[Effects and related mechanism of angiotensin-(1-7) on Toll-like receptor 4-mediated oxidative stress in human umbilical vein endothelial cells].
Objective:To explore the role and related mechanisms of angiotensin-(1-7)(Ang-(1-7)) on Toll-like receptor 4 (TLR4) mediated oxidized low-density lipoprotein(ox-LDL)-induced oxidative stress 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 μmol/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 μmol/L A-779 (Mas receptor) pretreated for 30 minutes, 1 μmol/L Ang-(1-7) pretreated for 30 minutes, then intervened with 75 mg/L ox-LDL), the ox-LDL+ A-779 group (1 μmol/L A-779 pretreated for 30 minutes, then intervened with 75 mg/L ox-LDL), the ox-LDL+ HTA125 group (10 μg/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 (TUNEL). 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-PCR) and Western blotting 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 (bothP<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 (bothP<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(bothP<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 (bothP<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,P<0.01)and ox-LDL+ HTA125 group(0.070±0.010,P<0.05)than in ox-LDL group, 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 (bothP<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(bothP<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 (bothP<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(bothP<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 (bothP<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(bothP<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 (bothP<0.01). The protein expression level of TLR4 was higher in ox-LDL group than in control group(0.18±0.02 vs. 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(bothP<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 (bothP<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.
Department of Cardiology, Second Hospitol of Shanxi Medical University, Taiyuan 030001, China.
W F
WF
Yan
J J
JJ
Xue
H Y
HY
Yang
B
B
Liang
Z M
ZM
Yang
D000803:Angiotensin I; D000804:Angiotensin II / Q000031:analogs & derivatives; D017209:Apoptosis; D002478:Cells, Cultured; D005452:Fluoresceins; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008077:Lipoproteins, LDL; D018384:Oxidative Stress; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D051197:Toll-Like Receptor 4
C092006:7-Ala-angiotensin (1-7); D005452:Fluoresceins; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C099252:oxidized low density lipoprotein; D000804:Angiotensin II; C029569:diacetyldichlorofluorescein; D000803:Angiotensin I; C118790:angiotensin I (1-7)
Angiotensins; Oxidative stress; Toll-like receptor 4