercially available corresponding reagent kits (Jiancheng Bioengineering Institute, Jiangsu, China) and a microplate reader (1510, Thermo Fisher, USA). One unit of enzyme activity was defined as the quantity of SOD required to inhibit the rate of reduction of cytochrome c by 50%. The absorbance change at 550 nm was monitored. SOD activity is expressed as units/mg protein. One unit of GSH-PX activity was defined as the GSH-PX in 1 mg protein that led to the decrease of 1 μmol/l glutathione (GSH) in the reactive system. GSH-PX activity was ex-

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pressed as units/mg protein. The CAT decomposition reaction of hydrogen peroxide can be terminated by adding ammonium molybdate, which generated a light yellow complex compound. The absorbance change at 405 nm to determine its generation can calculate the CAT activity. CAT activity was expressed as units/mg protein. T-AOC reflects the overall cellular endogenous antioxidative capability including both enzymatic and nonenzymatic antioxidants. All these antioxidants were estimated using the chromogenic reagent 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) with maximal absorbance at 414 nm. The T-AOC is expressed as units/mg protein. Protein analysis was performed by using BCA method according to manufacturer’s instruction. 2.7.2. Measurement of lipid peroxidation As an indicator of lipid peroxidation, the malondialdehyde (MDA) content in the brain was determined using the thiobarbituric acid method with maximal absorbance at 532 nm according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Jiangsu, China). The assay results were normalized to the protein concentration in each sample, and expressed as nmol/mg protein. 2.8. Cell death assay by flow cytometer After 48 h of HI injury, the hippocampus was taken out on ice, triturate and treated with trypsin for 15 min at 37 °C, after that, were rinsed three times with ice-cold PBS, and filtered through 400 mesh nylon net two times to remove large clumps of debris and make a single cell suspension. Later on, the cell suspension was stained with 5 μl of Annexin V-FITC staining solution at 4 °C for 15 min followed by 1 μl of a propidium (PI) staining solution at 4 °C for 5 min in the dark. After incubation for 15 min, 400 μl of binding buffer was added. Then, the samples were immediately analyzed by a researcher blind to the experimental groups using flow cytometer (BD, USA) and the data were analyzed by Cell Quest software. For each sample, data from 10,000 cells were recorded in list mode on logarithmic scales. There four quadrants represent dead cell, late injured cell, normal cell, early injured cell, respectively in flow cytometry results. The percentage of injured cells was calculated using 100 × (early + late) apoptotic cell number/total cell number. 2.9. Immunofluorescence analysis Each sample was subjected to observe the expression of Bax, Bcl2, and Caspase-3 by taking three consecutive coronal sections (5 μm) from the ischemic core region using a vibratome (Leica, Solms, Germany). Paraffin-embedded coronal brain sections were subjected to deparaffinization, rehydration, and underwent a microwave oven antigen retrieval (microwave method). The brain sections were incubated with the primary antibodies: Caspase-3, Bax and Bcl-2 (Caspase-3, 1:50; Bax, 1:50; Bcl-2, 1:50; Proteintech Group, USA) at 4 °C overnight. The next day, sections were washed with PBS and incubated for 1 h at 37 °C with FITC-labeled Goat Anti-Rabbit IgG (1:200; Proteintech Group, USA) followed by 4′,6-diamidino-2phenylindole (DAPI) for 5 min at room temperature. All samples were prepared in a single batch. Negative controls received an identical treatment except for the primary antibody and showed no positive signal. The fluorescence images of Bax, Bcl-2, and Caspase-3 in rat brains per section were randomly photographed (×400 and ×200) by a single investigator who was blind to sample identity using a confocal laser scanning microscope. 2.10. Western blot analysis The neonatal rats (n = 6, for each group) were decapitated and ischemic brains were rapidly collected. The prepared brain tissue was homogenized in 1:10 (w/v) ice-cold protein extraction buffer

