BDNF signaling after transient global cerebral ischemia in rats

Journal of Ethnopharmacology 252 (2020) 112612

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Angelica sinensis extract protects against ischemia-reperfusion injury in the hippocampus by activating p38 MAPK-mediated p90RSK/p-Bad and p90RSK/CREB/BDNF signaling after transient global cerebral ischemia in rats

T

Chin-Yi Chenga,b, Shung-Te Kaoc, Yu-Chen Leed,e,f,∗ a

School of Post-baccalaureate Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, 40402, Taiwan Department of Chinese Medicine, Hui-Sheng Hospital, 42056, Taichung, Taiwan School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, 40402, Taiwan d Department of Chinese Medicine, China Medical University Hospital, 40447, Taichung, Taiwan e Research Center for Chinese Medicine & Acupuncture, China Medical University, Taichung, 40402, Taiwan f Graduate Institute of Acupuncture Science, China Medical University, Taichung, 40402, Taiwan b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Global cerebral ischemia Angelica sinensis p38 mitogen-activated protein kinase 90-kDa ribosomal S6 kinase Phospho-Bad Brain-derived neurotrophic factor

Ethnopharmacological relevance: Angelica sinensis (Oliv.) Diels, commonly known as Dang Gui (DG), is one of the most popular traditional Chinese herbal medicines for the treatment of stroke. However, the effects of DG on transient global cerebral ischemia (GCI) and its precise mechanisms remain unclear. Aim of the study: This study aimed to investigate the effects of the DG extract on ischemia-reperfusion (I/R) injury in the hippocampus 7 d after transient GCI and to identify the potential mitogen-activated protein kinase (MAPK)-related signaling pathway in the hippocampus involved in the effects. Materials and methods: Rats were intragastrically administered DG at doses of 0.25 g/kg (DG-0.25g), 0.5 g/kg (DG-0.5g), or 1 g/kg (DG-1g) 1, 3, and 5 d after GCI. Results: DG-0.5g and DG-1g treatments effectively promoted hippocampal cornu ammonis 1 (CA1) neuronal survival. DG-0.5g and DG-1g treatments markedly increased phospho-p38 MAPK (p-p38 MAPK), phospho-90kDa ribosomal S6 kinase (p-p90RSK), cytosolic and mitochondrial phospho-Bad (p-Bad), phospho-cAMP response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), and p-CREB/BDNF expression; decreased 4-hydroxy-2-nonenal, cytochrome c (Cytc), and cleaved caspase-3 expression, and inhibited apoptosis in the hippocampal CA1 region. Pretreatment with a specific inhibitor of p38 MAPK, SB203580, completely blocked the effects of DG-1g on the expression of the aforementioned proteins. Conclusions: DG-0.5g and DG-1g treatments exerted neuroprotective effects on I/R injury by activating p38 MAPK-mediated p90RSK/p-Bad-induced anti-apoptotic-Cytc/caspase-3-related and p90RSK/CREB/BDNF survival signaling in the hippocampus 7 d after transient GCI.

1. Introduction Global cerebral ischemia (GCI) occurs when the blood supply to the brain is blocked or reduced, and it is usually caused by cardiac arrest or stroke (Cheng et al., 2012; Hu et al., 2013). The mechanisms of cerebral ischemic injury include inflammation, oxidative stress, necrosis, and apoptosis, which usually cause cognitive dysfunction (Bin et al., 2012). Hippocampal pyramidal neurons in the cornu ammonis 1 (CA1) region are particularly vulnerable following GCI (Arabian et al., 2015; Erfani et al., 2015). The hippocampus in the brain is involved in memory ∗

consolidation and learning (Wang et al., 2016a). GCI leads to memory deficits, cognitive impairment, and even vascular dementia (Cheng et al., 2012). In global cerebral ischemic injury, apoptosis in the hippocampal CA1 region is the major process of cell death and the major cause of memory impairment (Cho et al., 2015; Kim et al., 2014; Wang et al., 2016a). During GCI, apoptosis is a main mechanism of cell death and is regulated by several signaling pathways (Kenny et al., 2013). Mitogenactivated protein kinases (MAPKs) mediate intracellular signaling pathways associated with cell death and survival upon various stress

Corresponding author. Graduate Institute of Acupuncture Science, China Medical University, No.91, Hsueh-Shih Road, Taichung, 40402, Taiwan. E-mail address: [email protected] (Y.-C. Lee).

