High Glucose Effects On HUVEC For The Expression Of PAI-1, T-PA And The Intervention Role Of Tea Polyphenols

Objective: Diabetic macroangiopathy,one of the most commoncomplications of diabetes,has been the leading cause of acquiredatherosclerosis. Type-1Plasminogen activator inhibitor（PAI-1）,as a memberof the super family--Serine protease inhibitor,is the major physiologicalinhibitor of tissue-type plasminogenactivator and urokinase plasminogenactivator.As the clinical epidemiological survey reported,PAI-1in the plasmaof patients with atherosclerosis is increased,and high levels of PAI-1help tothe progress of plaque,which prompting PAI-1is closely related to the happenand the development of atherosclerosis[1]. Tea polyphenols is extracted fromthe tea,which is a kind of hydroxy phenolic compounds. The role of TP inprevent cardiovascular disease has been caused widespread attention,but theconcrete mechanism is not fully clear,especially the study of the TP how canprotect endothelial fibrinolytic function to prevent the AS is less more. In thisstudy,High sugar environmental induced the fibrinolytic function damage ofhuman umbilical vein endothelial cells (human umbilical vein endothelial cell,HUVEC),different concentrations of TP effect cultured HUVEC cells48hoursin high glucose, in order to detect the level of t-PA、PAI-1expression. Aimedto explore the protection of TP in vascular endothelial cell function andinfluence on fibrinolytic function in vitro experiment. It is possible to find newpathway to search for the prevention and treatment of diabeticmacroangiopathy by the mechanism of changes in expression of t-PA andPAI-1caused by high glucose.Content: Take aseptic umbilical cord, and after pancreatic enzymedigestion,HUVEC were cultivated by primary culture technology,whenachieved more than80%fusion,they were subcultured and taken3to5generations of cells into the next experiment. And the logarithmic phase-growth cells were randomly divided into nine groups: normal control（glucose5.5mmol/L,DMEM nutrient solution）;High glucose group（glucose30mmol/L,DMEM nutrient solution）;Hypertonic group （mannitol24.5mmol/L+glucose5.5mmol/L，DMEM nutrient solution）;TP controlgroups（TP concentration espectively is10mg/L、20mg/L、30mg/L,glucose5.5mmol/L， DMEM nutrient solution）， TP intervention groups （TPconcentration espectively is10mg/L、20mg/L、30mg/L,glucose30mmol/L，DMEM nutrient solution）. In addition to the high permeability in the controlgroups, each drug groups added each drug separately30minutes before thehigh sugar processing.4hole cells in each group. After adding drugs, theywere continued to develop after48hours,then detected mRNA and proteinexpression of t-PA and PAI-1in each group.Method:The original generation of culture and extend the cell culture;ELISA could determine the content of t-PA and PAI-1in cell supernatant；Viability of HRPEcells is evaluated by MTT assay; Cells extraction of totalRNA and protein; RT-PCR and western blot technologyResults:1Compared with normal control group,cell activity of high glucosegroup were reduced,which had statistical significance (P <0.05),but,there wasno statistical significance between normal control group and hypertonicgroup(P＞0.05）.2Compared with normal control group,HT1group cell activity weredecreased，HT3group increased the cell activity,and they all had statisticalsignificance (P<0.05);but there were no obvious difference between thenormal control group and the HT2group (P>0.05).3Compared with high glucose group,HT1group cell activity wasdecreased（P＜0.05）, HT2group cell vitality vigor increased but still lowerthan the normal control group （P＜0.05）,HT3cell vitality increasedsignificantly than high glucose group（P＜0.05），So choose20mg/LTP concentration (the concentration of sugar in thenormal cell vitality no effect and can reduce sugar environment effect on cell viability) for optimal experimental dose, The following experiments were allapplied this concentration.4Compared with normal control group, high glucose group significantlyincreased amount of PAI-1protein expression, and20mg/L TP can make highPAI-1protein expression of high glucose group dropped to normal, and theyall had statistically significant (P<0.05).But, hypertonic group did not have noobvious change.5Compared with normal control group,PAI-1mRNA level of highglucose group increased significantly,the HT2group was significantly reduced.The difference was statistically significant (P＜0.05). The high glucose groupt-PAmRNA level significantly lower than normal group (P<0.05), t-PAmRNAlevel between the HT2group and the normal group was no significantdifference (P>0.05); hypertonic group had no significant change (P>0.05).6Compared with normal control group,content of PAI-1in high glucosegroup increased significantly,while20μg/ml TP could make the content ofPAI-1in high glucose group down to normal,The difference was statisticallysignificant (P＜0.05);But the change of t-PA content was no statisticallysignificant.Conclusion:1High sugar environment can cause umbilical vein endothelial cellssecrete increased the expression of PAI-1activities, may be in the process ofdiabetes vascular lesions play an important role.2A certain concentration can reverse umbilical vein endothelialfibrinolytic dysfunctions caused by high sugar, it also can reduce theexpression of PAI-1, maintain PAI-1/t-the stability of PA, protect theumbilical vein endothelial cells. Tea polyphenols as a strong antioxidant, mostlikely play a positive role in the prevention and treatment of diabetes vascularlesions.