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Fig. 1. Protective effect of OMT against hypoxic-ischemic brain injury in neonatal rats. (A) TTC staining of representative coronal sections at 48 h after hypoxic–ischemic. The scale is shown on the left side of each TTC stained brain with 1 mm being the shortest interval. (B) Quantitative analysis of infarct volumes at 48 h after hypoxicischemic. Data are expressed as mean ± SEM (n = 6). ##p < 0.01, versus sham group, *p < 0.05, **p < 0.01 versus HI group.

using a glass homogenizer. Soluble proteins were collected and centrifuged at 12,000 g for 10 min at 4 °C. The protein concentration of the samples was determined by a BCA Protein Assay reagent kit. Equal amounts protein lysates (50 μg) in each group were separated by 12% sodium dodecyl sulfate–polyacrylamide gel

electrophoresis (SDS–PAGE) and subsequently transferred onto a nitrocellulose membrane (260 mA, 2 h). The membrane was then blocked with PBST containing 5% skim milk for 2 h at room temperature and then incubated with the primary rabbit monoclonal antibodies respectively overnight at 4 °C (Bax, 1:500; Bcl-2, 1:500;

Fig. 2. Effect of OMT post-treatment on histological alterations of ischemic cerebral cortex and hippocampus at 48 h after hypoxic-ischemic (hematoxylin–eosin staining ×400). (A–D) Ischemic cerebral cortex. (A) Sham group. (B) Vehicle group. (C) OMT 120 mg/kg group. (D) Nimodipine 1 mg/kg group. (E–H) Ischemic cerebral hippocampus CA3. (E) Sham group. (F) Vehicle group. (G) OMT 120 mg/kg group. (H) Nimodipine 1 mg/kg group. (I) Quantification of brain damage is shown as injury score. Data are expressed as mean ± SEM (n = 6). ##p < 0.01 versus sham group; **p < 0.01 versus HI group.

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Fig. 3. Ultrastructural changes in hippocampus neuron induced by hypoxic–ischemic and inhibition by OMT (×2500). (A) A representative nucleolus of sham group. (B) A representative nucleolus at 48 h after hypoxic–ischemic in HI group. (C) A representative nucleolus of administration with 120 mg/kg OMT in high-dose group. (D) A representative nucleolus in 1 mg/kg Nimodipine group.

Caspase-3, 1:1000; Proteintech Group, USA). The membranes were then washed and incubated with secondary antibody (anti-rabbit IgG,1:3000; Proteintech Group, USA). An anti-actin antibody (1:1000; Proteintech Group, USA) served as control. Protein bands were visualized with enhanced chemiluminescence reagents (ECL), and the signals densitometry were quantified by a observer blinded to the groups of the animals using a western blotting detection system (Quantity One, Bio-Rad Laboratories, USA). 2.11. Statistical analysis All analyses were performed using SPSS 17.0 Statistical Software (Chicago, IL). The data were presented as mean ± SEM. Statistical differences between more than two groups were assessed by using a one-way ANOVA followed by a post hoc test. Data of two groups

were analyzed by unpaired t test. A value of p < 0.05 was considered statistically significant.

3. Results 3.1. OMT provided neuroprotection after hypoxia-ischemia 3.1.1. TTC staining The cerebral infarct areas determined by TTC staining are illustrated in Fig. 1A, normal brain tissues appeared uniform red while the infarction region showed white. There were no remarkable cerebral infarct area in the sham-operated group rats, and pretreatment with OMT (30, 60 and 120 mg/kg) and Nimodipine group significantly reduced percentage of infarction to 18.20 ± 1.79% (p < 0.05),

Fig. 4. OMT attenuates oxidative stress after hypoxic-ischemic in neonatal rats. (A) Effect of OMT on the content of MDA at 48 h after hypoxic-ischemic. (B–E) Effect of OMT on the activities of SOD, GSH-Px, CAT, T-AOC at 48 h after hypoxic-ischemic. Data are expressed as mean ± SEM (n = 6). ##p < 0.01 versus sham group; **p < 0.01 versus HI group.