https://doi.org/10.1016/j.jep.2020.112612 Received 11 April 2019; Received in revised form 19 January 2020; Accepted 21 January 2020 Available online 24 January 2020 0378-8741/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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stimuli (Kim and Choi, 2010). The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade plays a vital role in promoting neuronal survival during focal and global brain ischemic injury (Wu et al., 2014; Xue et al., 2009). Under the cerebral ischemia and reperfusion (I/ R) condition, the activation of c-Jun NH2-terminal kinase (JNK) signaling in the hippocampal CA1 region causes neuronal apoptosis and cognitive impairment (Qi et al., 2016). The p38 MAPK signaling pathway plays a dual role in response to ischemic injury. Some studies have indicated that the activation of p38 MAPK in the hippocampus exacerbates inflammatory responses and impairs cognitive performance following GCI (Xuan et al., 2015; Yang et al., 2013), whereas other studies have found that the upregulation of p38 MAPK signaling exerts neuroprotective effects against apoptotic insults in the ischemic area in the subacute phase of focal cerebral ischemia (Cheng et al., 2016, 2017). Furthermore, ERK1/2 and p38 MAPK signaling facilitate the phosphorylation and activation of the downstream substrates, 90-kDa ribosomal S6 kinase (p90RSK) and cyclic adenosine monophosphate response element (CRE)-binding protein (CREB), to improve neuronal survival in the ischemic region in focal cerebral ischemia and GCI (Cheng et al., 2016, 2017; Jover-Mengual et al., 2007). Activated p90RSK phosphorylates Bad, a pro-apoptotic member of the Bcl-2 family, which subsequently binds to the chaperone protein 14-3-3 and inactivates its pro-apoptotic function, resulting in the inhibition of cytochrome c (Cytc) release from the mitochondria following cerebral ischemic injury (Koh, 2007). The transcription factor CREB, a downstream target of p90RSK, is activated by phosphorylation. PhosphoCREB subsequently binds to the CRE sequence, thereby inducing the CRE-mediated transcription of genes such as those for anti-apoptotic factors (such as Bcl-2) and brain-derived neurotrophic factor (BDNF) (Kitagawa, 2007; Liu et al., 2015). The activation of CREB in the hippocampal CA1 region protects against memory dysfunction in brain ischemic injury (Kitagawa, 2007). In response to stressful stimuli, an elevated Bax (pro-apoptotic protein)/Bcl-2 ratio or increased Bad expression facilitates the production of reactive oxygen species (ROS), which subsequently disrupt mitochondrial outer membrane integrity, leading to the release of Cytc and the activation of apoptosis (Arabian et al., 2015; Lei et al., 2015). As a neurotrophin, BDNF is involved in learning and memory processes. Thus, decreased BDNF expression in the hippocampus is implicated in the impairment of learning and memory following cerebral ischemic insults (Lee et al., 2016; Seo et al., 2014). In a study, the pharmacological upregulation of BDNF expression in the hippocampus suppressed apoptosis and restored memory deficits after GCI (Lee et al., 2016). Angelica sinensis (Oliv.) Diels, commonly known as Dang Gui (DG), is one of the most popular herbal medicines (Yi et al., 2009). In traditional Chinese medicine, DG has been widely used in the treatment of gynecological diseases (such as dysmenorrhea), cardiovascular diseases, and cerebrovascular diseases for centuries (Feng et al., 2012; Wu and Hsieh, 2011). In addition, the aqueous extract of DG can be orally administered daily at a dose range of 0.8–1.2 g/kg for the treatment of the patient with cerebrovascular diseases. The main active components of DG are polysaccharides, Z-ligustilide (3-butylidene-4,5-dihydrophthalide), and ferulic acid (4-hydroxy-3-methoxycinnamic acid) (Wu and Hsieh, 2011), which protected against ischemic injury and cognitive impairment by suppressing apoptosis and oxidative stress in the ischemic area in rodent models of cerebral ischemia (Ai et al., 2013; Cheng et al., 2016; Feng et al., 2012; Kuang et al., 2008; Lei et al., 2014). Pretreatment with the DG extract exerted anti-apoptotic effects against cerebral I/R injury by activating p38 MAPK-mediated signaling in the cortical penumbra in rats (Cheng et al., 2017). However, the effects of DG on I/R injury in the hippocampus in the subacute phase of transient GCI and precise mechanism remain unknown. Therefore, the main purpose of the present study was to evaluate the effects of DG on I/R injury in the hippocampus 7 d after transient GCI and to identify the potential precise MAPK-mediated signaling pathway

Table 1 Primary antibodies used for Western blotting in this study. Primary antibody

Dilution

Source

JNK phospho-JNK (p-JNK) ERK1/2 phospho-ERK1/2 (p-ERK) p38 MAPK phospho-p38 MAPK (p-p38 MAPK) Akt phospho-Akt (p-Akt) CREB phospho-CREB (p-CREB) Bcl-2 Bax phospho-p90RSK (p-p90RSK) Bad phospho-Bad (p-Bad) AIF Cleaved caspase-3 Actin (loading control) HSP60 (mitochondrial loading control)

1/1000 1/1000 1/1000 1/1000 1/1000 1/1000 1/1000 1/1000 1/1000 1/500 1/1000 1/1000 1/1000 1/1000 1/1000 1/1000 1/1000 1/5000 1/1000

CST (#9252) CST (#9251) CST (#9102) CST (#9101) CST (#9212) CST (#9211) CST (#4685) CST (#9271) CST (#9197) Millipore (DAM1482729) CST (#2876) CST (#2772) CST (#9344) CST (#9292) CST (#9291) CST (#4642) CST (#9664) NB(NB600-501) CST (#4870)

AIF, apoptosis-inducing factor; HSP60, heat shock protein 60; CST, Cell Signaling Technology; NB, Novus Biologicals. Table 2 Primary antibodies used for immunohistochemical analysis in this study. Primary antibody

Dilution

Source

Cytc Cleaved caspase-3 4-HNE BDNF

1/50 1/200 1/400 1/500

Proteintech (10993-1-AP) CST (#9664) JaICA(MHN-100P) Millipore (AB1779SP)

4HNE, 4-hydroxy-2-nonenal; CST, Cell Signaling Technology.

involved in the effects. 2. Materials and methods 2.1. Experimental animals Adult male 8-week old Sprague-Dawley rats weighting 300–350 g were purchased from Lasco Co. (Taiwan) and were used for this study. All rats were housed under standard conditions, which were set to 22 ± 2 °C and 55% relative humidity with a 12/12 h light/dark cycle. All study procedures were approved by the Institutional Animal Care and Use Committee of China Medical University (Permit Number: 2017–094). 2.2. DG extract preparation DG extract powder (Batch Number A1306801) used in this study was purchased from Chuang Song Zong (CSZ) Pharmaceutical Co., Ltd (Kaohsiung, Taiwan). The dried roots of DG were obtained from the province of Gansu, China, in March 2016. Appearance, microscopic, and DNA sequence identifications of the dried roots of DG were performed in CSZ Ligang Laboratory (Pingtung, Taiwan). According to the Chinese Pharmacopeia 2015 Edition, Taiwan Herbal Pharmacopeia 2nd Edition, and National Center for Biotechnology Information GenBank guidelines, the dried roots of DG were verified. All the results were authenticated by Professor Kun Chang Wu (College of Pharmacy, China Medical University, Taichung, Taiwan). A voucher specimen (A1306801) was preserved at CSZ Ligang Laboratory (Pingtung, Taiwan). DG extract powder was prepared as follows: The air-dried roots of DG were extracted by heating in water of 10 times of the herb weight for 1.5 h at 100 °C and then its aqueous extract and essential oil were collected. After concentrating the extract during decompression at 2

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Fig. 1. HPLC profiles of the (A) standard solution and (B) DG extract solution. AU, absorbance unit.

