The Effect And Mechanism Of DICAR On Angiogenesis After Cerebral Ischemia-reperfusion Injury

ObjectiveIschemic stroke(IS)is mostly caused by vascular occlusion,stenosis or rupture of the middle cerebral artery and its collateral circulation caused by a variety of predisposing factors,and then the blood flow of various cerebral arteries is blocked.Ischemia and hypoxia,patients mostly show transient or permanent neurological damage.IS accounts for 60%-80% of the total stroke cases,and has become one of the leading causes of death in adults worldwide,seriously affecting the quality of life of patients.So far,the clinical medical community still lacks effective theragy method that can reduce ischemia-induced nerve damage.Recombinant tissue plasmmogen activator(rt-PA)is currently the only one approved by China National Medical Products Administration that can treat ischemic stroke by dissolving blood clots and restoring blood reperfusion effective treatment.However,its application is limited due to the extremely narrow therapeutic time window and the need for rapid dosing in most patients within about 4.5 hours after the onset of ischemia.Induced angiogenesis can occur in the ischemic site of stroke animal models and clinical patients.The new blood vessels can further improve the supply of cerebral ischemia and improve neurological function,which is of great value in the : prognosis of IS.The density of newborns has an important relationship with patient prognosis.In the end,this study is mainly based on the research concept of angiogenesis to explore the pathophysiological mechanism of the treatment of IS.In this end,this study focuses on three aspects of IS treatment and mechanism based on the research concept of vascular neovascularization,in vivo and ex vivo experiments and biochemical analysis.MethodsPart 1: To investigate the effects of DICAR overexpression on cerebral ischemia-reperfusion-induced neurological damage and angiogenesis in mice.A midddle cerebral artery occlusion(MCAO)model was established with WT mice and DICAR-Tg mice to observe the expression changes of DICAR-Tg overexpression in cerebral ischemia-reperfusion injury.After 1 h and reperfusion for 24 h and 7 days of ischemia,TTC staining,longa and Eederson neurological injury scores,Morris water maze,and cerebral infarction volume were used to evaluate and compare.After 1h and 17 days of reperfusion in ischemia,the effects of DICAR on long-term neurological damage in stroke mice were evaluated by water maze experiment,pole rotation experiment,swimming speed and stage piercing experiment.Immunohistochemical behavior measured the expression of CD31 after cerebral ischemia and reperfusion,and Western blot measured the expression level of VEGFR-2,a protein associated with angiogenesis,to evaluate the effect of DICAR on OGD/R-induced angiogenesis.Part 2: DICAR regulates the molecular mechanism of proangiogenesis induced by cerebral ischemia-reperfusion injury.Establishment of oxyen and gluocse deprivation and reperfusion(OGD/R)model: the medium of hCMEC/D3 cells was replaced with glucose-free DMEM and incubated for 4 h at 37℃ in an anaerobic incubator with 95%N2 and 5% CO2 to simulate OGD damage,and hCMEC/D3 cells were transfected with hDICAR-JP followed by OGD 4h/R 24 h treatment,qPCR detection of VEGF,VEGF-A,VEGFR-2 expression,Western blot detection of VEGFR2 protein expression,Tube formation assay quantitative analysis to detect the density and nodes of in vitro neovascularization.Bioinformatics analysis software Circ Base,UCSC and ENCORI predict target miRNAs downstream of DICAR and downstream targets of miRNAs.ResultsPart 1: The expression level of DICAR detected by mouse brain IS1h/R24 h and IS1h/R7 d in mouse brain tissues was significantly increased.DICAR-Tg can significantly improve the neurological function score of ischemia-reperfusion mice during acute injury and reduce the volume of cerebral infarction.The results of water maze,pole climbing test,swimming experiment and platform piercing experiment showed that DICAR overexpression could significantly improve cognitive memory and motor function after 17 days of cerebral ischemia I/R,improve long-term neurological damage caused by ischemia-reperfusion,promote the expression of VEGFR-2 protein,and promote the new generation of brain microvessels in IS/R mice.Part 2: Transfection of hDICAR-JP,OGD 4h/R 24 h in vitro hCMEC/D3 cells can significantly inhibit OGD/R-induced cell death,promote the expression of VEGFR-2 protein,the expression of VEGF,VEGFR-2 and VEGFA genes,and the increase of the number and density of tubes.Using bioinformatics software Circ Base,UCSC,Targetscan and ENCORI,miR-361-3p may be a target miRNA downstream of DICAR,transfection of hDICAR-JP and miR-361-3p inhibitor in cells,and found that the role of hDICAR-JP in cells is reversed.Biotrust analysis predicts that PRMT1 may be a downstream target of miR-361-3p.Transfection of miR-361-3p mimic and PRMT1 overexpression vectors at the cellular level promotes the inhibitory effect of miR-361-3p mimic on PRMT1 protein expression,while miR-361-3p inhibitor reverses this result.ConclusionDICAR participated in the process of cerebral ischemia-reperfusion injury in mice,and overexpression can reduce the impact of reperfusion injury,and its mechanism may be through adsorption of miR-361-3p,indirectly regulating the expression of PRMT1,and playing a role in promoting angiogenesis in infarct region in ischemic stroke.This paper provides new ideas for exploring DICAR as a new biomarker and potential therapeutic target in cerebral ischemia-reperfusion injury.