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Fig. 5. The level of cell death in ischemic brain hippocampus neurons was measured by flow cytometer (1, 2, 3, and 4 quadrants represent dead neurons, late apoptotic neurons, normal neurons, early apoptotic neurons, respectively). (A–D) The results of apoptotic neurons in sham group, HI group, OMT (120 mg/kg) groups, and Nimodipine respectively. (E) The percentage of cell death in different groups at 48 h after hypoxic-ischemic. Data are expressed as mean ± SEM (n = 6). ##p < 0.01 versus sham group; *p < 0.05, **p < 0.01 versus HI group.

15.48 ± 1.54% (p < 0.01), 11.14 ± 1.16% (p < 0.01) and 9.96 ± 0.91% (p < 0.01) when compared to the HI group 21.92 ± 2.53% (Fig. 2B). 3.1.2. Histopathological examination The HE staining was used to examine histopathological changes of neonatal rat brain neurons, as shown in Fig. 2A–H. In the sham group, morphology of neurons in cortex and hippocampus CA3 region remained intact, the nucleus and cytoplasm were dyed clearly, and the distribution of neurons possessed regularity. However, histopathological scoring of brain injury confirmed the presence of severe brain damage to the ipsilateral hemisphere of the cerebral cortex and the hippocampus CA3 produced by HI. There was infarction involving almost all regions of the ipsilateral cerebral hemisphere (Fig. 2I, p < 0.01, brain damage >75%, score 5) in HI-treated rats. Neurons arrangement was disordered with loosened cytoplasms, in addition, interstitial edema, vacuolization, and karyopyknosis were observed in a large number of neurons in HI group. No damage was present in the contralateral cerebral hemisphere (data not shown). A remarkable reduction in the extension of injury was observed in the cerebral cortex and the hippocampus CA3 of OMT and Nimodipine post-treatment rats compared to HI-treated rats (Fig. 2I, p < 0.05). The damage was mostly confined to mild neuronal loss with the ipsilateral hemisphere being preserved in OMT and Nimodipine post-treatment rats (brain damage <25%, score ≤2). 3.1.3. Morphological evaluation Morphological changes observed by electron microscopy were showed as Fig. 3. Normal hippocampus neurons contained large oval nucleus with well-distributed chromatin, overt nucleolus, normal cell organelles, and the cytoplasm was abundant (Fig. 3A). After HI, hippocampus neurons showed severe damage. The majority of

nucleus were inordinate in shape with pyknotic chromatin, which indicated cell death, and swollen or vacuolated organelles emerged in HI group (Fig. 3B). However, after administration of OMT, morphology of hippocampus neurons had a varying degree of recovery, such as regular nucleus and slightly broken cell organelles. Moreover, the dead cell numbers were significantly diminished and normal neurons was markedly increased as well, especially in the OMT 120 mg/kg groups as well as Nimodipine groups (Fig. 3E, F). All of these results indicated that OMT treatment could alleviate brain injury induced by hypoxic-ischemic, in addition, these results revealed that the neuroprotective effect in the OMT 120 mg/ kg group is more obvious than that in OMT 30 and 60 mg/kg groups.

3.2. Effect of OMT on altered redox status induced by hypoxic-ischemic To demonstrate the effects of OMT on the oxidative stress induced by HI, MDA contents, SOD, GSH-Px, CAT, and T-AOC enzyme activities were measured after 48 h of HI injury. As shown in Fig. 4, the MDA content in the ischemic hemisphere was significantly increase in the HI group compared with the sham group (p < 0.01). After administration with OMT (120 mg/kg) and Nimodipine, MDA content decreased noticeably compared with HI group (Fig. 4A, p < 0.01). The results showed that SOD, GSH-Px, CAT, and T-AOC enzyme activities were significantly decreased in the HI group compared with the sham group (all p < 0.01). However, OMT (30, 60, 120 mg/kg) treatment groups remarkably restored their activities in a dose-dependent manner, especially at the dose of 120 mg/kg which had almost the same effects produced by Nimodipine (Fig. 4B–E).

