Procyanidine Exert A Great Effect On Expression Of VEGFE-2and Signaling Pathways In Endothelial Progenitor Cell Cultivated In High Concentration Of Glucose

Abstract: Objective: The rapid growth trend of diabetes all over theworld make human health problems face serious challenges in the21stcentury,which is more than expected.In2007,246million people may beattacked by diabetes mellitus disease.It has been estimated that the number willreach380million in2025, which accounts for about7.3%of the world’spopulation.At the same time, nearly308million people have abnormal glucosetolerance, which leads to the risk factors for type2diabetes. DM (DiabetesMellitus) is characterized by high blood glucose,abnormal glucose tolerancecaused by insulin resistance and/or pancreas function decline.Diabetic patientswith vascular complications are more easily led to disability or death,especially,30%~40%of the patients will be attacked by at least a kind ofvascular complication10years later.Many researches have found that DMvascular complications are closely associated with oxidative stress inendothelial cells which are originated from endothelial progenitor cells in thebone marrow.Some present studies reveal that high glucose environment hasgreat effects on the endothelial cell function.At the same time, the number ofendothelial progenitor cells in peripheral blood is obviously declining,whichsuggests that the function and number of endothelial progenitor cells have greatrelevance with diabetic vascular complications. It has been demonstrated thatprocyanidins and its low poly products have anti-inflammatory andanti-oxidative properties in most plants.The contents of procyanidine in berryplants are far more than other plants several times.Especially, the contents ofprocyanidine in grape seed is the most abundant. The experiment discusses thatProcyanidins and its low poly products have beneficial effects on function inEPCs with oxidative stress,which contributes to providing new theoreticalknowledge. Methods: Male SD rats are executed by cutting off the heads,immersed in75%alcohol for20minutes.Place rats on cleanworkbench，isolate the femur and tibia under sterile conditions,extract PBScontaining1％heparin with5ml Syringe to wash the marrow cavity untilflushing fluid is colorless.Collect all flushing fluid with high speedcentrifugation, and remove the supernatant,and collect cells in the bottom of thetubes.To separate bone marrow mononuclear cells in rats with Ficoll densitygradient centrifugation.Monocytes isolated are inoculated in6well-plate.Change cell medium every three days.Then,observe the cellmorphology under the inverted microscope.EPCs were identified by the methodof the double swallow of FITC-UEA-1/DiI-ac-LDL. EPCs are isolated by themethod of Ficoll density gradient centrifugation,and the proliferation andOxidative stress production was respectively detected by spectrophotometerand enzyme standard instrument in control group(5.5mmol/L+25mmol/Lmannitol) and high glucose group(30mmol/L). the proliferation of the EPCs aredetected from various OPC concentration group in different time points,in orderto select optimum concentration of procyanidins OPC (30u g/ml).Tubeformation capacity was respectively detected by the way of Matrigel matrix infour groups,including Con+30ug/ml OPC,Con group,HG+30ug/ml OPC andHG group. Finally MDA value and protein expression of VEGFR-2, AKT,IKB-a and NF-κB was respectively detected in the1d,3d,5d,7d. Results:Most of EPCs were roundness, shining brilliantly, and gradually began toadhere on the third day.Seven days later,some cells began to deform in spindleshape,and turned to the shape of paving stone.EPCs were identified by themethod of the double swallow of FITC-UEA-1/DiI-ac-LDL,and dualfluorescence positive cells were considered to be endothelial progenitorcells.Compared with Con group,apoptosis cells and oxidative stress productionwere more in HG group（P<0.05）.Tube formation capacity was found noobvious difference between Con+OPC and Con group（P>0.05）.The number of tube formation in HG+OPC group was more than HG group（P<0.05）. Therewere no differences of the protein expression of VEGFR-2, AKT, IKB-a,NF-κB and oxidative stress production between Con+OPC and Con group in1d,3d,5d,7d（P>0.05).Compared with HG group,oxidative stress productionsignificantly reduced（P<0.05）,while the protein expression of VEGFR-2,AKT,NF-κB obviously increased in HG+OPC group in1d,3d,5d,7d（P<0.05）, excluding the protein expression of IKB-a（P<0.05）.Conclusion:Compared with Con group, EPCs proliferation ability significantlyreduced,and MDA value obviously increased in HG group,which indicated thatEPCs produced excessive oxidative stress product (ROS) in high glucoseenvironment.Excessive oxidative stress product can lead to cell injury andinhibition of cells proliferation. Procyanidins can alleviate oxidative damage ofEPCs affected on high glucose, improve expression of VEGFR-2,and activateits downstream pathway to promote EPCs proliferation.