Mol Neurobiol DOI 10.1007/s12035-016-9731-7
Arachidonyl-2-Chloroethylamide Alleviates Cerebral Ischemia Injury Through Glycogen Synthase Kinase-3β-Mediated Mitochondrial Biogenesis and Functional Improvement Fuhai Bai 1 & Fan Guo 2 & Tao Jiang 1 & Haidong Wei 3 & Heng Zhou 1 & Hong Yin 2 & Haixing Zhong 1 & Lize Xiong 1 & Qiang Wang 1,4
Received: 24 July 2015 / Accepted: 19 January 2016 # Springer Science+Business Media New York 2016
Abstract Arachidonyl-2-chloroethylamide (ACEA), a highly selective agonist of cannabinoid receptor 1 (CB1R), has been reported to protect neurons in ischemic injury. We sought to investigate whether mitochondrial biogenesis was involved in the therapeutic effect of ACEA in cerebral ischemia. Focal cerebral ischemic injury was induced in adult male Sprague Dawley rats. Intraperitoneal injection of 1 mg/kg ACEA improved neurological behavior, reduced infarct volume, and inhibited apoptosis. The volume and numbers of mitochondria were significantly increased after ACEA administration. Expression of mitochondrial transcription factor A (Tfam), nuclear transcription factor-1 (Nrf-1), and cytochrome C oxidase subunit IV (COX IV) were also significantly upregulated in animals administered ACEA. One thousand nanomoles of ACEA inhibited mitochondrial dysfunction in primary rat cortical neurons exposed to oxygen-glucose deprivation (OGD). Furthermore, ACEA administration increased phosphorylation of glycogen synthase kinase-3β
Fuhai Bai and Fan Guo contributed equally to this work. * Lize Xiong [email protected] * Qiang Wang [email protected]
1
Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
2
Department of Radiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China
3
Department of Anesthesiology, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, China
4
Department of Anesthesiology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China
(GSK-3β) after reperfusion. Phosphorylation of GSK-3β induced mitochondrial biogenesis and preserved mitochondrial function whereas inhibition of phosphatidylinositol 3-kinase (PI3K) dampened phosphorylation of GSK-3β and reversed induction of mitochondrial biogenesis and function following ACEA administration. In conclusion, ACEA could induce mitochondrial biogenesis and improve mitochondrial function at the beginning of cerebral ischemia, thus alleviating cerebral ischemia injury. Phosphorylation of GSK-3β might be involved in the regulation of mitochondrial biogenesis induced by ACEA. Keywords Arachidonyl-2-chloroethylamide . Ischemic reperfusion injury . Mitochondrial biogenesis . Glycogen synthase kinase-3β . Cannabinoid receptor 1
Abbreviations ACEA Arachidonyl-2-chloroethylamide ATP Adenosine triphosphate CB1R Cannabinoid receptor 1 COX IV Cytochrome C oxidase subunit IV DMSO Dimethyl sulfoxide GSK-3β Glycogen synthase kinase-3β LDH Lactate dehydrogenase LY LY294002 MCAO Middle cerebral artery occlusion MDA Malondialdehyde mPTP Mitochondrial permeability transition pore NeuN Neuron-specific nuclear protein Nrf-1 Nuclear transcription factor-1 OGD Oxygen-glucose deprivation PI3K Phosphatidylinositol 3-kinase ROS Reactive oxygen species SD rats Sprague Dawley rats
Mol Neurobiol
TDZD-8 TEM Tfam TTC TUNEL Wrt ΔΨm/MMP
4-Benzyl-2-methyl-1,2,4-thiadiazolidine3,5-dione Transmission electron microscopy Mitochondrial transcription factor A 2,3,5-Triphenyltetrazolium chloride Terminal transferase biotinylated-dUTP nick end labeling Wortmannin Mitochondrial membrane potentials
Introduction Stroke remains one of the leading causes of long-term disability and death [1]. Activation of cell death signaling pathways, reactive oxygen species (ROS) generation, and calcium overload all contribute to brain injury after ischemic reperfusion injury [2]. Mitochondrial dysfunction plays a central role in these mechanisms. Mitochondria are highly dynamic organelles and continually undergo biogenesis, mitophagy, fission, and fusion [3]. Numerous studies have demonstrated that mitochondrial dynamics, particularly mitochondrial biogenesis, play an important role in ischemic injury [4, 5]. Therefore, targeting mitochondrial function represents a promising new therapeutic strategy for the treatment of cerebral ischemia injury. Mitochondrial transcription factor A (Tfam) and nuclear respiratory factor 1 (Nrf-1) are crucial for mitochondrial biogenesis. Nrf-1 controls transcription of nuclear-encoded mitochondrial genes essential for the electron transport chain and induces expression of Tfam [6]. Tfam, which is encoded in the nucleus, plays an important role in initiation of mtDNA replication and transcription of mitochondrial encoded genes [7]. Although many other mitochondrial transcription factors have been identified, Tfam and Nrf-1 appear to be the major regulators of mitochondrial biogenesis. Therefore, cellular levels of these proteins can be used to indicate mitochondrial biogenesis. Canabinoid receptor 1 (CB1R) is a G protein-coupled receptor expressed in neuronal plasma membranes and mitochondrial membranes. CB1R is evolved in neuronal metabolism and other important function [8]. Previous studies indicate that the marijuana-derivative cannabis Δ9-tetrahydrocannabinol could induce neuroprotection by up-regulating the CB1 receptor and maintained mitochondrial function [9, 10]. Other studies have also suggested that the CB1 receptor is involved in mitochondrial biogenesis in non-neural tissues [11]. The highly selective CB1R agonist arachidonyl-2chloroethylamide (ACEA) was reported to protect neurons via the anti-oxidative signaling pathway [12]. However, whether activation of the CB1R modulates mitochondrial biogenesis during neuroprotection against cerebral ischemic injury remains to be shown.
Glycogen synthase kinase-3 (GSK-3β) is a multifunctional molecule and plays important roles in cellular development, differentiation, inflammation, and tissue protection [13, 14]. GSK-3β is implicated in not only the protection of brain tissues in animals suffering from ischemia or brain trauma [15, 16] but also regulation of mitochondrial function and acted to restore impaired mitochondrial biogenesis [17, 18]. Furthermore, we previously observed that GSK-3β was involved in the protection of CB1 activation [19]. However, whether this molecule participates in ACEA regulation of mitochondrial biogenesis is still not clear. Based on these findings, this study was undertaken to investigate the role of mitochondrial biogenesis in the therapeutic activity of ACEA after cerebral ischemia. We also sought to investigate the role of GSK-3β in the therapeutic effect of ACEA to further elucidate the molecular mechanisms.