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Fig. 6. Effects of OMT on the expression of Caspase-3. (A) A representative photomicrographs of Caspase-3 immunofluorescence staining in the ischemic cortex (×400) and hippocampus CA3 (×200). (B) Representative western blot band of Caspase-3 activation in the ischemic brain at 48 h after hypoxic-ischemic. (C) Effect of OMT (120 mg/kg) on the Caspase-3 activation in HI neonatal rats at 48 h after hypoxic-ischemic. Data are expressed as mean ± SEM (n = 6). ##p < 0.01 versus sham group; *p < 0.05, **p < 0.01 versus HI group.

3.3. Effect of OMT on cell death in hippocampus after hypoxic-ischemic Cell death in hippocampus was evaluated using Annexin V-FITC/ PI double staining and flow cytometer. Annexin V-FITC-positive and PI negative cells were scored as early injured cells, whereas Annexin V-FITC/PI double-positive cells were considered to be in late injury. Cells that stained negative for both Annexin V-FITC and PI were considered viable (i.e. nonapoptotic) (Fig. 5A–D). In sham group, 0.94 ± 0.29% (a small amount of) injured cells were found in the left hippocampus. Compared to sham group, the percentage of injured cells was significantly increased to 80.59 ± 3.05% (p < 0.01) in HI group. However, HI with OMT (120 mg/ kg) and Nimodipine treatment group, the percentage of injured cells was reduced to 68.09 ± 5.51% (p < 0.05) and 56.52 ± 9.56% (Fig. 5E, p < 0.05). 3.4. OMT regulated the expression of apoptosis-related protein assessed by immunofluorescence and western blot Flow cytometry results indicate that OMT may reduce cell death in neonatal HI brain injury. We further determined which apoptosisrelated proteins may be involved in this effect. Caspase-3 has been reported as a major cause of brain injury after neonatal

stroke (Manabat et al., 2002). Bcl-2/Bax protein ratio is important in the mitochondrial apoptosis pathway (Gross et al., 1999). We examined the expression of Caspase-3, Bcl-2 and Bax in hippocampus CA3 and cortex region of rats (Figs. 6–8). In accordance with other studies (Manabat et al., 2002; Yu et al., 2012), we find that Caspase-3 and Bax protein expressions were significantly greater at 48 h after HI (Fig. 6C, p < 0.05; Fig. 7C, p < 0.01) while the expression of Bcl-2 protein was lower (Fig. 8C, p < 0.01) as well as a lower ratio of Bcl-2/Bax protein (Fig. 8D, p < 0.01) in the HI group compared with the sham group. In contrast, OMT (120 mg/kg) or Nimodipine post-treatment could significantly blocked the increase of Caspase 3 and Bax level (Fig. 6C, p < 0.05; Fig. 7C, p < 0.01), and remarkably increased Bcl-2 protein expression (Fig. 8C, p < 0.05), while the ratio of Bcl-2/Bax approximately returned to the control level compared with the HI group (Fig. 8D, p < 0.01). Consistent with the western blot results, immunofluorescence analysis revealed the expressions of Caspase-3 and Bax were significantly greater in ipsilateral hippocampus CA3 and cortex region of the HI group but were reduced by OMT (120 mg/ kg) or Nimodipine post-treatment (Figs. 6A, 7A). On the other hand, the expression of Bcl-2 was decreased in hippocampus CA3 and cortex region in the HI group, which were restored by OMT(120 mg/kg) and Nimodipine post-treatment, respectively (Fig. 8A).

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Fig. 7. Effects of OMT on the expression of Bax. (A) A representative photomicrographs of Bax immunofluorescence staining in the ischemic cortex (×400) and hippocampus CA3 (×200). (B) Representative western blot band of Bax activation in the ischemic brain at 48 h after hypoxic-ischemic. (C) Effect of OMT (120 mg/kg) on the Bax activation in HI neonatal rats at 48 h after hypoxic-ischemic. Data are expressed as mean ± SEM (n = 6). ##p < 0.01 versus sham group; *p < 0.05, **p < 0.01 versus HI group.

