Microvesicles Released By Human Embryonic Stem Cell Derived-neural Stem Cells Inhibit Apoptosis Of Cardiomyocyte In Vitro

BackgroundIschaemic heart disease?IHD?is one of the leading causes of death and disability worldwide.Timely reperfusion is the main treatment for IHD,which not only reduces infarct size but also prevents heart failure.However,the reperfusion process itself can induce myocardial cell death,which is referred to as myocardial reperfusion injury?MRI?.Myocardial reperfusion injury is caused by reactive oxygen species overproduction,energy metabolism disorders,neutrophil infiltration,calcium overload and vascular endothelial dysfunction,but there is still no effective treatment.Cell therapy is considered a viable option for treating myocardial reperfusion injury.Stem cell transplantation is an effective method that primarily improves damaged tissues by releasing autocrine and paracrine factors.However,major concerns such as teratoma formation,immune responses,difficulty harvesting cells,and limited cell proliferation and differentiation hinder the routine use of these cells as a treatment option in the clinic.Microvesicles?MVs?are a small class of extracellular vesicles that have become the key mediators of cell-to-cell communication.It has been reported that MVs derived from stem cells play an important role in MRI and are tissue functional materials for potential myocardial reperfusion injury repair.ObjectivesIn this study,we sought to explore the role and mechanism of MVs-derived neural stem cells derived from human embryonic stem cells?hESC-NSC-MVs?in inhibiting cardiomyocyte apoptosis.Methods?1?Differentiating from embryonic stem cells into neural stem cells,conditioned medium of hESC-NSC was collected,and microvesicles were extracted by differential centrifugation combined with ultracentrifugation.?2?Pre-incubation of hESC-NSC-MVs for 24 hours before H₂O₂ stimulation of cardiomyocytes,detection of apoptosis-related proteins:Bax and Bcl-2 by Western blot;The positive rate of Annexin V/PI in cardiomyocytes was detected by flow cytometry and the apoptotic rate was calculated.?3?pre-incubation with hESC-NSC-MVs for 24 hours before H₂O₂ stimulated cardiomyocytes,and detected the expression levels of autophagy-associated proteins:P62,LC3 and Beclin-1 in cardiomyocytes by Western blot; transmission electron microscopy and mRFP-GFP-Transfection of LC3 adenovirus to observe the number of autophagosomes in cardiomyocytes?4?The protein distribution was observed by SDS-PAGE electrophoresis and Coomassie blue staining of total protein in hESC-NSC-MVs,and the target protein was verified by Western blot.?5?Increasing the expression of HSP-70 in cardiomyocytes by thermal stimulation.Triptolide was used to inhibit the increased expression of HSP70,and then determined the effect of HSP-70 on cardiomyocyte apoptosis and autophagy.?6?The expression level of phosphorylated AKT in cardiomyocytes was detected by Western blot.Results?1?hESC-NSC-MVs inhibit appoptosis of HL-1 cardiomyocyte in vitro,and this inhibition is concentration-dependent.?2?hESC-NSC-MVs promote autophagy of HL-1 cardiomyocytes in vitro,and this promotion is concentration-dependent.?3?hESC-NSC-MVs are rich in HSP-70 and can increase the level of HSP-70 pin the target cells.?4?HSP-70 has the effect of inhibiting apoptosis of HL-1 cardiomyocytes in vitro.?5?HSP-70 has the effect of promoting autophagy of HL-1 cardiomyocytes in vitro.?6?hESC-NSC-MVs and HSP-70 can up-regulate the expression of phosphorylated AKT in HL-1 cardiomyocytes.ConclusionsOur results indicate that hESC-NSC-derived MVs promote autophagy by activating HSP-70 to activate AKT pathway,thereby inhibiting apoptosis of HL-1cardiomyocytes.However,this conclusion also needs to be verified in animals.