| Background:Stroke is characterized by high mortality and disability,and is one of the main factors threatening human health.The majority of stroke patients were ischemic stroke(IS),which accounted for 87%.IS refers to the lack of blood supply to the brain tissue due to the formation of thrombus or embolism,which leads to ischemia and hypoxia of nerve cells.Because the brain is a high-energy organ,it is sensitive to changes in the surrounding environment,and irreversible damage to nerve cells with the prolongation of ischemia and hypoxia.At present,the most effective treatment is thrombolytic therapy.But,thrombolytic therapy has a limited time window and is associated with the risk of bleeding,so the number of beneficiaries who can be treated by thrombolytic therapy is limited.Therefore,it is of great clinical significance to explore the mechanism of IS development and to find more effective therapeutic targets.Extracellular vesicles(EVs)are cells secreted nano-vesiculars that are encapsulated by phospholipid bilayers.In addition to transmitting information between adjacent cells,the most important feature is the ability to cross the blood-brain barrier,thereby completing the exchange of information between the blood and the central nervous tissue.EVs carry proteins,lipids and nucleic acids of donor cells.When taken up by the recipient cells,all substances in the EVs are released into the recipient cells,which then transmit the information of the donor cells to the recipient cells and regulate the recepient cells.Between the above functional characteristics,the role of EVs in central nervous system diseases has been extensively studied.Aims:Based on the OGD model of PC12 cells,proteomics and bioinformatics methods were used to analyze the changes of EVs protein composition during OGD,revealing the potential mechanism of damaged cell-derived EVs regulating receptor cells.Providing some theoretical support for understanding the effects of EVs derived from neuronal cells in the damaged area on the development of IS.Methods:1.Construction of the Oxygen glucose deprivation(ODD)model of PC12 cells.The OGD cell model was established by treating the PC12 cells with 0h,3h,6h and12 h in a combination of sugar-free medium and anaerobic culture environment(5%CO2 and 95% N2).The cells of OGD were cultured by CCK-8 and flow cytometry.Survival rate and apoptotic rate were analyzed to evaluate whether the model was successfully constructed.2.Separation,morphological observation and labeling protein detection of EVs.In this study,EVs were isolated from PC12 cell supernatants treated with OGD for0 h,3h,6h and 12 h by precipitant method.The morphology of EVs was observed by transmission electron microscopy.The surface markers of EVs were detected by Western blot.3.To investigate the effect of EVs derived from OGD treated PC12 cells on the norma PC12 cells.The EVs(10μor 20μg)from OGD 0h,3h,6h and 12 h were co-cultured with normal PC12 cells for 24 h,survival rate and apoptosis rate were detected by CCK-8 and flow cytometry.4.Identification of changes in protein composition in EVs derived from PC12 cells by OGD treatment.The peptides were fractionated by high performance liquid chromatography(HPLC),and the fractionated peptides were further separated by liquid chromatography-mass spectrometry(LC-MS).Identification analysis.5.Systematic analysis of functional changes in differentially expressed proteins in EVs.The proteins differentially expressed at different time points of OGD were systematically analyzed by means of Venn diagram,Gene Oncology(GO),KEGG,String and other bioinformatics methods.6.Interaction and quantitative analysis of differentially expressed proteins.In this study,the interaction network between proteins was analyzed by String database,and some differentially expressed proteins were quantitatively analyzed by parallel reaction monitoring(PRM).Results:1.With the prolongation of OGD treatment time,the survival rate of PC12 cells decreased gradually,and the apoptotic rate increased gradually,indicating that the degree of cell damage was gradually aggravated during OGD treatment,indicating that we successfully established OGD cell model.2.Morphological observation of EVs by transmission electron microscopy revealed that EVs have a size of about 100 nm and have typical bilayer membrane vesicles.Moreover,the expression levels of EVs-specific marker proteins CD9,CD63,CD81 and GAPDH did not change substantially during OGD.3.With the prolongation of OGD treatment of PC12 cells,the EVs derived from them could inhibit cell proliferation and promote cell apoptosis more significantly,and the effect was not concentration-dependent.4.MS-MS/LC identified a total of 1,650 proteins,of which 1377 can be quantified.Statistical analysis revealed that the number of differentially expressed proteins at 3h/Ctrl,6h/Ctrl,12h/Ctrl,6h/3h,12h/3h,and 12h/6h were 170,44,77,66,222,and 94,respectively.5.Venn diagram overlap results show that 3h/Ctrl and 6h/Ctrl differentially expressed proteins have high overlap,while 12h/Ctrl has low overlap with the former two.Further GO enrichment clustering and KEGG signaling pathway enrichment clustering revealed certain differences in organelle localization,functional classification and signaling pathways between differentially expressed proteins at different time points.6.Protein-protein interactions(PPI)results show that,in EVs from damaged cells,the interaction network of differentially expressed proteins can accuratelytransmit the changes about redox,sugar metabolism,coagulation and protein synthesis and degradation of different biological processes to the outward.7.PRM for different biological processes such as: glucose metabolism(Pgm1and Idh1),oxidative stress(Cat and Sod1),endocytosis(Cltc,Ctlb,Nrxn1),thrombosis(Vcl,Plat)Quantitative verification of representative molecules in vascularization(Mfge8,Igf2 r and Mmp2),protein folding and degradation(Tcp1,Cct protein family,Psmc protein family)showed results consistent with mass spectrometry.Conclusions:1.ODG treatment of PC12 cell-derived EVs can induce apoptosis of normal PC12 cells,and with the prolongation of OGD treatment time,the EVs-induced apoptosis ability is stronger.2.When OGD was treated for 3h and 6h,PC12 cells were in a relatively stable stress state,and the EVs differentially expressed proteins had higher overlap,which was consistent with the clinical thrombolytic therapy window.At 12 h,there was a significant difference between the differentially expressed proteins in the source EVs and 3h and 6h.3.OGD treatment of PC12 cell-derived EVs differentially expressed proteins involved in different biological processes such as glucose metabolism,redox and protein synthesis,folding and degradation.EVs may cause apoptosis of recipient cells by disrupting the biological processes of recipient cells.It is suggested that EVs derived from damaged nerve cells in the process of IS may be one of the mechanisms of damage to the nervous system.4.OGD treatment of PC12 cell-derived EVs showed significant changes in thrombin-related protein expression,high expression of proteins that promote thrombosis,and low expression of proteins that inhibit thrombosis.This suggests that damaged IS-derived EVs cross the blood-brain barrier into the blood system during IS,possibly further accelerating IS damage to the nervous system by promoting thrombosis.In summary,PC12 cells were in a relatively stable stress state in the early stage of OGD treatment,and there was a significant difference between the differentially expressed proteins in the source EVs and the early stage due to changes in cell status.Further bioinformatics analysis found that these differentially expressed proteins are involved in important biological processes such as regulation of glucose metabolism,redox,endocytosis,thrombosis,vascularization,protein synthesis,folding and degradation.In the process of IS,nerve cell-derived EVs in the damaged area may induce apoptosis by disturbing these biological processes of the recipient cells,further aggravating the progress of IS.At the same time,it is also possible to aggravate the damage of the nervous system to IS by promoting thrombosis.Therefore,during the development of IS,EVs secreted by cells in damaged areas may interact with different ways to aggravate the damage of IS to the nervous system. |
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