Materials and Methods Animals All animal procedures were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and were conducted according to the Fourth Military Medical University Guidelines for Animal Experimentation (Xi’an, China). Male Sprague Dawley rats (age 12 weeks; weight 250–350 g) were obtained from Experimental Animal Center of the Fourth Military Medical University. Upon arrival, rats were housed in the groups of two in standard breeding cages and maintained on a 12-h light/dark cycle (light–dark, 08:00–20:00 h), at 21 ± 2 °C, 60–70 % humidity, and with ad libitum access to water and food. All experiments were conducted during the first half of the light period (between 9:00 and 14:00 h).
Middle Cerebral Artery Occlusion Model and Drug Administration Transient focal cerebral ischemia was induced using the intraluminal filament model of middle cerebral artery occlusion (MCAO). In brief, rats were anesthetized with 3 % isoflurane. A heat blunted 6–0 nylon suture was inserted into the right common carotid artery and advanced through the internal carotid artery. The right external carotid artery and the common carotid artery were simultaneously ligated. The suture remained in position for 2 h during this arterial occlusion then was removed to allow subsequent reperfusion. In sham group animals, the vessels were visualized and cleared of connective tissues without inserting the suture.
Mol Neurobiol
Drug Administration Thirty minutes before MCAO, the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin was injected intraperitoneally at 0.6 mg/kg in 10 % Dimethyl sulfoxide (DMSO) [19, 20]. The vehicle group received the same volume of DMSO alone. Ten minutes before MCAO, another selective PI3K inhibitor, LY294002, was injected intracerebroventricularly. Anesthetized rats were secured in stereotaxic apparatus (Narishige, Tokyo, Japan), and the bregma point was exposed. The injection point was located 1.8 mm lateral and 1.5 mm posterior to the bregma in the right hemisphere. A hole was carefully drilled at this point, and a 25-μl Hamilton syringe (80630, Hamilton Co., Reno, NV, USA) was fixed using the stereotaxic apparatus and advanced vertically through this point to a depth of 4.0 mm beneath the dural surface. The right lateral ventricle was then infused with 10 μl 15 mM LY294002 in 50 % DMSO or 10 μl 50 % DMSO at a rate of 1 μl/min, and 10 μM dissolved in the DMSO for neurons [19, 21]. The needle was removed slowly to prevent reflux. One and a half hours after reperfusion, the selective GSK3β inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5dione (TDZD-8) was injected intraperitoneally at a dose of 1 mg/kg in 10 % DMSO [19] and 20 μM in cells [22]. All agents were obtained from Sigma-Aldrich (St. Louis, MO, USA). The selective CB1 receptor agonist, ACEA (Tocris Bioscience, Bristol, UK), was dissolved in 5 % DMSO. One hour after ischemic reperfusion injury, ACEA was administered intraperitoneally at 0.5, 1, or 2 mg/kg [23, 24]. Neurological Score Evaluation and Infarct Assessment Neurological deficit scores were assessed 72 h after reperfusion, in a blinded manner, according to an 18-point neurological scoring system, as previously reported [25]. After neurological score evaluation, the animals were decapitated under deep anesthesia and the brains were removed, frozen, and sectioned into six slices. Brain sections were stained with 2 % (w/v) 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, St. Louis, MO, USA) to evaluate infarct volume, as previously described [19]. Terminal Transferase Biotinylated-dUTP Nick End Labeling Twenty-four hours after reperfusion, neuronal apoptosis was assessed in the brain sections of five animals per group using in situ by terminal transferase biotinylated-dUTP nick end labeling (TUNEL) staining (Roche Applied Science, Penzberg, Germany). Briefly, sections were visualized by
light microscopy (×100 magnification) and the total number of positive cells in 0.10 mm2 pixels was counted [26]. Transmission Electron Microscopy The structure of cortical neuron mitochondria was assessed in three animals per group by transmission electron microscopy (TEM) 4 h after reperfusion. Vibratome sections from the penumbra were selected and transferred into phosphate buffer. The tissues were rinsed in buffer and postfixed with 1 % osmium tetroxide for 1 h, subjected to a graded ethanol dehydration, and infiltrated with a 50:50 mixture of propylene oxide and resin overnight. After 72 h, the tissues were embedded in resin, and 60 nm sections were cut and stained with 2 % uranyl acetate for 20 min and 0.5 % lead citrate for 5 min. The cortex ultrastructure was visualized using a Philips Tecnai 10 transmission electron microscope (Philips, Holland). All analyses were performed in a blinded and non-biased manner. For morphometric studies of mitochondria, 15 randomly selected areas per animal, which included large neuronal-like nuclei covering about one fourth of the visible image, were photographed at ×30,000 magnification and counted (three animals per group) [27]. Immunofluorescent Staining Co-localization of Nrf-1, Tfam, and cytochrome C oxidase subunit IV (COX IV) with neuron-specific nuclear protein (NeuN) was assessed by immunofluorescent staining. Sections were fixed in 4 % PFA then washed three times with phosphate-buffered saline (PBS) and incubated with antibodies directed toward Nrf-1, Tfam, or COX IV and NeuN (Life Technologies; 1:1000 dilution). Primary antibody staining was detected with a fluorescein isothiocyanate (FITC)-conjugated antibody (Kangwei; 1:200 dilution) or Cy3-conjugated antibody (Life Technologies; 1:500 dilution). Sections were visualized by fluorescence microscope (Olympus, Japan) [26]. Western Blot Analysis Rats were decapitated under deep anesthesia, and the hippocampus was rapidly separated and frozen at −80 °C until use. Membrane proteins were extracted from homogenized frozen tissues using a kit, according to the manufacturer’s instructions (Beyotime, Nantong, China). Protein concentration was estimated using a bicinchoninic acid (BCA) kit (Sigma, CA, USA), and 30 μg samples were loaded onto polyacrylamide-SDS gels. Gels were electrophoresed then transferred to PVDF membranes. Membranes were blocked with blocking buffer containing 3 % bovine serum albumin (Sigma) for 60 min and probed with antibodies directed toward Nrf-1 (Santa Cruz; 1:500 dilution), Tfam (Santa Cruz;
Mol Neurobiol
1:500 dilution), COX IV (Abcam; 1:1000 dilution), GSK-3β (Cell Signaling Technology; 1:500 dilutions), p-GSK-3β (Cell Signaling Technology; 1:500 dilutions), β-tubulin (Kangwei; 1:1000 dilution), or GAPDH (Kangwei; 1:1000 dilution) overnight at 4 °C. Primary antibody staining was detected with horseradish peroxidase-conjugated goat-antirabbit and rabbit-anti-goat antibodies (Beyotime, Nantong, China; 1:20,000 dilution). Each sample was immune-blotted in three independent experiments, and average optical density, relative to the internal standard (β-tubulin or GAPDH), was reported and analyzed (Bio-Rad Laboratories, Hercules, CA, USA) [26].
