Effect And Mechanism Of MCPIP-1 On The Umbilical Cord Blood Derived Endothelial Progenitor Cells In The Treatment Of Ischemic Stroke

Background:Ischemic stroke is a common disease that can cause disability or death and burden families and society.Stem cells have brought a new light to stroke treatment,but the unfavorable environment such as oxidative stress and inflammation in the brain after stroke leads to decreased survival of transplanted stem cells,which limits the application of stem cells.Human umbilical cord blood-derived endothelial colony forming cells(hUCB-ECFCs)have an in vivo vasculogenic advantage,and the aim of this study was to investigate the effects of monocyte chemoattractant proteininduced protein 1(Monocyte chemoattractant protein-induced protein 1(MCPIP1))in hUCB-ECFCs on cell proliferation,apoptosis,migration,ex vivo angiogenesis and cell survival after transplantation,and on the treatment of middle cerebral artery occlusion(MCAO)model in mice.occlusion MCAO)model and the mechanism of action.Methods:In vitro(Approval number:2017-SJWK-009):50 ml of umbilical cord blood was collected from full-term newborns,and monocytes were isolated by density gradient centrifugation.hUCB-ECFC was identified by flow cytometry using CD34,CD133,CD31,CD45,CD105,CD90 and VEGFR2 as the main markers,and divided into siRNA interference group(si-MCPIP1 group),siRNA control group(si-Negtive control group,i.e.,si-NC group),adenovirus overexpression group(Ad-OE-MCPIP1 group)and adenovirus null group(Ad-Negtive control group,i.e.,Ad-NC group),and ECFC group without any treatment(normal ECFC group).Migration ability was detected by scratch assay and Transwell assay;cell proliferation was detected by 5Ethynyl-2’-deoxyuridine(EDU)and CCK-8;cell apoptosis and apoptosis-related proteins BCL-XL and BAX were detected by flow cytometry and Western blot;cell proliferation was detected by in vitro stromal glue tube formation and In vitro stromal gel embolization and stromal gel embolization assays were performed to detect in vitro and in vivo angiogenesis;WB,solid phase microarray and TMT protein sequencing were used to detect HIF-la,Tie-2,CXCR4,SDF-1 and angiopoietin-1 angiogenesis-related protein expression and pathway changes.In vivo experiments(all animal experiments involved in this experiment followed the requirements of animal experimental ethical welfare guidelines,Approval number:LAEC-2022-116):MCAO model was prepared by internal carotid artery wire bolus method,and hUCBEPC was transfected with vacant lentivirus(LV-NC),knockdown MCPIP1 lentivirus(LV-sh-MCPIP1 lentivirus),which is Negtive control(LV-NC group),MCPIP1 interference group(LV-sh-MCPIP1 group),untreated MCAO+ECFC group,and MCAO+PBS negative control group.ECFC(3×105cells/1μLPBS)was obtained from each group of lateral ventricular transplantation,and brain tissue was stained with TTC at 24 hours after transplantation,and biopsy and neurological function scores were performed at 24 hours,3 days,5 days,7 days and 14 days,and brain tissue was collected at 7 days for CD31 and Ki67 as markers of vascular neogenesis,Nestin and Ki67 as neurogenesis markers were detected by immunofluorescence.changes in VEGF,HIF-la,angiopoietin-1,CD31 and nestin protein levels in serum and brain tissues were detected by ELISA and WB methods,respectively.Results:Compared with the NC group,MCPIP1 expression was significantly lower in the interference group(p<0.05),while it was significantly higher in the MCPIP1 overexpression group(p<0.01).The number of EDU-positive cells,the number of Transwell cells migrating and the number of branches,nodes and loops of stromal glue-forming tubes were higher in the siRNA knockdown MCPIP1 group than in the control group(p<0.05).In flow cytometry,the number of apoptotic cells in the MCPIP1 overexpression group was significantly higher than that in the control group(p<0.05),while there was no difference between the knockdown group and the control group;after lateral ventricular transplantation of ECFC in MCAO mice,TTC staining showed a reduction in the area of cerebral infarction,a decrease in neurological function scores,and an increase in angiogenic-related factors in serum and brain tissue;ki67 The number of ki67/CD31 and ki67/nestin immunofluorescent double-positive cells increased,and the survival time after transplantation was longer in the knockdown MCPIP group than in the control group.Conclusion:Knockdown of MCPIP1 increased ECFC proliferation,migration,angiogenesis and promoted cell survival after transplantation.hUCB-ECFC transplanted with knockdown of MCPIP1 in the lateral ventricles of MCAO mice significantly promoted neuroangiogenesis,reduced brain infarct volume and attenuated neurological impairment.