| Objective: To investigate the protective effects of flavones (calycosin and 2'-OH-3',4'-dimethoxyisoflavanone-7-O-β-D-glucopyranoside) extracted from Radix Astragali of Gansu province on vascular endothelial cells. The effects include anti-apoptosis, endocrine function and regulating the expressions of angiotensin-converting enzyme (ACE) / angiotensin-converting enzyme 2 (ACE2).Methods: Human umbilical vein endothelial cells (HUVECs) were clultured in vitro. The models of injuried endothelial cells were established and divided randomly into groups as followed: control, angiotensin II (AngII) (1×10-6mol·L-1), AngII (1×10-6mol·L-1+2'-OH-3',4'-dimethoxyisoflavanone-7-O-β-D-glucopyranoside(1μg·mL-1), AngII (1×10-6mol·L-1) + calycosin (10, 1, 0.1μg·mL-1). After 24 hours, morphology of apoptosis cells were studied by fluorescence microscope with Hoechst33258 / PI double staining and laser scanning confocal microscope (LSCM). Apoptosis ratio of AngII-induced HUVECs by flavones from Radix Astragali treatment and the expression of the apoptosis related gene Fas were obsereved by flow cytometry (FCM). Nitric oxide (NO) and endothelin-1 (ET-1) contents of the HUVECs cultured medium were measured by colorimetry and radiommunoassay. Changes in both protein and gene expression of ACE/ACE2 were detected with immunohistochemistry analysis and reverse transcription polymerase chain reaction (RT-PCR) technique respectively.Results: Compared to the control group, treatment of AngII-induced HUVECs with concentrations of 1×10-6mol·L-1 revealed that the apoptosis ratio was increased gradually (70.23±4.59 vs 12.48±2.50, P=0.000), the expression of the apoptosis-induced gene Fas was enhanced (96.43±1.23 vs 17.41±1.52, P=0.000). LSCM could distinguish apoptosis cells from normal cells. The induced apoptosis effects of AngII were inhibited by the treatment of calycosin (1μg·mL-1) and 2'-OH-3',4'-dimethoxyisoflavanone-7-O-β-D-glucopyranoside (1μg·mL-1) extracted from Radix Astragali of Gansu province (55.99±2.40 vs 70.23±4.59, P=0.001; 58.31 + 2.64 vs 70.23 + 4.59, P=0.002, respectively). Also the expression of Fas was decreased (73.73±2.15 vs 96.43±1.23, P=0.000; 76.63±1.67 vs 96.43±1.23, P=0.000, respectively). Meanwhile, calycosin (1μg·mL-1) and 2'-OH-3',4'-dimetho-xyisoflavanone-7-O-β-D-glucopyranoside (1μg·mL-1) inhibited ET-1 release (34.96+ 1.27 vs 60.28±0.93, P=0.000; 32.17±8.24 vs 60.28±0.93, P=0.036, respectively), while they increased NO contents (43.28±4.92 vs 27.96±5.04, P=0.011; 42.74±7.21 vs 27.96±5.04, P=0.014, respectively). Compared with control group, AngII (1×10-6mol·L-1) could promote both protein and gene expressions of ACE (133.44±2.43 vs 113.70±3.63, P=0.000; 0.83±0.03 vs 1.32±0.02, P=0.000, respectively) and inhibit these of expression of ACE2 in HUVECs significantly (94.36±7.88 vs 127.47±5.23, P=0.002; 0.78±0.04 vs 1.04±0.02, P=0.002, respectively). Calycosin induced a concentration dependent increasing both protein and gene expressions of ACE2 ( protein: 114.77±4.43 vs 94.36±7.88, P=0.030; 127.04±2.38 vs 94.36±7.88, P=0.006; 152.01±4.10 vs 94.36±7.88, P=0.001; mRNA: 1.02±0.01 vs 0.78±0.04, P=0.000; 1.14±0.03 vs 0.78±0.04, P=0.000; 1.27±0.02 vs 0.78±0.04, P=0.000, respectively), downregulating the expression of ACE (protein: 119.45±2.54 vs 133.44±2.43, P=0.001; 108.48±5.05 vs 133.44±2.43, P=0.000; 95.23±5.34 vs 133.44±2.43, P=0.000; mRNA: 1.21±0.01 vs 1.32±0.02, P=0.035; 1.10+0.01 vs 1.32±0.02, P=0.005; 1.21±0.01 vs 1.01±0.01, P=0.000). However, there were no statistic differences between the two kinds of flavones with the same concentration of above experiments (The two flavones's results of apoptosis ratio, expression of Fas, contents of NO, ET-1 in HUVECs were 55.99±2.40 vs 58.31±2.64, P=0.396; 73.73±2.15 vs 76.63±1.67, P=1.000; 34.96±1.27 vs 32.17±8.24, P=0.383; 43.28±4.92 vs 42.74±7.21, P=1.000, respectively).Conclusion: Flavones extracted from Radix Astragali of Gansu province have protective effects on HUVECs by inhibition of AngII-induced apoptosis, upregulating ET-1 and NO endocrine, downregulating of ACE and increasing the expressions of ACE2. The results suggested that flavones may play important roles in prevention and treatment of vascular disease.
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