Effect Of Betaine On Protection Of Human Umbilical Vein Endothelial Cells And Its Mechanisms

ObjectiveTo explore the effect of Betaine(Bet)on a model of injured human umbilical vascular endothelial cell (HUVEC) in vitro by H₂O₂;To discusse influence of Betaine on Bcl-2 and Bax expression;To observe the changes of SOD,MDA,nitrie oxide in HUVEC under oxidative damage,and to futher investigate the protective effects and its possible mechanisms of Betaine on HUVEC under oxidative damage. Provide informations for evaluation of BetaineMethodIn vitro perfusion and enzyme digestion method is used to culture HUVEC, And the cells are identified by morphological observation andâ…§: Ag Immunofluorescence staining.After the second generateon,select the cells in the exponential phase of growth,experiments were divided into five groups: (1) normal control; (2) H₂O₂ damage group: H₂O₂ (concentration 1mmol/L); (3) low dosage Betaine group: Betaine (9.3mg/L) + H₂O₂ (4) middling dosage Betaine group: Betaine (28mg/L) + H₂O₂; (5) high dosage Betaine group: Betaine (84mg/L) + H₂O₂; After the cells are treated for 4 hours, The shape of cells was observed in Phase contrast microscope and the cells viability is measured by the MTT assay. The cell apoptosis is determined by flow cytometric analysis and staining with AO and EB. Immunofluorescence and Immunohistochemical method are used to determine Bcl-2 and Bax expression. The activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) and nitrie oxide (NO) production were determined in cell medium and detemined in different ways.Results1 the cultured cells fused after inoculation for 5 to 7 days and assume typical'slabstone',â…§:Ag immunoreactivity showed 95% cell appeared flavovirens fluorescence .So the cells cultured in this method are proved to be HUVEC and fit for our study.2 Morphological observation show the cells shrieked and became circular in H₂O₂ damage group,so HUVECs are damaged obviously, while in groups with BET,condition of HUVECs is improved. The MTT assay Bet made these changes disappear or become less and depend on concentration of Bet.3 Flow cytometric analysis determine Bet reduce Apoptosis rate significantly and the result of cell staining with AO and EB is similar with that from FCM.4 Immunofluorescence staining show Bet made expression of Bcl-2 increase and expression of Bax decrease obviously by H₂O₂ damaged,the result of immunocytochemistry is similar with that from Immunofluorescence staining.5 Bet could increase the activity of SOD and decrease the content of MDA in HUVECs under H₂O₂,Bet could enhance the production of NO.Conclusion1 Bet could prevent apoptosis of HUVEC under H₂O₂ induced and protect its activity.2 The inhibition of Bet on apoptosis of HUVEC correlated with an increase Bcl-2 expression and decrease Bax expression.3 Bet could exhibit its protective effects against HUVEC injury by inereasing the activity of SOD and decreasinge the content of MDA in HUVECs under oxidative damage, Bet could protect the function of HUVEC by enhance the production of NO.