The Role And Mechanism Of Circulating Exo-miR-339-5p In The Regulation Of Vascular Endothelial Cell Function During Ischemic Stroke

Background:Ischemic stroke is an acute cerebrovascular disease with high morbidity,mortality and disability.Due to cerebral vascular blockage,local blood supply is insufficient,which eventually developed into brain function loss.At present,thrombolytic therapy is still mainly performed with tissue plasminogen activator,but it has some disadvantages such as narrow treatment time window and high risk of ischemia reperfusion injury.Endothelial cells,as the basic components of the blood-brain barrier,are essential for maintaining the blood-brain barrier and reducing neuroinflammation during cerebral ischemia.However,no ideal "endothelial protectant" has been found to deal with the vascular endothelial injury caused by ischemia.Therefore,finding effective vascular endothelial intervention strategies from a new perspective is one of the primary topics of prevention and treatment of ischemic stroke.In recent years,exosomes,as extracellular vesicles that can carry a large amount of biological information across the blood-brain barrier,were considered to poss great potential in the diagnosis and treatment of ischemic stroke.Therefore,as a research entry point,exosomes are of great significance in establishing effective treatment strategies by exploring potential molecular targets in the course of stroke.Objective:To find mi RNAs differentially expressed in circulating exosomes of patients with ischemic stroke.To demonstrate whether differentially expressed mi RNA can be an effective target for the treatment of ischemic stroke.Methods and results:1.Exosomes were purified from serum samples by overnight ultrafast centrifugation,followed by Transmission Electron Microscopy,Nanoparticle Tracking Analysis,and Western Blot.2.mi RNA was extracted from the serum exosomes of the ischemic stroke patients group and the control group by extraction kit.mi RNA sequencing and bioinformatics analysis was performed to find multiple mi RNAs with differential expression between the two groups.Rt-q PCR was used to further verify their expression in clinical samples and animal models,indicating that compared with the control group,mi R-339-5p was up-regulated in the serum exosomes of ischemic stroke patients and the infarcted brain tissues of Middle Cerebral Artery Occlusion(MCAO)mice.3.PKH67 staining showed that Oxygen and Glucose Deprivation(OGD)significantly enhanced communication between neurons and endothelial cells via exosomes.Transwell was used to co-culture of neurons and endothelial cells,and results showed that endothelial cells uptook more exo-mi R-339-5p from neurons under OGD model.4.Flow cytometry and TUNEL staining results showed that,compared with the control group,the apoptosis rate of endothelial cells overexpressed mi R-339-5p significantly decreased under the OGD model.Combined with Target Scan and other databases,we predicted the potential target genes of mi R-339-5p.Western Blot were used to confirm the negative regulation of mi R-339-5p on BCL2L11 and BAX.5.Rt-q PCR results showed that,under the OGD model,the expression of inflammatory cytokines in endothelial cells that overexpressed mi R-339-5p was significantly reduced compared with the control group.Conclusions:In patients with cerebral ischemia,mi R-339-5p in circulating exosomes was up-regulated.mi R-339-5p carried by exosomes can be absorbed by endothelial cells and played a protective role by negative regulation of pro-apoptotic proteins and proinflammatory factors.