The Neuroprotective Effects Of Naringenin On Cerebral Ischemia Reperfusion Injury:Focused Mitochondrial Dysfunction And Nrf2 Mediated Antioxidant Stress

ObjectiveCerebrovascular disease is a common disease,which with a high incidence,mortality,disability and ischemic cerebrovascular disease is the most common.The pathogenesis of ischemic cerebrovascular disease is complex,cross-linked by a variety of factors,involving energy metabolism disorders,oxidative stress injury,inflammatory response,excitatory amino acid toxicity,apoptosis,intracellular calcium overload,gene expression disorders,And eventually lead to neuroNAR apoptosis,blood destruction of cerebral infarction to form brain edema.Oxidative stress is an important mechanism involved in ischemia-induced neuroNAR injury.Oxidative stress injury caused by mitochondrial morphological structure damage,DNA mutation,adenylate synthesis is limited,oxidative stress product accumulation and other mitochondrial function damage is cerebral ischemia and reperfusion cell death after the core link.Therefore,focusing on antioxidant treatment and its sigNARing pathway,and anti-apoptotic material might be a new therapy for ischemic cerebrovascular disease therapy.Nuclear factor erythroid2-relatedfactor2(Nrf2)is a basic leucinezipper protein and aredox-sensitive transcription factor member of the Cap'n'Collar.Evidence demonstrated that Nrf2 and antioxidant response element pathway confer protection to a variety of oxidative stress related neurode generative insults in mammalian cells.Activity of Nrf2 isnegatively regulated by kelch-likeECH-associated protein 1(Keap1).Under basal conditions,Nrf2 is primarily localized in the cytoplasm and is associated with Keap1[12].This Keapl/Nrf2 complex is targeted for ubiquitin ation and proteasomal degradation,thereby maintaining Nrf2 activity in quiescence[13].In response to stress,Nrf2 is released from Keapl via phosphorylation and move to the nucleus to process sits transcriptioNAR activity[14].Therefore,discovery of new molecules that modulate the Nrf2/ARE pathway would hold promise for designing new strategies to treat stress-related diseases.Naringenin(NAR),a flavanone,is one of the major antioxidants present in citrus fruits,has been found to exhibit antioxidant,anti-carcinogenic and anti-mutagenic effects.Several studies have documented that NAR inhibit the growth of various cancer cells through the inhibition of cell proliferation and the activation of apoptosis.For the antioxidant effect,research indicated that NAR could reduce lactate dehydrogenase leakage and oxygen reactive species generation,increase mitochondrial membrane potential,and passivate activity of caspase 3/7 together with weakened DNA damage.However,the effect of NAR on the ischemic cerebrovascular disease,especially for ischemic stroke,remains unclear.To investigate whether the Naringenin has a neuroprotective effect on cerebral ischemia-reperfusion injury,SD rats were used as experimental subjects.The effects of oxygen and glucose deprivation/OGD/Rep model and rat middle cerebral artery ischemia-reperfusion(MACO)model.The protective effect of naringenin on cerebral ischemia-reperfusion injury and its relationship with mitochondrial dysfunction and Nrf2 were investigated from different angles in vitro and in animal models.Pathways to regulate the relationship between antioxidant stress.In this study,we investigated the mitochondrial structure and function of mitochondria after ischemia-reperfusion injury,and discussed the relationship between mitochondrial damage and oxidative stress.To clarify the appropriate concentration of pomelo peel has improved against oxidative stress injury.On the other hand,we deepened the Nrf2 sigNARing pathway to elucidate the neuroprotective effect of naringenin on Nrf2 pathway-mediated antioxidant stress,and to clarify the molecular mechanism of naringenin on neuroprotective effects of cerebral ischemia-reperfusion injury.Explore the new way of thinking for cerebrovascular disease prevention and control to provide a new therapeutic target.MethodsIn vitro studies:1.The neonatal SD rats were sacrificed under sterile conditions,and cultured in the neuroNAR culture medium.The epithelial nerve cells in the logarithmic growth phase were selected and picked up by immunofluorescence staining with NES-FITC and DAPI.2.The epithelial cells placed in ventilated hypoxic cell culture tank,slowly into the tank with 95%N2 and 5%C02 mixed gas to remove the origiNAR tank of air,thereby reducing the oxygen concentration.Positive pressure filled 30min after the formation of hypoxic environment,close the gas tank,remove the tank gas inlet and outlet pipe,placed in the incubator.After 90 min,the hypoxic cans were removed and the sugar-free Earle's solution was removed.The cells were kept in a culture medium and kept in a constant temperature cell incubator to establish a model of oxysaccharide deprivation/reperfusion injury.3.After treated with different concentrations of naringenin for 48 hours,MTT assay,Hoeschst33258 and Annexin V-FITC/PI double staining flow cytometry were used to detect the apoptosis of the epithelial nerve cells apoptosis.Detection of naringenin on ischemia-reperfusion injury oxidative stress