was concentrated to dryness, and finally dissolved in 10 mL of pure methanol as the test solution. The indicator components of the DG extract (ferulic acid and ligustilide) were analyzed using high performance liquid chromatography (HPLC) methods, as previously described (Cheng et al., 2017). Briefly, 20 μL of the standard solution or test solution was injected into a Waters HPLC system (Waters 2695, Waters Corp.). The mobile phase consisted of acetonitrile and 0.03% phosphoric acid solution. The gradient program was produced by increasing the ratio of acetonitrile from 5% to 70% and decreasing the ratio of phosphoric acid from 95% to 30% for 90 min at a flow rate of 1.0 mL/ min. The total run time was 95 min and the ultraviolet detection wavelength was set at 320 nm. The external standard method was employed for quantitative analysis.

lower temperature (< 50 °C), the concentrated extract, essential oil, and excipients (microcrystalline cellulose and corn starch) were loaded into the fluidized bed processor and granulated using top-spray fluidized bed coating techniques. Each gram of the DG extract powder contains 0.5 g of the DG extract, 0.4 g of microcrystalline cellulose, and 0.1 g of corn starch. In this study, DG extract powder was dissolved in double distilled water. The soluble fraction (supernatant) of the DG extract was collected by centrifugation at 1000×g for 10 min at 4 °C and the final concentration was maintained at 0.125 g/mL. 2.3. Analysis of indicators of the DG extract through high performance liquid chromatography The standards including ferulic acid [National Institutes for Food and Drug Control (NIFDC), China] with 99.6% purity and ligustilide (NIFDC, China) with 100% purity were accurately weighted and dissolved in pure methanol as the standard solution. Two grams of the DG extract powder was dissolved in 60 mL of pure methanol, and the solution was centrifuged at 9000 rpm for 10 min at 4 °C. The supernatant

2.4. Transient GCI The transient GCI model was established using the 4-vessel occlusion (4-VO) technique as previously described (Pulsinelli and Brierley, 1979). Briefly, on the first day, the rats were anesthetized with 3

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Fig. 2. Effects of DG treatment on hippocampal CA1 neuronal survival. (A) Representative images depict the surviving neurons in the hippocampal CA1 region in the Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups 7 d after transient GCI. (B) Representative image depicts a Nissl-stained coronal section of the hippocampus. The solid-line rectangle indicates the selected CA1 region in which the numbers of surviving neurons and immunopositive cells were measured. (C) The bar graph reveals the number of surviving neurons in the hippocampal CA1 region in the experimental groups (n = 5). Data are presented as mean ± SD. *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group. Arrows in (A) show surviving neurons. Scale bars in (A) and (B) are equal to 10 μm and 500 μm, respectively.

2.5.2. Nissl staining Seven days after transient GCI, the rats were sacrificed. Rats were transcardially perfused with 200 mL 0.9% saline and their brains were immediately removed, embedded in optimal cutting temperature medium in aluminum foil paper boxes, frozen at a temperature of −30 °C with dry ice, and cut in 15-μm thick to prepare brain tissue sections, as previously described (Beck et al., 2002). The brain sections were rinsed in phosphate-buffered saline (PBS) with Tween-20 (PBST) and post-fixed in 4% paraformaldehyde (PFA) at room temperature (RT). After incubation with cresyl echt violet solution (0.1%) for 5 min, the brain sections were rinsed quickly in distilled water, quickly dehydrated three times with absolute alcohol, dried, and mounted on a mounting medium (Assistant-Histokitt, Germany). The intact cells without any shrinkage were considered viable. The surviving neurons in the hippocampal CA1 region were counted in each of three 400 × magnification fields under a light microscope (Axioskop 40, Zeiss, Oerzen, Germany).

isoflurane (5% and 2% isoflurane for induction and maintenance, respectively) in oxygen and were subsequently placed in the prone position. A dorsal midline vertical skin incision was made to expose the first cervical vertebra. Both vertebral arteries were permanently electrocauterized by inserting an electrocautery needle into the right and left alar foramina of the first cervical vertebra. On the next day, the rats were anesthetized again and were placed in the supine position. Subsequently, a neck midline incision was made to expose the bilateral common carotid arteries (CCAs). After careful separation of the vagus nerve, the bilateral CCAs were occluded with vascular clips to induce ischemia. After 25 min of ischemia, the vascular clips were removed and reperfusion was allowed. The rats that lost their righting reflex and their pupils were dilated throughout the ischemic period were included. 2.5. Experiment A 2.5.1. Grouping The rats were randomly divided into five groups (n = 5/group): Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups. The rats in the DG-0.25g group were intragastrically administered 0.25 g/kg of DG 1, 3, and 5 d after transient GCI. Seven days after reperfusion, the rats were sacrificed. The rats in the DG-0.5g and DG-1g groups were conducted with the same procedures as those in the DG-0.25g group; however, the rats in the DG-0.5g and DG-1g groups were given DG at doses of 0.5 g/kg and 1 g/kg, respectively. The rats in the Control group were conducted with the same procedures as those in the DG-1g group, but they received saline solution instead of DG. The rats in the Sham group were conducted with the same procedure as those in the Control group, but their bilateral CCAs were not occluded.

2.6. Experiment B 2.6.1. Grouping The rats were randomly divided into five groups (n = 4–5/group): Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups. These groups were conducted with the same experimental procedures as those performed in Experiment A. 2.6.2. Western blot analysis Seven days after transient GCI, the rats were sacrificed and their brains were quickly removed. The two hemispheres of the rat brain 4

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Fig. 3. Effects of DG treatment on cytosolic MAPKs expression in the hippocampus. (A) Representative immunoblot images depict cytosolic p-JNK, JNK, p-ERK1/2, ERK1/2, p-p38 MAPK, and p38 MAPK expression in the hippocampus in the Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups 7 d after transient GCI. The ratios of (B) p-JNK/JNK, (C) p-ERK/ERK, and (D) p-p38 MAPK/p38 MAPK expression were measured in the hippocampus among the experimental groups. (n = 4–5). Cyto, cytosolic fraction. *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group.

were isolated, and the hippocampal tissues were carefully collected and homogenized on ice. The protein content of the cytosolic and mitochondrial fractions was measured using the Bio-Rad protein assay. Equal amounts (15 μg/well) of lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described previously (Hsiang et al., 2005). The separated proteins on the nitrocellulose (NC) membranes were incubated overnight at 4 °C with the primary antibodies mentioned in Table 1. After washing, the NC membranes were incubated with an anti-rabbit or anti-mouse secondary antibody (1:5000) for 1 h at RT. Band detection was performed using an enhanced chemiluminescence reagent (ECL-plus GE Healthcare).

