Effects Of Astragalus Membranaceus Injection On Endothelial Progenitor Cells From Human Peripheral Blood And Its P38 MAPK Signaling Pathways Mechanisms

Chapter One Effects of Astragalus Membranaceus Injection on Number and Activity of Endothelial Progenitor Cells from Human Peripheral BloodObjective:To investigate and evaluate the effects of Astragalus Membranaceus Injection(AMI)on the number of EPCs and the function of EPCs such as the capacity of migration,adhesion,in vitro vasculogenesis capacity,proliferation,differentiation and cell cycle.Methods.Total mononuclear cells(MNCs)were isolated from 10ml of peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin coated culture dishes.After 7 days culture,adherent cells were incubated with various concentrations of AMI(0.2mg/ml,2mg/ml,20mg/ml,200mg/ml)(in the medium as above) for 24hours or with 20mg/ml AMI for the respective time points(6 hours, 12 hours,24 hours and 48 hours).EPCs were characterized as adherent cells with double positive for DiLDL-uptake and lectin binding(by direct fluorescent staining)under a laser scanning confocal microscope(LSCM). EPCs were viewed with an inverted fluorescent microscope.EPCs were further documented by demonstrating the expression of KDR,CD133 and CD34 with flow cytometry.EPCs proliferation,migration and in vitro vasculogenesis activity were assayed by MTT assay,modified Boyden chamber assay and in vitro vasculogenesis kit,respectively.EPCs adhesion assay was performed by replating cells on fibronectin-coated dishes,then adherent cells were counted.EPCs differentiation and cell cycle were measured by flow cytometry.Results:(1)Effect of AMI on the nunbers of EPCs:AMI increased the number of isolated EPCs in both concentration and time-dependent manner.Apparent increase of EPCs number started at 2mg/ml and most evident at 20mg/ml(P＜0.01).It was also showed that concentration of 20mg/ml,AMI increase EPCs number after incubation for 6 hours and reached the maximum after incubation for 24 hours(P＜0.05 or 0.01)and decreased a little at 48 hours,though still significantly higher compared with control group(P＜0.01).(2)Effect of AMI on the migratory capacity of EPCs:AMI enhanced the migratory capacity of isolated EPCs followed the same concentration and time-dependent manner,maximum at 20mg/ml(P＜0.01).and the peak was reached after 24h incubation (P＜0.01)and decreased a little at 48 hours,but it was still significantly higher compared with control group(P＜0.01).(3)Effect of AMI on the adhesive capacity of EPCs:AMI improved the adhesive capacity of isolated EPCs in dose- and time-dependently(P＜0.01).The number of adhered cells increased after 6 hours(P＜0.05),with a peak at 24 hours(P＜0.01)and decreased a little at 48 hours,but it increased significantly compared with control group(P＜0.01).AMI had a peak effect at 20mg/ml concentration(P＜0.01).(4)Effect of AMI on EPCs in vitro vasculogenesis:In vitro vasculogenesis assay was used to investigate the ability of EPCs to participate in neovascularization,which is the most important activity of EPCs.AMI increased the number of tube-like structures in concentration-dependent manner,maximum at 20mg/ml(P＜0.01).The increase of tube-like structures number is in time-course manner at 20mg/ml,became apparent after 6 hours(P＜0.05) and reached the maximum after 24 hours(P＜0.01).Moreover,AMI made tube-like structures qualitatively different and more complex than control group.(5)Effect of AMI on the proliferative capacity of EPCs:AMI dose- and time-dependently improved EPCs proliferative activity.The proliferative capacity of EPCs improved after 6 hours(P＜0.01),with a peak after 24 hours(P＜0.01)at concentration of 20mg/ml(P＜0.01)and decreased a little at 48 hours,but it increased significantly compared with control group(P＜0.01).