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Overexpression of COX6B1 Protects Against I/R‑Induced Neuronal Injury in Rat Hippocampal Neurons

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Overexpression of COX6B1 Protects Against I/R‑Induced Neuronal Injury in Rat Hippocampal Neurons 4852 MOLECULAR MEDICINE REPORTS 19: 4852-4862, 2019 Overexpression of COX6B1 protects against I/R‑induced neuronal injury in rat hippocampal neurons SHAN YANG1, PENG WU2, JIANWEN XIAO3 and LI JIANG2 1Department of Pediatrics, Nanchuan People's Hospital Affiliated to Chongqing Medical University, Chongqing 408400; Departments of 2Neurology and 3Hematology, Children's Hospital Affiliated to Chongqing Medical University, Chongqing 400014, P.R. China Received June 15, 2018; Accepted March 27, 2019 DOI: 10.3892/mmr.2019.10144 Abstract. Cerebrovascular disease (CVD) is one of the leading the development of novel strategies for the prevention and causes of mortality worldwide. The role of cytochrome c treatment of CVD. oxidase subunit 6B1 (COX6B1) in the central nervous system remains unclear. The present study aimed to analyze the Introduction role of COX6B1 in rat hippocampal neurons extracted from fetal rats. The subcellular localization of the neuron‑specific Cerebrovascular disease (CVD) is a common disease affecting marker microtubule-associated protein 2 was detected by the nervous system and may be caused by various types of immunofluorescence assay. Cell viability was assessed using cerebrovascular injuries (1). Notably, CVD morbidity increased a cell counting kit, and the levels of apoptosis and cytosolic over the past years (2,3). In addition, CVD may affect the Ca2+ were analyzed by flow cytometry. The expression levels quality of life, and ~40% of patients with CVD present severe of the molecular factors downstream to COX6B1 were deter- disabilities (4). Ischemic cerebrovascular disease accounts for mined using reverse transcription-quantitative polymerase ~75% of all CVD cases and presents high disability, mortality chain reaction and western blotting. Reoxygenation following and recurrence rates (5). oxygen-glucose deprivation (OGD) decreased cell viability Treatments aimed to re‑establish blood flow and blood and the expression levels of COX6B1 in a time-dependent perfusion in the ischemic penumbra following cerebral isch- manner, and 60 min of reoxygenation was identified as the emia are necessary to improve the outcomes of patients with optimal time period for establishing an ischemia/reperfusion CVD (6,7). Previous studies demonstrated that metabolic (I/R) model. Overexpression of COX6B1 was demonstrated disorders, oxidative stress, neurotoxicity of the excitatory to reverse the viability of hippocampal neurons following I/R amino acids, inflammatory cell infiltration, cell apoptosis, treatment. Specifically, COX6B1 overexpression decreased microvascular neovascularization and additional factors are the cytosolic concentration of Ca2+ and suppressed neuronal involved in the development of cerebral ischemia-reperfusion apoptosis, which were increased following I/R treatment. (I/R) injury (8,9). The structure and the function of the Furthermore, overexpression of COX6B1 increased the vasculature may be severely impaired following I/R injury, protein expression levels of apoptosis regulator BCL‑2 and influencing the permeability of the blood‑brain barrier and mitochondrial cytochrome c (cyt c), and decreased the protein the mechanisms underlying vascular homeostasis; these expression levels of apoptosis regulator BCL2-associated processes may aggravate cerebral edema, leading to a decline X and cytosolic cyt c in I/R model cells. Collectively, the in the clinical outcomes of the patients (6,10,11). Therefore, present study results suggested that COX6B1 overexpression reducing reperfusion injury following cerebral infarction is an may reverse I/R‑induced neuronal damage by increasing the important topic of research. viability of neurons, by decreasing the cytosolic levels of Ca2+ Cytochrome c oxidase subunit 6B1 (COX6B1) is one of the and by suppressing apoptosis. These results may facilitate subunits of the cytochrome c oxidase (COX) and is expressed in numerous cell types, including yeast and HeLa cells (12). The human COX6B1 gene is located on chromosome 19q13; the coding sequence comprises 261 base pairs and the protein contains 87 amino acids (12-15). The principal role of COX6B1 Correspondence to: Dr Li Jiang, Department of Neurology, Children's Hospital Affiliated to Chongqing Medical University, is to connect two COX monomers to form a dimer, serving 136 Zhongshan Second Road, Chongqing 400014, P.R. China a role in the cell respiratory chain (16). Alterations in the E‑mail: [email protected] dimeric structure of the COX complex affects its function, and defects in the assembly of a functional dimer may cause severe Key words: cerebrovascular