DS + Control group; however, the occlusion of bilateral CCAs was not conducted.

2.7. Experiment C

2.7.3. Western blot analysis Seven days after transient GCI, the rats were sacrificed, and their brains were immediately removed for Western blot detection of p38 MAPK, p-p38 MAPK, CREB, p-CREB, p-p90RSK, Bad, and p-Bad (mentioned in Table 1) expression. Western blot assay was performed using the same protocols in Experiment B.

2.7.2. ICV injection of SB203580 or 1% DMSO The rats were anesthetized with isoflurane, and two burr holes were symmetrically made on both sides of the skull (3.0 mm posterior to the bregma, 2.0 mm lateral to the midline, and 3.5 mm below the cortical surface). Through each burr hole, the rats were ICV injected with 10 μL of a dilute solution of SB203580 (1 mM in 1% DMSO, #S1076 Selleckchem.com) or DMSO (1%). The solution was injected by using a 10-μL Hamilton syringe (Hamilton Company, Reno, NV, USA) fixed in the stereotactic frame.

2.7.1. Grouping The rats were randomly divided into four groups (n = 4–5/group): DS + Sham, DS + Control, DS + DG-1g, and SB20+DG-1g groups. The rats in the SB20+DG-1g group were conducted with the same experimental protocols as those in the DG-1g group performed in Experiment A, but they were intracerebroventricularly (ICV) injected with SB203580, a specific inhibitor of p38 MAPK, before bilateral CCA occlusion (BCCAO). The rats in the DS + DG-1g group were conducted with the same experimental protocols as those in the SB20+DG-1g group, but they were ICV injected with 1% dimethyl sulfoxide (DMSO) before BCCAO. The rats in the DS + Control group were conducted with the same experimental protocols as those in the Control group performed in Experiment A; however, they were ICV injected with 1% DMSO before BCCAO. The rats in the DS + Sham group were conducted with the same experimental protocols as those in the

2.8. Experiment D 2.8.1. Grouping The rats were randomly divided into six groups (n = 5/group): Sham, Control, DG-0.25g, DG-0.5g, DG-1g, and SB20+DG-1g groups. These groups were conducted with the same experimental procedures as those performed in Experiments A and C. 2.8.2. Immunohistochemical analysis Seven days after transient GCI, the rats were sacrificed; their brains 5

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Fig. 4. Effects of DG treatment on cytosolic p-p90RSK, p-Bad, Bad, p-CREB, and CREB expression in the hippocampus. (A) Representative immunoblot images depict cytosolic p-p90RSK, p-Bad, Bad, p-CREB, and CREB expression in the hippocampus in the Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups 7 d after transient GCI. The ratios of (B) p-p90RSK/actin, (C) p-Bad/Bad, and (D) p-CREB/CREB expression were measured in the hippocampus among the experimental groups (n = 4–5). *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group.

Jackson ImmunoResearch) secondary antibodies for 1 h at RT. The double-labeled sections were subsequently counterstained with 4′,6diamidino-2-phenylindole (DAPI, ab4139 abcam). The double-labeled cells in the selected hippocampal CA1 region were detected in a 200 × magnification field with a fluorescence microscope (CKX53, Olympus, Tokyo, Japan).

were immediately removed, and brain tissue sections were prepared as described in Section 2.5.2. The brain sections were incubated with primary antibodies (Table 2) overnight at 4 °C. Subsequent immunohistochemical (IHC) analysis was conducted, as described previously (Cheng et al., 2017). Cells with positive staining in the hippocampal CA1 region were counted in each of three 400 × magnification fields with a light microscope (Axioskop 40, Zeiss, Oerzen, Germany). The brain tissue sections from the Control group were not incubated with Cytc, cleaved caspase-3, or 4-HNE primary antibody and were used as negative controls. In addition, DG-1g-treated sections stained that were not incubated with the BDNF primary antibody were also used as a negative control.

2.9. Statistical analysis Data are expressed as mean ± standard deviation (SD). The data of the Sham, Control, and DG-treated groups were compared using oneway analysis of variance method and a post-hoc Scheffe test. P values less than 0.05 were considered statistically significant.

2.8.3. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay The serial brain tissue sections were used in the TUNEL assay. The brain tissue sections were first incubated with 20 μg/mL proteinase K (QIA33, Calbiochem, USA) for 20 min at RT, and the subsequent steps were performed as described previously (Cheng et al., 2014). TUNELpositive cells in the hippocampal CA1 region were calculated in each of three 400 × magnification fields with a light microscope.

3. Results 3.1. HPLC analysis of the DG extract In HPLC profile analysis, ferulic acid and ligustilide showed peaks at 33.6 and 76.5 min, respectively, for both the standard and DG extract solutions. In addition, the contents of ferulic acid and ligustilide in the DG extract were 0.25 and 0.01 mg/g, respectively (Fig. 1A and B).