Mitochondrial Function Analysis The effect of ACEA on mitochondrial function was assessed in vitro. Cortical neurons (n = 6) were exposed to oxygenglucose deprivation (OGD) for 1 h, then at the onset of reoxygenation incubated with culture medium containing vehicle (5 % DMSO) or the indicated concentration of ACEA. Control cells were cultured in drug-free medium. Cell viability was assessed by lactate dehydrogenase (LDH) release. To further evaluate the effect of GSK-3β mitochondrial function, 100 nM wortmannin, 10 μM LY294002, or 20 μM TDZD-8 was added at the onset of reoxygenation, as previously described [21, 22, 28]. The vehicle group received the same volume of DMSO. 1. Adenosine triphosphate (ATP) ATP levels were measured using a firefly luciferasebased ATP assay kit (Beyotime, China) according to the manufacturer’s instructions, on ice. Luminance was measured by monochromator microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The protein concentration was measured by BCA protein assay. Emitted light was linearly related to ATP concentration and measured using a microplate luminometer (GloMax® 96, Promega Corporation, WI 53711, USA). Data were normalized to the control group and expressed as percentage of control levels [29]. 2. Mitochondrial permeability transition pore (mPTP) opening Open mPTPs were detected using calcein–cobalt with a mPTP assay kit (Genmed Scientifics Inc., USA) according to the manufacturer’s directions. Briefly, during mPTP opening, calcein is released into cytosol where its fluorescence is quenched by cobalt chloride. Thus, changes in calcein fluorescence in the mitochondrial matrix reflected the degree of mPTP opening. Fluorescence intensity was measured by monochromator microplate reader using excitation at 488 nm and emission at 505 nm (Bio-Rad Laboratories, Hercules, CA, USA).
Mitochondrial protein concentration was measured using the BCA protein assay [30, 31]. 3. Malondialdehyde (MDA) The MDA level was measured using a commercial kit (Jiancheng Bioengineering Inc., Nanjing, China) according to the manufacturer’s instructions with minor modifications. Mitochondria lysate supernatant was incubated with the MDA reagent for 40 min at 95 °C. Absorbance at 532 nm was detected by Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Data were normalized to the control group and expressed as the percentage of control levels [32]. 4. Mitochondrial membrane potentials (ΔΨm) The ΔΨm was measured using a JC-1 kit according to the manufacturer’s instructions, using a specific fluorescent probe for ΔΨm. The cortical neurons were washed by warm PBS and incubated with JC-1 at a final concentration 2 μg/ml for 25 min at 37 °C. The images were captured by confocal laser scanning microscopy (Leica TCS SP5, Wetzlar, Germany) with both red and green channels using identical exposure settings [33]. 5. Reactive oxygen species (ROS) The production of ROS within mitochondria was measured using ROS Assay Kit (Applygen, Beijing, China) according to the manufacturer’s instructions. Briefly, the cortical neurons were incubated with 10 μM DCFH-DA for 20 min at 37 °C then washed three times with warm serum-free DMEM. Fluorescence was visualized by fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) [34].
Flow cytometry Analysis of Apoptosis Apoptosis was assessed using an Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA). Briefly, cells were washed twice in ice-cold PBS and resuspended in binding buffer at 1 × 106 cells/ ml. Cells were incubated with fluorescein-labeled annexin V and propidium iodide (PI) for 20 min. Staining was detected by flow cytometry at 525 and 575 nm, respectively, and analyzed using CellQuest (BD Bioscience, NJ, USA) [35]. Statistical Analysis SPSS 13.0 for Windows (SPSS Inc., Chicago, USA) was used to conduct statistical analyses. All values, except for neurological scores, were presented as the mean ± SD and were analyzed by one-way ANOVA. Group characteristics were compared using post hoc Student–Newman–Keuls tests. Neurological scores were
Mol Neurobiol
expressed as the median and were analyzed using Kruskal–Wallis tests followed by Mann–Whitney U tests with Bonferroni correction. P < 0.05 was considered statistically significant.
Results ACEA, a CB1R Agonist, Attenuated Cerebral Ischemic Injury To assess the therapeutic potential of highly selective CB1R agonist ACEA, we employed a rat model of cerebral ischemia. We recorded infarct sizes and neurological scores 72 h after reperfusion. In order to explore the effective dose of ACEA, we established different dosages and found that the infarct volume was lower in rats administered intraperitoneal injection of 1 or 2 mg/kg ACEA than that in model animals (F4, 25 = 18.60, P < 0.001, Fig. 1b). The graph shows means ± SDs; the number of animals in each group is six (1 mg P < 0.001 vs. MCAO group, 2 mg P < 0.001 vs. MCAO group). Data were analyzed using one-way ANOVA. Neurological scores were also higher in rats administered 1 or 2 mg/kg ACEA (F4,25 = 6.04, P = 0.002, Fig. 1c). The graph show means ± SDs; the number of animals in each group is six (1 mg P = 0.041 vs. MCAO group, 2 mg P = 0.004 vs. MCAO group), and data were analyzed using Kruskal– Wallis tests followed by Mann–Whitney U tests with Bonferroni correction. The effective dose 1 mg/kg ACEA was used for the following experiments. In brain sections of animals administered 1 mg/kg ACEA, the number of TUNEL-positive cells was significantly lower than in model animals (F3,16 = 79.90, P < 0.001, Fig. 1e). The graph shows means ± SDs; the number of animals in each group is five (P = 0.001 vs. MCAO group). Data were analyzed using one-way ANOVA.
ACEA Increased the Number and Volume of Mitochondria After Reperfusion The number and volume of cortical neuron mitochondria were assessed by TEM 4 h after reperfusion. As shown in Fig. 2a, b, the volume of mitochondria was significantly higher in MCAO model animals than in control animals. The volume and number of mitochondria were further increased in MCAO model animals administered ACEA (b1 , F 2,132 = 24.66, P < 0.001; b2, F2,132 = 26.10, P < 0.001, Fig. 2a, b). The graphs show means ± SDs; the number of animals in each group is three (b1 P = 0.003 vs. MCAO group, b2 P < 0.001 vs. MCAO group). Data were analyzed using one-way ANOVA.
