Effects Of Angiotensin-(1-7) On TLR4-mediated Oxidative Stress In Human Umbilical Vein Endothelial Cells And The Mechanisms

Objective:To explore the role and related mechanisms of angiotensin-(1-7)(Ang-(1-7)on Toll-like receptor 4(TLR4)-mediated oxidative stress induced by oxidized low-density lipoprotein(ox-LDL)in human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were cultured in vitro and divided into six groups: the control group(normal medium),the ox-LDL group(treated with 75 mg/L ox-LDL),the ox-LDL+Ang-(1-7)group(1 ?mol/L Ang-(1-7)pretreated for 30 minutes,then intervened with 75 mg/L ox-LDL),the ox-LDL+Ang-(1-7)+A-779 group(1 ?mol/L A-779(Mas receptor)pretreated for 30 minutes,1 ?mol/L Ang-(1-7)pretreated for 30 minutes,then intervened with 75 mg/L ox-LDL),the ox-LDL+A-779 group(1 ?mol/L A-779 pretreated for 30 minutes,then intervened with 75 mg/L ox-LDL),the ox-LDL+ HTA125 group(10?g/L HTA125(TLR4-blocking antibody)pretreated for 30 minutes,then intervened with 75 mg/L ox-LDL).The corresponding index was detected after 24 hours after intervention.Apoptosis of cells were detected by Annexin V-FITC / PI double staining flow cytometry and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUN EL).The generation of reactive oxygen species(ROS),products in oxidative stress,were detected by DCFH-DA staining.The mRNA and protein expression levels of NADPH oxidase 4(NOX4)and TLR4 were detected by real-time reverse transcription-polymerase chain reaction(RT-qPCR)and Western Blot analysis respectively.Results:(1)The results of Annexin V-FITC / PI double staining flow cytometry showed that the proportion of apoptotic cells was higher in ox-LDL group than in control group((21.18±1.40)% vs.(1.59±0.26)%,P<0.01),lower in ox-LDL+Ang-(1-7)group((7.42±1.07)%)and ox-LDL+HTA125 group((9.19±1.01)%)than in ox-LDL group(both P<0.01),higher in ox-LDL+Ang-(1-7)+A-779 group((19.91±1.30)%)and ox-LDL+A-779 group((20.47±0.95)%)than in ox-LDL+Ang-(1-7)group(both P<0.01).(2)The TUNEL results showed that the proportion of apoptotic cells was higher in ox-LDL group than in control group((10.83±0.77)% vs.(2.83±0.82)%,P<0.01),lower in ox-LDL+Ang-(1-7)group((3.66±0.54)%)and ox-LDL+HTA125 group((4.97±0.60)%)than in ox-LDL group(both P<0.01),higher in ox-LDL+Ang-(1-7)+A-779 group((10.69±0.62)%)and ox-LDL+A-779 group((11.43±0.42)%)than in ox-LDL+Ang-(1-7)group(both P<0.01).(3)ROS level was higher in ox-LDL group than in control group(0.093±0.014 vs.0.053±0.011,P<0.01),lower in ox-LDL+Ang-(1-7)group(0.063±0.011)and ox-LDL+HTA125 group(0.070±0.010)than in ox-LDL group(P<0.01,P<0.05),higher in ox-LDL+Ang-(1-7)+A-779 group(0.088±0.003)and ox-LDL+A-779 group(0.095±0.005)than in ox-LDL+Ang-(1-7)group(both P<0.01).(4)The mRNA expression level of NOX4 was higher in ox-LDL group than in control group(11.74±0.65 vs.1.00±0.00,P<0.01),lower in ox-LDL+Ang-(1-7)group(2.85±0.75)and ox-LDL+HTA125 group(5.57±0.52)than in ox-LDL group(both P<0.01),higher in ox-LDL+Ang-(1-7)+A-779 group(10.51±0.54)and ox-LDL+A-779 group(11.04±1.01)than in ox-LDL+Ang-(1-7)group(both P<0.01),higher in ox-LDL group than in control group(27.60±1.86 vs.1.00±0.00,P<0.01),lower in ox-LDL+Ang-(1-7)group(8.00±1.03)and ox-LDL+HTA125 group(14.83±0.97)than in ox-LDL group(both P<0.01),higher in ox-LDL+Ang-(1-7)+A-779 group(24.81±2.19)and ox-LDL+A-779 group(26.64±0.65)than in ox-LDL+Ang-(1-7)group(both P<0.01).(5)The protein expression level of NOX4 was higher in ox-LDL group than in control group(0.61±0.09 vs.0.23±0.02,P<0.01),lower in ox-LDL+Ang-(1-7)group(0.27±0.03)and ox-LDL+HTA125 group(0.22±0.02)than in ox-LDL group(both P<0.01),higher in ox-LDL+Ang-(1-7)+A-779 group(0.58±0.06)and ox-LDL+A-779 group(0.61±0.03)than in ox-LDL+Ang-(1-7)group(both P<0.01).The protein expression level of TLR4 was higher in ox-LDL group than in control group(0.18±0.02 ? 0.08±0.01,P<0.01),lower in ox-LDL+Ang-(1-7)group(0.07±0.01)and ox-LDL+HTA125 group(0.09±0.01)than in ox-LDL group(both P<0.01),higher in ox-LDL+Ang-(1-7)+A-779 group(0.18±0.02)and ox-LDL+A-779 group(0.20±0.02)than in ox-LDL+Ang-(1-7)group(both P<0.01).Conclusion:TLR4 mediated the ox-LDL induced injury in HUVECs,and Ang-(1-7)could attenuate ox-LDL induced injury in HUVECs by modulating the specific Mas receptors.