| The re-establishment of blood flow after IHD may further aggravate the tissue injury in the form of I/R injury.One of the critical events which contributes to myocardial I/R injury is intracellular calcium overload.IPC is a kind of powerful endogenous protective phenomenon defined as one or several episodes of short ischemia followed by reperfusion,generates profound protection against a subsequent sublethal ischemia.This protective phenomenon was discovered and confirmed to work perfectly in vascular endothelial cells too.Vascular endothelial cells are important barriers at the surface between blood and tissue,healthy and intact vascular endothelial cells may prevent or palliate the ischemia and I/R injury to cardiomyocytes.MVs are small vesicles released from the surface of activated or apoptotic cells which participate in various activities by carrying bioactive substances.whether the protection of IPC on I/R injury works by efficient MVs? We establish HPC model,which mimics IPC phenomenon in vivo,and intracellular calcium overload model,which represent I/R injury,to study the protective effect of HUVECs derived microvesicles induced by HPC on intracellular calcium overload H9c2 cardiomyocytes.Objective:To establish HPC model on HUVECs and do quantitative and morphology analysis of EMVs induced by HPC from HUVECs(HPC-EMVs).To investigate the effects of HPC-EMVs on normal and intracellular calcium overload and injuryed H9c2 cardiomyocytes.Methods:1.Establishment of stable hypoxic preconditioning model on HUVECs HUVECs 6×104 were seeded and cultured in 96 wells for 24 h then divided into Control,H/R and HPC groups.Control group was cultured in normal cell incubator and H/R group was treated by hypoxia for 12 h and reoxygenation for 4 h.HPC group was treated under short period of H/R,then followed by hypoxia for 12 h and reoxygenation for 4 h.Cell viability of each group was tested by MTT assay and proper cell density,time of hypoxic preconditioning and p H of hypoxic buffers were determined.2.The generation,quantitative and morphology analysis of HPC-EMVs HPC-EMVs were isolated by ultracentrifugation from the conditioned culture medium and proteins were quantified by BCA assay.Transmission electron microscopy(TME)analysis was used to detect the morphology of HPC-EMVs3.Establishment of intracellular calcium overload and injury model on H9c2 cardiomyocytes H9c2 cardiomyocytes 1×105 were seeded and cultured in 96 wells.Cell injury was induced by different concentrations of ionomycin,and whether or not 0.55μmol/L of ionomycin could induce intracellular calcium overload in H9c2 cardiomyocytes was verified.H9c2 cardiomyocytes 1×105 were seeded and cultured in 35 mm confocal dishes for 24 h.Cells were changed with 0.55 μmol/L of ionomycin diluted with DMEM and protected from light and cultured in incubator for 50 min then stained with Fluo-3AM diluted with DMEM.Fluorescence intensity of each groups was detected by laser scanning confocal microscope at an excitation wavelength of 488 nm.Photos of fluorescent H9c2 cardiomyocytes were analyzed by Image J and proper concentration of ionomycin was determined.Intracellular calcium overload and injury model on H9c2 cardiomyocytes was eatablished.4.Effects of HPC-EMVs on normal H9c2 cardiomyocytes H9c2 cardiomyocytes were randomly divided into Control,10,30,60 μg/m L HPC-EMVs groups.In Control group,culture medium was replaced by fresh DMEM without FBS.In HPC-EMVs groups,HPC-EMVs at different concentrations(10,30,60 μg/m L)were co-cultured with normal H9c2 cardiomyocytes for 4 h.Cell viability was tested by MTT assay.5.Effects of HPC-EMVs on intracellular calcium overload and injury H9c2 cardiomyocytes H9c2 cardiomyocytes were randomly divided into Control,0,10,30,60 μg/m L HPC-EMVs groups.In Control group,culture medium was replaced by fresh DMEM without FBS.In HPC-EMVs groups,HPC-EMVs at different concentrations(0,10,30,60 μg/m L)were pretreated with H9c2 cardiomyocytes for 4 h,then intracellular calcium overload was induced.First,fluorescent