ACEA Promoted Mitochondrial Biogenesis After Reperfusion Membrane proteins were extracted from homogenized rat cortex after cerebral ischemia, and expression of transcriptional factors associated with mitochondrial biogenesis was measured by western blotting. Semi-quantitative analysis of blots indicated that intraperitoneal injection of 1 mg/kg ACEA promoted expression of Nrf-1, Tfam, and COX IV. In animals administered ACEA, expression of Nrf-1 was significantly increased after 2 and 4 h, in comparison to model animals (F6,21 = 14.16, P < 0.001), expression of Tfam was significantly increased after 2, 4, and 24 h in comparison to model animals (F 6,21 = 12.55, P < 0.001), and expression of COX IV was significantly increased after 2 and 4 h in comparison to model animals (F6,21 = 10.32, P < 0.001, Fig. 2c–e). All summary graphs show means ± SDs; the number of animals in each group is four (Nrf-1 2 h P < 0.001 vs. MCAO 2 h group, Nrf-1 4 h P = 0.005 vs. MCAO 4 h group; Tfam 2 h P = 0.04 vs. MCAO 2 h group, Tfam 4 h P = 0.018 vs. MCAO 4 h group; Tfam 24 h P = 0.002 vs. MCAO 24 h group; COX IV 2 h P = 0.011 vs. MCAO 2 h group, COX IV 4 h P = 0.002 vs. MCAO 4 h group). Data were analyzed using one-way ANOVA. Co-localization of Nrf-1, Tfam, and COX IV with NeuN in the cortex was assessed by immunofluorescent staining 4 h after ischemia reperfusion. Cell-specific expression of Nrf-1, Tfam, and COX IV was elevated in rats administered ACEA (Fig. 2c–e). ACEA Increased Phosphorylation of GSK-3β After Reperfusion GSK-3β phosphorylation was increased in animals administered ACEA, in comparison to model animals, at 2 and 4 h after reperfusion (F4,15 = 10.76, P < 0.001, Fig. 3a). The graph show means ± SDs; the number of animals in each group is four (ACEA 2 h P = 0.042 vs. MCAO 2 h group, ACEA 4 h P = 0.007 vs. MCAO 4 h group). Data were analyzed by one-way ANOVA. GSK-3β Antagonists Dampened the Beneficial Effects of ACEA on Mitochondrial Biogenesis To further investigate whether the mitochondrial biogenesis induced by ACEA was GSK-3β-dependent, GSK-3β antagonists, wortmannin (wrt), and LY294002 (LY) were administered 30 and 10 min before MCAO, respectively. Expression of Nrf-1, COX IV, and Tfam was lower in animals administered wrt or LY than in animals treated with ACEA (Nrf-1 F6,21 = 22.70, P < 0.001; COX IV F6,
Mol Neurobiol Fig. 1 Effects of ACEA on infarct size and neurological scores in stroke rats. Representative TTC staining of the cerebral infarct (a), infarct size (b), and neurological scores (c) 72 h after reperfusion in the rats with 2 h of cerebral ischemia injury were presented (n = 6). Red area represented the live tissues while white area suggested dead or dying tissue. Representative terminal transferase biotinylateddUTP nick end labeling (TUNEL) staining (d, e). Black arrows indicate TUNEL-positive cells, scale bar = 20 μm. Data are presented as mean ± SD (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 versus MCAO group. Sham, fake operation, the vessels were visualized and cleared of connective tissues without inserting the suture; MCAO, rats exposed to middle cerebral artery occlusion for 2 h; ACEA, after MCAO 1 h rats were administered 1 mg/kg ACEA dissolved in DMSO; vehicle, after MCAO 1 h rats were only administered with DMSO
21 = 10.70, P < 0.001; Tfam F 6,21 = 12.86, P < 0.001, Fig. 3b). All summary graphs show means ± SDs; the number of animals in each group is four (Nrf-1, ACEA P = 0.007 vs. MCAO group, ACEA + wrt P = 0.045 vs. ACEA group, ACEA + LY P = 0.041 vs. ACEA group; COX IV, ACEA P = 0.01 vs. MCAO group, ACEA + wrt P = 0.002 vs. ACEA group, ACEA + LY P = 0.001 vs. ACEA group; Tfam, ACEA P = 0.003 vs. MCAO gro up, ACEA + w rt P = 0 .001 vs. ACEA grou p, ACEA + LY P = 0.001 vs. ACEA group). Data were analyzed by one-way ANOVA.
GSK-3β Phosphorylation Mimicked the Regulatory Effect of ACEA on Mitochondrial Biogenesis To test the role of GSK-3β in the protection effect of ACEA, 1.5 h after reperfusion the selective GSK-3β inhibitor TDZD8 (1 mg/kg) was injected intraperitoneally and phosphorylation of GSK-3β was assessed by western blot. Expression of Nrf-1, COX IV, and Tfam were elevated in animals administered TDZD-8, in comparison to MCAO group (Nrf-1, F4, 15 = 15.21, P < 0.001; COX IV, F 4,15 = 16.82, P < 0.001; Tfam, F4,15 = 8.19, P = 0.001, Fig. 3c). All summary graphs
Mol Neurobiol
Fig. 2 Effects of ACEA on mitochondrial ultrastructure and expression of mitochondrial biogenesis proteins after reperfusion. a, b Representative transmission electron microphotographs showed mitochondrial ultrastructure in the penumbra 4 h after reperfusion. Mitochondrial ultrastructures are indicated by black arrows. Data are mean ± SD (n = 3). Scale bars = 0.5 μm. Western blot analysis and
immunofluorescent staining of Nrf-1 (c), Tfam (d), and COX IV (e) in the sham, MCAO, and ACEA groups. Data are presented as mean ± SD (n = 4). NeuN was presented in green in c and e and red in d. Scale bars = 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001 versus MCAO group. Nrf-1 nuclear respiratory factor 1, Tfam mitochondrial transcription factor A, COX IV cytochrome C oxidase subunit IV
show means ± SDs; the number of animals in each group is four (Nrf-1 P = 0.036 vs. MCAO group, COX IV P < 0.001
vs. MCAO group, Tfam P = 0.007 vs. MCAO group). Data were analyzed using one-way ANOVA.