(6)Effect of AMI on EPCs cell cycle:Effect of AMI on cell cycle became apparent at 2mg/ml concentration.Flow cytometry analysis showed that S-phase and G₂/M-phase rate was increased but G₀/G₁-phase rate was decreased(P＜0.05or0.01),AMI had a peak effect at 20mg/ml concentration(P＜0.01).(7)Effect of AMI on EPCs differentiation:Effect of AMI on EPCs differentiation became apparent at 2mg/ml concentration,the relative proportion of EPCs expressing CD14 and CD64 was decreased and the cells expressing the endothelial marker von Willebrand factor was increased as compared with that in control group(P＜0.05or0.01),AMI had a peak effect at 20mg/ml concentration(P＜0.01).Conclusions:(1)AMI increases the quantity of EPCs in a dose- and time-dependent manner.(2)AMI improves the activity of proliferation and migration,adhesive capacity and In vitro vasculogenesis capacity of EPCs in dose- and time-dependent manner.(3)AMI affects cell cycle of EPCs,increases the S-phase and G₂/M-phase rate and decreases the G₀/G₁-phase rate in a dose- dependent manner.(4)AMI affects differentiation of EPCs,coincubation of AMI with the EPCs reduced the relative proportion of cells expressing the monocytic marker proteins CD14 and CD64,increased the relative proportion of cells expressing the endothelial marker von Willebrand factor,suggesting that AMI prevents progenitor cell differentiation toward the macrophage lineage and thereby facilitates endothelial cell differentiation. Chapter Two Effects of Astragalus membranaceus Injection on p38 MAPK Signaling Pathways in Cultured Endothelial Progenitor Cells from Human Peripheral Blood with High GlucoseObjective:To investigate the effects of Astragalus Membranaceus Injection(AMI)on the p38 mitogen-activated protein kinase(p38 MAPK) in cultured endothelial progenitor cells(EPCs)from human peripheral blood with high glucose.Thus to study the influence and it's mechanism of high glucose and AMI on EPCs quantity and activity.Methods:EPCs were incubated with various concentrations of glucose(5mM,10mM,20mM,30mM)for 24hours and with 30mM glucose for the various time point(10min,30min,60min,120min). Simultaneously,EPCs pre-incubated with various concentrations of AMI (5mg/ml,10mg/ml,20mg/ml,50mg/ml)for 24hours and with 20 mg/ml AMI for the differently time point(6 hours,12 hours,24 hours,48 hours),and then all groups were incubated with 30mM glucose for 60min.EPCs were viewed with an inverted fluorescent microscope.EPCs proliferation and differentiation were assayed by MTT assay and flow cytometry.Expression of p38 MAPK and phospho- p38 MAPK were measured using Western Blot.Results:(1)High glucose decreases the number of EPCs in a dose-and time-dependent manner.In addition,glucose dose- and time-dependently impaired EPCs proliferative and differentiative capacity (P＜0.05or0.01).Pre-incubated with AMI and specific p38-inhibitor SB203580 could increase EPCs quantity and improves EPCs proliferative and differentiative capacity(P＜0.01).(2)Incubation of EPCs with high glucose dose- and time-dependently increased p38-phosphorylation.The maximum of p38 activity was obtained at 60 minutes of incubation (P＜0.01).(3)Pre-incubation of the EPCs with AMI inhibited p38 phosphorylation caused by high glucose in a dose- and time-dependent manner(P＜0.01).Conclusion:(1)High glucose decreased the number of EPCs and impaired the capacity of proliferation and differentiation toward the endothelial cell lineage in a dose- and time-dependent manner.(2)Preincubated with AMI and specific p38-inhibitor SB203580 increased the number and improved the capacity of proliferation and differentiation of EPCs cultured with high glucose.(3)Incubation of EPCs with high glucose dose- and time-dependently increased p38-phosphorylation.AMI inhibited p38 phosphorylation caused by high glucose in a dose- and time-dependent manner.It may be one of the mechanism of AMI on EPCs number and activity.