2.8.4. Immunofluorescence double staining The brain sections were incubated with 5% bovine serum albumin in PBST for 30 min at RT and were subsequently incubated with mouse anti-p-CREB (1/100 dilution, DAM 1482729 Millipore) and rabbit antiBDNF (1/200 dilution, ab108319 abcam) antibodies overnight at 4 °C. After being washed with PBST, the brain sections were simultaneously incubated with DyLight 594-conjugated AffiniPure goat anti-mouse IgG (red, 1/200 dilution, Jackson ImmunoResearch) and DyLight 488conjugated AffiniPure goat anti-rabbit IgG (Green, 1/200 dilution,

3.2. Effects of DG treatment on neuronal survival The numbers of surviving neurons and immunopositive cells within the solid-line rectangle in the hippocampal CA1 region in Fig. 2B were calculated (three 400 × magnification fields). The number of surviving neurons in the hippocampal CA1 region was markedly decreased in the Control group in comparison with the Sham group (P < 0.05) and was markedly increased in the DG-0.5g and DG-1g groups in comparison 6

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Fig. 5. Effects of DG treatment on mitochondrial AIF, p-Bad, and Bad expression and cytosolic AIF and cleaved caspase-3 expression in the hippocampus. (A) Representative immunoblot images depict mitochondrial AIF, p-Bad, and Bad expression and cytosolic AIF and cleaved caspase-3 expression in the hippocampus in the Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups 7 d after transient GCI. The ratios of (B) mitochondrial AIF/HSP60, (C) mitochondrial p-Bad/Bad, (D) cytosolic AIF/actin, and (E) cytosolic cleaved caspase-3/actin were measured in the hippocampus among the experimental groups (n = 4–5). Mito, mitochondrial fraction. *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group.

upregulated in the DG-0.5g (2.4-fold, 2.6-fold, and 2.7-fold, respectively) and DG-1g (2.8-fold, 3.1-fold, and 2.6-fold, respectively) groups in comparison with the Control group 7 d after transient GCI (all P < 0.05; Fig. 4A–D). However, no marked differences were observed in the ratios of cytosolic p-CREB/CREB, p-p90RSK/actin, and p-Bad/ Bad expression between the Control and DG-0.25g groups (P > 0.05).

with the Control group (both P < 0.05; Fig. 2A and C) 7 d after transient GCI. However, no marked differences were observed in the number of surviving neurons between the Control and DG-0.25g groups (P > 0.05). 3.3. Effects of DG treatment on the cytosolic expression of MAPKs Western blot assay showed no marked differences in the ratios of cytosolic p-JNK/JNK and p-ERK/ERK expression in the hippocampus among the Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups 7 d after transient GCI (all P > 0.05; Fig. 3A–C). The ratio of cytosolic pp38 MAPK/p38MAPK expression was markedly downregulated in the Control group (0.4-fold) in comparison with the Sham group (P < 0.05) and was markedly upregulated in the DG-0.5g (2.4-fold) and DG-1g (2.6-fold) groups in comparison with the Control group (both P < 0.05; Fig. 3A and D). However, no marked differences were observed in the ratio of cytosolic p-p38MAPK/p38MAPK expression between the Control and DG-0.25g groups (P > 0.05).

3.5. Effects of DG treatment on the mitochondrial expression of apoptosisinducing factor (AIF), p-Bad, and Bad and the cytosolic expression of AIF and cleaved caspase-3 No marked differences were observed in the ratios of mitochondrial and cytosolic AIF/actin expression in the hippocampus among the Sham, Control, DG-0.25g, DG-0.5g, and DG-1g groups 7 d after transient GCI (all P > 0.05; Fig. 5A, B, and 5D). The ratio of mitochondrial p-Bad/Bad expression was markedly downregulated in the Control group (0.3-fold) in comparison with the Sham group (P < 0.05) and was markedly upregulated in the DG-0.5g group (2.7-fold) and DG-1g group (3.2-fold) in comparison with the Control group (both P < 0.05; Fig. 5A and C). By contrast, the ratio of cytosolic cleaved caspase-3/ actin expression was markedly upregulated in the Control group (3.4fold) in comparison with the Sham group (P < 0.05) and was markedly downregulated in the DG-0.5g (0.4-fold) and DG-1g (0.3-fold) groups in comparison with the Control groups (both P < 0.05; Fig. 5A and E). However, no marked differences were observed in the ratios of mitochondrial p-Bad/Bad and cytosolic cleaved caspase-3/actin

3.4. Effects of DG treatment on the cytosolic expression of p-CREB, CREB, p-p90RSK, p-Bad, and Bad The ratios of cytosolic p-p90RSK/actin, p-Bad/Bad, and p-CREB/ CREB expression in the hippocampus were markedly downregulated in the Control group (0.5-fold, 0.4-fold, and 0.4-fold, respectively) in comparison with the Sham group (all P < 0.05) and were markedly 7

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Fig. 6. Effects of DS + DG-1g and SB20+DG-1g treatments on cytosolic p-p38 MAPK, p38 MAPK, p-p90RSK, p-Bad, Bad, p-CREB, and CREB expression and mitochondrial p-Bad and Bad expression in the hippocampus. (A) Representative immunoblot images depict cytosolic p-p38 MAPK, p38 MAPK, p-p90RSK, p-Bad, Bad, p-CREB, and CREB expression and mitochondrial p-Bad and Bad expression in the hippocampus in the DS + Sham, DS + Control, DS + DG-1g, and SB20+DG1g groups 7 d after transient GCI. The ratios of (B) cytosolic p-p38 MAPK/p38 MAPK, (C) cytosolic p-p90RSK/actin, (D) cytosolic p-Bad/Bad, (E) cytosolic p-CREB/ CREB, and (F) mitochondrial p-Bad/Bad expression were measured in the hippocampus among the experimental groups (n = 4–5). *P < 0.05 in comparison with the DS + Sham group; #P < 0.05 in comparison with the DS + Control group.

DG-1g groups in comparison with the Control group 7 d after transient GCI (all P < 0.05; Fig. 7A, B, 7E, 7F, 8A, 8B, 8D, and 8E). However, no marked differences were observed in the numbers of Cytc-, cleaved caspase-3-, 4HNE-, and TUNEL-positive cells among the Control, DG0.25g, and SB20+DG-1g groups (all P > 0.05).

expression between the Control and DG-0.25g (both P > 0.05). 3.6. Effects of DS + DG-1g and SB20+DG-1g treatments on the cytosolic expression of p-p38 MAPK, p38 MAPK, p-CREB, CREB, p-p90RSK, p-Bad, and Bad, and the mitochondrial expression of p-Bad and Bad The ratios of cytosolic p-p38 MAPK/p38 MAPK, p-p90RSK/actin, pBad/Bad, and p-CREB/CREB expression and mitochondrial p-Bad/Bad expression in the hippocampus were markedly downregulated in the DS + Control group (0.4-fold, 0.6-fold, 0.5-fold, 0.4-fold, and 0.4-fold, respectively) in comparison with the DS + Sham group and were markedly upregulated in the DS + DG-1g group (2.1-fold, 2.2-fold, 2.2fold, 2.6-fold, and 3.4-fold, respectively) in comparison with the DS + Control group 7 d after transient GCI (all P < 0.05; Fig. 6A–F). However, no marked differences were observed in the ratios of cytosolic p-p38 MAPK/p38 MAPK, p-p90RSK/actin, p-Bad/Bad, and p-CREB/ CREB expression and mitochondrial p-Bad/Bad expression between the DS + Control and SB20+DG-1g groups (all P > 0.05).