Mol Neurobiol
Fig. 3 GSK-3β phosphorylation participated in the effect of ACEA of mitochondrial biogenesis after reperfusion. a Western blot indicating expression of phosphorylated and unphosphorylated GSK-3β (Ser-9) in rats. Data are presented as mean ± SD (n = 4). *P < 0.05, ** P
Arachidonyl-2-Chloroethylamide Alleviates Cerebral Ischemia Injury Through Glycogen Synthase Kinase-3β-Mediated Mitochondrial Biogenesis and Functional Improvement Fuhai Bai 1 & Fan Guo 2 & Tao Jiang 1 & Haidong Wei 3 & Heng Zhou 1 & Hong Yin 2 & Haixing Zhong 1 & Lize Xiong 1 & Qiang Wang 1,4
Received: 24 July 2015 / Accepted: 19 January 2016 # Springer Science+Business Media New York 2016
Abstract Arachidonyl-2-chloroethylamide (ACEA), a highly selective agonist of cannabinoid receptor 1 (CB1R), has been reported to protect neurons in ischemic injury. We sought to investigate whether mitochondrial biogenesis was involved in the therapeutic effect of ACEA in cerebral ischemia. Focal cerebral ischemic injury was induced in adult male Sprague Dawley rats. Intraperitoneal injection of 1 mg/kg ACEA improved neurological behavior, reduced infarct volume, and inhibited apoptosis. The volume and numbers of mitochondria were significantly increased after ACEA administration. Expression of mitochondrial transcription factor A (Tfam), nuclear transcription factor-1 (Nrf-1), and cytochrome C oxidase subunit IV (COX IV) were also significantly upregulated in animals administered ACEA. One thousand nanomoles of ACEA inhibited mitochondrial dysfunction in primary rat cortical neurons exposed to oxygen-glucose deprivation (OGD). Furthermore, ACEA administration increased phosphorylation of glycogen synthase kinase-3β
Fuhai Bai and Fan Guo contributed equally to this work. * Lize Xiong [email protected] * Qiang Wang [email protected]
1
Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
2
Department of Radiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China
3
Department of Anesthesiology, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, China
4
Department of Anesthesiology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China
(GSK-3β) after reperfusion. Phosphorylation of GSK-3β induced mitochondrial biogenesis and preserved mitochondrial function whereas inhibition of phosphatidylinositol 3-kinase (PI3K) dampened phosphorylation of GSK-3β and reversed induction of mitochondrial biogenesis and function following ACEA administration. In conclusion, ACEA could induce mitochondrial biogenesis and improve mitochondrial function at the beginning of cerebral ischemia, thus alleviating cerebral ischemia injury. Phosphorylation of GSK-3β might be involved in the regulation of mitochondrial biogenesis induced by ACEA. Keywords Arachidonyl-2-chloroethylamide . Ischemic reperfusion injury . Mitochondrial biogenesis . Glycogen synthase kinase-3β . Cannabinoid receptor 1
Abbreviations ACEA Arachidonyl-2-chloroethylamide ATP Adenosine triphosphate CB1R Cannabinoid receptor 1 COX IV Cytochrome C oxidase subunit IV DMSO Dimethyl sulfoxide GSK-3β Glycogen synthase kinase-3β LDH Lactate dehydrogenase LY LY294002 MCAO Middle cerebral artery occlusion MDA Malondialdehyde mPTP Mitochondrial permeability transition pore NeuN Neuron-specific nuclear protein Nrf-1 Nuclear transcription factor-1 OGD Oxygen-glucose deprivation PI3K Phosphatidylinositol 3-kinase ROS Reactive oxygen species SD rats Sprague Dawley rats
Mol Neurobiol
TDZD-8 TEM Tfam TTC TUNEL Wrt ΔΨm/MMP
4-Benzyl-2-methyl-1,2,4-thiadiazolidine3,5-dione Transmission electron microscopy Mitochondrial transcription factor A 2,3,5-Triphenyltetrazolium chloride Terminal transferase biotinylated-dUTP nick end labeling Wortmannin Mitochondrial membrane potentials
Introduction Stroke remains one of the leading causes of long-term disability and death [1]. Activation of cell death signaling pathways, reactive oxygen species (ROS) generation, and calcium overload all contribute to brain injury after ischemic reperfusion injury [2]. Mitochondrial dysfunction plays a central role in these mechanisms. Mitochondria are highly dynamic organelles and continually undergo biogenesis, mitophagy, fission, and fusion [3]. Numerous studies have demonstrated that mitochondrial dynamics, particularly mitochondrial biogenesis, play an important role in ischemic injury [4, 5]. Therefore, targeting mitochondrial function represents a promising new therapeutic strategy for the treatment of cerebral ischemia injury. Mitochondrial transcription factor A (Tfam) and nuclear respiratory factor 1 (Nrf-1) are crucial for mitochondrial biogenesis. Nrf-1 controls transcription of nuclear-encoded mitochondrial genes essential for the electron transport chain and induces expression of Tfam [6]. Tfam, which is encoded in the nucleus, plays an important role in initiation of mtDNA replication and transcription of mitochondrial encoded genes [7]. Although many other mitochondrial transcription factors have been identified, Tfam and Nrf-1 appear to be the major regulators of mitochondrial biogenesis. Therefore, cellular levels of these proteins can be used to indicate mitochondrial biogenesis. Canabinoid receptor 1 (CB1R) is a G protein-coupled receptor expressed in neuronal plasma membranes and mitochondrial membranes. CB1R is evolved in neuronal metabolism and other important function [8]. Previous studies indicate that the marijuana-derivative cannabis Δ9-tetrahydrocannabinol could induce neuroprotection by up-regulating the CB1 receptor and maintained mitochondrial function [9, 10]. Other studies have also suggested that the CB1 receptor is involved in mitochondrial biogenesis in non-neural tissues [11]. The highly selective CB1R agonist arachidonyl-2chloroethylamide (ACEA) was reported to protect neurons via the anti-oxidative signaling pathway [12]. However, whether activation of the CB1R modulates mitochondrial biogenesis during neuroprotection against cerebral ischemic injury remains to be shown.
Glycogen synthase kinase-3 (GSK-3β) is a multifunctional molecule and plays important roles in cellular development, differentiation, inflammation, and tissue protection [13, 14]. GSK-3β is implicated in not only the protection of brain tissues in animals suffering from ischemia or brain trauma [15, 16] but also regulation of mitochondrial function and acted to restore impaired mitochondrial biogenesis [17, 18]. Furthermore, we previously observed that GSK-3β was involved in the protection of CB1 activation [19]. However, whether this molecule participates in ACEA regulation of mitochondrial biogenesis is still not clear. Based on these findings, this study was undertaken to investigate the role of mitochondrial biogenesis in the therapeutic activity of ACEA after cerebral ischemia. We also sought to investigate the role of GSK-3β in the therapeutic effect of ACEA to further elucidate the molecular mechanisms.
