Abstract:

Objective To study the effect of angiopoietin-1 （Ang-1） and tyrosine kinase receptor 2 （Tie2） inhibitor on glucose transportation in the human umbilical vein endothelial cells （HUVECs） cultured under high glucose conditions， and to clarify its mechanism. Methods The HUVECs were cultured in high glucose （30 mmol·L?1） in vitro and treated with 0， 200， 500， 1 000， and 2 000 μg·L?1 Ang-1 and 0， 2 500， 5 000， and 7 500 nmol·L?1 Tie2 inhibitor； cell counting kit-8 （CCK-8） method was used to detect the cell activity to screen the optimal concentrations of Ang-1 and Tie2 inhibitor. Glucose kit was used to detect the glucose level in the supernatant of the HUVECs after Ang-1 intervention. The HUVECs were randomly divided into blank control group （NG group）， high glucose group （HG group）， HG+Tie2 inhibitor group （HG+In-Tie2 group）， HG+Ang-1 group， HG+Ang-1+Tie2 inhibitor group （HG+Ang-1+In-Tie2 group）， and HG+Ang-1+phosphatidylinositol 3-kinase（PI3K） inhibitor group （HG+Ang-1+LY294002 group）. 5-Ethynyl-2'-deoxyuridine （EdU） method was used to detect the proliferation activities of the cells in various groups； YO-PRO-1/PI method was used to detect the apoptotic rates of the cells in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of Ang-1 mRNA and Tie2 mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of Tie2， glucose transporter 1 （GLUT1）， and glucose transporter 4 （GLUT4） proteins and the ratios of phosphorylated PI3K （p-PI3K）/PI3K and phosphorylated protein kinase B （p-AKT）/AKT in the cells in various groups. Results The CCK-8 assay results showed that compared with 0 μg·L?1 Ang-1 group， the activity of the HUVECs was significantly increased after treated with 200 μg·L?1 Ang-1 for 48 h （P<0.01）； compared with 0 nmol·L?1 Tie2 inhibitor group， the activity of the HUVECs was significantly decreased after treated with 2 500、 5 000 and 7 500 nmol·L?1 Tie2 inhibitor （P<0.01）； the optimal concentrations of Ang-1 and Tie2 inhibitor were 200 μg·L?1 and 2 500 nmol·L?1， respectively. Compared with NG group， the glucose level in the supernatant of the HUVECs in HG group was significantly increased （P<0.01）； compared with HG group， the glucose level in the supernatant of the HUVECs in Ang-1 group was significantly decreased （P<0.01）. The EdU assay results showed that compared with NG group， the proliferation activity of the HUVECs in HG group was significantly decreased （P<0.01）； compared with HG group， the proliferation activity of the HUVECs in HG+In-Tie2 group was significantly decreased （P<0.01）， and the proliferation activity of the HUVECs in HG+Ang-1 group was significantly increased （P<0.01）； compared with HG+Ang-1 group， the proliferation activities of the HUVECs in HG+Ang-1+In-Tie2 group and HG+Ang-1+LY294002 group were significantly decreased （P<0.01）. The YO-PRO-1/PI assay results showed that compared with NG group， the apoptotic rate of the HUVECs in HG group was significantly increased （P<0.01）； compared with HG group， the apoptotic rate of the HUVECs in HG+In-Tie2 group was significantly increased （P<0.01）， and the apoptotic rate of the HUVECs in HG+Ang-1 group was significantly decreased （P<0.01）； compared with HG+Ang-1 group， the apoptotic rates of the HUVECs in HG+Ang-1+In-Tie2 group and HG+Ang-1+LY294002 group were significantly increased （P<0.01）. The RT-qPCR results showed that compared with NG group， the expression levels of Ang-1 mRNA and Tie2 mRNA in the HUVECs in HG group and HG+In-Tie2 group were significantly decreased （P<0.01）； compared with HG group， the expression levels of Ang-1 mRNA and Tie2 mRNA in HG+ In-Tie2 group were significantly decreased （P<0.01）， and the expression levels of Ang-1 mRNA and Tie2 mRNA in the HUVECs in HG+Ang-1 group were significantly increased （P<0.05）； compared with HG+Ang-1 group， the expression levels of Ang-1 mRNA and Tie2 mRNA in the HUVECs in HG+Ang-1+In-Tie2 group and HG+Ang-1+LY294002 group were significantly decreased （P<0.05 or P<0.01）. The Western blotting results showed that compared with NG group， the expression level of Tie2 protein in the HUVECs in HG group was significantly decreased （P<0.01）， and the expression levels of GLUT1 and GLUT4 proteins were significantly increased （P<0.01）； compared with HG group， the expression levels of Tie2， GLUT1， and GLUT4 proteins in the HUVECs in HG+In-Tie2 group were significantly decreased （P<0.01）， the expression level of Tie2 protein in the HUVECs in HG+Ang-1 group was significantly increased （P<0.01）， and the expression levels of GLUT1 and GLUT4 proteins were significantly decreased （P<0.01）； compared with HG+Ang-1 group， the expression levels of Tie2， GLUT1， and GLUT4 proteins in the HUVECs in HG+Ang-1+In-Tie2 group and HG+Ang-1+LY294002 group were significantly decreased （P<0.01）. Compared with NG group， the p-PI3K/PI3K and p-AKT/AKT ratios in the HUVECs in HG group were significantly increased （P<0.01）； compared with HG group， the p-PI3K/PI3K and p-AKT/AKT ratios in the HUVECs in HG+In-Tie2 group were significantly decreased （P<0.01）， and the p-PI3K/PI3K and p-AKT/AKT ratios in the HUVECs in HG+Ang-1 group were significantly decreased （P<0.01）； compared with HG+Ang-1 group， the p-PI3K/PI3K and p-AKT/AKT ratios in the HUVECs in HG+Ang-1+In-Tie2 group and HG+Ang-1+LY294002 group were significantly decreased （P<0.01）. Conclusion Ang-1 down-regulates the expressions of GLUT1 and GLUT4 in the HUVECs cultured under high glucose conditions； the binding of Ang-1 to Tie2 may down-regulate GLUT1 and GLUT4 via the PI3K/AKT signaling pathway to participate in the glucose transportation in the HUVECs cultured under high glucose conditions.

Key words: Diabetic nephropathy, Angiopoietin-1, Glucose transporter protein, Tyrosine kinase receptor 2, Phosphatidylinositol 3-kinase, Protein kinase B