3.8. Effects of DG treatment on BDNF expression

3.7. Effects of DG treatment on Cytc, cleaved caspase-3, 4-HNE, and TUNEL expression

p-CREB/BDNF double-labeled cells within the solid-line square in the selected hippocampal CA1 region were detected (one 200 × magnification field, Fig. 10B). The number of p-CREB/BDNF double-labeled cells in the selected hippocampal CA1 region was markedly downregulated in the Control group in comparison with the Sham group (P < 0.05) and was markedly upregulated in the DG-0.5g and DG-1g groups in comparison with the Control group 7 d after transient GCI

The number of BDNF-positive cells in the hippocampal CA1 region was markedly downregulated in the Control group in comparison with the Sham group (P < 0.05) and was markedly upregulated in the DG0.5g and DG-1g groups in comparison with the Control group 7 d after transient GCI (both P < 0.05; Fig. 9A and C). However, no marked differences were observed in the number of BDNF-positive cells among the Control, DG-0.25g, and SB20+DG-1g groups (P > 0.05). 3.9. Effect of DG treatment on the expression of p-CREB/BDNF doublelabeled cells

The numbers of Cytc-, cleaved caspase-3-, 4HNE-, and TUNEL-positive cells in the hippocampal CA1 region (Fig. 2B) were markedly upregulated in the Control group in comparison with the Sham group (all P < 0.05) and were markedly downregulated in the DG-0.5g and 8

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Fig. 7. Effects of DG treatment on Cytc and cleaved caspase-3 expression in the hippocampal CA1 region. Representative images depict (A) Cytc and (B) cleaved caspase-3 expression in the hippocampal CA1 region in the Sham, Control, DG-0.25g, DG-0.5g, DG-1g, and SB20+DG-1g groups 7 d after transient GCI. Representative images (C) and (D) show the negative controls for Cytc and cleaved caspase-3 antibodies, respectively. The bar graphs reveal the numbers of (E) Cytcand (F) cleaved caspase-3-positive cells in the hippocampal CA1 region among the experimental groups (n = 4–5). *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group. Arrows in (A) and (B) show Cytc- and cleaved caspase-3-positive cells, respectively. Scale bars in (B) and (D) are equal to 10 μm.

apoptosis occurred predominantly in the hippocampal CA1 region, and DG-0.5g and DG-1g treatments effectively downregulated cerebral I/Rinduced apoptosis in the subacute phase of GCI. Based on these findings, we first deduce that DG-0.5g and DG-1g treatments effectively protect hippocampal CA1 neurons following global cerebral ischemic injury. Furthermore, the effects of DG on I/R injury are at least partially due to the downregulation of neuron apoptosis in the hippocampal CA1 region 7 d after transient GCI. MAPK-mediated signaling pathways are involved in the regulation of apoptosis in the progression of cerebral ischemia (Niizuma et al., 2010). JNK activated by oxidative stress causes the apoptosis of the neurons in the hippocampal CA1 region following global brain ischemia (Zhang et al., 2003). By contrast, the upregulation of ERK1/2 exerts neuroprotective effects against neuronal apoptosis by decreasing Bax expression in the hippocampus after GCI (Zhao et al., 2014). The p38 MAPK-mediated pathway plays a dual role in the regulation of apoptosis in different models of cerebral ischemia (Cheng et al., 2017; Nozaki et al., 2001). Yang et al. (2013) reported that p38 MAPK activation triggered caspase-3-mediated apoptosis in the hippocampus by downregulating Bcl-2 expression and upregulating Bax expression 7 d after global cerebral hypoperfusion (Yang et al., 2013). By contrast, Zhu et al. (2014) showed that hypoxic preconditioning exerted neuroprotective effects against apoptosis by activating p38 MAPK-mediated signaling in the hippocampus in the subacute phase of transient GCI

(both P < 0.05; Fig. 10A and C). No significant differences were observed in the number of p-CREB/BDNF double-labeled cells among the Control, DG-0.25g, and SB20+DG-1g groups (P > 0.05). 4. Discussion In this study, we evaluated the effects of DG on the survival of neurons in the hippocampal CA1 region in a transient GCI model established using the 4-VO technique, which caused significant neuronal death in the CA1 layer of the dorsal hippocampus 7 d after transient GCI in a previous study (Sadelli et al., 2017). Studies have demonstrated that the hippocampus plays a crucial role in learning and memory formation, and selective neuronal death in the hippocampal CA1 region is closely related to memory impairment following transient GCI (Badr et al., 2016; Lu et al., 2016; Yang et al., 2017). Our results showed that the administration of DG at doses of 0.5 g/kg (DG-0.5g) and 1 g/kg (DG-1g), but not 0.25 g/kg (DG-0.25g), effectively promoted neuronal survival in the hippocampal CA1 region 7 d after transient GCI. Accumulating evidence has shown that apoptosis is the main mechanism of hippocampal CA1 neuronal death, which exacerbates memory loss during global cerebral ischemic injury (Ji et al., 2014; Wang et al., 2016b). Neuronal apoptosis in the hippocampal CA1 region is present as early as 2 d and reaches a peak 3–7 d after GCI (Arabian et al., 2015; Cheng et al., 2012). Our TUNEL assay also revealed that neuronal 9