intensity of Ca2+ of 60 μg/m L HPC-EMVs group was detected.Cell viability was tested by MTT assay.Evidence for H9c2 apoptosis was detected by LDH and Caspase 3 activity measurement.Apoptotic rates were observed by Hoechst 33258 staining.Bcl-2/Bax expression was assessed by western blot.Results:1.Establishment of successful hypoxic preconditioning model on HUVECs HUVECs were conducted a short period of hypoxia by 95% N2-5% CO2 with p H6.8 hypoxic buffer for 10 min followed by reperfusion for 15 min,then added equivalent p H 5.8 hypoxic buffer,HUVECs generated profound protection against a subsequent long-time hypoxia for 12 h and reperfusion for 4 h.The cell viability of HPC groups were significantly higher than H/R groups(93.9 ± 6.9% vs 79.6 ± 5.7%,P<0.01),which confirmed the success of establishment of hypoxic preconditioning model on HUVECs.2.Isolation and identification of HPC-EMVs Cultural supernatant was centrifugated at 2,700 g,4 ℃ for 20 min to remove cell debris.Supernatant was subjected to a centrifugation at 33,000 rpm,4 ℃ for148 min to pellet HPC-EMVs.HPC-EMVs proteins were quantified at 0.304 ± 0.06μg/μL.TME analysis was used to detect the morphology of HPC-EMvs.As results,HPC-EMVs displayed a spherical shape under TEM,with a size ranged from 100 to1000 nm.3.Establishment of successful intracellular calcium overload and injury model on H9c2 cardiomyocytes Compared to Control group,0.55 μmol/L ionomycin significantly reduced cell viability(63.2 ± 4.7% vs 100 ± 0%,P<0.001).So we chose 0.55 μmol/L ionomycin to verity level of intracellular calcium overload on H9c2 cardiomyocytes.Compared to Control group,0.55 μmol/L ionomycin significantly improved the mean fluorescence intensity of intracellular calcium in H9c2 cardiomyocytes(38.57 ±0.83 a.u.vs 24.28 ± 0.90 a.u.,P<0.001).4.Pro-apoptotic effects of HPC-EMVs on normal H9c2 cardiomyocytes Compared to Control group,10 μg/m L HPC-EMVs slightly decreased the cell viability of H9c2 cardiomyocytes(97.3 ± 2.8% vs 100 ± 0%),while 30,60 μg/m L HPC-EMVs dose-dependently lowered the cell viability(91.9 ± 1.4%,77.4 ± 3.1%vs 100 ± 0%,P<0.001).5.Anti-apoptotic effects of HPC-EMVs on intracellular calcium overload and injury H9c2 cardiomyocytes Analysis of fluorescence intensity showed that 60 μg/m L HPC-EMVs remarkablely decreased H9c2 intracellular calcium overload(30.96 ± 1.19 a.u.vs38.57 ± 0.83 a.u.,P<0.001)than HPC-EMVs 0 group.Intracellular calcium overload was induced by ionomycin.Compared to HPC-EMVs 0 group,pretreatment with 30,60 μg/m L HPC-EMVs increased the cell viability(70.3 ± 4.3%,74.4 ± 5.6% vs 63.2 ± 4.7%,P<0.001).Moreover,LDH leakage in HPC-EMVs 30 and 60 group significantly reduced(157.24 ± 16.51 U/L,145.01 ± 5.86 U/L vs 178.93 ± 12.86 U/L,P<0.05,P<0.001).Hoechst 33258 staining results revealed a reduction of cell fragmentation and nuclear agglomeration with the increase of HPC-EMVs concentration.Meanwhile,Caspase 3 activity in HPC-EMVs30,60 groups was significantly decreased(314.27 ± 9.53 U/μg,304.25 ± 10.49 U/μg vs 365.44 ± 9.54 U/μg,P<0.001).Western blot results showed that the expression of Bcl-2/Bax were significantly increased in HPC-EMVs 60 group.Conclusion:1.Stable HPC model on HUVECs was established.2.The pelleted vesicles were analyzed by TME and displayed a spherical shape with a size ranged from 100 to 1000 nm which confirmed the pelleted vesicles were HPC-EMVs.3.Stable intracellular calcium overload and injury model on H9c2 cardiomyocytes was established.4.Compared to Control group,30,60 μg/m L HPC-EMVs dose-dependently lowered the cell viability and increased LDH activity of normal H9c2 cardiomyocytes.5.Compared to HPC-EMVs 0 group,60 μg/m L HPC-EMVs significantly decreased fluorescence intensity of intracellular calcium overload H9c2.30,60μg/m L HPC-EMVs dose-dependently increased the cell viability while decreased LDH and Caspase 3 activity of intracellular calcium overload H9c2 cardiomyocytes,60 μg/m L HPC-EMVs increased Bcl-2/Bax expression. |
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