The Role Of BRCA1 Protein In Cerebral Ischemia-reperfusion Injury And Its Underlying Mechanisms

[Background and objectives]There is a large generation of reactive oxygenation species(ROS)following cerebral ischemia-reperfusion.Oxidative stress damage plays an important role in the pathological process of ischemic stroke.Redox imbalance is related to the clinical outcome of acute ischemic stroke,but the underlying mechanisms remain elusive.Therefore,a better understanding of oxidative stress injury after cerebral ischemia-reperfusion may provide clues for finding therapeutic targets against ischemic stroke.Breast cancer susceptibility protein 1(BRCA1)can modulate oxidative stress and reduce intracellular ROS production.Overexpression of BRCA1 in breast cancer cell line can promote the expression of NRF2 and the genes containing antioxidant response element in the promoter,such as NQO1,GPX3 and SOD1.Besides,BRCA1 can repair DNA damage lesion caused by ROS.BRCA1 is expressed in neurons in the hippocampus.Loss funtion of BRCA1 leads to increased DNA damage,limited neurite growth,and aggravated cognitive dysfunction.The expression of BRCA1 is upregulated after myocardial infarction.However,the role of endogenous BRCA1 in early brain injury and long-term neurofunctional recovery following cerebral ischemia-reperfusion has not been fully elucidated.The present study aimed to investigate the role of BRCA1 in the pathogenesis of cerebral ischemia-reperfusion injury and the underlying mechanisms.[Methods]In this study,the middle cerebral artery occlusion(MCAO)model and oxygen glucose deprivation-reoxygenation(OGD/R)model were used as ischemic stroke models in vivo and in vitro.The expression pattern and cellular location of BRCA1 was detected by western blotting,real-time PCR,and immunoflourescence staining.Genetic approaches were conducted to increase BRCA1 expression by lentivirus delivery.The infarct volume and edema volume were detected by TTC staining.Neurological deficits were studied by mNSS.Spontaneous activities were assessed by open filed test.Cognitive function was evaluated by Morris Water Maze test.Neuronal apoptosis was evaluated by TUNEL and PI/hoechst 33342 staining.The oxidative stress injury,the DNA damage and the number of granule cells were assessed by immunoflourescence staining.The levels of ROS,SOD and MDA were detected by ELISA kits.The expressions of molecules in p53-associated apoptosis pathway,4-HNE,DNA damage and repair-related proteins,molecules in NRF2/ARE antioxidant pathway and synaptogenesis-related proteins were detected by western blotting.Co-immunoprecipitation and GST pull-down assay were applied to evaluate whether there is a direct association between BRCA1 and NRF2,and detect the specific binding site.Dual luciferase reporter gene and real-time PCR assay were used to evaluate whether BRCA1 gene could bind to the promoter of NRF2 and regulate the expression of genes in NRF2/ARE pathway.[Results]BRCA1 was expressed in neurons in the peri-infact zone.The temporal expression pattern of BRCA1 was increased first and then decreased following cerebral ischemia-reperfusion.Primary neuron,microglia and astrocyte were cultured and performed with OGD/R.The results demostrated that BRCA1 was mainly expressed in neurons and the expression pattern was similar to the results in vivo.Upregulation of BRCA1 by lentivirus transfection could attenuate cerebral ischemia-reperfusion injury.Overexpression of BRCA1 inhibited p53-associated apoptosis pathway,reduced neuronal apoptosis,decreased oxidative stress positive cells,suppressed the production of ROS and the end product of lipid peroxidation,increased SOD activity and protein expressions of Ku70 and Ku80.Overexpression of BRCA1 reduced infarct volume and edema volume,and attenuated neurological deficits.In vitro,OGD/R-induced neuronal apoptosis,intracellular ROS production and DNA damage were inhibited after transfected with lentivirus.The luciferase reporter gene system confirmed that BRCA1 gene can bind to the promoter of NRF2 gene.Real-time PCR demonstrated that overexpression of BRCA1 can promote the expression of NRF2 and downstream genes including HO-1,NQO1,GPX4,GCLC and SOD2.Co-immunoprecitation and GST pull-down illustrated that BRCA1 can bind with NRF2 via its BRCT domain,thus promoting the expression of molecules in NRF2/ARE pathway.In addition,overexpression of BRCA1 can improve spontaneous activity and cognitive function of MCAO mice through upregulating the expression of synaptogenesis proteins and the density of DCX immunofluorescence in CA1.[Conclusions]The expression pattern of BRCA1 was first increased and then decreased after cerebral ischemia-reperfusion injury.BRCA1 was expressed on neurons.Overexpression of BRCA1 could protect early brain injury.Mechanistically,BRCA1 could interact with NRF2 via its BRCT domain.The cross-talk between BRCA1 and NRF2 acitvated NRF2/ARE signaling pathway and thus inhibited ROS production and lipid peroxidation.Ehanced BRCA1 expression promoted damaged DNA repair through non-homologous end jointing pathway.In addition,upregulation of BRCA1 could promote neurological recovery by potentiating synaptic plasticity in the hippocampus.