Astragaloside IV Attenuates Angiotensin II-Induced Inflammatory Responses in Endothelial Cells: Involvement of Mitochondria

Figures

• 

  Figure 1. Effect of astragaloside IV (AS-IV) on the viability of human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with AS-IV (3, 10, or 30 µM) for the indicated times. Cell viability was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results are expressed as means ± SE. *p < 0.05 compared with the corresponding control group.

• 

  Figure 2. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced production of proinflammatory cytokines/chemokines including TNF-α (A), IL-6 (B), and MCP-1 (C) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h. The ELISA assay was used to measure the levels of proinflammatory cytokines/chemokines in the culture medium. Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared with HUVECs exposed to Ang II alone.

• 

  Figure 3. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced ICAM-1 (A) and VCAM-1 (B) expression in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h. The cell-based ELISA assay was used to determine the expression of ICAM-1 and VCAM-1 in HUVECs. Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared with HUVECs exposed to Ang II alone.

• 

  Figure 4. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced monocyte adhesion to endothelial cells (A and B) and the activation of NF-κB (C) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h or 30 min. Subsequently, HUVECs were coincubated with fluorescence-labeled human monocytes for 30 min or the NF-κB activity in HUVECs was assayed by the use of a nonradioactive ELISA-based assay kit. (A) Fluorescent images of monocyte adhesion to HUVECs were visualized with a fluorescence microscope at 485/535 nm. Magnification, × 200. HUVECs were incubated with (a) a vehicle (control), (b) Ang II alone, Ang II with AS-IV at the concentration of (c) 3, (d) 10, or (e) 30 µM before coincubation with fluorescence-labeled monocytes. (B) Fluorescence intensity of monocyte attachment to HUVECs was quantified using a spectrofluorometer. (C) The activities of NF-κB in HUVECs were assayed using a specific TransAM NF-κB p65 transcription factor assay kit. Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared…

• 

  Figure 5. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced changes in mitochondrial membrane potential (MMP) (A and B) and ATP levels (C) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h. Subsequently, HUVECs were subjected to JC-1 staining or the measurement of ATP levels in HUVECs using a commercial assay kit as described in Materials and Methods. (A) Fluorescent images of HUVECs stained with JC-1 were visualized with a fluorescence microscope. Magnification, × 200. HUVECs were incubated with (a) a vehicle (control), (b) Ang II alone, Ang II with AS-IV at the concentration of (c) 3, (d) 10, or (e) 30 µM before being stained with JC-1. (B) Fluorescence intensities of green and red signals were quantified for each image using ImageJ software. The relative MMP was calculated as the ratio of red (J-aggregate) to green (J-monomer). Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared with HUVECs exposed to Ang II alone.

• 

  Figure 6. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced production of malondialdehyde (MDA) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h. The levels of MDA in HUVECs were measured using a commercial assay kit as described in Materials and Methods. Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared with HUVECs exposed to Ang II alone.

• 

  Figure 7. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced production of intracellular reactive oxygen species (A and B) and mitochondrial superoxide (C and D) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h, and subsequently subjected to DCFH-DA or MitoSOX Red staining. (A and C) Fluorescent images of HUVECs stained with DCFH-DA or MitoSOX Red were visualized with a fluorescence microscope at 488/525 nm or 510/580 nm. Magnification, × 200. HUVECs were incubated with (a) a vehicle (control), (b) Ang II alone, Ang II with AS-IV at the concentration of (c) 3, (d) 10, or (e) 30 µM before being stained with DCFH-DA or MitoSOX Red. (B and D) Fluorescence intensity of HUVECs stained with DCFH-DA or MitoSOX Red was quantified for each image using ImageJ software. Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared with HUVECs exposed to Ang II alone.

• 

  Figure 8. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced decreases in the activities of total SOD (A), Mn-SOD (B), and CuZn-SOD (C) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h. The activities of total SOD, Mn-SOD, and CuZn-SOD in HUVECs were measured using a commercial assay kit as described in Materials and Methods. Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared with HUVECs exposed to Ang II alone.

• 

  Figure 9. Effect of astragaloside IV (AS-IV) on angiotensin II (Ang II)-induced decreases in the activities of complexes I (A) and III (B) in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were preincubated with or without AS-IV (3, 10, or 30 µM) 1 h before incubation with Ang II (1 µM) for 12 h. The activities of complexes I and III in HUVECs were measured using a commercial assay kit as described in Materials and Methods. Results are expressed as means ± SE. *p < 0.05 compared with the control group; #p < 0.05 and ##p < 0.01 compared with HUVECs exposed to Ang II alone.