disease, cytochrome c oxidase subunit diseases (17,18). A previous study demonstrated that genetic 6B1, hippocampal neurons, apoptosis mutations in the conserved regions of COX6B1 may lead to mitochondrial encephalopathy (16). Mitochondrial diseases are frequently associated with mutations in or an absence of YANG et al: ROLES OF COX6B1 IN CVD 4853 COX (19). Additionally, previous studies reported that abnormal incubated with anti-microtubule-associated protein 2 (MAP2) alternations of COX6B1 markedly affect the function of COX, antibody (1:100; cat. no. ab32454; Abcam, Cambridge, UK) at potentially leading to the occurrence of cerebromyopathy, 4˚C for 24 h, and washed with PBS three times. Subsequently, hydrocephalus and other diseases (16,17). COX6B1 protein the cells were incubated with goat anti‑rabbit fluorescein isothio- may be associated with the development of the central nervous cyanate (FITC)-conjugated immunoglobulin G (IgG; 1:5,000; system (16). Previous studies reported that COX6B1 may protect cat. no. ab6717; Abcam) at 37˚C for 1 h. Following incubation, the myocardium against I/R injury by regulating mitochondrial the cells were washed with PBS three times, and subsequently function (20,21); however, whether COX6B1 protects hippo- incubated with DAPI (Thermo Fisher Scientific, Inc.) at room campal neurons against I/R injury has not been reported, and temperature for 10 min. Stained samples were observed under an the molecular mechanisms underlying the role of COX6B1 in MF43 fluorescence microscope (magnification, x200 and x400; the development of the nervous system remains unclear. Micro-shot Technology Limited, Guangzhou, China). In the present study, a model of I/R injury was constructed using rat hippocampal neurons. Additionally, the molecular Oxygen‑glucose deprivation/reoxygenation (OGD/R) in vitro mechanisms underlying the role of COX6B1 following I/R model establishment. Hippocampal neurons (1x106 cells/ml) injury in hippocampal neurons was investigated. were cultured in neurobasal medium containing 2% B-27 supplement at 37˚C with 5% CO2 for 10 days and cells were Materials and methods exposed to OGD by replacing the neurobasal medium with Earle's balanced salt solution (EBSS) without glucose. The cells Animals. A total of 6 pregnant Sprague‑Dawley (SD) rats at were maintained in an anaerobic chamber (YQX‑II; Shanghai gestational day 18 (age, 2 months; weight, 180-200 g) were CIMO Medical Instrument Co., Ltd., Shanghai, China) with obtained from Guangdong Medical Laboratory Animal Center 95% N2 and 5% CO2 for 60 min at 37˚C. Subsequently, cells (Foshan, China) and maintained at 22±1˚C, 40‑70% humidity, were subjected to reoxygenation treatment (incubation in with free access to food and water, under a 12-h light/dark neurobasal medium in an incubator with 5% CO2 at 37˚C) for cycle. The animal experiments were approved by The Ethics 10, 30 or 60 min prior to further experimentation. Committee of The Nanchuan People's Hospital Affiliated to Chongqing Medical University (Chongqing, China). Cell viability analysis. Following the aforementioned OGD/R treatment, a Cell Counting Kit-8 (CCK-8) was performed to Extraction of hippocampal neurons. Pregnant SD rats were determine the viability of cells following OGD and OGD/R anesthetized by ether, and 12 fetal rats were extracted. The fetal treatment. The hippocampal neurons (2x103 cells/well) were rats were transferred to Petri dishes containing 75% ethanol and seeded into 96-well plates and maintained in an incubator the heads were removed using a guillotine (cat. no. 7950; Ugo for 24 h at 37˚C. Following incubation, 10 µl CCK‑8 reagent Basile SRL, Gemonio, Italy). Subsequently, the hippocampi (Beijing Solarbio Science & Technology Co., Ltd., Beijing, were dissected and digested with 2.5 g/l trypsin (Beyotime China) was added into each well. Cells were transferred into Institute of Biotechnology, Haimen, China) for 15 min at 37˚C. the incubator and maintained for 2 h at 37˚C. A microplate Dulbecco's modified Eagle's medium/Ham's F-12 nutrient reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was mixture (DMEM/F12; Gibco; Thermo Fisher Scientific, Inc., used to detect the absorbance at 450 nm. Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) was added Cell transfection. The pcDNA3.1(+)-empty vector (EV) to the tissues and gently agitated. Cell suspension was filtered and pcDNA3.1(+)‑COX6B1 vector were purchased from through a 400-µm nylon mesh sieve, centrifuged at 1,000 x g GenomeDitech Co., Ltd., (Shanghai, China). Cells were seeded for 5 min and resuspended in DMEM/F12 at a density of (2x105 cells/well) into 6-well plates, serum-starved overnight, 1x106 cells/ml. The neurons obtained were cultured in an and subsequently transfected with COX6B1 vector (20 nM) ® incubator under a humidified atmosphere containing 5% CO2 using Lipofectamine 3000
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