Protection On The Injury Of Endothelial Cells In Vitro By Flavones Of Radix Astragali From Gansu Province

Objective: To investigate the protective effects by flavones (formononetin and calycosin-7-O-β-D-glucopyranoside) of Radix astragali from Gansu province on angiotensin II(AngII)-induced injury of human umbilical vein endothelial cells (HUVECs) in vitro.Methods: Human umbilical vein endothelial cells(HUVECs) were cultured in vitro, MTT colorimetric assay was used to detect the survival rate of influence from formononetin(0.01, 0.1, 1.0, 10mg·L^-1) and calycosin-7-O-β-D-glucopyranoside (0.01, 0.1, 1.0,10 mg·L^-1) to the cultured HUVECs, Hoechst 33258 dyeing was used to observe AngⅡ -induced nuclear morphological changes and the apoptosis of HUVECs was detected by flow cytometry and Laser-Scanning Confocal Microscope. The expression change of Fas was observed.The lactate dehydrogenase (LDH) leakage from HUVECs were investigated by spectrophotometer. Supernatant nitric oxide (NO) and endothelin-1 (ET-1 ) contents in HUVECs were measured by NO assay kitand radioimmunoassay correspondingly.Results: It was found that incubation with AngII(10'6 mol-L^') for 24h increased HUVECs apoptosis(67.97±7.82 vs 2.69±0.25, P=0.000);formononetin(1.0mg-L^K lOmg-L"1) and calycosin-7-0-f}-D-glucopyranoside{\ .OmgL" K 10mg-L l) signify-cantly increased the survival rate of HUVECs (0.7025±0.0091 vs 0.6843±0.0057, i^O.005, 0.7128±0.0062 vs 0.6843±0.0057, P=0.000, 0.7003±0.0081 vs 0.6843 ±0.0057, P=0.012, 0.7095±0.0067 vs 0.6843±0.0057, P=0.000, respectively). There were no significant differences of the cell survival rate between formononetin (l.Omg-L"1) andformononetin(\OmgL^l) and the same as the results of calycosin-7-0-P-D-glucopyranoside (1.0mg-L \ lOmg-L" '). Formononetin(\.OmgL" ') and calycosin-7-0-fl-D-glucopyranoside(1.0mg-L^]) significantly decreased Angll-induced HUVECs' apoptosis (42.13±5.55 v^ 2.69±0.25, P=0.000, 47.17±5.81 vs 2.69±0.25, P=0.002) and decreased the expression of Fas(65.63±5.01 vs 79.90±6.56, P=0.011, 69.07±6.50 vs 79.90±6.56, P=0.037). Meanwhile, formononetin{\.Qmg-lTx) and calycosin-7-0-fi-D-glucopyranoside(1.0mgL^') decreased LDH leakage (480.28 ± 102.35 vs 1543.85±411.78, P=0.000, 471.58±70.43 vs 1543.85±411.78, P=0.000) and ET1 releases(46.92±5.41 vs 81.45±8.9, P^O.000, 47.89±8.56 vs 81.45±8.9, P=0.000) as well as increased NO releases(37.34±5.01 vs 27.33±5.63, P=0.017, 36.32±5.12 vs 27.33±5.63, P=0.028);There were no significant differences between formononetin (l.Omg-LT1) and calycosin-7-0-fi-D-glucopyranoside (1.0mg-L"1) of above experi- ments. Two flavones, results of MTT, apoptosis, expression of Fas, contents of NO, ET-1 and LDH in HUVECs were 0.7025±0.0091 vs 0.7003±0.0081, P=0.707, 42.13±5.55 vs 47.17±5.81, P^O.304, 65.63±5.01 vs 69.07±6.50, P=0A5l, 37.34±5.01 vs 36.32±5.12, P=0.m, 46.92±5.41 v^ 47.89±8.56, P=0.852, 480.28 ±102.35 vs 471.58±70.43, ?=0.956, respectively.Conclusion: Angiotensin II may be involved in the injuring HUVECs directly and causing endothelial dysfunction. Flavones separated from Radix astragali of Gansu province has precisely protective effects on angiotensin Il-induced HUVECs injury, which suggested that falvone may play a role in the prevention and treatmentof vascular diseases. There were no significant difference of protection from AngH-induced endothelial cell injury between the same concentration flavones formononetin and calycosin-7-O-fi-D-glucopyranoside.