4. Discussion Hypoxic-ischemic brain damage (HIBD) during the neonatal period is the leading causes of neonatal death and nervous system development disorders throughout the world (Komur et al., 2014). There is evidence that the pathogenesis of petrinatal HIBD was complex, involving energy failure, free radical damage, increased intracellular calcium, excitotoxicity, and caspase-dependent cell death in the immature brain (Johnston et al., 2011; Northington et al., 2001; Vasiljevic et al., 2011). Heretofore, however, there are still no effective pharmacological strategies available for HIBD, accordingly, the study on rescuing brain tissue in HIBD is an important area. OMT, a Chinese natural herb, has proved to possess a variety of pharmacological properties and been used for the treatment of many diseases (Cui et al., 2011). As a potential candidate for treating HIBD in newborns, the current investigation evaluated the effects of OMT using an well-established neonatal rat HI model (Rice et al., 1981), which has gained wide acceptance in studying basic physiology, brain injury mechanisms, and therapeutic interventions after perinatal brain damage (Martin et al., 2013). These animals were chosen because the brain tissue of a 7-day-old rat is histologically similar to that of a 32–34 week gestation human fetus or infants (Vannucci and Vannucci, 2005). Infarct volume plays an important role in evaluating the validity of cerebrovascular drugs in the treatment of ischemic brain diseases. In our preliminary experiments, brain infarct volume was measured at 24, 48 and 72 h after the HI insult, and

we observed that point-in-time of 48 h indicated moderate tissue damage and OMT post-treatment dramatically increased the surviving brain volume, with optimal effects at 48 h after HI (data not shown). Therefore, we decided to use this point-in-time in the following studies. In our current study, OMT significantly decreased the percentage of brain infarct volume at 48 h after HI in neonatal rats. In addition, the degree of ischemic damage was observed by HE staining, this method commonly be employed to identify the histopathology changes and link the development of HIBD, however, the size of the ischemia affected regions and neuronal necrosis were significantly decreased by OMT post-treatment. Another important observation was electron microscopy, the data showed that hippocampus neurons displayed prominent morphological injuries after HI, nevertheless these morphological damages were mitigated in the OMT post-treatment group. Therefore, the histological and morphological results discussed earlier suggested that OMT has a protective effect following HIBD. To understand the possible mechanisms underlying neuroprotective effects of OMT, we evaluated its effects on altered redox status and cell death in hippocampus. It has long been known that oxidative stress and reactive oxygen radicals, such as superoxide anions, hydroxyl radicals and hydrogen peroxide, which constantly produced ROS, play important roles in the pathogenesis of neonatal HIBD (Saito et al., 2005). Normally, there is a balance between the formation of ROS and its consumption by the

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Fig. 8. Effects of OMT on the expression of Bcl-2. (A) A representative photomicrographs of Bcl-2 immunofluorescence staining in the ischemic cortex (×400) and hippocampus CA3 (×200). (B) Representative western blot band of Bcl-2 activation in the ischemic brain at 48 h after hypoxic-ischemic. (C–D) Effect of OMT (120 mg/kg) on the Bcl-2 activation and the ratio of Bcl-2/Bax in HI neonatal rats at 48 h after hypoxic-ischemic. Data are expressed as mean ± SEM (n = 6). ##p < 0.01 versus sham group; *p < 0.05, **p < 0.01 versus HI group.

endogenous antioxidant systems. HI causes over-production of ROS in mitochondria, which causes oxidative damage to lipids, proteins, and DNA, cannot be handled by the endogenous anti-oxidant systems because of low activities of anti-oxidative enzymes (Yao et al., 2014; Zhang et al., 2014). In recent years, a parallel relationship has been found between increases in plasma malondialdehyde (MDA) levels, which is a toxic final product of lipid peroxidation and a sensitive marker of oxidative stress, and the intensity of HIBD (Komur et al., 2014). In addition, numerous studies (Chen and Huang, 2009; Dong et al., 2013; Peng et al., 2015) have suggested that endogenous antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) can provide substantial protection against ROS. In the present study, the results also revealed that OMT post-treatment for HI neonatal rats antagonized the HI induced increase in brain MDA levels and the decrease in SOD, GSH-PX, CAT, T-AOC activity, which suggested that OMT could