on cells caused by poisoning,leading to decreased cell viability and apoptosis play a protective role.4.The apoptotic proteins such as capsase-3,bax,bcl-2 and Cyt C protein and gene expression levels were detected by Western blot and fluorescence quantitative PCR after treated with different concentrations of naringenin for 48 hours.To investigate the protective effect of naringenin on apoptosis of epithelial nerve cells which induced by glucose deprivation/reperfusion.5.The mitochondrial morphological structure of the epithelial nerve cells was observed by immunohistochemistry after 48 hours of intervention with different concentrations of naringenin.The kit was used to detect mitochondrial membrane potential and mitochondrial adenosine Acid content changes.To further understand the mitochondrial structure and function injury caused by cell oxidative stress,clear whether the element of pomelo peel element for the detection of pomelo peel on ischemia-reperfusion injury oxidative stress cause toxic to cells,led to the decrease of the cell vitality and protective cell apoptosis.6.Ormal epithelial nerve cells adherent culture oxygen deprivation/reperfusion after processing,sugar with different concentrations of pomelo peel intervention treatment after 48 h,detecting reactive molecules ROS cell oxidative stress and with chemical colorimetry the detection of intracellular MDA and GSH levels and SOD activity.To further understand the mitochondrial structure and function injury caused by cell oxidative stress,clear whether the element of pomelo peel element for the detection of pomelo peel on ischemia-reperfusion injury oxidative stress cause toxic to cells,led to the decrease of the cell vitality and protective cell apoptosis.7.siRNA-Nrf2,OENrf2 interference Nrf2 expression,oxygen deprivation/reperfusion sugar processing,to give 80 microns pomelo peel treatment after 48 h,determined by MTT method and Annexin V-FITC/PI double flow cytometry to detect cell apoptosis.Tested nerve cells after Nrf2 gene expression and silence,pomelo,whether by Nrf2 pathway caused by ischemia-reperfusion injury oxidatie stress to cells poison,led to the decrease of the cell vitality and protective cell apoptosis.8.siRNA-Nrf2,OENrf2 interference Nrf2 expression,oxygen deprivation/reperfusion sugar processing,to give 80 microns pomelo peel treatment after 48 h,detection of oxidative stress reactive molecules in cells ROS,MDA and GSH levels and SOD activity.9.siRNA-Nrf2,OENrf2 interference Nrf2 expression,oxygen deprivation/reperfusion sugar processing,to give 80 microns pomelo peel treatment after 48 h,Western blot and EMSA electrophoretic migration testing Nrf2 protein transfer condition and the rest ARE combined.10.siRNA-Nrf2,OENrf2 interference Nrf2 expression,oxygen deprivation/reperfusion sugar processing,to give 80 microns pomelo peel treatment after 48 h,Western blot and fluorescence quantitative PCR detection Nrf2,Keap1,HO-1 and NOQ1 protein and gene expression level.In vivo experiment:1.Using the method of line switch to establish the rat middle cerebral artery ischemia reperfusion(MCAO)animal model,and give no concentration pomelo peel treatment,postoperative neurobehavioral scores,TTC dyeing,brain water content determination and HE staining.After observation of pomelo peel,pretreatment of ischemic nerve protective effect.2.Using the method of line switch to establish the rat middle cerebral artery ischemia reperfusion(MCAO)animal model,and give no concentration pomelo peel treatment after 72 h,electron microscope scanning each mitochondrial morphological structures,the kits in the mitochondrial membrane potential and mitochondrial adenosine content changes.Pomelo peel pigment on ischemia/reperfusion rats caused by mitochondrial structure and function of tissue damage,the influence of the energy metabolism disorder.3.Using the method of line switch to establish the rat middle cerebral artery ischemia reperfusion(MCAO)animal model,and give no concentration pomelo peel treatment after 72 h,ROS,MDA and GSH level in the brain tissue and SOD activity.To further understand the mitochondrial structure and function injury caused by cell oxidative stress,clear whether the element of pomelo peel element for the detection of pomelo peel on ischemia-reperfusion injury oxidative stress cause toxic to cells,led to the decrease of the cell vitality and protective cell apoptosis.4.Using the method of line switch to establish the rat middle cerebral artery ischemia reperfusion(MCAO)animal model,and give no concentration pomelo peel treatment after 72 h,TUNEL method to observe the apoptosis of brain tissue,westren bloting method to observe the brain Nrf2,Keap1,HO-1,NQO1 protein expression,fluorescent polymerase chain reaction(PCR)to detect Nrf2,Keap1,HO-1,NQO1 gene expression,while detecting the DNA of Nrf2 combined with vigor and Keap1 nitroso situation.Observation of pomelo peel element of ischemia-reperfusion injury rats brain tissue Nrf2 pathway mediated anti oxidative stress effects and its possible mechanism.ConclusionNaringenin has protective effect on the nerve cells during the pathogenesis of ischemia-reperfusion injury,which is related to the mitochondrial dysfunction and Nrf2-mediated antioxidant stress.