Materials and Methods Animals All animal procedures were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and were conducted according to the Fourth Military Medical University Guidelines for Animal Experimentation (Xi’an, China). Male Sprague Dawley rats (age 12 weeks; weight 250–350 g) were obtained from Experimental Animal Center of the Fourth Military Medical University. Upon arrival, rats were housed in the groups of two in standard breeding cages and maintained on a 12-h light/dark cycle (light–dark, 08:00–20:00 h), at 21 ± 2 °C, 60–70 % humidity, and with ad libitum access to water and food. All experiments were conducted during the first half of the light period (between 9:00 and 14:00 h).
Middle Cerebral Artery Occlusion Model and Drug Administration Transient focal cerebral ischemia was induced using the intraluminal filament model of middle cerebral artery occlusion (MCAO). In brief, rats were anesthetized with 3 % isoflurane. A heat blunted 6–0 nylon suture was inserted into the right common carotid artery and advanced through the internal carotid artery. The right external carotid artery and the common carotid artery were simultaneously ligated. The suture remained in position for 2 h during this arterial occlusion then was removed to allow subsequent reperfusion. In sham group animals, the vessels were visualized and cleared of connective tissues without inserting the suture.
Mol Neurobiol
Drug Administration Thirty minutes before MCAO, the selective phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin was injected intraperitoneally at 0.6 mg/kg in 10 % Dimethyl sulfoxide (DMSO) [19, 20]. The vehicle group received the same volume of DMSO alone. Ten minutes before MCAO, another selective PI3K inhibitor, LY294002, was injected intracerebroventricularly. Anesthetized rats were secured in stereotaxic apparatus (Narishige, Tokyo, Japan), and the bregma point was exposed. The injection point was located 1.8 mm lateral and 1.5 mm posterior to the bregma in the right hemisphere. A hole was carefully drilled at this point, and a 25-μl Hamilton syringe (80630, Hamilton Co., Reno, NV, USA) was fixed using the stereotaxic apparatus and advanced vertically through this point to a depth of 4.0 mm beneath the dural surface. The right lateral ventricle was then infused with 10 μl 15 mM LY294002 in 50 % DMSO or 10 μl 50 % DMSO at a rate of 1 μl/min, and 10 μM dissolved in the DMSO for neurons [19, 21]. The needle was removed slowly to prevent reflux. One and a half hours after reperfusion, the selective GSK3β inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5dione (TDZD-8) was injected intraperitoneally at a dose of 1 mg/kg in 10 % DMSO [19] and 20 μM in cells [22]. All agents were obtained from Sigma-Aldrich (St. Louis, MO, USA). The selective CB1 receptor agonist, ACEA (Tocris Bioscience, Bristol, UK), was dissolved in 5 % DMSO. One hour after ischemic reperfusion injury, ACEA was administered intraperitoneally at 0.5, 1, or 2 mg/kg [23, 24]. Neurological Score Evaluation and Infarct Assessment Neurological deficit scores were assessed 72 h after reperfusion, in a blinded manner, according to an 18-point neurological scoring system, as previously reported [25]. After neurological score evaluation, the animals were decapitated under deep anesthesia and the brains were removed, frozen, and sectioned into six slices. Brain sections were stained with 2 % (w/v) 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, St. Louis, MO, USA) to evaluate infarct volume, as previously described [19]. Terminal Transferase Biotinylated-dUTP Nick End Labeling Twenty-four hours after reperfusion, neuronal apoptosis was assessed in the brain sections of five animals per group using in situ by terminal transferase biotinylated-dUTP nick end labeling (TUNEL) staining (Roche Applied Science, Penzberg, Germany). Briefly, sections were visualized by
light microscopy (×100 magnification) and the total number of positive cells in 0.10 mm2 pixels was counted [26]. Transmission Electron Microscopy The structure of cortical neuron mitochondria was assessed in three animals per group by transmission electron microscopy (TEM) 4 h after reperfusion. Vibratome sections from the penumbra were selected and transferred into phosphate buffer. The tissues were rinsed in buffer and postfixed with 1 % osmium tetroxide for 1 h, subjected to a graded ethanol dehydration, and infiltrated with a 50:50 mixture of propylene oxide and resin overnight. After 72 h, the tissues were embedded in resin, and 60 nm sections were cut and stained with 2 % uranyl acetate for 20 min and 0.5 % lead citrate for 5 min. The cortex ultrastructure was visualized using a Philips Tecnai 10 transmission electron microscope (Philips, Holland). All analyses were performed in a blinded and non-biased manner. For morphometric studies of mitochondria, 15 randomly selected areas per animal, which included large neuronal-like nuclei covering about one fourth of the visible image, were photographed at ×30,000 magnification and counted (three animals per group) [27]. Immunofluorescent Staining Co-localization of Nrf-1, Tfam, and cytochrome C oxidase subunit IV (COX IV) with neuron-specific nuclear protein (NeuN) was assessed by immunofluorescent staining. Sections were fixed in 4 % PFA then washed three times with phosphate-buffered saline (PBS) and incubated with antibodies directed toward Nrf-1, Tfam, or COX IV and NeuN (Life Technologies; 1:1000 dilution). Primary antibody staining was detected with a fluorescein isothiocyanate (FITC)-conjugated antibody (Kangwei; 1:200 dilution) or Cy3-conjugated antibody (Life Technologies; 1:500 dilution). Sections were visualized by fluorescence microscope (Olympus, Japan) [26]. Western Blot Analysis Rats were decapitated under deep anesthesia, and the hippocampus was rapidly separated and frozen at −80 °C until use. Membrane proteins were extracted from homogenized frozen tissues using a kit, according to the manufacturer’s instructions (Beyotime, Nantong, China). Protein concentration was estimated using a bicinchoninic acid (BCA) kit (Sigma, CA, USA), and 30 μg samples were loaded onto polyacrylamide-SDS gels. Gels were electrophoresed then transferred to PVDF membranes. Membranes were blocked with blocking buffer containing 3 % bovine serum albumin (Sigma) for 60 min and probed with antibodies directed toward Nrf-1 (Santa Cruz; 1:500 dilution), Tfam (Santa Cruz;
Mol Neurobiol
1:500 dilution), COX IV (Abcam; 1:1000 dilution), GSK-3β (Cell Signaling Technology; 1:500 dilutions), p-GSK-3β (Cell Signaling Technology; 1:500 dilutions), β-tubulin (Kangwei; 1:1000 dilution), or GAPDH (Kangwei; 1:1000 dilution) overnight at 4 °C. Primary antibody staining was detected with horseradish peroxidase-conjugated goat-antirabbit and rabbit-anti-goat antibodies (Beyotime, Nantong, China; 1:20,000 dilution). Each sample was immune-blotted in three independent experiments, and average optical density, relative to the internal standard (β-tubulin or GAPDH), was reported and analyzed (Bio-Rad Laboratories, Hercules, CA, USA) [26].
