Protective Effect And Mechanism Of RIPC Serumal Exosomes On Ischemia/Hypoxia Tolerance Of Cerebral Nerves

Objective Human remote ischemic preconditioning model,human endothelial cell HMEC-1 ischemic preconditioning model and SH-SY5 Y cell oxygen and glucose deprivation model were used in this study.The main neuroprotective substances in RIPC were explored by analyzing the differences in gene expression between cells and exosomes after exosomes were isolated,The source and potential way of protective substances were clarified.Method(1)Male college volunteers were recruited for ischemic preconditioning using a remote ischemic preconditioning trainer and venous blood and serum were collected from the elbow.Serum exosomes were isolated by differential centrifugation.Exosome morphology,size and contents were detected and identified using methods such as chip sequencing and transmission electron microscopy.(2)OGD treatment after exosomes are incubated with SH-SY5 Y cells.Detect the effects of RIPC exosomes on ischemic tolerance and genomic methylation of nerve cells by Westren Blot,Real-Time PCR,MTS,COBRA,and bioinformatics prediction,and identify the main protective substances and interactions of RIPC exosomes Relationships and protection mechanisms.(3)Mir-126 was overexpressed by lentivirus in sh-sy5 y cells,and the relationship between mir-126 and genomic methylation was clarified by combining bioinformatics prediction results.(4)HMEC-1 was trained in ischemic preconditioning,and the expression level of miR-126 in cells and exosomes was analyzed using Real-Time PCR to determine the source of RIPC exosomes.Results(1)The results of transmission electron microscopy and Westren Blot showed that the hypervelocity centrifugation method could successfully isolate serum exosomes and ensure the purity and homogeneity of exosomes.(2)Exosomes were co-incubated with SH-SY5 Y cells for 4h,and the exosomes were observed to enter SH-SY5 Y cells by laser confocal microscopy.(3)After co-incubation of exosomes with SH-SY5 Y cells for 24 hours,intracellular global methylation levels were significantly reduced,DNMT m RNA was down-regulated,and DNMT3 A and DNMT3 B protein expressions were reduced.This phenomenon is caused by the high expression of miR-126 in RIPC exosomes combined with DNMT3 B m RNA.(4)Lentivirus was used to overexpress miR-126 in SH-SY5 Y cells.The expression of DNMT1 and DNMT3 A was up-regulated in SH-SY5 Y cells,the DNMT3 B gene was significantly lower expressed,and the degree of cellular methylation was decreased.(5)Compared with the OGD group,after adding RIPC exosomes to SH-SY5 Y cells,the vitality was significantly restored.The levels of pro-apoptotic proteins such as P53 and Bax were down-regulated,and the expressions of anti-apoptotic proteins such as Bcl-2 were up-regulated.And the global methylation level of SH-SY5 Y cell is reduced.(6)When miR-126-rich exosomes were added during normal culture or OGD treatment,the expression of VEGF gene in SH-SY5 Y cells was significantly up-regulated.When miR-126 was overexpressed in cells,the expression of VEGF in cells was significantly inhibited.(7)Hypoxic / ischemic preconditioning treated HMEC-1 cells,miR-126 expression in cells and media exosomes were up-regulated almost twice.Conclusions RIPC can stimulate the microvascular endothelial cells of the limb to secrete miR-126-rich exosomes.After these exosomes enter the nerve center with the blood,miR-126 can inhibit the expression of DNMT3 B in nerve cells and affect the DNA methylation in nerve cell.This may regulate hypoxic/ ischemic tolerance-related genes expresseing to exert neuroprotective effects.