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Fig. 8. Effect of DG treatment on 4-HNE and TUNEL expression in the hippocampal CA1 region. Representative images depict (A) 4-HNE and (B) TUNEL expression in the hippocampal CA1 region in the Sham, Control, DG-0.25g, DG-0.5g, DG-1g, and SB20+DG-1g groups 7 d after transient GCI. Representative image (C) shows the negative controls for the 4-HNE antibody. The bar graphs reveal the numbers of (D) 4-HNE- and (E) TUNEL-positive cells in the hippocampal CA1 region among the experimental groups (n = 4–5). *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group. Arrows in (A) and (B) show 4HNE- and TUNEL-positive cells, respectively. Scale bars in (B) and (C) are equal to 10 μm.

transition pore opening and preserved mitochondrial outer membrane integrity, leading to the suppression of Cytc release, and this process occurred independently of Bcl-2-related signaling in in vitro (Roy et al., 2009) and in vivo (Cheng et al., 2017) models. Studies have shown that the pharmacological activation of p38 MAPK/p90RSK signaling exerted neuroprotective effects against caspase-3-mediated apoptosis in the penumbral cortex by upregulating p-Bad-dependent Bcl-2 (Cheng et al., 2016) or p-Bad itself (Cheng et al., 2017) in the subacute phase of focal cerebral ischemia. In addition, in global cerebral ischemic injury, increased p-Bad expression promoted memory recovery by inhibiting neuronal apoptosis in the hippocampal CA1 region (Koh et al., 2006; Lei et al., 2015). Our immunoblotting results revealed that cytosolic pp90RSK expression and cytosolic and mitochondrial p-Bad expression were markedly downregulated in the hippocampus after global cerebral ischemic injury. DG-0.5g and DG-1g treatments effectively restored the aforementioned proteins 7 d after transient GCI. Based on these results, we suggest that DG treatment effectively stabilizes the mitochondrial outer membrane by enhancing p-Bad expression in the hippocampus. Furthermore, the p38 MAPK-mediated anti-apoptotic effects of DG are most likely attributed to the activation of p90RSK/p-Bad-related signaling 7 d after transient global cerebral ischemic injury. The transcription factor CREB, another downstream target of p90RSK, is associated with neuronal survival and differentiation and axon growth, and it regulates the expression of various genes and

(Zhu et al., 2014). Our Western blot results revealed that the expression of phosphorylated p38 MAPK was markedly reduced in the hippocampus following cerebral ischemic insults. DG-0.5g and DG-1g treatments effectively rescued the decreased level of p-p38 MAPK expression induced by I/R injury 7 d after GCI. Our results also revealed that DG treatment did not affect the expression of p-JNK or p-ERK1/2 in the hippocampus. These findings further indicate that DG-0.5g and DG-1g treatments protect hippocampal neurons from apoptosis, possibly by activating p38 MAPK-, but not JNK- or ERK1/2-mediated signaling 7 d after global cerebral I/R injury. Growing evidence has shown that activated p38 MAPK or ERK1/2 phosphorylates p90RSK, which subsequently triggers the phosphorylation of Bad in the ischemic area in the acute (Koh, 2008; Yu et al., 2018) and subacute (Cheng et al., 2016, 2017) phases of cerebral ischemia. Bad, a BH3-only pro-apoptotic protein, binds to the anti-apoptotic Bcl-2 (Bcl-xL) protein and induces Bax translocation from the cytosol to the mitochondria, leading to the activation of Cytc-initiated caspase cascades (Koh, 2008). However, the phosphorylation of Bad promotes interactions between p-Bad and 14-3-3 protein, which prevents the combination of Bad with anti-apoptotic proteins such as Bcl-2 and BclxL at the mitochondrial outer membrane, thereby inhibiting the Baxinduced Cytc/caspase-3-mediated apoptotic pathway (Yu et al., 2018). Some studies have reported that the upregulated expression of mitochondrial p-Bad effectively inhibited mitochondrial permeability 10

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Fig. 9. Effect of DG treatment on BDNF expression in the hippocampal CA1 region. (A) Representative images depict BDNF expression in the hippocampal CA1 region in the Sham, Control, DG-0.25g, DG-0.5g, DG-1g, and SB20+DG-1g groups 7 d after transient GCI. Representative image (B) shows the negative controls for the BDNF antibody. (C) The bar graph reveals the number of BDNF-positive cells in the hippocampal CA1 region among the experimental groups (n = 4–5). *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group. Arrows in (A) show BDNF-positive cells. Scale bars in (A) and (B) are equal to 10 μm.

not alter the levels of cytosolic and mitochondrial AIF expression among the experimental groups. These findings indicate that the neuroprotective effects of DG on global cerebral I/R injury are due to the activation of p38 MAPK/p90RSK/p-Bad signaling, which preserves mitochondrial outer membrane integrity, thereby attenuating oxidative stress-induced Cytc/caspase-3-mediated apoptosis in the hippocampus. Thus, the effects of DG against apoptosis are possibly attributed to the regulation of p38 MAPK/p90RSK/p-Bad- and p90RSK/CREB-, but not AIF-, mediated signaling pathway 7 d after transient GCI. BDNF, a downstream target of CREB, is involved in neurogenesis, synaptic plasticity, and cell survival and plays a pivotal role in learning and memory (Boneva and Yamashima, 2012; Yamada et al., 2002). BDNF expression in the hippocampus is positively correlated with the enhancement of hippocampal CA1 neuronal survival after GCI (Zhang et al., 2018). Previous studies have shown that BDNF exerts protective effects against memory deficits by increasing hippocampal CA1 neuronal survival in the subacute phase of GCI (Lee et al., 2016; Zhang et al., 2018). Our IHC and immunofluorescence (IF) double staining results revealed that the expression levels of BDNF and p-CREB/BDNF were markedly downregulated in the hippocampal CA1 region; DG-0.5g and DG-1g treatments effectively upregulated the expression levels of BDNF and p-CREB/BDNF 7 d after transient GCI. Based on these results, we propose that the effects of DG on I/R injury are most likely attributed to the activation of p38 MAPK-mediated p90RSK/p-Bad and p90RSK/CREB/BDNF signaling in the hippocampus 7 d after transient GCI. To verify the precise role of p38 MAPK in the effects of DG on I/R injury following transient GCI, pretreatment with SB203580 (an inhibitor of p38 MAPK) was conducted in the DG-1g group (SB20+DG-1g group). Immunoblotting, IHC, and IF double staining revealed that SB20+DG-1g treatment effectively abrogated the effects of DG-1g on the activation of p38 MAPK/p90RSK signaling. Subsequently, in the SB20+DG-1g group, the p-Bad-related anti-4-HNE/Cytc/caspase-3mediated apoptotic and CREB/BDNF signaling pathways were suppressed in the hippocampus in the subacute phase of global cerebral ischemic injury. These finding indicate that the neuroprotective effects