Mitochondrial Function Analysis The effect of ACEA on mitochondrial function was assessed in vitro. Cortical neurons (n = 6) were exposed to oxygenglucose deprivation (OGD) for 1 h, then at the onset of reoxygenation incubated with culture medium containing vehicle (5 % DMSO) or the indicated concentration of ACEA. Control cells were cultured in drug-free medium. Cell viability was assessed by lactate dehydrogenase (LDH) release. To further evaluate the effect of GSK-3β mitochondrial function, 100 nM wortmannin, 10 μM LY294002, or 20 μM TDZD-8 was added at the onset of reoxygenation, as previously described [21, 22, 28]. The vehicle group received the same volume of DMSO. 1. Adenosine triphosphate (ATP) ATP levels were measured using a firefly luciferasebased ATP assay kit (Beyotime, China) according to the manufacturer’s instructions, on ice. Luminance was measured by monochromator microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The protein concentration was measured by BCA protein assay. Emitted light was linearly related to ATP concentration and measured using a microplate luminometer (GloMax® 96, Promega Corporation, WI 53711, USA). Data were normalized to the control group and expressed as percentage of control levels [29]. 2. Mitochondrial permeability transition pore (mPTP) opening Open mPTPs were detected using calcein–cobalt with a mPTP assay kit (Genmed Scientifics Inc., USA) according to the manufacturer’s directions. Briefly, during mPTP opening, calcein is released into cytosol where its fluorescence is quenched by cobalt chloride. Thus, changes in calcein fluorescence in the mitochondrial matrix reflected the degree of mPTP opening. Fluorescence intensity was measured by monochromator microplate reader using excitation at 488 nm and emission at 505 nm (Bio-Rad Laboratories, Hercules, CA, USA).
Mitochondrial protein concentration was measured using the BCA protein assay [30, 31]. 3. Malondialdehyde (MDA) The MDA level was measured using a commercial kit (Jiancheng Bioengineering Inc., Nanjing, China) according to the manufacturer’s instructions with minor modifications. Mitochondria lysate supernatant was incubated with the MDA reagent for 40 min at 95 °C. Absorbance at 532 nm was detected by Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Data were normalized to the control group and expressed as the percentage of control levels [32]. 4. Mitochondrial membrane potentials (ΔΨm) The ΔΨm was measured using a JC-1 kit according to the manufacturer’s instructions, using a specific fluorescent probe for ΔΨm. The cortical neurons were washed by warm PBS and incubated with JC-1 at a final concentration 2 μg/ml for 25 min at 37 °C. The images were captured by confocal laser scanning microscopy (Leica TCS SP5, Wetzlar, Germany) with both red and green channels using identical exposure settings [33]. 5. Reactive oxygen species (ROS) The production of ROS within mitochondria was measured using ROS Assay Kit (Applygen, Beijing, China) according to the manufacturer’s instructions. Briefly, the cortical neurons were incubated with 10 μM DCFH-DA for 20 min at 37 °C then washed three times with warm serum-free DMEM. Fluorescence was visualized by fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) [34].
Flow cytometry Analysis of Apoptosis Apoptosis was assessed using an Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA). Briefly, cells were washed twice in ice-cold PBS and resuspended in binding buffer at 1 × 106 cells/ ml. Cells were incubated with fluorescein-labeled annexin V and propidium iodide (PI) for 20 min. Staining was detected by flow cytometry at 525 and 575 nm, respectively, and analyzed using CellQuest (BD Bioscience, NJ, USA) [35]. Statistical Analysis SPSS 13.0 for Windows (SPSS Inc., Chicago, USA) was used to conduct statistical analyses. All values, except for neurological scores, were presented as the mean ± SD and were analyzed by one-way ANOVA. Group characteristics were compared using post hoc Student–Newman–Keuls tests. Neurological scores were
Mol Neurobiol
expressed as the median and were analyzed using Kruskal–Wallis tests followed by Mann–Whitney U tests with Bonferroni correction. P < 0.05 was considered statistically significant.
Results ACEA, a CB1R Agonist, Attenuated Cerebral Ischemic Injury To assess the therapeutic potential of highly selective CB1R agonist ACEA, we employed a rat model of cerebral ischemia. We recorded infarct sizes and neurological scores 72 h after reperfusion. In order to explore the effective dose of ACEA, we established different dosages and found that the infarct volume was lower in rats administered intraperitoneal injection of 1 or 2 mg/kg ACEA than that in model animals (F4, 25 = 18.60, P < 0.001, Fig. 1b). The graph shows means ± SDs; the number of animals in each group is six (1 mg P < 0.001 vs. MCAO group, 2 mg P < 0.001 vs. MCAO group). Data were analyzed using one-way ANOVA. Neurological scores were also higher in rats administered 1 or 2 mg/kg ACEA (F4,25 = 6.04, P = 0.002, Fig. 1c). The graph show means ± SDs; the number of animals in each group is six (1 mg P = 0.041 vs. MCAO group, 2 mg P = 0.004 vs. MCAO group), and data were analyzed using Kruskal– Wallis tests followed by Mann–Whitney U tests with Bonferroni correction. The effective dose 1 mg/kg ACEA was used for the following experiments. In brain sections of animals administered 1 mg/kg ACEA, the number of TUNEL-positive cells was significantly lower than in model animals (F3,16 = 79.90, P < 0.001, Fig. 1e). The graph shows means ± SDs; the number of animals in each group is five (P = 0.001 vs. MCAO group). Data were analyzed using one-way ANOVA.
ACEA Increased the Number and Volume of Mitochondria After Reperfusion The number and volume of cortical neuron mitochondria were assessed by TEM 4 h after reperfusion. As shown in Fig. 2a, b, the volume of mitochondria was significantly higher in MCAO model animals than in control animals. The volume and number of mitochondria were further increased in MCAO model animals administered ACEA (b1 , F 2,132 = 24.66, P < 0.001; b2, F2,132 = 26.10, P < 0.001, Fig. 2a, b). The graphs show means ± SDs; the number of animals in each group is three (b1 P = 0.003 vs. MCAO group, b2 P < 0.001 vs. MCAO group). Data were analyzed using one-way ANOVA.
