Abstract： The purpose of this study was to investigate the relationship between the edaravone protective effects on neurons and Nrf2 signaling molecules in cerebral ischemia-reperfusion injury. In vivo experiments were performed by establishing a middle cerebral artery occlusion model in SD rats, and in vitro experiments were performed using hydrogen peroxide (H2O2) to damage PC12 cells to establish an oxidative stress model. The pathological status of the brain was detected by TTC, HE and Nissl staining. Reactive oxygen species (ROS), malondialdehyde (MDA) contents, and superoxide dismutase (SOD) activity were measured to reflect the level of oxidative stress. In addition, Hoechst 33342 staining and measurement of mitochondrial membrane potential (MTP) were used to investigate the cell-level damage. Nrf2 expression was determined using immunohistochemistry and Western blotting. Nrf2 knockout PC12 cells were constructed to confirm the role of Nrf2 signaling molecules in inhibiting oxidative stress-induced injury. The results suggest that after edaravone administration, both animal-level and TTC staining confirmed that cerebral ischemia-reperfusion injury (CIRI) rats had reduced cerebral infarcted areas (P <0.001), ROS and MDA levels decreased (P<0.01), SOD activity increased (P<0.01); at the cell level, apoptotic cells decreased (P<0.05), MTP increased (P<0.01), ROS and MDA levels decreased, and the SOD activity increased (P <0.01); at the molecular level, immunohistochemistry and Western blotting showed that the Nrf2 protein level was increased compared with the control group. H2O2 induced damage in Nrf2-knockout PC12 cell was aggravated and the therapeutic effect of edaravone was significantly weakened. In summary, Nrf2 plays a key role in edaravone in reducing cerebral ischemia-reperfusion-induced oxidative stress injury.
[1]Vos T, Barber RM, Bell B, et al.Global,regional,and national incidence,prevalence,and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries,1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[J].Lancet, 2015, 386(9995):743-800[2]Hofmeijer J, van Putten MJ.Ischemic cerebral damage: an appraisal of synaptic failure[J].Stroke, 2012, 43(2):607-615[3]Khanna A, Kahle KT, Walcott BP, et al.Disruption of ion homeostasis in the neurogliovascular unit underlies the pathogenesis of ischemic cerebral edema[J].Transl Stroke Res, 2014, 5(1):3-16[4] Sun C, Fukushi Y, Wang Y, et al.Astrocytes protect neurons in the hippocampal CA3 against ischemia by suppressing the intracellular Ca2+ overload[J]. Front Cell Neurosci, 2018, 12: 280[5]Yang J, Liu, M, Zhou, J, et al.Edaravone for acute intracerebral haemorrhage[J].Cochrane Database Syst Rev, 2011, 16(2):C-D007755[6] MacDougall G, Anderton RS, Mastaglia FL, et al.Mitochondria and neuroprotection in stroke: Cationic arginine-rich peptides (CARPs) as a novel class of mitochondria-targeted neuroprotective therapeutics[J]. Neurobiol Dis, 2019, 121: 17-33[7] Song Y, Ding W, Bei Y, et al.Insulin is a potential antioxidant for diabetes-associated cognitive decline via regulating Nrf2 dependent antioxidant enzymes[J]. Biomed Pharmacother, 2018, 104: 474-484[8]Cook AL, Vitale AM, Ravishankar S, et al.NRF2 activation restores disease related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson's disease[J].PLoS One, 2011, 6(7):e21907-[9]Ramsey CP, Glass CA, Montgomery MB, et al.Expression of Nrf2 in neurodegenerative diseases[J].J Neuropathol Exp Neurol, 2007, 66(1):75-85[10]Sarlette A, Krampfl K, Grothe C, et al.Nuclear erythroid 2-related factor 2-antioxidative response element signaling pathway