| Stroke is known as it’s high related mortality and disability,which cause catastrophic consequent among public healthcare of human being.There are two types of bedrock in terms of it’s nosogenesis:ischemia and hemorrhage.Cerebral ischemia remain above 75% of overall stroke incidence,and after the onset of cerebral ischemia,the inevitable blood reperfusion would exacerbate cerebral infarction,which is named CIRI.Thrombolysis by using plasminogen activator is the most efficient and approved therapy for those acute ischemic stroke patients.However,the narrow therapeutic window limites its clinical application.Thus,new drugs and agents are urgently required for the treatment of CIRI caused by ischemic stroke.Coumarins are derived from a group of considerably important heterocyclic compounds that exist in natural,and they are often used in certain drugs based on it’s various chemical structure and showing a variety of biological activities.Many of coumarins compounds have been found as natural antioxidants,among of these compounds,Biscoumarins derivatives received a more considerable attention because their special molecular structures.Those compounds exert diverse biological properties through modification of their structure by linking different substituents on the central methylene.Oxidative and antioxidant process coexists in cells,they tend to be balanced in physiological condition.When cells are damaged,this process would be broken and cells were under oxidative stress,causing reactive oxygen species accumulation,which exhausted endogenous antioxidant factor,consequently led irreversible damage to cells.Central nervous system is vulnerable to oxidative stress damage that caused by CIRI,maintain the redox balance of the central nervous system is crucial to curb IS.【Objective】 1.Potential neuroprotective compounds were selected from a series of novel bicoumarin that synthesized and authenticated by our laboratory;2.After target compounds were screened out,the neuroprotective effect of the compound based on CIRI induced by MCAO were studied,including detailed studies in vitro and in vivo;3.To determine the effect of target compound on ROS,antioxidant enzyme mRNA and other antioxidant indicators in OGD neurons;4.To reveal the effect of target compound on the endogenous antioxidant pathway Nrf2/keap1 signaling and its specific mechanism and to clarify the specific mode and bonding site of the compound molecule with the structure of antioxidant regular protein Keap1;5.To observe the protective effect of target compound on the mitochondria of neurons,and protective mechanism of the compound on the mitochondria of neurons under oxidative stress.【Method】 1.The cortical tissue of fetus from pregnant mice was separated by microoperation,and the primary neurons were selectively cultured by using the special neuron culture medium and B27 for subsequent experimental study.2.Through nuclear magnetic resonance hydrogen spectrum methods to verify and ensure the purity of compounds.Then,based on antioxidant capacity,we screened out a compound which showed antioxidative effects on neurons which is under OS.Then we established OGD model and MCAO model to further verify the reliability of the compound’s nerve protective effect.3.CCk-8 assay was used to determine the stability of compound cells and the range of cells survival rate.PI/Hoechst staining was used to study the influence of the compound on necrosis of OGD neurons.q-RCR was used to detect the influence of the compound on mRNA of neuronic antioxidant enzyme and content of MDA.4.Animal experiments were conducted to study the protective effect of the compound in vivo,cerebral infarction volume,neurobehavioral score and cerebral edema were detected,and pathological change were detected by HE and NISIL staining.5.Western blot,immunohistochemistry,immunofluorescence were used to study the effect of the compound on oxidation Nrf2/Keap1 signaling pathway,and molecular docking technology was used to study the specific binding mode between the compound and target protein Keap1.6.JC-1 staining,transmission electron microscopy and ATP level were measured to detect the changes of the function of the mitochondria of OGD neurons under OS with the intervention of the compound.【Result】 1.In the early stage of this study,a series of novel bicoumarin derivatives with coumarin nucleus were synthesized,from which we screened compounds with potential antioxidant capcacity,and we further studied the effects and mechanism of the target compound on CIRI.2.Compounds with antioxidant potential were screened by using oxidative damage model of neurons.The results indicated that COM 3 significantly increased the expression of two antioxidant enzymes,SOD and GSH-Px,among six candidates compounds.3.To further explore the protective effect,after CIRI,of COM 3 on cellular and animal level,the results showed that the protective effect of COM 3 at 10 μg/ml concentration on primary cultured neurons induced by OGD injury was the optimal,which also alleviated the neuronal necrosis and apoptosis after OGD.At 10mg/kg concentration,intraperitoneal injection of COM 3 had the most efficiency protective effect on C57BL/6 mice with MCAO injury,and COM 3 reduced the cerebral infarction volume and improved the neurobehavioral score in MCAO mice.4.In further mechanism research,molecular docking results showed that COM 3 relying on three hydrogen bonds and two π-π action to occupy Nrf2 and Keap1 protein binding sites,which affected the Keap1 in term of the ability of combination with Nrf2,and this bonding effect finally performed an inhibition of the activity of Keap1,promoted Nrf2 nuclear transcription,the level of antioxidant enzymes,and reduced ROS accumulation within the neurons,which was also showed in Western Blot test.REDOX regulation of neurons metabolism returned to the physically normal level.5.In order to further explored the neuroprotective mechanism of COM 3,the experimental results indicated that COM 3 reduced mitochondrial membrane potential,increased cells ATP level,improved the mitochondrial morphology of neurons,and alleviated OS of mitochondrial.【Conclusion】A novel type of biscoumarins named COM 3,which is synthesised and screened by our own laboratory,alleviated the OS caused by CIRI in central nervous system and promoted mitochondrial function of neurons against OGD.These type of biscoumarins build a theoretical foundation for future research that intervene the nervous system by using independent synthetic componds to protect against CIRI,and provide a new idea for the independent researches and development of biscoumarins derivatives as new type drugs for IS treating. |
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