proteins such as anti-apoptotic factors (Bcl-2 and Bcl-xL) and neurotrophic factors (nerve growth factor and BDNF) in response to ischemic stimulation (Cheng et al., 2016; Kitagawa, 2007; Zhao et al., 2012). Previous studies have shown that phosphorylated CREB expression in the cytosol and nucleus are positively related; this finding indicates that the activation of CREB results in the transcription of downstream genes in the nucleus following transient cerebral ischemia (Cheng et al., 2016, 2017). The CREB-related signaling pathway is involved in hippocampal synaptic plasticity and plays a pivotal role in learning and memory functions in mammals (Wu et al., 2015). The upregulation of p-CREB and Bcl-2 expression in the hippocampus protects against memory deficits by inhibiting caspase-3-mediated apoptosis in the CA1 region after global cerebral ischemic injury (Han et al., 2013; Wu et al., 2015). In addition, in global cerebral I/R injury, AIF is a caspase-independent death effector that was released from the mitochondria into the nucleus, leading to large-scale DNA fragmentation, and the pharmacological inhibitor of AIF effectively inhibited apoptosis in the hippocampal CA1 region (Niimura et al., 2006). In response to ischemic stress, the production of ROS is increased, and the increased ROS directly attack cellular constituents such as lipids, proteins, and nucleic acids, which cause oxidative damage in the ischemic area (Popa-Wagner et al., 2013). ROS promote mitochondrial outer membrane permeabilization through lipid peroxidation, whereas decreasing ROS production after cerebral I/R injury can preserve the integrity of mitochondrial outer membrane, preventing apoptosis in the ischemic region (Kowaltowski et al., 2001; Liang et al., 2013). Previous studies have demonstrated that the downregulated expression of 4-HNE, a marker of lipid peroxidation, in the hippocampal CA1 region effectively attenuates memory impairment in the subacute phase of transient GCI (Raz et al., 2010; Wang et al., 2013). Our results revealed that cytosolic p-CREB expression was markedly decreased in the hippocampus, and DG-0.5g and DG1g treatments effectively restored p-CREB expression in the subacute phase of transient GCI. Furthermore, the expression of Cytc, cleaved caspase-3, and 4-HNE was markedly upregulated in the hippocampal CA1 region. DG treatment effectively attenuated the expression of these proteins 7 d after global cerebral I/R injury. However, DG treatment did 11

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Fig. 10. Effect of DG treatment on p-CREB/BDNF expression in the hippocampal CA1 region. (A) Representative merged images show the expression of p-CREB(red), BDNF- (green), p-CREB/BDNF- (yellow), and DAPI-(nuclear counterstain, blue) positive cells in the hippocampal CA1 region in the Sham, Control, DG-0.25g, DG-0.5g, DG-1g, and SB20+DG-1g groups 7 d after transient GCI. (B) Representative image depicts a DAPI-stained coronal section of the hippocampus. The solid-line square indicates the selected CA1 region in which the number of p-CREB/BDNF double-labeled cells was calculated. (C) The bar graph reveals the number of p-CREB/ BDNF double-labeled cells in the hippocampal CA1 region among the experimental groups (n = 4–5). *P < 0.05 in comparison with the Sham group; #P < 0.05 in comparison with the Control group. Arrows in (A) show the p-CREB/BDNF double-labeled cells. Scale bars in (A) and (B) are equal to 50 μm and 500 μm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

of DG on global cerebral I/R injury are due to the activation of p38 MAPK/p90RSK signaling. Furthermore, the effects of DG against apoptosis are attributed to the activation of p90RSK/p-Bad-related antiCytc/caspase-3-mediated apoptotic and p90RSK/CREB/BDNF signaling in the hippocampus 7 d after transient GCI.

Author contributions

5. Conclusions

Declaration of competing interest

Chin-Yi Cheng and Yu-Chen Lee designed the experiments. Chin-Yi Cheng performed the experiments, analyzed the data and wrote the manuscript. Shung-Te Kao participated in the conception and design of the study.

The authors have no conflicts of interest to declare.

DG-0.5g and DG-1g treatments effectively rescued hippocampal CA1 neurons from global cerebral I/R injury by activating p38 MAPK/ p90RSK signaling. Subsequently, the effects of DG on promoting p90RSK/p-Bad signaling contribute to the preservation of mitochondrial integrity and the prevention of oxidative stress-induced Cytc/ caspase-3-mediated apoptosis. The p90RSK-induced neuronal survival effects of DG were mediated through CREB/BDNF signaling. Thus, DG treatment protected against cerebral I/R injury by activating p38 MAPK-mediated p90RSK/p-Bad anti-apoptotic and p90RSK/CREB/ BDNF survival signaling in the hippocampus 7 d after GCI. The effects of the A. sinensis extract on I/R injury indicate that A. sinensis is a promising therapeutic strategy for the subacute phase of global cerebral ischemic injury. However, further investigation is required to explore the precise mechanisms of A. sinensis for GCI for future clinical applications.

Acknowledgments This study was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 106-2320-B-039-029-) and China Medical University Hospital (DMR-108-177), Taichung, Taiwan. We thank Miss Shu-Tuan Chiang for kindly helping in DG extract preparation. References Ai, S., Fan, X., Fan, L., Sun, Q., Liu, Y., Tao, X., Dai, K., 2013. Extraction and chemical characterization of Angelica sinensis polysaccharides and its antioxidant activity. Carbohydr. Polym. 94 (2), 731–736.

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