Down-Regulation Of ER/PI3K/AKT Pathway Mediating High Glucose Induced Dysfunction Of Human Umbilical Vein Endothelial Cells

Objective:Diabetes mellitus（DM）and Cardiovascular Disease（CVD）are both the leading causes of death worldwide.DM is associated with CVD,hyperglycemia,hyperlipidemia and insulin resistance induce by diabetes are major risk factors of developing vascular complications,and the abnormal of blood glucose,blood lipids caused by CVD may lead to type 2 DM.The harmfulness of DM is the complication caused by DM,including CVD,Osteoporosis,diabetic nephropathy and retinopathy,and more than 75%of DM patients died from CVD.Therefore,the key to cure DM is to deal with the vascular diseases complication of DM.The morbidity of CVD is lower in premenopausal women than men,the cardiovascular protection of estrogen is the major factor leading to the sex difference of the morbidity of CVD,estrogen resist the damage of glucose by regulate the proliferation,differentiation,migration and the production of NO in endothelial cells through estrogen receptor（ER）.Thus,hormone replacement therapy always used as the first choice in cure CVD.However,the cardiovascular protective effect of estrogen has decreased significantly in DM patients.The objective of this study is to research the effects of estrogen on human umbilical vein endothelial cells function in the expression change of ER.Methods:（1）HUVECs were respectively exposed to high glucose（33mmol/L）and E₂(1×10^-9mol/L)for 48h,the expression of ER was detected by immunofluorescence staining.（2）Western blot was used to detect the effects of high glucose on the expression of ER,PI3K,p-AKT.The cells were treated with glucose for 48 h at different concentrations of 5.5 mmol/L,11 mmol/L,22 mmol/L,33mmol/L,44 mmol/L and different time of 1 day,2 days,3 days,4 days,5 days at concentration of 33mmol/L.（3）To estimate whether high glucose affects the function of HUVECs by inhibiting ER/PI3K/AKT pathway.Cells were respectively exposed to high glucose（33mmol/L）,E₂(1×10^-9mol/L),high glucose with E₂,high glucose with ER inhibitor ICI182780（10n M）,high glucose with ICI182780 and E₂,high glucose with PI3K inhibitor LY294002（10μM）,high glucose with LY294002 and E₂for48h.CCK-8 assay,DCFH-DAassay,Annexin V-FITC/PI staining and DAF-FM DA assay are used to detect cell proliferation,the level of ROS,cell apoptosis and NO generation.The expression of ER,PI3K,p-AKT was detected by Western blot.Results:（1）High glucose inhibit the expression of ERαand ERβsignificantly,and E₂eliminate the inhibitory effect of high glucose on ER.（2）High glucose inhibit the expression of ER、PI3K、p-AKT in a dose-and time-dependent manner,the expression of ERα,ERβ,PI3K,p-AKT are both increased at the present of E₂,and reduced after adding ER inhibitor ICI182780.PI3K inhibitor LY294002 inhibit the promotion of E₂on PI3K,p-AKT.（3）There are obviously decrease of NO,cell viability,and increase of ROS,cell apoptosis in endothelial cell treated with high glucose at concentrations of 33mmol/L;E₂increase the viability of endothelial cells,the generation of NO and inhibit apoptosis and ROS;the promotion of E₂on cells deficiency in the present of ICI182780 and LY294002.Conclusion:High glucose inhibits the of generation NO and increase the levels of ROS leaading to the decrease of cell vitality and the increase of apoptosis in HUVECs.Thedamage effects of high glucose was induced by down-regulating ER/PI3K/AKT pathway.E₂activate the ER/PI3K/AKT pathway and recove endothelial cells functioning under condition of high glucose.