alleviate HIBD in neonatal rats at least partly due to increasing antioxidant enzyme activities and decreasing lipid peroxide. Increasing data suggested that cell death mechanism plays a prominent role in the progress of ischemic brain injury in neonatal rodents and humans than in adult brain ischemia (Zhu et al., 2005, 2007). Inhibition of apoptosis, a form of programmed cell death, has been taken as a novel and promising therapeutic direction for neuronal rescue following neonatal HI or stroke (Northington et al., 2005). It was therefore thought to be worthwhile to explore the effect of OMT on apoptosis after HI in neonatal rats. Current evidence suggests that in neonatal HI injury, both the extrinsic and intrinsic cell death pathways may be activated (Zhang et al., 2004), and caspase3, a key mediator of cell death, has been identified as the converging point for both death signals (Zhang et al., 2004). Cerebral hypoxic-ischemia can promote a series of pathological changes in neurons such as mitochondrial membrane

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depolarization and permeability transition pore opening, which result in ectopic expression of the death promoter Bax from the cytosol to the mitochondria, triggers cytochrome c release from mitochondria to cytosol. Release of cytochrome c into the cytosol leads to the formation of the apoptosome, a complex composed of apoptoticprotease activating factor-1(Apaf-1), procaspase-9, and ATP. Then, the apoptosome activates procaspase-9, which is followed by the activation of procaspase-3, ultimately cell death occurs (Abas et al., 2010; Wang et al., 2014). Especially, as members of the Bcl-2 family of proteins, Bax and Bcl-2 proteins serve as a class of apoptosisregulators at its early stages. Moreover, Bcl-2 is an anti-apoptotic protein that counteracts the pro-apoptotic effects of Bax. Above all, an appropriate ratio of Bax/Bcl-2 can keep homeostatic state in cells and ensure cell survival (Chen and Huang, 2009). In the present experiment, we demonstrated that OMT with the dose of 120 mg/kg could significantly suppress expression of Caspase-3 and Bax but concurrently increase Bcl-2 expression and the ratio of Bcl-2/Bax in HI neonatal rats brain, which strongly supported the notion that OMT has an anti-cell death activity as reported recently (Dong et al., 2011; Hong-Li et al., 2008; Jiang et al., 2005; Xiao et al., 2014; Zhao et al., 2008). These observations indicated that OMT has protective effect on HI induced cell death, likely through inhibiting the expression of Caspase-3 protein and increasing the ratio of Bcl-2/Bax. It still needs to be considered that, in our studies of flow cytometer and immunofluorescence, we did not differentiate if these cells were neuronal or glial cells and the chosen planes of immunofluorescence represent a part of the brain. On the other hand, the planes were selected according to the expected insult site and neuroprotective effect of OMT should similarly affect both types of cells. In addition, the increasing number of researches demonstrated that OMT could induce apoptosis in many tumor cell lines (Liu et al., 2014; Wei et al., 2014; Wu et al., 2014). The dual regulation and control of OMT on apoptosis may be due to its differential effects on dividing cells and non-dividing cells. It is worth noting that a weakness of the finding is that multiple pathways are involved in the cell death process after HIBD and the detailed neurobiological and cellular mechanisms underlying OMT treatment need to be further investigated. Future study will test different time courses including posttreatment for OMT in the neonatal rat HI model and evaluate its therapeutic widow and the long-term neurodevelopmental effect for neonatal stroke. 5. Conclusion This study demonstrated that post-treatment with OMT has shown neuroprotective effects on HIBD in neonatal rats. OMT has significantly reduced infarct volume and the percentage of cell death, and ameliorated histopathology and morphological injury, at least in part by increasing antioxidant enzyme activities, reducing lipid peroxide, as well as decreasing the expression of Caspase-3 protein and increasing the ratio of Bcl-2/Bax. Therefore, the present study raises the possibility that OMT may be used as a potential neuroprotective agent for the treatment of neonatal HIBD in the future clinical trials. Acknowledgments The authors gratefully acknowledge the financial supported by the Natural Science Foundation of Ningxia Province (Grant No. NZ13138). Conflict of interest The authors declare that they have no conflicts of interest.

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