ACEA Promoted Mitochondrial Biogenesis After Reperfusion Membrane proteins were extracted from homogenized rat cortex after cerebral ischemia, and expression of transcriptional factors associated with mitochondrial biogenesis was measured by western blotting. Semi-quantitative analysis of blots indicated that intraperitoneal injection of 1 mg/kg ACEA promoted expression of Nrf-1, Tfam, and COX IV. In animals administered ACEA, expression of Nrf-1 was significantly increased after 2 and 4 h, in comparison to model animals (F6,21 = 14.16, P < 0.001), expression of Tfam was significantly increased after 2, 4, and 24 h in comparison to model animals (F 6,21 = 12.55, P < 0.001), and expression of COX IV was significantly increased after 2 and 4 h in comparison to model animals (F6,21 = 10.32, P < 0.001, Fig. 2c–e). All summary graphs show means ± SDs; the number of animals in each group is four (Nrf-1 2 h P < 0.001 vs. MCAO 2 h group, Nrf-1 4 h P = 0.005 vs. MCAO 4 h group; Tfam 2 h P = 0.04 vs. MCAO 2 h group, Tfam 4 h P = 0.018 vs. MCAO 4 h group; Tfam 24 h P = 0.002 vs. MCAO 24 h group; COX IV 2 h P = 0.011 vs. MCAO 2 h group, COX IV 4 h P = 0.002 vs. MCAO 4 h group). Data were analyzed using one-way ANOVA. Co-localization of Nrf-1, Tfam, and COX IV with NeuN in the cortex was assessed by immunofluorescent staining 4 h after ischemia reperfusion. Cell-specific expression of Nrf-1, Tfam, and COX IV was elevated in rats administered ACEA (Fig. 2c–e). ACEA Increased Phosphorylation of GSK-3β After Reperfusion GSK-3β phosphorylation was increased in animals administered ACEA, in comparison to model animals, at 2 and 4 h after reperfusion (F4,15 = 10.76, P < 0.001, Fig. 3a). The graph show means ± SDs; the number of animals in each group is four (ACEA 2 h P = 0.042 vs. MCAO 2 h group, ACEA 4 h P = 0.007 vs. MCAO 4 h group). Data were analyzed by one-way ANOVA. GSK-3β Antagonists Dampened the Beneficial Effects of ACEA on Mitochondrial Biogenesis To further investigate whether the mitochondrial biogenesis induced by ACEA was GSK-3β-dependent, GSK-3β antagonists, wortmannin (wrt), and LY294002 (LY) were administered 30 and 10 min before MCAO, respectively. Expression of Nrf-1, COX IV, and Tfam was lower in animals administered wrt or LY than in animals treated with ACEA (Nrf-1 F6,21 = 22.70, P < 0.001; COX IV F6,
Mol Neurobiol Fig. 1 Effects of ACEA on infarct size and neurological scores in stroke rats. Representative TTC staining of the cerebral infarct (a), infarct size (b), and neurological scores (c) 72 h after reperfusion in the rats with 2 h of cerebral ischemia injury were presented (n = 6). Red area represented the live tissues while white area suggested dead or dying tissue. Representative terminal transferase biotinylateddUTP nick end labeling (TUNEL) staining (d, e). Black arrows indicate TUNEL-positive cells, scale bar = 20 μm. Data are presented as mean ± SD (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 versus MCAO group. Sham, fake operation, the vessels were visualized and cleared of connective tissues without inserting the suture; MCAO, rats exposed to middle cerebral artery occlusion for 2 h; ACEA, after MCAO 1 h rats were administered 1 mg/kg ACEA dissolved in DMSO; vehicle, after MCAO 1 h rats were only administered with DMSO
21 = 10.70, P < 0.001; Tfam F 6,21 = 12.86, P < 0.001, Fig. 3b). All summary graphs show means ± SDs; the number of animals in each group is four (Nrf-1, ACEA P = 0.007 vs. MCAO group, ACEA + wrt P = 0.045 vs. ACEA group, ACEA + LY P = 0.041 vs. ACEA group; COX IV, ACEA P = 0.01 vs. MCAO group, ACEA + wrt P = 0.002 vs. ACEA group, ACEA + LY P = 0.001 vs. ACEA group; Tfam, ACEA P = 0.003 vs. MCAO gro up, ACEA + w rt P = 0 .001 vs. ACEA grou p, ACEA + LY P = 0.001 vs. ACEA group). Data were analyzed by one-way ANOVA.
GSK-3β Phosphorylation Mimicked the Regulatory Effect of ACEA on Mitochondrial Biogenesis To test the role of GSK-3β in the protection effect of ACEA, 1.5 h after reperfusion the selective GSK-3β inhibitor TDZD8 (1 mg/kg) was injected intraperitoneally and phosphorylation of GSK-3β was assessed by western blot. Expression of Nrf-1, COX IV, and Tfam were elevated in animals administered TDZD-8, in comparison to MCAO group (Nrf-1, F4, 15 = 15.21, P < 0.001; COX IV, F 4,15 = 16.82, P < 0.001; Tfam, F4,15 = 8.19, P = 0.001, Fig. 3c). All summary graphs
Mol Neurobiol
Fig. 2 Effects of ACEA on mitochondrial ultrastructure and expression of mitochondrial biogenesis proteins after reperfusion. a, b Representative transmission electron microphotographs showed mitochondrial ultrastructure in the penumbra 4 h after reperfusion. Mitochondrial ultrastructures are indicated by black arrows. Data are mean ± SD (n = 3). Scale bars = 0.5 μm. Western blot analysis and
immunofluorescent staining of Nrf-1 (c), Tfam (d), and COX IV (e) in the sham, MCAO, and ACEA groups. Data are presented as mean ± SD (n = 4). NeuN was presented in green in c and e and red in d. Scale bars = 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001 versus MCAO group. Nrf-1 nuclear respiratory factor 1, Tfam mitochondrial transcription factor A, COX IV cytochrome C oxidase subunit IV
show means ± SDs; the number of animals in each group is four (Nrf-1 P = 0.036 vs. MCAO group, COX IV P < 0.001
vs. MCAO group, Tfam P = 0.007 vs. MCAO group). Data were analyzed using one-way ANOVA.
Mol Neurobiol
Fig. 3 GSK-3β phosphorylation participated in the effect of ACEA of mitochondrial biogenesis after reperfusion. a Western blot indicating expression of phosphorylated and unphosphorylated GSK-3β (Ser-9) in rats. Data are presented as mean ± SD (n = 4). *P < 0.05, ** P
