The Research Of Endothelial Cell-derived Exosome In Neuroprotection After Ischemia Reperfusion Injury

Objectives:Remote ischemia postconditioning(RIP)in the treatment of cerebral ischemia reduces the ischemia-reperfusion(I/R)injury.However,the mechanism was less known.In the present study,based on what we did before,we hypothesize that the protective effect of remote ischemia postconditioning on neurological damage was mediated by exosome-released by endothelial cells to study the effect of exsomes after ischemia /reperfusion injury in SH-SY5 Y cells.Materials and methods:First,we established the right middle cerebral artery occlusion/reperfusion with remote ischemia postconditioning on rats.Use TTC staining,HE staining and immunohistochemical staining to evaluate the injury of brain.Second,establish the I/R injury cell model including human umbilical vein endothelial cells(HUVECs)and SH-SY5 Y cells,and evaluate the injury of cell by flow cytometry.We assessed markers of exosomes(CD63,HSP70 and TSG101)by immunohistochemistry,Western blot and flow cytometry.Exosomes were extracted from both animal serum and HUVECs culture medium,and identified by electron microscopy.Third,the role of endothelial cell-derived exosome in proliferation,apoptosis,cell cycle,migration and invasion of I/R SH-SY5 Y cells were evaluated.In addition,apoptosis-related molecules including Caspase-3,Bax and Bcl-2 were further detected.Results:1.Successfully establish the right middle cerebral artery occlusion/reperfusion with remote ischemia postconditioning(RIP)on rats.Evaluate the injury of brain after24 hours of reperfusion.a.The results of TTC staining showed that the infarct volume of cerebral ischemic infusion in RIP group was significantly lower than that in Model group,and no infarct volume in sham operation group.b.The results of H & E staining showed that the anatomical structure of the hippocampus in the sham operation group was complete,full and no apoptosis.The cell death and apoptotic cells in Model group were significantly higher than RIP group.c.Immunohistochemical staining showed that the apoptotic rate of hippocampus in Sham group was significantly lower in RIP group and Model group,and the apoptosis rate of RIP group was lower than Model group.2.Successfully establish the I/R injury cell model including human umbilical vein endothelial cells(HUVECs)and SH-SY5 Y cells and I / R time was 6h / 24 h.Supernatant centrifugation was used to extract the exosome from both of animal serum and HUVECs culture supernatant.a.The results of immunohistochemical staining showed that there was no significant change in the expression of CD63,HSP70 and TSG101 in the hippocampus of Sham,Model and RIP groups.b.The results of transmission electron microscopy and particle size analysis showed that the shape of the exosomes was round or oval,and the diameter was between 40 and 100 nm.The number of exosomes in the RIP group was significantly higher than that in the model group.c.The protein of CD63,HSP70 and TSG101 were all expressed in serum of each group.But expression of these protein in RIP group was significantly higher than that in Sham and Model group.The results of flow cytometry also showed the same results.3.CCK8 and Edu test results suggest that HUVECs derived exosomes can effectively reduce the effect of I / R injury on the viability and proliferation in SH-SY5 Y cells.4.The effect of HUVECs on the apoptosis and cell cycle of SH-SY5 Y cells after I /R injury:a.Immunofluorescence,flow cytometry,Hochest 33258 showed that the apoptosis of I / R was significantly increased after I / R injury.After adding Exosome can effectively reduce the apoptosis;b.Flow cytometry showed that the ratio of SH-SY5 Y cells were significantly increased in the G0 / G1 phase after I / R injury,and the ratio of S phase cells was significantly decreased;compared with SH-SY5 Y I / R group,the ratio of cells in the group treated with exosome decreased in G0 / G1 phase,and S phase cells increased.5.HUVECs derived exosomes can effectively restore invasion and migration ability of SH-SY5 Y cell after I / R injury.6.q PCR and Western blot showed that the expression of Bax and Caspase-3 in I / R cells was significantly increased and the expression of Bcl-2 was significantly decreased after I / R injury.After treated with Exosome,the expression of Bax and Caspase-3 expression levels were downregulated and Bcl-2 expression levels were upregulated.Conclusions:1?RIP significantly reduced the apoptosis changes of brain hippocampal tissue and infarct size of brain.2?RIP increases exosomes and expression levels of CD63,HSP70 and TSG101 in plasma.3?HUVECs-derived exosome significantly suppressed the I/R-induced cell cycle arrest and apoptosis,and-recovery cell proliferation,migration and invasion in SH-SY5 Y nerve cell after I/R injury.HUVECs-derived exosome significantly increased expression of anti-apoptosis-related gene.That suggested that exosome is expected to become one of the effective treatment of stroke.