| Ischemic heart disease(IHD)is one of the most important diseases that threaten human health and the morbidity and mortality of IHD are high.Vascular endothelial dysfunction,which functions as the beginning of myocardial ischemic injury,is closely related to the occurrence and development of IHD.At present,ischemic preconditioning(IPC)is expected to relieve the myocardial ischemic injury.It was found that the count of microvesicles(MVs)derived from myocardial tissue of IPC-rats was significantly increased,and MVs exerted anti-injury effects on myocardial ischemic injury.MVs participate in intercellular communication among different types of cells in the whole cardiovascular system.MVs carry active moleculars that regulate cellular functions and most of these MVs,which play an important role in IPC resistance to myocardial ischemic injury,are derived from endothelial cells.However,there has been no report about the effects of MVs derived from endothelial cells induced by hypoxia preconditioning(HPC)on cardiomyocytes.In this study,we used human umbilical vein endothelial cells(HUVECs)induced by HPC to release MVs,and investigated the effects of MVs on normal H9c2 cardiomyocytes and apoptotic-related proteins.We aimed to explore new directions to study the protective effects of IPC in myocardial I/R injury.Objective:In this study,HPC model was established by using HUVECs at the cellular level.HPC was used to induce the secretion of MVs from HUVECs,to explore the effects of HPC-MVs on normal H9c2 cells and the expression of Caspase 3,Bcl-2 and Bax.Methods:1.Establishment of HUVECs HPC modelThe normal HUVECs were cultured into 96-well plates for 24 h and divided into Control group,H/R(hypoxia/reoxygenation)and HPC group.Control group was normally cultured.H/R group was cultured by hypoxia for 12 h and reoxygenation for 4 h.HPC group was treated with different pre-oxygenation/reoxygenation conditions,then followed by hypoxia for 12 h and reoxygenation for 4 h.The cell viability of each group was tested by MTT assay.Proper cells density,the pH of the hypoxic solution in pre-hypoxia/reoxygenation and the time of pre-hypoxia/reoxygenation were determined to establish the HPC model.2.The release,qualitative and quantitative analysis of MVs induced by HPC from HUVECsHPC-EMVs were obtained from the culture supernatant of HUVECs induced by HPC through two-step centrifugation.The protein of HPC-EMVs was quantified by BCA assay.Partial suspensions were used to observe the ultrastructure of MVs by transmission electron microscopy.3.Effects of HPC-EMVs on normal H9c2 cellsThe H9c2 cells 1×10~5 were cultured into 96-well plates for 24 h.The cells were divided into Control group,10,30 and 60?g/mL HPC-EMVs groups.The Control group was cultured in DMEM medium without FBS for 4 h;in HPC-EMVs groups,H9c2 were incubated with 10,30,60?g/mL HPC-EMVs diluted with DMEM for 4 h.Cell viability and injury were measured by MTT assay and the activity of lactate dehydrogenase(LDH)in cell culture medium was tested by colorimetric method respectively.Morphological changes of H9c2 cells were observed by Hoechst 33258 staining.To detection of apoptosis-related proteins,Caspase 3 activity was determined by colorimetry and the expression of Bcl-2 and Bax in H9c2 cells were detected by Western blot.Result:1.HUVECs HPC model was successfully established.HUVECs 6×10~4 were cultured in 96-well plates,HPC group was added 100?L pH 6.8 hypoxia solution into each well,the cells were treated by pre-hypoxia for 10 min/reoxygenation for 15 min,then the same amount of pH 5.8 hypoxia solution was added into the wells,hypoxia for 12 h/reoxygenation for 4 h.Compared with H/R group,the cells viability of HPC group was significantly increased(94.83±2.55%vs 75.98±4.54%,P<0.01),and the model was stable and reproducible.2.The release,qualitative and quantitative analysis of MVs induced by HPC from HUVECsHPC induced HUVECs to release MVs successfully,known as HPC-EMVs.The protein of HPC-EMVs was determined by BCA method and quantified at0.298±0.045?g/?L.Under the transmission electron microscope,HPC-EMVs were observed in circular or elliptical membranous vesicle-like structure and the diameters of MVs were 100-1000 nm.3.Effects of HPC-EMVs on normal H9c2 cellsCompared with Control group,the cells viability of HPC-EMVs 30 and HPC-EMVs 60?g/mL were significantly decreased(90.43±1.21%,73.19±2.11%vs 100±0%,P<0.01 or P<0.001),while the activity of LDH in HPC-EMVs 30 and HPC-EMVs 60?g/mL group were significantly increased(132.97±4.92,157.38±6.94 vs 116.23±6.88,P<0.001),the activity of LDH in HPC-EMVs 10?g/mL group reduced slightly compared with Control group(114.25±6.31 vs 116.23±6.88).From the Hoechst 33258 staining,with the increasing concentration of HPC-EMVs,fragmented or condensed nuclei could be observed,the numbers of cells debris and apoptotic cells were increased.Compared with Control group,the activity of Caspase 3 in HPC-EMVs 60?g/mL group was significantly elevated(307.29±9.25 vs 140.04±10.37,P<0.01),compared with HPC-EMVs 10 and 30?g/mL groups,the activity of Caspase 3 in HPC-EMVs 60 group was significantly increased(142.51±10.55,182.78±6.58vs 307.29±9.25,P<0.001).Compared with Control group,the ratio of Bcl-2/Bax decreased significantly with the increasing concentration of HPC-EMVs.Conclusion:1.The model of HPC of HUVECs was successfully established in this experiment.2.MVs were generated from HUVECs induced by the method of hypoxia solution pH 6.8/5.8 and pre-hypoxia 10 min/reoxygenation 15 min.The concentration of HPC-EMVs protein was 0.298±0.045?g/?L.Under the transmission electron microscope,HPC-EMVs were observed in circular or elliptical membranous vesicle-like structure and ranged from 100 to 1000 nm.3.HPC-EMVs decreased the cells viability of normal H9c2 cells and increased LDH leakage and HPC-EMVs 60?g/mL had the most significant effects.HPC-EMVs reduced the ratio of Bcl-2/Bax significantly,which suggested that the apoptosis mechanisms of HPC-EMVs may be involved in down-regulating the ratio of Bcl-2/Bax. |
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