Cyclosporin-A reduced the cytotoxicity of propranolol in HUVECs via p38 MAPK signaling

Figures

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  Figure 1. CyA reduced propranolol-induced growth inhibition. (A) HUVECs were treated with DMSO (control) or propranolol (10–200 μg/mL) for 24 hours. Cell viability was measured by MTT assay, and results were expressed in the percentage of viable cells. (B) HUVECs were treated with propranolol or CyA (10 μg/mL) combined with propranolol (82.277 μg/mL) for 24 hours. Cell viability was measured by MTT assay. ∗P < .05 and ∗∗P < .01. DMSO = dimethyl sulfoxide, HUVECs = human umbilical vein endothelial cells.

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  Figure 2. CyA decreases propranolol-induced mitochondrial oxidative stress in HUVECs. (A) The level of ROS of HUVECs treated with propranolol (82.277 μg/mL) and CyA (10 μg/mL) for 24 hours was detected. (B) Cells were treated with DMSO (control), propranolol, and CyA (10 μM) for 24 hours. Apoptosis assay using flow cytometry after staining with annexin V-FITC/propidium iodide (PI). Representative scatter plots of PI (Y-axis) versus annexin V (X-axis). ∗∗P < .01 and ∗∗∗P < .001. HUVECs = human umbilical vein endothelial cells, ROS = reactive oxygen species.

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  Figure 3. Effect of CyA and propranolol combination on p38 MAPK pathway. (A) Cells were treated with DMSO (control), propranolol (82.277 μg/mL), and or CyA (10 μg/mL) for 24 hours. After treatment, cell extracts were prepared and subjected for Western blot analysis. Expression of p38, c-caspase3 was measured in HUVECs. GAPDH was used as an internal loading control. (B) Intensities of protein signals were normalized to GAPDH. ∗∗P < .01 and ∗∗∗P < .001. DMSO = dimethyl sulfoxide, HUVECs = human umbilical vein endothelial cells, MAPKs = mitogen-activated protein kinases.

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  Figure 4. CyA affects HUVECs to propranolol via activating the p38 signaling pathway. (A) HUVECs were pretreated with p38 inhibitor sb203580 (10 μM), then treated with 82.277 μg/mL propranolol and/or CyA (10 μg/mL) for 24 hours. The level of p-MKK3, p-MKK6, P38MAPK, NQO1, Bax, and cleaved caspase3 was detected by Western blot analysis (WB). Intensities of proteins signals were normalized to GAPDH. Results are representative of three different experiments. Values are mean ± SE. ∗∗P < .01 and ∗∗∗P < .001. HUVECs = human umbilical vein endothelial cells.

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  Figure 5. HUVECs were pretreated with p38 inhibitor SB203580 (10 μM), then treated with propranolol (82.277 μg/mL) and/or CyA (10 μg/mL) for 24 hours. MTT detected the inhibition rate of cell. Values are mean ± SE of three measurements. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. HUVECs = human umbilical vein endothelial cells.

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  Figure 6. CyA affects HUVECs apoptosis and ROS level to propranolol via activating the p38 signaling pathway. (A) HUVECs were pretreated with p38 inhibitor sb203580 (10 μM), then treated with propranolol (82.277 μg/mL) and/or CyA (10 μg/mL) for 24 hours. The level of ROS was detected. (B) HUVECs were pretreated with p38 inhibitor sb203580, then treated with propranolol (82.277 μg/mL) and/or CyA (10 μg/mL) for 24 hours. The apoptosis was detected using flow cytometry after staining with annexin V-FITC/propidium iodide. Representative scatter plots of PI (Y-axis) versus annexin V (X-axis) were shown. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. HUVECs = human umbilical vein endothelial cells.