in motor cortex and spinal cord in amyotrophic lateral sclerosis[J].J Neuropathol Exp Neurol, 2008, 67(11):1055-1062[11] Raina R, Sen D.Can crosstalk between DOR and PARP reduce oxidative stress mediated neurodegeneration? [J]. Neurochem Int, 2018, 112: 206-218[12]Kikuchi K, Kawahara KI, Miyagi N, et al.Edaravone: A new therapeutic approach for the treatment of acute stroke[J].Med Hypotheses, 2010, 75(6):583-585[13] Song Y, Bei Y, Xiao Y, et al.Edaravone, a free radical scavenger, protects neuronal cells’ mitochondria from ischemia by inactivating another new critical factor of the 5-lipoxygenase pathway affecting the arachidonic acid metabolism[J]. Brain Res, 2018, 1690: 96-104[14]Gao F, Wang S, Guo Y, et al.Protective effects of repetitive transcranial magnetic stimulation in a rat model of transient cerebral ischaemia: a microPET study[J].Eur J Nucl Med Mol Imaging, 2010, 37(5):954-961[15]Kapadia R, Tureyen K, Bowen KK, et al.Decreased brain damage and curtailed inflammation in transcription factor CCAATenhancer binding protein beta knockout mice following transient focal cerebral ischemia[J].J Neurochem, 2006, 98(6):1718-1731[16] Li C, Che LH, Ji TF, et al.Effects of the TLR4 signaling pathway on apoptosis of neuronal cells in diabetes mellitus complicated with cerebral infarction in a rat model[J]. Sci Rep, 2017, 7: 43834[17]Song Y, Wu ZC, Ding W, et al.NF-κB in mitochondria regulates PC12 cell apoptosis following lipopolysaccharide-induced injury[J].J Zhejiang Univ Sci B, 2018, 19(6):425-435[18]Chen B, Yue R, Yang Y, et al.Protective effects of (E)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeine against hydrogen peroxide-induced injury in PC12 cells[J].Neurochem Res, 2015, 40(3):531-541[19]Wang H, Naghavi M, Allen C, et al.Global,regional,and national life expectancy,all-cause mortality,and cause-specific mortality for 249 causes of death,1980-2015: a systematic analysis for the Global Burden of Disease Study 2015[J].Lancet, 2016, 388(10053):1459-1544[20]Salim S.Oxidative stress and the central nervous system[J].J Pharmacol Exp Ther, 2017, 360(1):201-205[21]Ozkul A, Akyol A, Yenisey C, et al.Oxidative stress in acute ischemic stroke[J].J Clin Neurosci, 2007, 14(11):1062-1066[22]Sies H.Oxidative stress: oxidants and antioxidants[J].Exp Physiol, 1997, 82(2):291-295[23]Circu ML, Aw TY.Reactive oxygen species,cellular redox systems,and apoptosis[J].Free Radic Biol Med, 2010, 48(6):749-762[24] Liao L, Shi J, Jiang C, et al.Activation of anti-oxidant of curcumin pyrazole derivatives through preservation of mitochondria function and Nrf2 signaling pathway[J]. Neurochem Int, 2019, 125: 82-90[25]Mancuso M, Coppede F, Migliore L, et al.Mitochondrial dysfunction,oxidative stress and neurodegeneration[J].J Alzheimers Dis, 2006, 10(1):59-73[26]Friedman JR, Nunnari J.Mitochondrial form and function[J].Nature, 2014, 505(7483):335-343[27] Kalogeris T, Bao Y, Korthuis RJ.Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning[J]. Redox Biol, 2014, 2: 702-714[28]Kobayashi A, Kang MI, Okawa H, et al.Oxidative stress sensor Keap1 functions as an adaptor for Cul3- based E3 ligase to regulate proteasomal degradation of Nrf2[J].Mol Cell Biol, 2004, 24(16):7130-7139[29]de Vries HE, Witte M, Hondius D, et al.Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease?[J].Free Radic Biol Med, 2008, 45(10):1375-1383[30]Dong J, Sulik KK, Chen SY.Nrf2-mediated transcriptional induction of antioxidant response in mouse embryos exposed to ethanol in vivo: implications for the prevention of fetal alcohol spectrum disorders[J].Antioxid Redox Signal, 2008, 10(12):2023-2033
